Colon Cancer, Chemotherapy, & Antioxidants

Chemotherapy and AntioxidantsIn the previous three issues of Avenues, we reported on the use of antioxidants along with chemotherapy as they apply to prostate, breast, and lung cancer patients. In this issue, we turn our focus to colon cancer.

In most cases, colon cancer treatment involves chemotherapy. However, toxicity and tumor cell drug resistance are notable drawbacks to this treatment.

Although not commonly addressed in clinical consultation, scientific evidence suggests that combining certain chemotherapy treatments with specific antioxidants at defined dosages can improve drug effectiveness or may reduce side effect severity.

This issue is important because it has long been the opinion of most practicing oncologists that antioxidants should not be used concurrently with chemotherapy as it was believed that the combination might inhibit chemotherapy effectiveness. This reluctance stems, in part, from the fact that some chemotherapy drugs work by strongly promoting oxidation. This is especially the case for the class of chemotherapy drugs called anthracyclines (Adriamycin and epirubicin), the alkylating agents (chlorambucil, cyclophosphamide, thiotepa, and busulfan), and the platinum drugs (cisplatin and carboplatin). Antioxidants, by definition, inhibit oxidation, so it was believed that antioxidants would prevent these chemotherapy drugs from working properly.

The controversy around using antioxidants together with chemotherapy is not based on many studies that show an adverse effect of adding antioxidants to chemotherapy. Rather this controversy is based on several studies that show how depleting glutathione, which is a natural antioxidant in the body, can enhance the treatment effect of chemotherapy. (Meijer, Mulder et al. 1990; Mans, Schuurhuis et al. 1992; Doyle, Ross et al. 1995; Versantvoort, Broxterman et al. 1995; Zaman, Lankelma et al. 1995; Kurokawa, Nishio et al. 1997; Lee, Park et al. 2004) Although this has led many oncologists to believe that this means all antioxidants should not be combined with chemotherapy, there are numerous laboratory and human studies showing how combining chemotherapy with antioxidants can indeed be helpful.

In this systematic review, we searched for every paper on the combination of antioxidants and chemotherapy in colon cancer and found only one laboratory (and no human) studies demonstrating a harmful effect, which was the combination of the antioxidant N-acetyl cysteine with paclitaxel. (Alexandre, Nicco et al. 2006)

Chemotherapy drugs that cause high levels of “oxidative stress” are thought to rely in part on oxidative stress to kill cancer cells, but other effects of that oxidative stress may also be getting in the way of the effectiveness of the chemotherapy. This is because oxidative stress slows cell replication. However, chemotherapy relies on fast cancer cell replication to be effective because it is during the moment of cell replication that chemotherapy kills cancer cells. (Conklin 2000) One approach to addressing this problem is the addition of certain antioxidants at specific dosages to lessen oxidative stress, thus making the chemotherapy treatment more effective (Perumal, Shanthi et al. 2005).

The interaction between chemotherapy and antioxidants is more complex than simply promoting and inhibiting oxidative stress. There are several mechanisms by which chemotherapy drugs function and antioxidants also have a number of different effects on the body. Each antioxidant has a different interaction with chemotherapy and this effect can change based upon the dosage used. Because the chemotherapy-antioxidant debate often focuses almost exclusively on oxidative stress, in this paper we broaden that discussion to include the many mechanisms by which antioxidants help in the fight against colon cancer.

The question is really not whether antioxidants should be used in combination with chemotherapy but rather which should be used and at what dosages.

PURPOSE OF THIS PAPER
In this evidence-based review article, we discuss the results of current research showing how antioxidants may enhance or, in some cases, inhibit the therapeutic action of specific chemotherapy drugs used in the treatment of colon cancer. Some of these antioxidants may also reduce chemotherapy side effects or inhibit chemotherapy resistance in colon cancer cells. Finally, some of these antioxidants have been found to be useful for restoring the natural antioxidants in the body, which are often depleted after the completion of chemotherapy.

HOW NUTRIENT DEPLETION FROM CHEMOTHERAPY CAN OCCUR
Chemotherapy can cause nutrient depletion from two major side effects. One is nausea and vomiting, making it more difficult for the patient to maintain adequate nutrient intake. Another is toxicity to cells in the gastrointestinal tract, making it more difficult for the intestines to adequately absorb nutrients. Antioxidants are not the only nutrients to become depleted; vitamins, minerals, amino acids, and fatty acids may also be compromised, especially in patients who have suffered these side effects for a prolonged amount of time.

METHODS
We searched for clinical or laboratory data published in peer-reviewed medical journals, conducted by cancer researchers in universities and medical research facilities around the world. Some of these studies are still in early stages and include only laboratory or animal data while others have advanced to include human volunteers.

We organized these data into the major categories of specific chemotherapy drugs. Within each section for a specific drug are found the research on combinations of that drug with various antioxidants, grouped by the name of the antioxidant in alphabetical order. We also point out specifically which studies were conducted in a laboratory (i.e. using cancer cell cultures), which were conducted using animals, and which were conducted with human volunteers. As each antioxidant appears in the paper for the first time, we provide some introduction to the antioxidant including what food sources naturally contain it, other common applications in clinical use, and typical dosages. The dosages given are not necessarily appropriate for all patients and should be individualized with practitioner guidance.

MECHANISMS OF ANTIOXIDANTS IN CANCER THERAPY
There are various mechanisms by which antioxidants play a roll in cancer therapy.

AMP-activated protein kinase (AMPK)
AMPK is an enzyme that helps inhibit the growth of some cancers (including colon cancer) when it is activated. AMPK can be activated by exercise and fasting, and by genes that suppress tumors. (Shaw, Kosmatka et al. 2004; Luo, Saha et al. 2005)

Antioxidants that are known to activate AMPK and suppress growth of colon cancer include genistein (Hwang, Ha et al. 2005), green tea or the compound found in green tea called EGCG, (Hwang, Ha et al. 2007) selenium, (Hwang, Kim et al. 2006) and capsaicin, which is found in chili peppers. (Kim, Hwang et al. 2007)

BCL-2 Family
The proteins in the BCL-2 family can determine whether a cell lives or dies. The following are proteins in this family:
1) BCL-2 and BCL-XL are proteins that promote cell survival.
2) BH3-only proteins can sense stress in a cell and can send a signal for the cell to die.
3) Bax and Bak are proteins that, when activated, lead to cell death. In the development of colon cancer, there are frequently mutations in the genes encoding for Bax. (Rupnarain, Dlamini et al. 2004; Woerner, Kloor et al. 2005; Fernandez-Luna 2007; Adams and Cory 2007)

The antioxidant genistein activates Bax. (Hwang, Ha et al. 2005) While the antioxidants N-acetyl cysteine and vitamin E and the chemotherapy agent 5-fluorouracil do not activate Bax when used alone, when either antioxidant is combined with 5-fluorouracil, Bax expression doubles, meaning that the combination is superior to chemotherapy used alone. (Adeyemo, Imtiaz et al. 2001)

Caspase-3
Caspase-3 is an enzyme that promotes cell death. (Porter and Janicke 1999) The above-mentioned activation of Bax and Bak (from the BCL-2 family) precede activation of caspases such as caspase-3. (Fernandez-Luna 2007)

Increase in caspase-3 activation in colon cancer cells may be caused by antioxidants such as curcumin, (Jaiswal, Marlow et al. 2002) diallyl sulfide (from garlic), (Sriram, Kalayarasan et al. 2008) and the synthetic antioxidant edavarone. (Kokura, Yoshida et al. 2005) Increasing caspase-3 activation means that this enzyme can help control cancer cell growth.

Cyclooxygenase-2 (COX-2)
COX-2 is an enzyme that is involved in the development of colon cancer. COX-2 is also involved in resistance to cell death. COX-2 is correlated with angiogenesis and tumor growth and metastasis. Note that COX-2 is also correlated with normal angiogenesis, such as wound healing. For colon cancer patients, it is beneficial to inhibit COX-2. For example, long-term use of COX-2 inhibitors, which are non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, decreases the risk of colorectal cancer and can regress colorectal polyps in patients with familial adenomatous polyposis. (Bakhle 2001; Sinicrope 2006)

Examples of antioxidants that inhibit COX-2 in colon cancer include curcumin, (Du, Jiang et al. 2006) genistein, (Hwang, Ha et al. 2005) n-3 PUFA from fish oils, and EGCG from green tea. (Hwang, Ha et al. 2007)

c-Myc
c-Myc is a protein that plays an essential role in the regulation of healthy cell growth. Misregulation of this protein occurs in many cancers including colon cancer. (Wierstra and Alves 2008; Robson, Pelengaris et al. 2006)

Curcumin can suppress colon cancer by regulating c-Myc activity. (Jaiswal, Marlow et al. 2002) DHA from fish oil together with 5-fluorouracil can cause colon cancer cell death, which involves c-Myc protein regulation. (Calviello, Di Nicuolo et al. 2005)

Growth Factor Receptors
Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER-2), and insulin-like growth factor 1 receptor (IGF-1R) are all receptors that are involved in normal cell division and growth. However, when genetic mutations lead to overexpression or overactivity, they are associated with uncontrolled cell division and cancer. (Sibilia, Kroismayr et al. 2007) New cancer therapies are being developed that target these receptors. (Press and Lenz 2007) Cetuximab (Erbitux) is one of these new therapies used in the treatment of metastatic colorectal cancer and this drug works by inhibiting EGFR. The antioxidant curcumin decreases expression and activation of EGFR, HER-2, and 1GF-1R. (Patel, Sengupta et al. 2008)

Glucose Transporter Protein 1 (Glut-1)
Glut-1 is a transporter protein that transports glucose into tissues. It is present in normal cells, but is overly expressed in tumors because tumors require more glucose to meet their increased metabolic needs. In colorectal cancers, expression of Glut-1 has been associated with decreased survival time and more advanced tumors. (Fogt, Wellmann et al. 2001)

The antioxidants EGCG from green tea (Hwang, Ha et al. 2007) and genistein from soy (Hwang, Ha et al. 2005) both decreased Glut-1 in colon cancer cells.

Glutathione S-transferase pi (GSTpi)
GSTpi is part of a family of glutathione S-transferase. Low glutathione S-transferase has been correlated with colon cancer risk. Glutathione S-transferase levels can be increased by eating fruits and vegetables (Grubben, Nagengast et al. 2001) and curcumin. (Chauhan 2002)

Conversely, GSTpi has also been associated with doxorubicin chemotherapy resistance in colon cancer cells. (Takahashi and Niitsu 1994) Lower levels of GSTpi allow doxorubicin to more effectively kill colon cancer cells. Agaricus bisporus lectin, found in the ordinary white button mushroom, is an inhibitor of GSTpi and can increase the effectiveness of several chemotherapy agents in colon cancer. (Goto, Kamada et al. 2002)

Nuclear factor kappa B (NF-kappaB)
Activation of NF-kappaB, a protein complex, is not favorable in cancer treatment. It leads to cellular events that promote inflammation, cell proliferation, angiogenesis, metastasis, and discourages cell death. NF-kappaB is associated with cancer risk, poor prognosis, and contributes to chemotherapy resistance. (Lee, Jeon et al. 2007; Sethi, Sung et al. 2008)

NF-kappaB is activated by many chemotherapeutic compounds and is also inhibited by antioxidants including curcumin, (Hatcher, Planalp et al. 2008) the synthetic antioxidant edavarone, (Kokura, Yoshida et al. 2005) and saponins extracted from ginseng. (Choo, Sakurai et al. 2008) Although diallyl sulfide extracted from garlic increases expression of NF-kappaB, it also showed strong inhibition of cancer cell growth. (Sriram, Kalayarasan et al. 2008)

p53 and p21
p53 is a protein our cells can produce to help suppress the growth of tumors, including colon cancer, and often becomes activated when the DNA has mutations. However, viruses or even other genes can inactivate p53. The gene encoding for p53 may be mutated, particularly in families with a high incidence of cancer, leaving cells more vulnerable to unregulated growth.

p21 is a related protein that is activated by p53 and is also involved in suppressing tumor growth. (Maddika, Ande et al. 2007)

Activation of p53 and p21 in colon cancer has been demonstrated with genistein from soy (Hwang, Ha et al. 2005) and vitamin E. (Chinery, Brockman et al. 1997)

Prostaglandins
Prostaglandins, a group of hormone-like chemicals, promote colon cancer cell growth and are generated by Cox-2 (see Cox-2 on previous page). (Pai, Nakamura et al. 2003)

EGCG from green tea, (Hwang, Ha et al. 2007) genistein from soy (Hwang, Ha et al. 2005) and selenium (Hwang, Kim et al. 2006) can all decrease prostaglandins.

Vascular Endothelial Growth Factor
Vascular Endothelial Growth Factor (VEGF) is an important protein involved in developing a new blood supply such as vasculogenesis (formation of new blood vessels when there are no pre-existing ones) and angiogenesis (formation of new blood vessels from pre-existing ones). In the case of cancer, most tumors require a more extensive blood supply to bring nutrition to support rapid growth. (Veeravagu, Hsu et al. 2007)

EGCG from green tea decreases VEGF. (Hwang, Ha et al. 2007)

5-Fluorouracil

ALPHA-MANGOSTIN
Alpha-Mangostin is a compound found in the pericarp (the tissue surrounding the seed) of the mangosteen fruit. Mangosteen is a tropical evergreen tree that originates from the Malay Archipelago between mainland Southeastern Asia and Australia. Alpha-Mangostin is a powerful antioxidant and has antibiotic, antiviral, and anti-inflammatory activity.

» Mangosteen: Typical doses of mangosteen extract (in capsule or juice form) range from 400 mg to 60,000 mg per day.

In a laboratory study, when alpha-mangostin was combined with 5-fluorouracil (at a concentration of 2.5 µM each), the growth suppression of human colon cancer cells was significantly stronger than 5-fluorouracil treatment alone (at a higher dose of 5 µM). (Nakagawa, Iinuma et al. 2007)

AVEMAR
Avemar is a fermented wheat germ extract developed in Hungary. It supports healthy immune function, inhibits cell proliferation and cell adhesion, enhances apoptosis, and has antioxidant activity.

» Avemar: For an adult of average weight (approximately 155 lbs), a typical daily dose is 9 g, ideally one hour before eating. Patients with a body weight of 200 lbs or more should consume two 9 g doses per day, ideally one hour before breakfast and dinner.

In an animal study using mice with colon cancer, avemar significantly enhanced the anti-metastatic effect of 5-fluorouracil and dacarbazine. Combined treatment decreased toxicity such as weight loss compared to chemotherapy agents administered alone. (Hidvegi, Raso et al. 1999)

CURCUMIN
Curcumin is a polyphenol and is an extract of the Indian curry spice plant turmeric. Curcumin is known for its anti-tumor, antioxidant, anti-amyloid, and anti-inflammatory properties. It also promotes healthy bile excretion and healthy platelet function.

» Curcumin: The best supplements contain curcumin at 75% or higher concentration. Typical doses range from 500 mg to 2,000 mg daily. Take with meals, as curcumin can cause stomach upset when taken on an empty stomach. Bioavailability and potency are increased when combined with bioperine, an extract from black pepper.

In a laboratory study, curcumin was used in combination with 5-fluorouracil in the treatment of human colon cancer cells. This combination proved synergistic. Additionally, COX-2 protein expression was reduced almost six-fold in the combined treatment. (Du, Jiang et al. 2006)

A second laboratory study confirmed the same finding, that curcumin in combination with 5-fluorouracil markedly inhibited the growth of colon cancer cells in comparison to 5-fluorouracil alone. (Kim, Park et al. 2005)

D-GLUCARATE
D-glucarate is a botanical extract from grapefruit, apples, oranges, broccoli, and brussel sprouts. It supports the internal detoxification system called glucuronidation, which makes foreign substances easier to remove from the body. For dietary supplementation, it is combined with calcium to form calcium d-glucarate.

» Calcium D-glucarate: Typical doses range from 50 to 1,000 mg per day.

In a laboratory study with colon tumors from rats, 5-fluorouracil was combined with D-glucarate, which enhanced its effectiveness. (Schmittgen, Koolemans-Beynen et al. 1992)

DIALLYL DISULFIDE (DADS)
Diallyl disulfide (DADS) is a major organosulfur compound found in garlic (Allium sativum) oil.

» Garlic: Typical doses range from 5 to 15 grams per day of whole, fresh garlic and 4,000 to 8,000 micrograms per day of allicin extract.

In an animal study, DADS did not change the effectiveness of 5-fluorouracil treatment in mice with colon tumors. However, the combined treatment significantly reduced chemotherapy-induced side effects such as depressed leukocyte counts, decreased spleen weight, and elevated plasma urea. (Sundaram and Milner 1996)

DOCOSAHEXAENOIC ACID (DHA)
Omega-3 fatty acids come from several different sources and in several different forms. Sources include blue-green algae, fish oil, and eggs. The most active in cancer care are docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). It is used as a supplement in clinical practice for many therapeutic uses, some of which include cancer, heart disease, depression, diabetes, and multiple sclerosis.

» Omega 3 Polyunsaturated fatty acids (PUFA, from fish oil including DHA/EPA): Typical dosage range is from 1,000 mg to 10,000 mg daily. When finding a fish oil supplement, it is important to identify brands that can provide assurance that they’ve tested for heavy metals such as mercury.

DHA enhances colon cancer cell death of four different cell lines together with 5-fluorouracil according to a laboratory study. Concentrations of 5-fluorouracil and DHA used in the study were lower than concentrations typically achieved in patients who are treated with these two agents. 5-fluorouracil inhibited BCL-2 and BCL-XL (types of proteins that enhance tumor cell growth) and induced overexpression of c-Myc. DHA markedly increased these effects, working synergistically with 5-fluorouracil. (Calviello, Di Nicuolo et al. 2005)

EICOSAPENTAENOIC ACID (EPA)
In an animal study using mice, EPA increased the tumor growth inhibition by 5-fluorouracil. It also prevented weight loss normally induced by 5-fluorouracil. (Wynter, Russell et al. 2004)

EPIGALLOCATECHIN-3-GALLATE (EGCG) & GREEN TEA EXTRACT
Epigallocatechin-3-gallate (EGCG) is the principal polyphenol found in green tea.

» EGCG: One cup of green tea contains between 10 and 400 mg of polyphenols depending on the source, amount of leaves used, and steeping time. EGCG may be conveniently obtained from extracts. A good product contains 725 mg, standardized to 98% polyphenols, 45% of which is EGCG.

In a laboratory study, EGCG in combination with 5-fluorouracil or etoposide, reduced colon cancer cell growth more effectively than either 5-fluorouracil or etoposide alone. This study found that EGCG strongly activated AMPK (an enzyme that helps inhibit the growth of some cancers), inhibited COX-2, decreased VEGF (vascular endothelial growth factor), and the glucose transporter Glut-1. (Hwang, Ha et al. 2007)

In a case report series, seven patients with familial polyposis underwent surgery and then received follow up preventive treatment with 5-fluorouracil suppositories and green tea extract. No rectal cancer developed in any of these patients. (Ichikawa, Takahashi et al. 1998)

FISH OIL (DHA/EPA)
In a laboratory study using colon cancer cells, the combined treatment of 5-fluorouracil with fish oil-based lipid emulsion led to significantly more growth inhibition when compared to either treatment alone. (Jordan and Stein 2003)

» Fish Oil: Typical dosages of fish oil range from 1,000 mg to 10,000 mg daily.

GENISTEIN
Genistein is an isoflavone found in legumes, especially soybeans. Isoflavones are antioxidants that counteract the damaging effects of free radicals in body tissues. Isoflavones, such as genistein, also have anti-angiogenic effects, blocking the formation of new blood vessels needed to support the growth of tumors.

» Genistein: A good product will use organic non-GMO genistein. To achieve anti-tumor effects, the target daily dose, based on animal studies and calculations for similar human dosage, is 1500 mg. The recommended dose for furtherresearch is between 100 mg and 1100 mg. (Boik 2001) One cup of soy milk will contain on average about 45 mg of genistein and the other related isoflavones.

When genistein was used together with 5-fluorouracil in the treatment of colon cancer cells in a laboratory study, cell death of colon cancer cells was significantly increased when compared to 5-fluorouracil alone. Investigating the mechanism of this synergistic combination revealed increased activation of AMPK, up-regulation of p53, p21, and Bax, as well as abrogation of the up-regulated state of COX-2 and Glut-1 normally induced by 5-fluorouracil, all of which are ideal for maximum treatment effect. (Hwang, Ha et al. 2005)

GLUTAMINE
Glutamine is a nonessential amino acid. It is necessary for rapidly dividing cells including the intestines and immune system.

» Glutamine: Typical doses range from 500 to 1000 mg per day.

A double blind, randomized controlled trial was conduced to assess whether oral glutamine could prevent intestinal complications of treatment with 5-fluorouracil and folinic acid. Seventy colorectal patients who had not received any chemotherapy prior to the study were enrolled and received either chemotherapy with placebo or chemotherapy with 18 g per day of oral glutamine. Glutamine treatment started five days prior to the beginning of chemotherapy and continued for fifteen days after completion of chemotherapy. The study only covered one chemotherapy cycle, which was given daily for five days.

The reduction of intestinal absorption was more marked in the placebo group and reduction of intestinal permeability was also higher in the placebo group. Average diarrhea was less in the glutamine group and also did not last as long. In the placebo group, one patient developed grade-4 diarrhea (defined as physiological consequences requiring intensive care or haemodynamic collapse) while no patients in the glutamine group developed grade-4 diarrhea. Patients were instructed to take loperamide tablets upon experiencing diarrhea. Patients in the glutamine group consumed less loperamide tablets than the placebo group. This trial thus demonstrated that glutamine protected intestinal mucosa from chemotherapy induced damage. (Daniele, Perrone et al. 2001)

GLUTATHIONE
Glutathione is one of the most powerful and important natural antioxidants produced in the body.

» Glutathione: Typical dosage ranges between 50 mg and 600 mg daily. N-acetyl cysteine is the pre-cursor of glutathione and is more efficiently absorbed. When taking Glutathione or N-acetyl cysteine, combine with three times as much vitamin C. Vitamin C prevents these amino acids from being oxidized in the body and ensures their ability to act as antioxidants.

In a clinical trial with 33 gastric cancer patients and 17 colon cancer patients, intravenous glutathione (1,200 mg per day) was tested in combination with 5-fluorouracil, FT-207 (a different configuration of fluorouracil), and tegafur suppositories. In gastric cancer patients who received glutathione, 5-fluorouracil concentrations in tumors were significantly higher than in patients who did not receive glutathione. However, this effect was not observed in colon cancer patients. (Kitajima, Ikeda et al. 1987)

A laboratory study found that cellular glutathione levels naturally became elevated in human colon cancer cells exposed to 5-fluorouracil for 24 hours. The study found that pretreatment of cancer cells with 5-fluorouracil enhanced the uptake of methotrexate and suggested that the increase in glutathione may have a role in the enhanced methotrexate uptake and cytotoxicity of both chemotherapy drugs. (Chen, Chen et al. 1995)

LENTINAN
Lentinan is a polysaccharide derived from the edible Japanese shiitake mushroom. It possesses immunostimulating antitumor properties.

» Shiitake mushroom extracts: Typical doses range from 100 to 400 mg per day.

In a randomized controlled trial testing lentinan in combination with 5-fluorouracil and mitomycin C or tegafur, a survival advantage was found in advanced or recurrent gastrointestinal cancer patients receiving lentinan. This effect was significant in gastric cancer patients. In colorectal cancer patients, there was also a survival advantage. (Taguchi, Furue, et al. 1985)

N-ACETYL CYSTEINE
N-acetyl cysteine is an efficiently absorbed and used form of the amino acid, L-cysteine. L-cysteine, L-glutamic acid, and glycine are the three amino acids that form glutathione, which is one of the most important and powerful antioxidants in the body.

» N-acetyl cysteine: Typical dosages range between 600 and 1,800 mg per day.

In a laboratory study, N-acetyl cystine augmented apoptosis in combination with 5-fluorouracil in colorectal cancer cell lines. This study found a possible mechanism with induced expression of the pro-apoptotic protein Bax in the treatment combining the two therapies. It was found that neither 5-fluorouracil nor N-acetyl cysteine alone induced an increase in Bax expression, however when combined, Bax expression doubled. (Adeyemo, Imtiaz et al. 2001)

NOTOGINSENG FLOWER EXTRACT
Also known as tianqi and sanqi, notoginseng is a very commonly used Chinese herb for regulating the blood, improving immune function, decreasing fatigue, and improving cognitive, sexual and physical performance.

» Notoginseng: Typical doses range between 250 mg and 1,500 mg daily.

In a laboratory study, notoginseng flower extract significantly increased the anti-proliferation effect of 5-fluorouracil in human colorectal cancer cells. Used as a single agent, 5-fluorouracil inhibited the growth of colon cancer cells by 31.1% and when combined with the notoginseng flower extract, this effect was increased to 59.4%. (Wang, Luo et al. 2007)

URIDINE
Uridine is a nucleoside that can be extracted from sugarcane.

» Uridine: A supplement called NucleomaxX contains 6 g of nucleosides (including uridine) from an extract of sugarcane. The recommended dose is 3 sachets per day, dissolved in water for three consecutive days, once every month.

An animal study used mice with colon cancer both sensitive and resistant to 5-fluorouracil that were treated with the chemotherapy agents 5-fluorouracil and leucovorin. Treatment with uridine (3,500 mg per kg) allowed the use of higher doses of 5-fluorouracil, which improved the antitumor effect on mice that had tumors resistant to 5-fluorouracil. (Nadal, van Groeningen et al. 1989)

VITAMIN E
Vitamin E includes several related compounds: Tocopherols and tocotrienols, each of which have four subtypes of alpha, beta, gamma, and delta. Previously, only alpha-tocopherol was considered important, however each type has unique contributions to health. The best dietary sources of vitamin E are considered to be unrefined, cold-pressed vegetable oils (such as wheat germ, sunflower seed, and olive oils) and raw or sprouted seeds, nuts, and grains.

» Vitamin E: Avoid synthetic vitamin E, such as alpha-tocopherol or succinate. Seek out the mixed tocopherols, including tocopherols and tocotrienols. Typical dosage ranges from 50 IU to 800 IU daily.

In a laboratory study, vitamin E augmented apoptosis in combination with 5-fluorouracil in colorectal cancer cell lines. This study found a possible mechanism with induced expression of the pro-apoptotic protein named Bax in the treatment combining the two therapies. It was found that neither 5-fluorouracil nor vitamin E alone induced an increase in Bax expression, however when combined, Bax expression doubled. (Adeyemo, Imtiaz et al. 2001)

In a laboratory and animal study, the combination of vitamin E and 5-fluorouracil significantly enhanced colorectal cancer tumor growth inhibition in vitro and in vivo. The mechanism by which vitamin E caused apoptosis was by induction of p21, a powerful inhibitor of the cell cycle. (Chinery, Brockman et al. 1997)

Cyclophosphamide

EICOSAPENTAENOIC ACID (EPA)
In an animal study using mice, EPA from fish oil increased the tumor growth inhibition by cyclophosphamide. (Wynter, Russell et al. 2004)

FOLFOX

CURCUMIN
In a laboratory study, curcumin significantly enhanced the inhibition of growth in two different colon cancer cell lines in combination with the FOLFOX chemotherapy regimen. FOLFOX chemotherapy includes leucovorin, 5-fluorouracil, and oxaliplatin. The combination treatment with curcumin decreased activation and expression of epidermal growth factor receptor (EGFR), HER-2 (also known as ErbB-2), and insulin-like growth factor-1 receptor (IGF-1R) and their downstream effectors Akt and cycloxygenase-2. (Patel, Sengupta et al. 2008)

GOSHAJINKIGAN (NIU CHE SHEN QI WAN)
Goshajinkigan is the Japanese name (Niu Che Shen Qi Wan is the Chinese name) for an herbal formula that is used in the treatment of diabetic neuropathy. In a case report from Japan, a 57-year-old woman with advanced stage IV colon cancer underwent surgery and after the operation was given the chemotherapy regimen called FOLFOX 6. The patient took two courses of chemotherapy and grade-2 neurotoxicity developed. The patient was then given the goshajinkigan herbal formula from the third chemotherapy course and the severity of neurotoxicity reduced to grade-1. (Mamiya, Kono et al. 2007)

Gemcitabine

DOCOSAHEXAENOIC ACID (DHA) & EICOSAPENTAENOIC ACID (EPA)
In an animal study using mice, EPA and DHA had no effect on the antitumor effect of gemcitabine. (Wynter, Russell et al. 2004)

Interleukin-2

MELATONIN
Melatonin is a hormone that is released from the pineal gland in the evening and promotes normal sleep; its secretion diminishes significantly with age. It is known to help maintain cell health and many people take it to improve sleep. It is also known to reduce metastasis in cancer patients. In most published studies, melatonin shows a beneficial effect, although it has been reported that in a small proportion of people, melatonin can paradoxically cause sleep disturbance. In others, there can be residual daytime drowsiness, which is usually resolved by using a lower dose.

» Melatonin: Typical dosages range from 1 mg to 20 mg. If aiming for a high dosage, one should start with 1 mg and increase the dosage slowly by 1 mg every 3 to 7 days. The ideal is to achieve peak blood levels of melatonin at about 2am. To do so, one can take the melatonin at bedtime, ideally between 9pm and 10pm.

A clinical trial included 50 metastatic colorectal cancer patients who either had progressed or did not respond to treatment with 5-fluorouracil and folates. Patients were randomized to receive either interleukin-2 (3 million IU per day subcutaneously for 6 days a week for 4 weeks) and melatonin (40 mg per day orally) or supportive care. A partial response was achieved in 3 out of 25 patients treated with interleukin-2 and melatonin whereas no tumor regression occurred in patients receiving only supportive care. Survival at one year was significantly higher in patients treated with interleukin-2 and melatonin (9 out of 25) in comparison to patients receiving only supportive care (3 out of 25). (Barni, Lissoni et al. 1995)

Another clinical trial enrolled 14 metastatic colorectal cancer patients pretreated with 5-fluorouracil. Patients were given low dose interleukin-2 (3 million IU per day for 6 days a week once per day subcutaneously for 4 weeks) with melatonin (50mg per day orally at 8pm). Thirteen patients were evaluable: no tumor regression was seen and stabilized disease was achieved in four patients for a median duration of five months. The other nine patients progressed. (Barni, Lissoni et al. 1992)

Irinotecan

AGARICUS BISPORUS LECTIN
Agaricus bisporus lectin is the active component found in the table mushroom or button mushroom, one of the most widely cultivated mushrooms in the world.

In a laboratory study, agaricus bisporus lectin increase the sensitivity of human colon cancer cells to the treatment of irinotecan, cisplatin, and doxorubicin. No effect was observed when combined with etoposide and 5-fluorouracil. The mechanism thought to underlie this synergism was that agaricus bisporus lectin is an inhibitor of the nuclear transport of glutathione S-transferase (GST) pi. GST pi was found to accumulate in cancer cells in response to chemotherapy and may contribute to drug resistance. (Goto, Kamada et al. 2002)

EDAVARONE
Edavarone is a synthetic antioxidant. We report its use here to demonstrate how pharmaceutical companies are investigating antioxidants (that can be patented) in combination with chemotherapy, while physicians continue warning about natural antioxidant use.

» Edavarone: Edavarone (trade name Radicut) is a free-radical scavenger manufactured in Japan and used primarily in the treatment of acute stroke.

In a laboratory study, edaravone enhanced the treatment effect of irinotecan in human colon cancer cells. The mechanism included inhibition of NF-kappaB and also enhanced activation of caspase-3. Irinotecan also activated caspase-3 and in contrast to edaravone activated NF-kB. Edaravone scavenged the reactive oxygen species induced by irinotecan and, according to conventional wisdom and the current stance on antioxidants in combination with chemotherapy, this should mean edavarone would decrease treatment effect of ironotecan. To the contrary, however, the combination is synergistic, meaning there was a beneficial enhancement of the therapeutic effect of irinotecan. The study continued in vivo, where irinotecan combined with edaravone reduced subcutaneous tumor growth and pulmonary metastases more than irinotecan treatment alone. (Kokura, Yoshida et al. 2005)

MELATONIN
A clinical trial investigated combined treatment of irinotecan and melatonin. Thirty metastatic colorectal cancer patients, who had progressed after at least one chemotherapy treatment including 5-fluorouracil, were randomized to treatment with irinotecan alone or irinotecan (125 mg per m2 weekly, administered intravenously) plus melatonin (20 mg per day taken orally, in the evening) for nine consecutive weeks of treatment. There was no complete response. Partial responses occurred in 2 out of 16 patients receiving ironotecan alone and in 5 out of 14 patients also receiving melatonin. Stable disease occurred in 5 out of 16 patients receiving ironotecan alone and in 7 out of 14 patients also receiving melatonin. Disease control in patients receiving ironotecan alone was 7 out of 16 and 12 out of 14 in patients also receiving melatonin. This is a significantly higher percentage of disease control for patients receiving a combination of ironotecan and melatonin. Diarrhea of grade 3-4 occurred in 6 out of 16 patients treated with irinotecan alone and in 4 out of 14 patients treated with irinotecan plus melatonin and required a 50% dose reduction. Disease control was similar in patients who received full dose of chemotherapy and those receiving a reduced dose. (Cerea, Vaghi et al. 2003)

SELENIUM
Selenium is an essential trace mineral in the body and is found in variable amounts in food depending on the soil content of selenium. Brazil nuts are the single best food source of selenium. One of its roles in the body is as an antioxidant and it is most widely known as a cancer preventive.

» Selenium (mineral): The US adult Tolerable Upper Intake Level (UL) is 400 micrograms a day and the Lowest Observed Adverse Effects Level (LOAEL) for adults is about 900 micrograms daily. There are several different forms of selenium. Se-Methylselenocysteine is a highly bioavailable form because it is not incorporated within a protein such as the form selenomethionine. We recommend getting selenium either in the organically bound forms such as of Se-Methylselenocysteine or a combination of selenium compounds with L-selenomethionine, sodium selenate, selenodiglutathione, and Se-methylselenocysteine.

An animal study using mice with colon cancer types that are sensitive and resistant to chemotherapy tested the selenium compounds 5-methylselenocysteine and seleno-L-methionine together with various chemotherapy agents including irinotecan. Selenium was highly protective against toxicity induced by various chemotherapy agents including 5-fuorouracil, cisplatin, oxaliplatin, paclitaxel, and doxorubicin. The effect of selenium on treatment effectiveness was measured only with irinotecan. In combination with the maximum tolerated dose of irinotecan, selenium (administered seven days prior to chemotherapy and continuing throughout chemotherapy treatment) significantly increased the cure rates of mice with colon cancer tumors that are sensitive and resistant to irinotecan. For example, in tumors resistant to irinotecan treatment with ironotecan alone lead to a cure rate of 0%. When combined with selenium, the cure rate rose to 20%. In tumors that were sensitive to ironotecan, whereas treatment with ironotecan alone showed a cure rate of 20%, irinotecan combined with selenium showed a cure rate of an astonishing 100%. Selenium combined with chemotherapy treatment was also effective in preventing weight loss. (Cao, Durrani et al. 2004)

Oxaliplatin, 5-Fluorouracil, and Leucovorin

CALCIUM GLUCONATE & MAGNESIUM SULFATE
Calcium gluconate is a mineral that is found naturally in foods such as almonds, sesame seeds, dark green leafy vegetables, milk, and yogurt. Calcium (Ca) is needed for many bodily functions, especially bone formation. Calcium can bind to other minerals and aid in removal from the body. Calcium gluconate is used to prevent and treat calcium deficiencies.

Magnesium (Mg) is a mineral that is present in many foods, including almonds, cashews, soybeans, buckwheat, and wheat bran. Magnesium helps maintain normal nerve and muscle function, keeps the heart rhythm steady, keeps bones strong, and supports the immune system. Magnesium sulfate is one form of magnesium supplements.

A retrospective cohort study from France included 161 colorectal cancer patients. 65 patients received chemotherapy alone and 96 patients received intravenous infusions of calcium gluconate and magnesium sulfate (1 g each) delivered over 15 minutes prior to chemotherapy. Chemotherapy treatment consisted of oxaliplatin, 5-fluorouracil, and leucovorin. The FOLFOX 4 regimen (oxaliplatin 85 mg per m2 every two weeks), FOLFOX 6 (oxaliplatin 100 mg per m2 every two weeks), and FUFOX (130 mg per m2 every three weeks) were used. Tumor response and toxicity were evaluated. Treatment efficacy was evaluated three months after the beginning of treatment. The number of patients receiving FUFOX regimen were similar, 42 receiving CaMg and 43 receiving chemotherapy alone. The other regimens had unequal numbers of patients either receiving CaMg or not, therefore this study only collected data on treatment efficacy in the FUFOX group.

Treatment efficacy: Both the number of treatment cycles and the mean cumulative oxaliplatin doses were higher in patients treated with CaMg compared to patients receiving chemotherapy alone, however the difference was not statistically significant. After 12 cycles of chemotherapy, the percentage of patients remaining in treatment was greater in patients receiving CaMg. This was seen most pronounced in patients receiving the FOLFOX 6 regimen where 36% of patients remained receiving CaMg and 11% remained receiving chemotherapy alone. The response rate was 45% in patients receiving CaMg and 35% in patients receiving chemotherapy alone.

No CaMg induced toxicity was observed. At the end of treatment, 20% of patients receiving CaMg had neuropathy compared to 45% of patients receiving chemotherapy alone. The percentage of patients who withdrew from treatment due to any toxicity was 33% in patients receiving CaMg and 51% in patients receiving chemotherapy alone. The percentage of patients who withdrew due to neurotoxicity was only 4% in patients receiving CaMg and 31% in patients receiving chemotherapy alone. The percentage of patients who withdrew due to grade 3 chronic neurotoxicity was 0% in patients receiving CaMg and 12% in patients receiving chemotherapy alone. With regard to chronic neuropathy at the end of treatment, 65% of patients receiving CaMg had no neuropathy and only 37% receiving chemotherapy alone did not have neuropathy. The duration of neuropathy equal or greater than 2 was longer in patients receiving chemotherapy alone. For example, patients on the FOLFOX 4 chemotherapy regimen receiving CaMg had neuropathy lasting two months, and patients receiving chemotherapy alone for 12 months. Chronic grade 3 neuropathy at the end of treatment was 8% in patients receiving CaMg and 20% in patients receiving chemotherapy alone. Grade 3 neuropathy is defined by the National Cancer Institute as “paresthesia interfering with function, severe objective sensory loss” and in the case specific to oxaliplatin as “paresthesia, dysesthesia causing functional impairment.”

Other chemotherapy-induced toxicities were also mitigated. Diarrhea greater or equal to grade 2 occurred in only 10% percent of chemotherapy cycles for patients receiving CaMg, and 27% for patients receiving chemotherapy alone. Asthenia greater or equal to grade 2 occurred in only 13% percent of chemotherapy cycles for patients receiving CaMg and 41% for patients receiving chemotherapy alone. Stable weight was 92% in patients receiving CaMg and only 75% in patients receiving chemotherapy alone. (Gamelin, Boisdron-Celle et al. 2004)

An encouraging case report emerged a few years later showing how a protocol using calcium gluconate and magnesium sulfate helped a woman continue chemotherapy. A 40-year-old woman had 4 out of 6 weeks of one cycle of the FLOX chemotherapy regimen (500 mg per m2 5-fluorouracil weekly, 500 mg per m2 folinic acid and 85 mg per m2 oxaliplatin). During the first infusion of oxaliplatin, she experienced severe nausea and vomiting as well as a headache and loss of vision in her right eye, which resolved in 24 hours. The administration of the second dose of oxaliplatin was extended to three hours, however she developed severe chest pain, headache and loss of vision, for which she received diphenhydramine and hydrocortisone and recovered in the emergency room with intravenous support.

Then the patient was referred to Yale New Haven Hospital (Yale University School of Medicine) where she received intravenous calcium gluconate 1 g and magnesium sulfate 1 g prior to and following the FOLFOX 6 chemotherapy regimen. Prior to chemotherapy she also received famotidine, diphenhydramine, ondansetron, and decadron. The patient did not develop any of the side effects that she experienced during the prior treatments with oxaliplatin. She successfully completed nine cycles of FOLFOX 6 using the same medications mentioned previously. (Wrzesinski, McGurk et al. 2007)

GLUTATHIONE
A randomized, double-blind, placebo-controlled clinical trial was conducted to assess glutathione in the prevention of neurotoxicity induced by oxaliplatin-based chemotherapy. Fifty-two patients with advanced colorectal cancer were enrolled: 26 patients received chemotherapy (oxaliplatin, 5-fluorouracil, and leucovorin) with normal saline solution (as a placebo) and 26 patients received the same chemotherapy with glutathione (1,500 mg per m2). Evaluations of neurotoxicity were performed at baseline and after 4, 8, and 12 cycles of treatment.

After the fourth chemotherapy cycle, 7 out of 26 patients in the GSH group had neuropathy (grade 1-2) and 11 out of 26 patients in the placebo group had neuropathy (grade 1-2). After the eighth cycle, 9 out of 21 evaluable patients in the GSH group had neuropathy (grade 1-2). The nineteen evaluable patients in the placebo group didn’t fare as well, with 10 patients having grade 1-2 neuropathy and 5 patients having grade 3-4 neuropathy. After twelve cycles, 8 out of 10 evaluable patients in the GSH group had grade 1-2 neuropathy and one patient had grade 3 neuropathy. In the placebo group, 2 out of 8 evaluable patients had grade 1-2 neuropathy and 6 patients had grade 3-4 neuropathy.

After eight cycles, 11 out of 19 patients in the placebo group and only 2 out of 21 patients in the GSH group had grade 2-4 neuropathy. After 12 cycles, 8 out of 8 patients in the placebo arm and only 3 out of 10 in the GSH arm had grade 2-4 neuropathy. Sural sensory nerve conduction was reduced significantly in the placebo group but not in the GSH group. The overall response rate to treatment was slightly higher in the GSH group at 26.9% and was 23.1% in the placebo arm, thus showing no reduction in the efficacy of chemotherapy treatment. This study provides evidence that GSH is promising as a therapy for the prevention of oxaliplatin-induced neuropathy, and that GSH does not interfere with the anti-tumor effect of this chemotherapy regimen. (Cascinu, Catalano et al. 2002; Arnould, Hennebelle et al. 2003)

N-ACETYL CYSTEINE
Fourteen stage III colon cancer patients were enrolled in a clinical trial to assess whether oral N-acetyl cysteine could reduce neurotoxicity induced by a chemotherapy regimen including oxaliplatin, 5-fluorouracil, and low dose leucovorin. Five patients (group-A) received a combination of chemotherapy and N-acetyl cysteine (1,200 mg). Nine patients (group-B) received chemotherapy alone. Evaluations were made at the onset of treatment and after 4, 8, and 12 treatment cycles. After four cycles of chemotherapy, 40% in group-A had grade 1 neuropathy compared to 77.8% in group-B. After eight cycles of chemotherapy, 60% in group-A had grade 1 neuropathy and none had a higher grade of neuropathy. In contrast, patients in group-B began to develop higher grades of neuropathy, with 44.4% still at grade 1, but 55.6% now progressed to grade 2 neuropathy. After twelve cycles of chemotherapy, 60% in group-A had grade 1 neuropathy and 20% had grade 2 neuropathy. In group-B, 11.1% had grade 1 neuropathy, 55.6% had grade 2 neuropathy, and 33.3% developed grade 3 neuropathy. Therefore patients receiving N-acetyl cysteine developed significantly less neuropathy. (Lin, Lee et al. 2006)

Tegafur

N-ACETYL CYSTEINE
N-acetyl cysteine did not significantly alter the activity of tegafur in colon cancer cells. However, N-acetyl cysteine did reduce the effectiveness of a very new drug that is a derivative of tegafur, called butyroyloxymethyl-tegafur derivative 3. (Engel, Nudelman et al. 2008)

POLYSACCHARIDE-K (PSK)
Polysaccharide-K (PSK) is extracted from a mushroom called turkey tail. Other names include Trametes versicolor and Coriolus versicolor (Latin), yun zhi (Chinese), and kawaratake (Japanese). It is commonly used to boost immune health and often used with cancer patients.

» PSK: Typical doses for cancer patients range between 2 to 6 g.

In an animal study, the chemotherapy combination of tegafur (Uracil) (UFT) plus leucovorin (LV) was weakly effective with colon cancer. The addition of PSK did not make the anti-tumor treatment any more or less effective. The same was true for Lewis lung cancer. However, in Meth A sarcoma, PSK did enhance this chemotherapy regimen. (Katoh and Ooshiro 2007)

Paclitaxel

COMBINATIONS TO AVOID: N-ACETYL CYSTEINE
In an animal study using mice with colon cancer, N-acetyl cysteine interfered with the anti-tumor activity of paclitaxel when used in combination. However N-acetyl cysteine did prevent toxicity to blood cells due to paclitaxel. Because this study reports that N-acetyl cysteine can reduce effectiveness of paclitaxel, we recommend not using this combination. (Alexandre, Nicco et al. 2006)

Pemetrexed

FOLIC ACID AND VITAMIN B12
Vitamin B12, also known as cobalamins, can be found in various dietary sources, including liver, meat, eggs, milk, and saltwater fish.

» Vitamin B12: Typical adult doses range from 1 to 3 micrograms per day.

Folic acid, also known as vitamin B9, is found in dietary sources such as dark leafy green vegetables, liver, egg yolk, various beans, and peas.

» Vitamin B9: Typical adult doses range from 300 micrograms to no more than 900 micrograms per day.

In the earlier stages of developing pemetrexed as a treatment for cancer (mostly used for malignant pleural mesothelioma and non-small cell lung cancer, but having activity against colon cancer as well), life threatening toxicities were encountered. It was soon discovered that supplementation with folic acid and vitamin B12 could minimize these toxicities when included in the treatment regimen. (Curtin and Hughes 2001; Scagliotti and Novello 2003)

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REFERENCES AND SELECT STUDY ABSTRACTS

Adams, J. M. and S. Cory (2007). “Bcl-2-regulated apoptosis: mechanism and therapeutic potential.” Curr Opin Immunol 19(5): 488-96.

Apoptosis is essential for tissue homeostasis, particularly in the hematopoietic compartment, where its impairment can elicit neoplastic or autoimmune diseases. Whether stressed cells live or die is largely determined by interplay between opposing members of the Bcl-2 protein family. Bcl-2 and its closest homologs promote cell survival, but two other factions promote apoptosis. The BH3-only proteins sense and relay stress signals, but commitment to apoptosis requires Bax or Bak. The BH3-only proteins appear to activate Bax and Bak indirectly, by engaging and neutralizing their pro-survival relatives, which otherwise constrain Bax and Bak from permeabilizing mitochondria. The Bcl-2 family may also regulate autophagy and mitochondrial fission/fusion. Its pro-survival members are attractive therapeutic targets in cancer and perhaps autoimmunity and viral infections.

Adeyemo, D., F. Imtiaz, et al. (2001). “Antioxidants enhance the susceptibility of colon carcinoma cells to 5-fluorouracil by augmenting the induction of the bax protein.” Cancer Lett 164(1): 77-84.

5 Fluorouracil (5 FU), the most effective systemic chemotherapeutic agent in the management of advanced colorectal carcinoma acts by inducing apoptosis. Response rates, approximately 20% is improved by folinic acid. This study investigates similar modulation of 5 FU-induced apoptosis by oxidant quenching. A five-fold reduction of intracellular oxidant levels by antioxidants N-acetylcysteine and vitamin E did not induce apoptosis, it however augmented pro-apoptotic bax protein expression, and apoptotic response to a non-toxic dose of 5 FU in the colorectal cancer cell lines colo 201 and colo 205. This suggests that reduction of intracellular levels of reactive oxygen species enhance susceptibility to 5 FU (apoptotic stimuli) by augmentation of bax expression.

Alexandre, J., C. Nicco, et al. (2006). “Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir.” J Natl Cancer Inst 98(4): 236-44.

BACKGROUND: Anticancer drugs act by increasing intracellular hydrogen peroxide levels. Mangafodipir, a superoxide dismutase (SOD) mimic with catalase and glutathione reductase activities, protects normal cells from apoptosis induced by H2O2. We investigated its and other oxidative stress modulators’ effects on anticancer drug activity in vitro and in vivo. METHODS: Cell lysis and intracellular reactive oxygen species levels were assessed in vitro in human leukocytes from healthy subjects and in murine CT26 colon cancer cells. Cells were exposed to the chemotherapeutic agents paclitaxel, oxaliplatin, or 5-fluorouracil, either in the presence or absence of mangafodipir and other oxidative stress modulators. Cell viability was evaluated by the methylthiazoletetrazolium assay. The effects of mangafodipir and other oxidative stress modulators on peripheral blood counts and on tumor growth were studied in BALB/c mice that were implanted with CT26 tumors and treated with 20 mg/kg paclitaxel. Survival of BALB/c mice infected with Staphylococcus aureus was also examined by treatment group. Statistical tests were two-sided. RESULTS: In vitro lysis of leukocytes exposed to paclitaxel, oxaliplatin, or 5-fluorouracil in combination with mangafodipir was decreased by 46% (95% confidence interval [CI] = 44% to 48%), 30.5% (95% CI = 29% to 32%), and 15% (95% CI = 10% to 20%), compared with lysis of cells treated with anticancer agent alone. Mangafodipir also statistically significantly enhanced in vitro anticancer drug cytotoxicity toward CT26 cancer cells. In vivo, mangafodipir protected mice against paclitaxel-induced leukopenia. Moreover, the survival rate of mice infected with S. aureus and treated with paclitaxel was higher when mangafodipir was also administered (survival: 3 of 17 versus 14 of 17, P < .001). In addition, mangafodipir amplified the inhibitory effect of paclitaxel on CT26 tumor growth in mice. CONCLUSIONS: Mangafodipir decreased hematotoxicity and enhanced cytotoxicity of anticancer agents.

Arnould, S., I. Hennebelle, et al. (2003). “Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines.” Eur J Cancer 39(1): 112-9.

Oxaliplatin (L-OHP) is a new platinum analogue that has shown antitumour activity against colon cancer both in vitro and in vivo and is now used in the chemotherapeutic treatment of metastatic colon and rectal cancer. L-OHP like cisplatin (CDDP), is detoxified by glutathione (GSH)-related enzymes and forms platinum (Pt)-DNA adducts lesions that are repaired by the nucleotide excision repair system (NER). We investigated the cytotoxicity and the pharmacology of L-OHP and CDDP on a panel of six colon cell lines in vitro. We showed that GSH and glutathione S-transferase (GST) activity were not correlated to oxaliplatin cytotoxicity. Pt-DNA adducts formation and repair were correlated with CDDP, but not with L-OHP cytotoxicity. The determination of ERCC1 and XPA expression, two enzymes of the NER pathway, by reverse transcriptase-polymerase chain reaction (RT-PCR), demonstrated that ERCC1 expression was predictive of L-OHP sensitivity (r(2)=0.67, P=0.02) and XPA level after oxaliplatin exposure was also correlated to L-OHP IC(50) (r(2)=0.5; P=0.04). The knowledge of such correlations could help predict the sensitivity of patients with colon cancer to L-OHP.

Bakhle, Y. S. (2001). “COX-2 and cancer: a new approach to an old problem.” Br J Pharmacol 134(6): 1137-50.

Barni, S., P. Lissoni, et al. (1995). “A randomized study of low-dose subcutaneous interleukin-2 plus melatonin versus supportive care alone in metastatic colorectal cancer patients progressing under 5-fluorouracil and folates.” Oncology 52(3): 243-5.

Chemotherapy with 5-fluorouracil (5-FU) and folates represents the first-line standard therapy for metastatic colorectal cancer, whereas at present there is no conventional second-time treatment. Because of its importance in generating an effective anticancer immune response, interleukin-2 (IL-2) could constitute a new promising therapy of advanced colon cancer. Generally, IL-2 may determine tumor regressions in colon cancer only when it is given at high toxic doses. Our preliminary studies have shown that the pineal hormone melatonin may amplify IL-2 activity, which becomes active also at low doses in several tumor histotypes. On the basis, we have performed a clinical trial to evaluate the impact of low-dose IL-2 plus melatonin on the survival time in metastatic colon cancer, which progressed in response to 5-FU plus folates. The study included 50 metastatic colorectal cancer patients, who did not respond or progressed after initial response to first-line chemotherapy with 5-FU and folates. Patients were randomized to receive supportive care alone or low-dose subcutaneous IL-2 (3 million IU/day for 6 days/week for 4 weeks) plus melatonin (40 mg/day orally). No spontaneous tumor regression occurred in patients receiving supportive care alone. A partial response was achieved in 3/25 patients treated with immunotherapy. Percent survival at 1 year was significantly higher in patients treated with immunotherapy than in those treated with supportive care alone (9/25 vs. 3/25, p < 0.05). This study suggests that low-dose subcutaneous IL-2 plus melatonin may be effective as a second-line therapy to induce tumor regression and to prolong percent survival at 1 year in metastatic colorectal cancer patients progressing under 5-FU and folates.

Barni, S., P. Lissoni, et al. (1992). “Neuroimmunotherapy with subcutaneous low-dose interleukin-2 and the pineal hormone melatonin as a second-line treatment in metastatic colorectal carcinoma.” Tumori 78(6): 383-7.

On the basis of our previous preliminary data which showed that the pineal hormone melatonin (MLT) may potentiate IL-2 activity and reduce the dose of IL-2 required to determine an effective host antitumor response, we performed a clinical study with low-dose IL-2 given once/day subcutaneously (3 million IU/day for 6 days/week for 4 weeks) in association with MLT (50 mg/day orally at 8.00 p.m. every day) as a second-line therapy in metastatic colorectal cancer patients pretreated with 5-fluorouracil. Evaluable patients were 13/14, and most of them showed disseminated liver metastases. No objective tumor regression was seen. A stabilization of disease was achieved in 4/13 patients (median duration 5+ months), and the other 9 patients progressed. Mean number of lymphocytes and eosinophils significantly increased during the treatment. Moreover, the mean increase in lymphocyte number was significantly higher in patients with stable disease than in those with progressive disease, whereas there was no difference as regards eosinophils. Serum levels of neopterin and tumor necrosis factor (TNF) significantly increased during therapy, and TNF increase was correlated to the side effects rather than to the control of cancer development. This study shows that neuroimmunotherapy with low-dose interleukin-2 and MLT, even though capable of determining an evident expansion of immune cells involved in host antitumor response, does not seem to be effective in terms of tumor regression in metastatic colon cancer patients pretreated with 5-fluorouracil.

Calviello, G., F. Di Nicuolo, et al. (2005). “Docosahexaenoic acid enhances the susceptibility of human colorectal cancer cells to 5-fluorouracil.” Cancer Chemother Pharmacol 55(1): 12-20.

PURPOSE: Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells. METHODS: When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 microM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (< or =10 microM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo. RESULTS: Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU. CONCLUSION: Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.

Cao, S., F. A. Durrani, et al. (2004). “Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human tumor xenografts.” Clin Cancer Res 10(7): 2561-9.

PURPOSE: Studies were carried out in athymic nude mice bearing human squamous cell carcinoma of the head and neck (FaDu and A253) and colon carcinoma (HCT-8 and HT-29) xenografts to evaluate the potential role of selenium-containing compounds as selective modulators of the toxicity and antitumor activity of selected anticancer drugs with particular emphasis on irinotecan, a topoisomerase I poison. EXPERIMENTAL DESIGN: Antitumor activity and toxicity were evaluated using nontoxic doses (0.2 mg/mouse/day) and schedule (14-28 days) of the selenium-containing compounds, 5-methylselenocysteine and seleno-L-methionine, administered orally to nude mice daily for 7 days before i.v. administration of anticancer drugs, with continued selenium treatment for 7-21 days, depending on anticancer drugs under evaluation. Several doses of anticancer drugs were used, including the maximum tolerated dose (MTD) and toxic doses. Although many chemotherapeutic agents were evaluated for toxicity protection by selenium, data on antitumor activity were primarily obtained using the MTD, 2 x MTD, and 3 x MTD of weekly x4 schedule of irinotecan. RESULTS: Selenium was highly protective against toxicity induced by a variety of chemotherapeutic agents. Furthermore, selenium increased significantly the cure rate of xenografts bearing human tumors that are sensitive (HCT-8 and FaDu) and resistant (HT-29 and A253) to irinotecan. The high cure rate (100%) was achieved in nude mice bearing HCT-8 and FaDu xenografts treated with the MTD of irinotecan (100 mg/kg/week x 4) when combined with selenium. Administration of higher doses of irinotecan (200 and 300 mg/kg/week x 4) was required to achieve high cure rate for HT-29 and A253 xenografts. Administration of these higher doses was possible due to selective protection of normal tissues by selenium. Thus, the use of selenium as selective modulator of the therapeutic efficacy of anticancer drugs is new and novel. CONCLUSIONS: We demonstrated that selenium is a highly effective modulator of the therapeutic efficacy and selectivity of anticancer drugs in nude mice bearing human tumor xenografts of colon carcinoma and squamous cell carcinoma of the head and neck. The observed in vivo synergic interaction is highly dependent on the schedule of selenium.

Cascinu, S., V. Catalano, et al. (2002). “Neuroprotective effect of reduced glutathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial.” J Clin Oncol 20(16): 3478-83.

PURPOSE: We performed a randomized, double-blind, placebo-controlled trial to assess the efficacy of glutathione (GSH) in the prevention of oxaliplatin-induced neurotoxicity. PATIENTS AND METHODS: Fifty-two patients treated with a bimonthly oxaliplatin-based regimen were randomized to receive GSH (1,500 mg/m(2) over a 15-minute infusion period before oxaliplatin) or normal saline solution. Clinical neurologic evaluation and electrophysiologic investigations were performed at baseline and after four (oxaliplatin dose, 400 mg/m(2)), eight (oxaliplatin dose, 800 mg/m(2)), and 12 (oxaliplatin dose, 1,200 mg/m(2)) cycles of treatment. RESULTS: At the fourth cycle, seven patients showed clinically evident neuropathy in the GSH arm, whereas 11 patients in the placebo arm did. After the eighth cycle, nine of 21 assessable patients in the GSH arm suffered from neurotoxicity compared with 15 of 19 in the placebo arm. With regard to grade 2 to 4 National Cancer Institute common toxicity criteria, 11 patients experienced neuropathy in the placebo arm compared with only two patients in the GSH arm (P =.003). After 12 cycles, grade 2 to 4 neurotoxicity was observed in three patients in the GSH arm and in eight patients in the placebo arm (P =.004). The neurophysiologic investigations (sural sensory nerve conduction) showed a statistically significant reduction of the values in the placebo arm but not in the GSH arm. The response rate was 26.9% in the GSH arm and 23.1% in the placebo arm, showing no reduction in activity of oxaliplatin. CONCLUSION: This study provides evidence that GSH is a promising drug for the prevention of oxaliplatin-induced neuropathy, and that it does not reduce the clinical activity of oxaliplatin.

Cerea, G., M. Vaghi, et al. (2003). “Biomodulation of cancer chemotherapy for metastatic colorectal cancer: a randomized study of weekly low-dose irinotecan alone versus irinotecan plus the oncostatic pineal hormone melatonin in metastatic colorectal cancer patients progressing on 5-fluorouracil-containing combinations.” Anticancer Res 23(2C): 1951-4.

Recent advances in immunobiological knowledge have suggested the possibility of enhancing the therapeutic activity of various chemotherapeutic agents by a concomitant administration of anti-oxidant drugs and/or immunomodulating neurohormones. In particular, the pineal neurohormone melatonin (MLT), which is able to exert both antioxidant and immunomodulating effects, has been proven to enhance the efficacy of various chemotherapeutic drugs, namely cisplatin, anthracyclines and 5-fluorouracil, whereas at present there are no data about its possible influence on cytotoxic drugs effective in the treatment of colon cancer other than 5-fluorouracil, such as irinotecan (CPT-11). The present study was performed to evaluate the influence of a concomitant administration of MLT on CPT-11 therapeutic activity in metastatic colorectal cancer. The study included 30 metastatic colorectal cancer patients progressing after at least one previous chemotherapeutic line containing 5-fluorouracil, who were randomized to be treated with CPT-11 alone or CPT-11 plus MLT. According to a weekly low-dose schedule, CPT-11 was given i.v. at 125 mg/m2/week for 9 consecutive weeks. MLT was administered orally at 20 mg/day during the dark period of the day. No complete response was observed. A partial response (PR) was achieved in 2 out of 16 patients treated with CPT-11 alone and in 5 out of 14 patients concomitantly treated with MLT. Moreover, a stable disease (SD) was obtained in 5 out of 16 patients treated with CPT-11 alone and in 7 out of 14 patients treated with CPT-11 plus MLT. Therefore, the percent of disease-control achieved in patients concomitantly treated with MLT was significantly higher than that observed in those treated with chemotherapy alone (12 out of 14 vs 7 out of 16, p < 0.05). The only important toxicity was diarrhoea grade 3-4, which occurred in 6 out of 16 patients treated with CPT-11 alone and in 4 out of 14 patients treated with CPT-11 plus MLT, which required a 50% dose reduction. However, taken together, patients treated with CPT-11 at 50% of the planned dose showed a percent of disease control comparable to that achieved in patients who had no dose reduction (6 out of 10 vs 13 out of 20). This preliminary study shows that the efficacy of weekly low-dose CPT-11 in pretreated metastatic colorectal cancer patients may be enhanced by a concomitant daily administration of the pineal hormone MLT, according to the results previously reported for other chemotherapeutic agents. Moreover, since the dose reduction of CPT-11 does not influence its efficacy, the dose of CPT-11 for successive studies might be not greater than 70 mg/m2.

Chauhan, D. P. (2002). “Chemotherapeutic potential of curcumin for colorectal cancer.” Curr Pharm Des 8(19): 1695-706.

Colorectal cancer is one of the leading causes of cancer deaths in the Western world. More than 56,000 newly diagnosed colorectal cancer patients die each year in the United States. Available therapies are either not effective or have unwanted side effects. Epidemiological data suggest that dietary manipulations play an important role in the prevention of many human cancers. Curcumin the yellow pigment in turmeric has been widely used for centuries in the Asian countries without any toxic effects. Epidemiological data also suggest that curcumin may be responsible for the lower rate of colorectal cancer in these countries. Curcumin is a naturally occurring powerful anti-inflammatory medicine. The anticancer properties of curcumin have been shown in cultured cells and animal studies. Curcumin inhibits lipooxygenase activity and is a specific inhibitor of cyclooxygenase-2 expression. Curcumin inhibits the initiation of carcinogenesis by inhibiting the cytochrome P-450 enzyme activity and increasing the levels of glutathione-S-transferase. Curcumin inhibits the promotion/progression stages of carcinogenesis. The anti-tumor effect of curcumin has been attributed in part to the arrest of cancer cells in S, G2/M cell cycle phase and induction of apoptosis. Curcumin inhibits the growth of DNA mismatch repair defective colon cancer cells. Therefore, curcumin may have value as a safe chemotherapeutic agent for the treatment of tumors exhibiting DNA mismatch repair deficient and microsatellite instable phenotype. Curcumin should be considered as a safe, non-toxic and easy to use chemotherapeutic agent for colorectal cancers arise in the setting of chromosomal instability as well as microsatellite instability.

Chen, M. F., L. T. Chen, et al. (1995). “Effect of 5-fluorouracil on methotrexate transport and cytotoxicity in HT29 colon adenocarcinoma cells.” Cancer Lett 88(2): 133-40.

Exposure of the human colon adenocarcinoma HT29 cells to different concentrations of 5-fluorouracil (5FU) for 24 h resulted in a dose-dependent increase in the cellular glutathione (GSH) level which remained elevated up to 72 h following 5FU treatment. Pretreatment of HT29 cells with 5FU was found to enhance the uptake of methotrexate (MTX) as compared to control cells. Administration of MTX 24 h after 5FU was found to be synergistic, whereas administration of MTX 24 h before 5FU or together with 5FU was not found to be synergistic. The results of this study suggest that the increase in cellular GSH level as a result of 5FU pretreatment may play a role in the enhancement of MTX uptake and the cytotoxicity of both drugs in HT29 cells.

Chen, M. F., L. T. Chen, et al. (1995). “5-Fluorouracil cytotoxicity in human colon HT-29 cells with moderately increased or decreased cellular glutathione level.” Anticancer Res 15(1): 163-7.

Little is known whether diet or certain components in the diet can modulate the efficacy of 5-fluorouracil (5FU) in patients with colon carcinoma. Glutathione (GSH), an important antioxidant and anticarcinogen, is present in many foods in varying amounts. This study examined whether a moderately increased or decreased cellular GSH level had any effect on the growth of human colon adenocarcinoma cells HT-29 and on the cytotoxic activity of 5FU in these cells. GSH and buthionine sulfoximine were used to enhance or reduce the GSH level respectively in these cells. A 34% increase in cellular GSH level had no effect on the growth of HT-29 cells, nor on the cytotoxic activity of 5FU as determined by the MTT colorimetric assay and cell counts. A 50% reduction in the cellular GSH level was found to enhance 5FU cytotoxicity by 20% to 31% as determined by the MTT colorimetric assay, depending on the 5FU concentration. This study shows that a moderate change in the GSH level in HT-29 cells had little or no effect on the cells’ growth, but a decrease in cellular GSH level slightly enhanced the cytotoxic activity of 5FU in these cells.

Chinery, R., J. A. Brockman, et al. (1997). “Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPbeta.” Nat Med 3(11): 1233-41.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Five-fluorouracil (5FU) remains the single most effective treatment for advanced disease, despite a response rate of only 20%. Herein, we show that the antioxidants pyrrolidinedithiocarbamate and vitamin E induce apoptosis in CRC cells. This effect is mediated by induction of p21WAF1/CIP1, a powerful inhibitor of the cell cycle, through a mechanism involving C/EBPbeta (a member of the CCAAT/enhancer binding protein family of transcription factors), independent of p53. Antioxidants significantly enhance CRC tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin) and in vivo (5FU). Thus, chemotherapeutic agents administered in the presence of antioxidants may provide a novel therapy for colorectal cancer.

Choo, M. K., H. Sakurai, et al. (2008). “A ginseng saponin metabolite suppresses tumor necrosis factor-alpha-promoted metastasis by suppressing nuclear factor-kappaB signaling in murine colon cancer cells.” Oncol Rep 19(3): 595-600.

SC-514, an inhibitor of IkappaB kinase beta (IKKbeta), blocked the TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) as well as the TNF-alpha-promoted metastasis of murine colon adenocarcinoma cells. We investigated the effect of 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1), a main intestinal bacterial metabolite of ginseng, on the NF-kappaB-dependent metastasis. M1 was effective in suppressing the TNF-alpha-induced activation of NF-kappaB, expression of matrix metalloprotease-9 (MMP-9), migration and invasion. The TNF-alpha-evoked increase in lung and liver metastasis of colon carcinoma was also abrogated by treatment with M1 in vitro. These results suggest that ginseng has potential to suppress inflammation-related metastasis by downregulating the NF-kappaB signaling pathway.

Conklin, K. A. (2000). “Dietary antioxidants during cancer chemotherapy: impact on chemotherapeutic effectiveness and development of side effects.” Nutr Cancer 37(1): 1-18.

Curtin, N. J. and A. N. Hughes (2001). “Pemetrexed disodium, a novel antifolate with multiple targets.” Lancet Oncol 2(5): 298-306.

Pemetrexed disodium is a potent new antifolate which inhibits many folate-dependent reactions that are essential for cell proliferation. Its primary target is thymidylate synthase but it also inhibits folate-dependent enzymes involved in purine synthesis. Cells that are resistant to antifolates are generally less resistant to pemetrexed, irrespective of the mechanism of resistance. Pemetrexed has shown good activity in preclinical models with human tumour cells and xenografts. In the majority of clinical trials of pemetrexed, the dose-limiting toxic effect is neutropenia; other side-effects are mostly gastrointestinal. Preclinical studies indicate that the toxic effects of pemetrexed can be reduced by dietary folate, resulting in an improved therapeutic index. Low folate status is also associated with higher levels of toxicity in patients. As a single agent pemetrexed has shown good activity against non-small-cell lung cancer, squamous-cell carcinoma of head and neck, colon cancer, and breast cancer, and it appears to be particularly active in combination with cisplatin against non-small-cell lung cancer and mesothelioma. Phase II and III studies are underway.

Daniele, B., F. Perrone, et al. (2001). “Oral glutamine in the prevention of fluorouracil induced intestinal toxicity: a double blind, placebo controlled, randomised trial.” Gut 48(1): 28-33.

BACKGROUND: 5-Fluorouracil (FU) in association with folinic acid (FA) is the most frequently used chemotherapeutic agent in colorectal cancer but it often causes diarrhoea. Animal and human studies suggest that glutamine stimulates intestinal mucosal growth. AIM: To determine if oral glutamine prevents changes in intestinal absorption (IA) and permeability (IP) induced by FU/FA. METHODS: Seventy chemotherapy naive patients with colorectal cancer were randomly assigned to oral glutamine (18 g/day) or placebo before the first cycle of FU (450 mg/m(2)) and FA (100 mg/m(2)) administered intravenously for five days. Treatment was continued for 15 days, starting five days before the beginning of chemotherapy. IA (D-xylose urinary excretion) and IP (cellobiose-mannitol test) were assessed at baseline and four and five days after the end of the first cycle of chemotherapy, respectively. Patients kept a daily record of diarrhoea, scored using the classification system of the National Cancer Institute (Bethesda, Maryland, USA). Duration of diarrhoea was recorded and the area under the curve (AUC) was calculated for each patient. RESULTS: Baseline patient characteristics and basal values of IP and IA tests were similar in the two arms. After one cycle of chemotherapy, the reduction in IA (D-xylose absorption) was more marked in the placebo arm (7.1% v 3. 8%; p=0.02); reduction of IP to mannitol was higher in the placebo arm (9.2% v 4.5%; p=0.02); and urinary recovery of cellobiose was not different between the study arms (p=0.60). Accordingly, the cellobiose-mannitol ratio increased more in the placebo arm (0.037 v 0.012; p=0.04). Average AUC of diarrhoea (1.9 v 4.5; p=0.09) and average number of loperamide tablets taken (0.4 v 2.6; p=0.002) were reduced in the glutamine arm. CONCLUSIONS: Glutamine reduces changes in IA and IP induced by FU and may have a protective effect on FU induced diarrhoea.

Doyle, L. A., D. D. Ross, et al. (1995). “An etoposide-resistant lung cancer subline overexpresses the multidrug resistance-associated protein.” Br J Cancer 72(3): 535-42.

Du, B., L. Jiang, et al. (2006). “Synergistic inhibitory effects of curcumin and 5-fluorouracil on the growth of the human colon cancer cell line HT-29.” Chemotherapy 52(1): 23-8.

The synergistic effect of combination treatment with COX-2 inhibitors and chemotherapy may be another promising therapy regimen in the future treatment of colorectal cancer. Curcumin, a major yellow pigment in turmeric which is used widely all over the world, inhibits the growth of human colon cancer cell line HT-29 significantly and specifically inhibits the expression of COX-2 protein. However, the worldwide exposure of populations to curcumin raised the question of whether this agent would enhance or inhibit the effects of chemotherapy. In this report, we evaluated the growth-inhibitory effect of curcumin and a traditional chemotherapy agent, 5-FU, against the proliferation of a human colon cancer cell line (HT-29). The combination effect was quantitatively determined using the method of median-effect principle and the combination index. The inhibition of COX-2 expression after treatment with the curcumin-5-FU combination was also evaluated by Western blot analysis. The IC(50) value in the HT-29 cells for curcumin was 15.9 +/- 1.96 microM and for 5-FU it was 17.3 +/- 1.85 microM. When curcumin and 5-FU were used concurrently, synergistic inhibition of growth was quantitatively demonstrated. The level of COX-2 protein expression was reduced almost 6-fold after the combination treatment. Our results demonstrate synergism between curcumin and 5-FU at higher doses against the human colon cancer cell line HT-29. This synergism was associated with the decreased expression of COX-2 protein.

Engel, D., A. Nudelman, et al. (2008). “Novel Prodrugs of Tegafur that Display Improved Anticancer Activity and Antiangiogenic Properties.” J Med Chem 51(2): 314-23.

New and more potent prodrugs of the 5-fluorouracyl family derived by hydroxymethylation or acyloxymethylation of 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1 H,3 H)-pyrimidinedione (tegafur, 1) are described. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity, and anticancer activities in vitro and in vivo, superior to those of tegafur. 5-Fluoro-1-(tetrahydro-2-furanyl)-2,4(1 H,3 H)-pyrimidinedione (tegafur, 1), the oral prodrug of 5-FU, has been widely used for treatment of gastrointestinal malignancies with modest efficacy. The aim of this study was to develop and characterize new and more potent prodrugs of the 5-FU family derived by hydroxymethylation or acyloxymethylation of tegafur. Comparison between the effect of tegafur and the new prodrugs on the viability of a variety of cancer cell lines showed that the IC 50 and IC 90 values of the novel prodrugs were 5-10-fold lower than those of tegafur. While significant differences between the IC 50 values of tegafur were observed between the sensitive HT-29 and the resistant LS-1034 colon cancer cell lines, the prodrugs affected them to a similar degree, suggesting that they overcame drug resistance. The increased potency of the prodrugs could be attributed to the antiproliferative contribution imparted by formaldehyde and butyric acid, released upon metabolic degradation. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity and anticancer activities in vitro and in vivo, superior to those of tegafur.

Fernandez-Luna, J. L. (2007). “Apoptosis regulators as targets for cancer therapy.” Clin Transl Oncol 9(9): 555-62.

Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions between proteins of the Bcl-2 family. A death signal is transmitted through the BH3-only proteins to Bax and Bak which in turn permeabilise the outer mitochondrial membrane allowing the release of apoptogenic factors, which triggers activation of cell-deathpromoting caspases. These proteolytic enzymes are tightly controlled by members of the inhibitor of apoptosis (IAP) family. Activation of the caspase cascade via cell death receptors also represents a key apoptotic pathway in both normal and tumour cells. Basic knowledge of these apoptosis regulators provides the basis for novel therapeutic strategies aimed at promoting tumour cell death or enhancing susceptibility to apoptotic inducers. This review focuses on these strategies.

Fogt, F., A. Wellmann, et al. (2001). “Glut-1 expression in dysplastic and regenerative lesions of the colon.” Int J Mol Med 7(6): 615-9.

Monosaccaride transporter proteins are responsible for transmembrane transport of monosaccarides into cells. Glucose transporter protein 1 (Glut-1) is most prevalent in the cell membranes of erythrocytes and facilitates transport of glucose in tissues with barrier functions, i.e. blood brain barrier. Expression of Glut-1 in malignant tumors is increased due to increased metabolic need of the proliferating cell populations. In colorectal adenomas and carcinomas, membranous expression of Glut-1 has been associated with higher grade of tumors and decreased survival time. We studied the expression of Glut-1 in dysplastic proliferations of the colon which included sporadic adenomas and dysplasia associated lesions (DALM) in patients with ulcerative colitis and reactive/regenerative proliferations of the colon, including non-dysplastic chronic colitis, acute colitis and ischemia. Two patterns of Glut-1 expression were detected. Most adenomas and DALMs showed at least focal membranous expression of Glut-1. In addition a second staining pattern was recognized which consisted of prominent supranuclear dots. This pattern of staining was not only seen in adenomas and DALM but also in non-dysplastic areas immediately surrounding sporadic adenomas, in regenerative chronic colitis and in areas surrounding acute inflammation. Areas away from dysplasia did not show any positive staining for Glut-1. We conclude that two distinct patterns of Glut-1 expression may be found in colonic epithelial proliferation: membranous staining, associated with dysplasia, and, heretofore not described, supranuclear staining which may be related to Glut-1 expression secondary to expression of specific growth factors and not necessarily related to dysplasia.

Gamelin, L., M. Boisdron-Celle, et al. (2004). “Prevention of oxaliplatin-related neurotoxicity by calcium and magnesium infusions: a retrospective study of 161 patients receiving oxaliplatin combined with 5-Fluorouracil and leucovorin for advanced colorectal cancer.” Clin Cancer Res 10(12 Pt 1): 4055-61.

PURPOSE: Oxaliplatin is active in colorectal cancer. Sensory neurotoxicity is its dose-limiting toxicity. It may come from an effect on neuronal voltage-gated Na channels, via the liberation one its metabolite, oxalate. We decided to use Ca and Mg as oxalate chelators. EXPERIMENTAL DESIGN: A retrospective cohort of 161 patients treated with oxaliplatin + 5-fluorouracil and leucovorin for advanced colorectal cancer, with three regimens of oxaliplatin (85 mg/m(2)/2w, 100/2w, 130/3w) was identified. Ninety-six patients received infusions of Ca gluconate and Mg sulfate (1 g) before and after oxaliplatin (Ca/Mg group) and 65 did not. RESULTS: Only 4% of patients withdrew for neurotoxicity in the Ca/Mg group versus 31% in the control group (P = 0.000003). The tumor response rate was similar in both groups. The percentage of patients with grade 3 distal paresthesia was lower in Ca/Mg group (7 versus 26%, P = 0.001). Acute symptoms such as distal and lingual paresthesia were much less frequent and severe (P = 10(-7)), and pseudolaryngospasm was never reported in Ca/Mg group. At the end of the treatment, 20% of patients in Ca/Mg group had neuropathy versus 45% (P = 0.003). Patients with grade 2 and 3 at the end of the treatment in the 85 mg/m(2) oxaliplatin group recovered significantly more rapidly from neuropathy than patients without Ca/Mg. CONCLUSIONS: Ca/Mg infusions seem to reduce incidence and intensity of acute oxaliplatin-induced symptoms and might delay cumulative neuropathy, especially in 85 mg/m(2) oxaliplatin dosage.

Goto, S., K. Kamada, et al. (2002). “Significance of nuclear glutathione S-transferase pi in resistance to anti-cancer drugs.” Jpn J Cancer Res 93(9): 1047-56.

Recent study has shown that nuclear glutathione S-transferase (GST) pi accumulates in cancer cells resistant to doxorubicin hydrochloride (DOX) and may function to prevent nuclear DNA damage caused by DOX (Goto et al., FASEB J., 15, 2702 – 2714 (2001)). It is not clear if the amount of nuclear GSTpi increases in response to other anti-cancer drugs and if so, what is the physiological significance of the nuclear transfer of GSTpi in the acquisition of drug-resistance in cancer cells. In the present study, we employed three cancer cell lines, HCT8 human colonic cancer cells, A549 human lung adenocarcinoma cells, and T98G human glioblastoma cells. We estimated the nuclear transfer of GSTpi induced by the anti-cancer drugs cisplatin (CDDP), irinotecan hydrochloride (CPT-11), etoposide (VP-16) and 5-fluorouracil (5-FU). It was found that: (1) Nuclear GSTpi accumulated in these cancer cells in response to CDDP, DOX, CPT-11, VP-16 and 5-FU. (2) An inhibitor of the nuclear transport of GSTpi, edible mushroom lectin (Agaricus bisporus lectin, ABL), increased the sensitivity of the cancer cells to DOX and CDDP, and partially to CPT-11. Treatment with ABL had no apparent effect on the cytotoxicity of VP-16 and 5-FU. These results suggest that inhibitors of the nuclear transfer of GSTpi have practical value in producing an increase of sensitivity to DOX, CDDP and CPT-11.

Grubben, M. J., F. M. Nagengast, et al. (2001). “The glutathione biotransformation system and colorectal cancer risk in humans.” Scand J Gastroenterol Suppl(234): 68-76.

Evidence for a protective role of the glutathione biotransformation system in carcinogenesis is growing. However, most data on this system in relation to colorectal cancer originate from animal studies. Here we review the human data. In humans, a significant association was found between glutathione S-transferase (GST) activity in the mucosa along the gastrointestinal tract and the corresponding tumour incidence. Low activity was correlated with high tumour incidence and vice versa. Also, in normal colonic mucosa, GST activity is lower in patients at risk of colon cancer than in healthy controls and therefore interventions which increase the glutathione detoxification capacity may reduce cancer incidence. Consumption of vegetables and fruit is associated with a lower risk of colorectal cancer. Human intervention studies showed that (components from) vegetables induced colonic glutathione detoxification capacity. Such an effect could contribute to a lower colon cancer risk, but further data are needed. The human GSTs consist of four main classes–alpha (A), mu (M), pi (P) and theta (T)–each of which is divided into one or more isoforms. Functional polymorphisms are known for the GST genes M1, P1 and T1 and they all lead to less active enzymes compared to the wild-type gene products. However, studies that compared these GST polymorphisms in relation to colon cancer risk were not conclusive with respect to an increased or decreased risk of a particular genotype. Diet or medication can also influence the expression levels of specific isoenzymes and the effect of such interventions on cancer risk deserves more attention.

Hatcher, H., R. Planalp, et al. (2008). “Curcumin: From ancient medicine to current clinical trials.” Cell Mol Life Sci.

Curcumin is the active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa). Curcumin has a surprisingly wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity. The pleiotropic activities of curcumin derive from its complex chemistry as well as its ability to influence multiple signaling pathways, including survival pathways such as those regulated by NF-kappaB, Akt, and growth factors; cytoprotective pathways dependent on Nrf2; and metastatic and angiogenic pathways. Curcumin is a free radical scavenger and hydrogen donor, and exhibits both pro- and antioxidant activity. It also binds metals, particularly iron and copper, and can function as an iron chelator. Curcumin is remarkably non-toxic and exhibits limited bioavailability. Curcumin exhibits great promise as a therapeutic agent, and is currently in human clinical trials for a variety of conditions, including multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and Alzheimer’s disease.

Hidvegi, M., E. Raso, et al. (1999). “MSC, a new benzoquinone-containing natural product with antimetastatic effect.” Cancer Biother Radiopharm 14(4): 277-89.

An orally applicable fermentation product of wheat germ containing 0.04% substituted benzoquinone (MSC) has been invented by Hungarian chemists under the trade name of AVEMAR. Oral administration (3 g/kg body weight) of MSC enhances blastic transformation of splenic lymphocytes in mice. The same treatment shortens the survival time of skin grafts in a co-isogenic mouse skin transplantation model, pointing to the immune-reconstructive effect of MSC. A highly significant antimetastatic effect of MSC has been observed in three metastasis models (3LL-HH, B16, HCR-25). The antimetastatic effect of MSC–besides the immune-reconstitution–may also be due to its cell adhesion inhibitory, cell proliferation inhibitory, apoptosis enhancing, and antioxidant characteristics, also observed in our in vitro experiments. It is even more noteworthy that combined treatment with MSC and one of the following antineoplastic agents (5-FU and DTIC)–both in wide use in every day clinical practice–exhibited a significantly enhanced antimetastatic effect in appropriate metastasis models (established from C38 mouse colon carcinoma and B16 mouse melanoma respectively) as compared to the effect elicited by any component of these therapeutic compositions (MSC + 5-FU and MSC + DTIC) administered alone. The results show that the fermented wheat germ extract (MSC) has more than an additive effect and synergistically enhanced the metastasis inhibitory effect of both antineoplastic agents studied till now. It is also worthy of mention that the synchronous treatment with MSC profoundly decreased the toxic side effects of the applied antineoplastic agents (decreased weight loss etc). Based on the biological effects of MSC–shown to be non-toxic by subacute toxicology studies–this product may be used as an adjuvant in the therapy of malignant neoplasia and other diseases caused by or following immune-deficiency.

Hwang, J. T., J. Ha, et al. (2007). “Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway.” Cancer Lett 247(1): 115-21.

EGCG [(-)epigallocatechin-3-gallate], a green tea-derived polyphenol, has been shown to suppress cancer cell proliferation, and interfere with the several signaling pathways and induce apoptosis. Practically, there is emerging evidence that EGCG has a potential to increase the efficacy of chemotherapy in patients. We hypothesized that EGCG may exert cell cytotoxicity through modulating AMPK (AMP-activated protein kinase) followed by the decrease in COX-2 expression. EGCG treatment to colon cancer cells resulted in a strong activation of AMPK and an inhibition of COX-2 expression. The decreased COX-2 expression as well as prostaglandin E(2) secretion by EGCG was completely abolished by inhibiting AMPK by an AMPK inhibitor, Compound C. Also, the activation of AMPK was accompanied with the reduction of VEGF (vascular endothelial growth factor) and glucose transporter, Glut-1 in EGCG-treated cancer cells. These findings support the regulatory role of AMPK in COX-2 expression in EGCG-treated cancer cells. Furthermore, we have found that reactive oxygen species (ROS) is an upstream signal of AMPK, and the combined treatment of EGCG and chemotherapeutic agents, 5-FU or Etoposide, exert a novel therapeutic effect on chemo-resistant colon cancer cells. AMPK, a molecule of newly defined cancer target, was shown to control COX-2 in EGCG-treated colon cancer cells.

Hwang, J. T., J. Ha, et al. (2005). “Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways.” Biochem Biophys Res Commun 332(2): 433-40.

5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemo-resistance remains as a major obstacle in clinical settings. In the present study, HT-29 colon cancer cells were markedly sensitized to apoptosis by both 5-FU and genistein compared to the 5-FU treatment alone. There is an emerging evidence that genistein, soy-derived phytoestrogen, may have potential as a chemotherapeutic agent capable of inducing apoptosis or suppressing tumor promoting proteins such as cyclooxygenase-2 (COX-2). However, the precise mechanism of cellular cytotoxicity of genistein is not known. The present study focused on the correlation of AMPK and COX-2 in combined cytotoxicity of 5-FU and genistein, since AMPK is known as a primary cellular homeostasis regulator and a possible target molecule of cancer treatment, and COX-2 as cell proliferation and anti-apoptotic molecule. Our results demonstrated that the combination of 5-FU and genistein abolished the up-regulated state of COX-2 and prostaglandin secretion caused by 5-FU treatment in HT-29 colon cancer cells. These appear to be followed by the specific activation of AMPK and the up-regulation of p53, p21, and Bax by genistein. Under same conditions, the induction of Glut-1 by 5-FU was diminished by the combination treatment with 5-FU and genistein. Furthermore, the reactive oxygen species (ROS) was found as an upstream signal for AMPK activation by genistein. These results suggested that the combination of 5-FU and genistein exert a novel chemotherapeutic effect in colon cancers, and AMPK may be a novel regulatory molecule of COX-2 expression, further implying its involvement in cytotoxicity caused by genistein.

Hwang, J. T., Y. M. Kim, et al. (2006). “Selenium regulates cyclooxygenase-2 and extracellular signal-regulated kinase signaling pathways by activating AMP-activated protein kinase in colon cancer cells.” Cancer Res 66(20): 10057-63.

Epidemiologic and experimental evidences indicate that selenium, an essential trace element, can reduce the risk of a variety of cancers. Protection against certain types of cancers, particularly colorectal cancers, is closely associated with pathways involving cyclooxygenase-2 (COX-2). We found that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, mediates critical anticancer effects of selenium via a COX-2/prostaglandin E(2) signaling pathway. Selenium activated AMPK in tumor xenografts as well as in colon cancer cell lines, and this activation seemed to be essential to the decrease in COX-2 expressions. Transduction with dominant-negative AMPK into colon cancer cells or application of cox-2(-/-)-negative cells supported the evidence that AMPK is an upstream signal of COX-2 and inhibits cell proliferation. In HT-29 colon cancer cells, carcinogenic agent 12-O-tetradecanoylphorbol-13-acetate (TPA) activated extracellular signal-regulated kinase (ERK) that led to COX-2 expression and selenium blocked the TPA-induced ERK and COX-2 activation via AMPK. We also showed the role of a reactive oxygen species as an AMPK activation signal in selenium-treated cells. We propose that AMPK is a novel and critical regulatory component in selenium-induced cancer cell death, further implying AMPK as a prime target of tumorigenesis.

Ichikawa, D., T. Takahashi, et al. (1998). “[Postoperative management of the preserved rectal segment in patients with familial polyposis: the use of 5-fluorouracil suppositories and green tea extract to inhibit tumor growth].” Nippon Geka Gakkai Zasshi 99(6): 391-5.

We report the clinical details of seven patients with familial polyposis. They underwent subtotal colectomy with ileorectostomy, and were treated with 5-fluorouracil suppositories and green tea extract after surgery. Some regression of the polyps in the preserved rectal segment was observed, and no rectal cancer developed in any of these patients.

Jaiswal, A. S., B. P. Marlow, et al. (2002). “Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.” Oncogene 21(55): 8414-27.

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

Jordan, A. and J. Stein (2003). “Effect of an omega-3 fatty acid containing lipid emulsion alone and in combination with 5-fluorouracil (5-FU) on growth of the colon cancer cell line Caco-2.” Eur J Nutr 42(6): 324-31.

BACKGROUND: In this study we examined the effects of a fish oil-based lipid emulsion (FO) rich in omega-3 fatty acids, which is used in humans as a component of parenteral nutrition, on the growth of the colon cancer cell line Caco-2. AIM OF THE STUDY: The aim of the present study was to investigate whether the FO influences growth and chemosensitivity of the colon cancer cell line Caco-2. FO was tested alone and in combination with the anticancer drug 5-fluorouracil (5-FU). METHODS: Cell numbers were determined with crystal violet staining, cell cycle distribution was assessed using a flow cytometer and apoptosis was visualized by staining nuclei with diamino-phenylindole hydrochloride. RESULTS: FO inhibited growth of Caco-2 cells in a time and dose dependent manner. FO treatment evoked apoptosis as confirmed by cell morphology. Cell cycle analysis identified an accumulation of cells in the G(2)/M phase after incubation with FO. The combined treatment of the cells with FO and 5-FU resulted in a significant enhancement of the growth inhibition seen after exposure to either substance alone. Treatment of the cells with 5-FU specifically blocked the cell cycle in the S phase. The combined treatment of 5-FU with FO showed a further increase in the accumulation of cells in the S phase. CONCLUSIONS: In conclusion, FO has a potent antiproliferative effect on Caco-2 cells, at least in part, due to a decrease in the progression of the cell cycle and the induction of apoptosis. The combination of FO with 5-FU results in an additive growth inhibitory effect.

Katoh, R. and M. Ooshiro (2007). “Enhancement of antitumor effect of tegafur/uracil (UFT) plus leucovorin by combined treatment with protein-bound polysaccharide, PSK, in mouse models.” Cell Mol Immunol 4(4): 295-9.

We evaluated the antitumor effect of combined therapy with tegafur/uracil (UFT) plus leucovorin (LV) (UFT/LV) and protein-bound polysaccharide, PSK, in three mouse models of transplantable tumors. UFT/LV showed antitumor effect against Meth A sarcoma, and the antitumor effect was enhanced when PSK given concomitantly. UFT/LV showed antitumor effect to Lewis lung carcinoma and PSK alone also showed antitumor effect at high dose, but a combination of UFT/LV and PSK resulted in no enhanced antitumor effect. Colon 26 carcinoma was weakly responsive to UFT/LV, and no enhancement of antitumor effect was found even PSK was used in combination. In conclusion, while the effect of PSK varies depending on tumor, combined use of UFT/LV and PSK may be expected to augment the antitumor effect.

Kim, K. H., H. Y. Park, et al. (2005). “[The inhibitory effect of curcumin on the growth of human colon cancer cells (HT-29, WiDr) in vitro].” Korean J Gastroenterol 45(4): 277-84.

BACKGROUND/AIMS: The effects of curcumin on the growth of human colon cancer cell lines, HT-29 and WiDr cells were examined and the effects of 5-fluorouracil (5-FU) were also studied. METHODS: The growth of HT-29 and WiDr cells were examined by counting cell number on two and four days treatment with 1-40 microm of curcumin, and 0.1 microg/mL, 0.3 microg/mL of 5-FU. The reversibility of curcumin was examined on one day to seven days treatment with 10 microm curcumin after seeding to 2 x 10(4) cells/well. To examine the inhibitory effects of curcumin, cell cycle analysis was done on the HT-29 cells after four days treatment with 20 microm curcumin. RESULTS: Curcumin inhibited the growth of HT-29 and WiDr cells in a dose-dependent fashion. The growth rate of the group in which curcumin was removed by media change 24 hours after the treatment of curcumin was not different from that of control group. Curcumin combined with 5-FU markedly inhibited the growth of HT-29 and WiDr cells compared to curcumin or 5-FU alone. After four days treatment of HT-29 cells with 20 microm curcumin, the fraction of cells in G2-M phase was 35.3% in curcumin group, much higher than 13.8% of the control group. CONCLUSIONS: Curcumin significantly inhibited the growth of HT-29 and WiDr cells in a dose-dependent, reversible fashion.

Kim, Y. M., J. T. Hwang, et al. (2007). “Involvement of AMPK signaling cascade in capsaicin-induced apoptosis of HT-29 colon cancer cells.” Ann N Y Acad Sci 1095: 496-503.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is activated during ATP-depleting metabolic states, such as hypoxia, heat shock, oxidative stress, and exercise. As a highly conserved heterotrimeric kinase that functions as a major metabolic switch to maintain energy homeostasis, AMPK has been shown to exert as an intrinsic regulator of mammalian cell cycle. Moreover, AMPK cascade has emerged as an important pathway implicated in cancer control. In this article, we have investigated the effects of capsaicin on apoptosis in relation to AMPK activation in colon cancer cell. Capsaicin-induced apoptosis was revealed by the presence of nucleobodies in the capsaicin-treated HT-29 colon cancer cells. Concomitantly, the activation of AMPK and the increased expression of the inactive form of acetyl-CoA carboxylase (ACC) were detected in capsaicin-treated colon cancer cells. We showed that both capsaicin and 5′-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an AMPK activator possess the AMPK-activating capacity as well as apoptosis-inducing properties. Evidence of the association between AMPK activation and the increased apoptosis in HT-29 colon cancer cells by capsaicin treatment, and further findings of the correlation of the activated AMPK and the elevated apoptosis by cotreatment of AICAR and capsaicin support AMPK as an important component of apoptosis, as well as a possible target of cancer control.

Kitajima, M., Y. Ikeda, et al. (1987). “[The effect of tegafur suppository and glutathione in patients with gastric and colonic cancer with special reference to the histopathological anticancer effect].” Gan To Kagaku Ryoho 14(11): 3131-9.

The purpose of these studies is to evaluate the anticancerous effects of tegafur suppository and Glutathione (GSH) as enhanced drug and compare the difference of the histopathological effects and tissue concentration of 5-FU and FT-207 in gastric and colon cancer. Thirty three patients with gastric cancer and 17 with colon cancer were treated by tegafur suppository at a dose of 1,500 mg/day and GSH, 1,200 mg/day intravenously. These patients were clinically divided into two groups, one of which was treated with tegafur suppository (Group A) and another administered with suppository and GSH (Group B). Five-fluorouracil was measured by GC-MF method and FT-207 was also done by HPLC method. After surgery, relationship between tissue concentration of 5-FU and histopathological effects were investigated. In gastric cancer, 5-FU concentration in cancer tissue was significantly kept high level in cancer tissue in patients treated with tegafur and GSH (Group B). However, there was no same results in colon cancer. These results seemed to be the difference of organ specificity. According to histopathological studies, well differentiated adenocarcinoma including papillary carcinoma were markedly effective compared to poorly differentiated adenocarcinoma in both cancer. This difference of anticancerous effect was supposed to be microangiographically different of microvascular architecture and quantity of anticancerous agents in tumor.

Kokura, S., N. Yoshida, et al. (2005). “The radical scavenger edaravone enhances the anti-tumor effects of CPT-11 in murine colon cancer by increasing apoptosis via inhibition of NF-kappaB.” Cancer Lett 229(2): 223-33.

The transcription factor NF-kappaB is reportedly activated by anti-cancer chemotherapeutic compounds in many cancer cell lines and NF-kappaB activation is one mechanism by which tumors become resistant to apoptosis. Antioxidants have been reported to serve as potent NF-kB inhibitors. In this study, we investigated the ability of edaravone to enhance apoptosis induced by CPT-11 through inhibition of NF-kB. In vitro, SN38, the active metabolite of CPT-11, induced activation of NF-kB, the production of intracellular reactive oxygen species, the activation of caspase-3, and apoptosis in colon26 cells. Pretreatment with edaravone scavenged the SN38-produced reactive oxygen species, and inhibited the SN38-induced activation of NF-kB. Moreover, edaravone enhanced the activation of caspase-3, and the level of apoptosis induced by SN38. In vivo, the combination of edaravone with CPT-11 reduced subcutaneous tumor growth and number of pulmonary metastases more effectively than CPT-11 alone. These results demonstrate that the combination of edaravone with CPT-11 may constitute a new strategy for treating primary and metastatic colon cancer.

Kurokawa, H., K. Nishio, et al. (1997). “Effect of glutathione depletion on cisplatin resistance in cancer cells transfected with the gamma-glutamylcysteine synthetase gene.” Jpn J Cancer Res 88(2): 108-10.

Lee, C. H., Y. T. Jeon, et al. (2007). “NF-kappaB as a potential molecular target for cancer therapy.” Biofactors 29(1): 19-35.

Nuclear factor kappaB (NF-kappaB), a transcription factor, plays an important role in carcinogenesis as well as in the regulation of immune and inflammatory responses. NF-kappaB induces the expression of diverse target genes that promote cell proliferation, regulate apoptosis, facilitate angiogenesis and stimulate invasion and metastasis. Furthermore, many cancer cells show aberrant or constitutive NF-kappaB activation which mediates resistance to chemo- and radio-therapy. Therefore, the inhibition of NF-kappaB activation and its signaling pathway offers a potential cancer therapy strategy. In addition, recent studies have shown that NF-kappaB can also play a tumor suppressor role in certain settings. In this review, we focus on the role of NF-kappaB in carcinogenesis and the therapeutic potential of targeting NF-kappaB in cancer therapy.

Lee, C. S., S. Y. Park, et al. (2004). “Effect of change in cellular GSH levels on mitochondrial damage and cell viability loss due to mitomycin c in small cell lung cancer cells.” Biochem Pharmacol 68(9): 1857-67.

Luo, Z., A. K. Saha, et al. (2005). “AMPK, the metabolic syndrome and cancer.” Trends Pharmacol Sci 26(2): 69-76.

The fuel-sensing enzyme 5′-AMP-activated protein kinase (AMPK) has a major role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise, changes in fuel availability and the adipocyte-derived hormones leptin and adiponectin. Recent studies indicate that abnormalities in cellular lipid metabolism are involved in the pathogenesis of the metabolic syndrome, possibly because of dysregulation of AMPK and malonyl-CoA, a closely related molecule. As we discuss in this article, several findings also point to a link between AMPK and the growth and/or survival of some cancer cells. Thus, it has been demonstrated recently that the tumor suppressor LKB1 is a kinase that has a major role in phosphorylating and activating AMPK, and that another tumor suppressor, tuberous sclerosis complex 2, is phosphorylated and activated by AMPK. In addition, other studies indicate that mammalian homolog of target of rapamycin (mTOR), which has been implicated in the pathogenesis of insulin resistance and many types of cancer, is inhibited by AMPK.

Maddika, S., S. R. Ande, et al. (2007). “Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy.” Drug Resist Updat 10(1-2): 13-29.

The partial cross-utilization of molecules and pathways involved in opposing processes like cell survival, proliferation and cell death, assures that mutations within one signaling cascade will also affect the other opposite process at least to some extent, thus contributing to homeostatic regulatory circuits. This review highlights some of the connections between opposite-acting pathways. Thus, we discuss the role of cyclins in the apoptotic process, and in the regulation of cell proliferation. CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted both for their apoptosis-regulating capacity and also for their effect on the cell cycle progression. The PI3-K/Akt cell survival pathway is shown as regulator of cell metabolism and cell survival, but examples are also provided where aberrant activity of the pathway may contribute to the induction of apoptosis. Myc/Mad/Max proteins are shown both as a powerful S-phase driving complex and as apoptosis-sensitizers. We also discuss multifunctional proteins like p53 and Rb (RBL1/p107, RBL2/p130) both in the context of G1-S transition and as apoptotic triggers. Finally, we reflect on novel therapeutic approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells.

Mamiya, N., T. Kono, et al. (2007). “[A case of neurotoxicity reduced with goshajinkigan in modified FOLFOX6 chemotherapy for advanced colon cancer].” Gan To Kagaku Ryoho 34(8): 1295-7.

In performing FOLFOX (infusional 5-FU/LV with oxaliplatin) for advanced colorectal cancer, neurotoxicity of oxaliplatin (L-OHP) is the serious dose limiting factor. On the other hand, goshajinkigan is recently considered as an effective agent for the neurotoxicity of taxanes in Japan.We have applied goshajinkigan (TJ 107) for a case of advanced colon cancer with mFOLFOX 6, and experienced a reduction in numbness, the adverse effect of LOHP. A 57-year-old woman with descending colon cancer (H 1, P 3, Stage IV) underwent hemicolectomy D 2, rt.colectomy, bilateral oophorectomy, cholecystectomy and transverse colonostomy. After operation, mFOLFOX 6 was applied. In order to reduce the neurotoxicity of L-OHP, TJ 107 was used together from the third course. The severities of neurotoxicity before and after administration of TJ 107 were grade 2 and 1,respectively. TJ 107 could reduce or prevent the neurotoxicity of L-OHP.

Mans, D. R., G. J. Schuurhuis, et al. (1992). “Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.” Eur J Cancer 28A(8-9): 1447-52.

Meijer, C., N. H. Mulder, et al. (1990). “The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line.” Br J Cancer 62(1): 72-7.

Nadal, J. C., C. J. van Groeningen, et al. (1989). “Schedule-dependency of in vivo modulation of 5-fluorouracil by leucovorin and uridine in murine colon carcinoma.” Invest New Drugs 7(2-3): 163-72.

The effect of leucovorin (LV) given in various doses and schedules on the in vivo antitumor activity and toxicity of 5-fluorouracil (5FU) was studied in two murine colon cancer lines, i.e., Colon 26 (relatively resistant to 5FU) and Colon 38 (5FU sensitive), maintained in Balb-c and C57B1/6 mice, respectively. Mice were treated weekly with 5FU at the maximum tolerated dose, alone and in combination with LV. In Colon 26, neither simultaneous administration of 5FU and LV nor 5FU combined with delayed administration of LV potentiated the antitumor activity of 5FU. LV given twice – 1 hr before (50 mg/kg) and then together (50 mg/kg) with 5FU (100 mg/kg) – gave significantly better delay of tumor growth of both tumor lines than 5FU did alone (100 mg/kg). No differences were found after a total LV dose of 100 or 200 mg/kg. Delayed administration of uridine (3500 mg/kg) allowed the use of higher 5FU doses, which improved the antitumor effect on Colon 26. Systemic toxicity led to moderate weight loss in treated mice, but was comparable for mice treated with 5FU alone or combined with LV. Hematological toxicity consisted of moderate leukopenia (nadir 40%), which was observed with the most active schedule and was less severe than with 5FU alone. This schedule did not cause thrombocytopenia, but after discontinuation the thrombocyte count showed an overshoot. Addition of uridine to this schedule reduced hematological toxicity only slightly. It is concluded that LV potentiated the antitumor activity of 5FU against two solid tumor lines, i.e., a relatively resistant and a sensitive murine colon carcinoma, and that toxicity was moderate.

Nakagawa, Y., M. Iinuma, et al. (2007). “Characterized mechanism of alpha-mangostin-induced cell death: caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells.” Bioorg Med Chem 15(16): 5620-8.

alpha-Mangostin, a xanthone from the pericarps of mangosteen (Garcinia mangostana Linn.), was evaluated for in vitro cytotoxicity against human colon cancer DLD-1 cells. The number of viable cells was consistently decreased by the treatment with alpha-mangostin at more than 20 microM. The cytotoxic effect of 20 microM alpha-mangostin was found to be mainly due to apoptosis, as indicated by morphological findings. Western blotting, the results of an apoptosis inhibition assay using caspase inhibitors, and the examination of caspase activity did not demonstrate the activation of any of the caspases tested. However, endonuclease-G released from mitochondria with the decreased mitochondrial membrane potential was shown. The levels of phospho-Erk1/2 were increased in the early phase until 1h after the start of treatment and thereafter decreased, and increased again in the late phase. On the other hand, the level of phospho-Akt was sharply reduced with the process of apoptosis after 6h of treatment. Interestingly, the level of microRNA-143, which negatively regulates Erk5 at translation, gradually increased until 24h following the start of treatment. We also examined the synergistic growth suppression in DLD-1 cells by the combined treatment of the cells with alpha-mangostin and 5-FU which is one of the most effective chemotherapeutic agents for colorectal adenocarcinoma. The co-treatment with alpha-mangostin and 5-FU, both at 2.5 microM, augmented growth inhibition compared with the treatment with 5 microM of alpha-mangostin or 5 microM 5-FU alone. These findings indicate unique mechanisms of alpha-mangostin-induced apoptosis and its action as an effective chemosensitizer.

Pai, R., T. Nakamura, et al. (2003). “Prostaglandins promote colon cancer cell invasion; signaling by cross-talk between two distinct growth factor receptors.” Faseb J 17(12): 1640-7.

Colorectal cancer is the second most frequent cancer in the Western world, often lethal when invasion and/or metastasis occur. In addition to hepatocyte growth factor (HGF), colon cancer invasion may be driven by prostaglandins, especially the E2 series (PGE2), generated by the cyclooxygenase-2 (Cox-2) enzyme. While concentration of PGE2 as well as expression of Cox-2, HGF receptor (c-Met-R), epidermal growth factor receptor (EGFR), and beta-catenin are all dramatically increased in colon cancers and implicated in their growth and invasion, the precise role of PGE2 in the latter process remains unclear. Here we provide evidence that PGE2 transactivates c-Met-R (contingent upon functional EGFR), increases tyrosine phosphorylation and nuclear accumulation of beta-catenin, and induces urokinase-type plasminogen activator receptor (uPAR) mRNA expression. This is accompanied by increased beta-catenin association with c-Met-R and enhanced colon cancer cell invasiveness. Inactivation of EGFR and c-Met-R significantly reduced PGE2-induced cancer cell invasiveness. Clinical relevance of these findings is confirmed by our immunohistochemical studies demonstrating that cancer cells in the invasive front overexpress Cox-2, c-Met-R, and beta-catenin. Our findings explain a functional relationship between prostaglandins, EGFR, and c-Met-R in colon cancer growth and invasion.

Patel, B. B., R. Sengupta, et al. (2008). “Curcumin enhances the effects of 5-fluorouracil and oxaliplatin in mediating growth inhibition of colon cancer cells by modulating EGFR and IGF-1R.” Int J Cancer 122(2): 267-73.

Curcumin (diferuloylmethane), which has been shown to inhibit growth of transformed cells, has no discernible toxicity and achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but with limited success. The present investigation was, therefore, undertaken to examine whether curcumin in combination with conventional chemotherapeutic agent(s)/regimen will be a superior therapeutic strategy for colorectal cancer. Indeed, results of our in vitro studies demonstrated that curcumin together with FOLFOX produced a significantly greater inhibition (p < 0.01) of growth and stimulated apoptosis (p < 0.001) of colon cancer HCT-116 and HT-29 cells than that caused by curcumin, 5-FU, curcumin + 5-FU or FOLFOX. These changes were associated with decreased expression and activation (tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72-100%) and IGF-1R (67%) as well as their downstream effectors such as Akt and cycloxygenase-2 (51-97%). Furthermore, while these agents produced a 2-3-fold increase in the expression of IGF-binding protein-3 (IGFBP-3), curcumin together with FOLFOX caused a 5-fold increase in the same, when compared to controls. This in turn led to increased sequestration of IGF by IGFBP-3 rendering IGF-1 unavailable for binding to and activation of IGF-1R. We conclude that the superior effects of the combination therapy of curcumin and FOLFOX are due to attenuation of EGFRs and IGF-1R signaling pathways. We also suggest that inclusion of curcumin to the conventional chemotherapeutic agent(s)/regimen could be an effective therapeutic strategy for colorectal cancer.

Perumal, S. S., P. Shanthi, et al. (2005). “Augmented efficacy of tamoxifen in rat breast tumorigenesis when gavaged along with riboflavin, niacin, and CoQ10: effects on lipid peroxidation and antioxidants in mitochondria.” Chem Biol Interact 152(1): 49-58.

Porter, A. G. and R. U. Janicke (1999). “Emerging roles of caspase-3 in apoptosis.” Cell Death Differ 6(2): 99-104.

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.

Press, M. F. and H. J. Lenz (2007). “EGFR, HER2 and VEGF pathways: validated targets for cancer treatment.” Drugs 67(14): 2045-75.

Targeted therapies are rationally designed to interfere with specific molecular events that are important in tumour growth, progression or survival. Several targeted therapies with anti-tumour activity in human cancer cell lines and xenograft models have now been shown to produce objective responses, delay disease progression and, in some cases, improve survival of patients with advanced malignancies. These targeted therapies include cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody; gefitinib and erlotinib, EGFR-specific tyrosine kinase inhibitors; trastuzumab, an anti-human EGFR type 2 (HER2)-related monoclonal antibody; lapatinib, a dual inhibitor of both EGFR- and HER2-associated tyrosine kinases; and bevacizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody.On the basis of preclinical and clinical evidence, EGFR, HER2 and VEGF represent validated targets for cancer therapy and remain the subject of intensive investigation. Both EGFR and HER2 are targets found on cancer cells, whereas VEGF is a target that acts in the tumour microenvironment. Clinical studies are focusing on how to best incorporate targeted therapy into current treatment regimens and other studies are exploring whether different strategies for inhibiting these targets will offer greater benefit. It is clear that optimal use of targeted therapy will depend on understanding how these drugs work mechanistically, and recognising that their activities may differ across patient populations, tumour types and disease stages, as well as when and how they are used in cancer treatment. The results achieved with targeted therapies to date are promising, although they illustrate the need for additional preclinical and clinical study.

Robson, S., S. Pelengaris, et al. (2006). “c-Myc and downstream targets in the pathogenesis and treatment of cancer.” Recent Patents Anticancer Drug Discov 1(3): 305-26.

The c-Myc oncoprotein is a master regulator of genes involved in diverse cellular processes. Situated upstream of signalling pathways regulating cellular replication/growth as well as apoptosis/growth arrest, c-Myc may help integrate processes determining cell numbers and tissue size in physiology and disease. In cancer, this ‘dual potential’ allows c-Myc to act as its own tumour suppressor. Evidently, given that deregulated expression of c-Myc is present in most, if not all, human cancers (Table 1) and is associated with a poor prognosis, by implication these in-built ‘failsafe’ mechanisms have been overcome. To explore the complex activity of c-Myc and its potential as a therapeutic target ‘post-genome era’ technologies for determining global gene expression alongside advanced new models for the study of tumourigenesis in vivo have proved invaluable. Thus, many recent studies have provided encouragement for the therapeutic targeting of c-Myc in cancer and have revealed new protein targets for manipulating aspects of c-Myc activity. The remarkable regression of even advanced and genetically unstable tumours, seen following deactivation of c-Myc in various models is particularly exciting. This review will discuss what is known about the role of c-Myc in growth deregulation and cancer and will conclude with a discussion of the most promising recent developments in Myc-targeted therapeutics.

Rupnarain, C., Z. Dlamini, et al. (2004). “Colon cancer: genomics and apoptotic events.” Biol Chem 385(6): 449-64.

Colon cancer is the third most common cancer globally. The risk of developing colon cancer is influenced by a number of factors that include age and diet, but is primarily a genetic disease, resulting from oncogene over-expression and tumour suppressor gene inactivation. The induction and progression of the disease is briefly outlined, as are the cellular changes that occur in its progression. While colon cancer is uniformly amenable to surgery if detected at the early stages, advanced carcinomas are usually lethal, with metastases to the liver being the most common cause of death. Oncogenes and genetic mutations that occur in colon cancer are featured. The molecules and signals that act to eradicate or initiate the apoptosis cascade in cancer cells, are elucidated, and these include caspases, Fas, Bax, Bid, APC, antisense hTERT, PUMA, 15-LOX-1, ceramide, butyrate, tributyrin and PPARgamma, whereas the molecules which promote colon cancer cell survival are p53 mutants, Bcl-2, Neu3 and COX-2. Cancer therapies aimed at controlling colon cancer are reviewed briefly.

Scagliotti, G. V. and S. Novello (2003). “Pemetrexed and its emerging role in the treatment of thoracic malignancies.” Expert Opin Investig Drugs 12(5): 853-63.

Pemetrexed (Alimta); Eli Lilly and Co., Indianapolis, IN, USA) is a unique multitargeted antifolate that inhibits at least three enzymes, thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase. This novel drug is being evaluated in a comprehensive clinical programme for use in both front-line and second-line therapies. It has shown broad activity in a number of solid tumours, including colon cancer, breast cancer, lung cancer, head and neck, cervical cancer and others. While a number of antifolates have been evaluated in clinical trials, further development has been stopped or delayed by the occurrence of life-threatening toxicities. Similar trends were also initially observed with pemetrexed as well, but investigators later showed that these toxicities could be minimised with folic acid and vitamin B(12) supplementation included in the treatment regimen. Preliminary data indicate that this supplementation does not hamper drug efficacy in most tumour types and in many cases, supplemented patients exhibit improved clinical outcome. Here, the current data for pemetrexed in treating thoracic malignancies are reviewed, with special focus on malignant pleural mesothelioma and non-small cell lung cancer.

Schmittgen, T. D., A. Koolemans-Beynen, et al. (1992). “Effects of 5-fluorouracil, leucovorin, and glucarate in rat colon-tumor explants.” Cancer Chemother Pharmacol 30(1): 25-30.

In a previous study, we showed that 5-fluorouracil (FU) is active against the dimethylhydrazine-induced colon tumor in rats; a 7-day infusion of FU at 30 mg/kg daily produced 85% tumor-free cures. The present study examined the effects of FU alone and in combination with leucovorin (LV) or D-glucarate (GT) using an ex vivo system that maintained the growth of the rat colon-tumor explants on collagen gels. The labeling index (LI) was determined by the incorporation of [3H]-thymidine and autoradiography. The mean LI of the untreated control was 64.8% +/- 19.8%. The IC50, IC90, and IC95 values following a 7-day exposure to FU were 0.36, 0.75, and 1.22 microM, respectively. In comparison, the steady-state FU concentration required to produce 67% tumor-free cures in rats following a 7-day infusion is 1.54 microM. LV alone did not produce any antiproliferative effect at concentrations as high as 10 microM. The addition of LV at concentrations of 0.001-10 microM did not significantly reduce the IC50 of FU. The lack of effect of LV may have been due to tissue saturation with folate provided in the culture medium. GT alone reduced the tumor LI by 20%-30% at concentrations of 0.1-10 microM. GT enhanced the effect of FU. As compared with FU alone, the addition of GT at concentrations of 0.1 and 1.0 microM reduced the IC50 of FU by 47% and 60% to 0.21 and 0.16 microM, respectively. Assessment of the potentiation of the inhibitory effect of FU by GT using two-way analysis of variance and the isobologram method indicated a significant synergistic interaction between FU and GT. This interaction occurred within the FU concentration range of 0.08 and 0.4 microM. In summary, these data indicate that (a) the IC values for FU are comparable in tumor explants and in rats, suggesting that the effects in cultured tumors reflect those in intact animals; (b) GT alone showed antitumor activity, albeit relatively minor as compared with FU; (c) FU and GT exhibited synergistic activity, which was most pronounced at FU concentrations that produced submaximal activity (less than 30% inhibition of tumor LI); and (d) GT and LV had different effects on the growth inhibition by FU, suggesting that GT acts by a mechanism different from the thymidylate synthase-directed effect of FU and LV.

Sethi, G., B. Sung, et al. (2008). “Nuclear factor-kappaB activation: from bench to bedside.” Exp Biol Med (Maywood) 233(1): 21-31.

Nuclear factor-kappaB (NF-kappaB) is a proinflammatory transcription factor that has emerged as an important player in the development and progression of malignant cancers. NF-kappaB targets genes that promote tumor cell proliferation, survival, metastasis, inflammation, invasion, and angiogenesis. Constitutive or aberrant activation of NF-kappa is frequently encountered in many human tumors and is associated with a resistant phenotype and poor prognosis. The mechanism of such persistent NF-kappaB activation is not clear but may involve defects in signaling pathways, mutations, or chromosomal rearrangements. Suppression of constitutive NF-kappaB activation inhibits the oncogenic potential of transformed cells and thus makes NF-kappaB an interesting new therapeutic target in cancer.

Shaw, R. J., M. Kosmatka, et al. (2004). “The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress.” Proc Natl Acad Sci U S A 101(10): 3329-35.

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive. Here we present biochemical and genetic evidence indicating that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. We show that LKB1 directly phosphorylates Thr-172 of AMPKalpha in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, we show that LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress. On the basis of these results, we propose a model to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics.

Sibilia, M., R. Kroismayr, et al. (2007). “The epidermal growth factor receptor: from development to tumorigenesis.” Differentiation 75(9): 770-87.

The epidermal growth factor receptor (EGFR) is activated by many ligands and belongs to a family of tyrosine kinase receptors, including ErbB2, ErbB3, and ErbB4. These receptors are de-regulated in many human tumors, and EGFR amplification, overexpression, and mutations are detected at a high frequency in carcinomas and glioblastomas, which are tumors of epithelial and glial origin, respectively. From the analysis of EGFR-deficient mice, it seems that the cell types mostly affected by the absence of EGFR are epithelial and glial cells, the same cell types where the EGFR is found to be overexpressed in human tumors. Therefore, it is important to define molecularly the function of EGFR signaling in the development of these cell types, because this knowledge will be of fundamental importance to understand how aberrant EGFR signaling can lead to tumor formation and progression. A molecular understanding of the pathways that control the development of a given tissue or cell type will also provide the basis for developing better combination therapies targeting different key components of the EGFR signaling network in the respective cancerous cells. Here, we will review the current knowledge, mostly derived from the analysis of genetically modified mice and cells, about the function of the EGFR in specific organs and tissues and in sites where the EGFR is found to be overexpressed in human tumors.

Sinicrope, F. A. (2006). “Targeting cyclooxygenase-2 for prevention and therapy of colorectal cancer.” Mol Carcinog 45(6): 447-54.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that regulates prostaglandin synthesis and is overexpressed at sites of inflammation and in several epithelial cancers. A causal link for COX-2 in epithelial tumorigenesis was shown in genetically manipulated animal models of colon and breast carcinoma. Studies have elucidated the regulation of COX-2 expression and have identified EP receptors through which prostanoids exert their biological effects. Mechanistic studies indicated that COX-2 is involved in apoptosis resistance, angiogenesis, and tumor cell invasiveness, which appear to contribute to its effects in tumorigenesis. Furthermore, forced COX-2 expression has been shown to suppress apoptosis by modulating the level of death receptor 5 (DR5) and this effect was reversed by a COX inhibitor. COX enzymes are targets for cancer prevention as shown by the observation that nonselective COX and selective COX-2 inhibitors have been reported to effectively prevent experimental colon cancer and can regress colorectal polyps in patients with familial adenomatous polyposis. This review will focus on the role of COX-2 as a target for the prevention and treatment of human colorectal cancer.

Sriram, N., S. Kalayarasan, et al. (2008). “Diallyl sulfide induces apoptosis in Colo 320 DM human colon cancer cells: involvement of caspase-3, NF-kappaB, and ERK-2.” Mol Cell Biochem 311(1-2): 157-65.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Diallyl sulfide (DAS), an organosulfur component of garlic has been known for its chemopreventive activities against various cancers and also in recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. Thus the present study was focused to elucidate the anticancerous effect and the mode of action of DAS against Colo 320 DM colon cancer cells. DAS induced apoptosis in Colo 320 DM cells was revealed by flow cytometer analysis and phosphatidyl serine exposure. DAS also promoted cell cycle arrest substantially at G2/M phase in Colo 320 DM cells. The production of reactive oxygen intermediates, which were examined by 2,7-dichlorodihydrofluorescein diacetate (H(2)DCF-DA), increased with time, after treatment with DAS. The activities of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were decreased upon DAS treatment, which shows the antiproliferative and the cytotoxic effects, respectively. The expression of NF-kappaB was upregulated in DAS treated cells, compared to normal cells. Further, DAS promoted the expression of caspase-3 and suppression of Extracellular Regulatory Kinase-2 (ERK-2) activity in Colo 320 DM cells that was determined by Western blot analysis. In conclusion, DAS increased the production of ROS, caused cell cycle arrest, decreased cell proliferation and induced apoptosis in Colo 320 DM cells. Thus, this study put forward DAS as a drug that can possibly be used to treat cancers.

Sundaram, S. G. and J. A. Milner (1996). “Diallyl disulfide suppresses the growth of human colon tumor cell xenografts in athymic nude mice.” J Nutr 126(5): 1355-61.

The present studies examined the anti-proliferative effects of diallyl disulfide (DADS) on the growth of human colon tumor cell line, HCT-15, xenografts in 6-wk-old female NCr nu/nu mice with an initial body weight of 20-22 g. Intraperitoneal injection of 1 mg DADS thrice weekly reduced tumor volume by 69% (P < 0.05) without apparent ill consequences such as altered growth of the host. Providing this quantity of DADS intragastrically also inhibited growth of the HCT-15 tumor. At equivalent DADS dosages, intraperitoneal treatment was proportionately more effective (P < 0.05) in reducing tumor growth than gastric intubation. Tumor inhibition caused by DADS (0.5 mg thrice weekly) was similar to that occurring with 5-fluorouracil (5-FU) treatment (0.5 mg thrice weekly). Combining DADS and 5-FU was no more effective in inhibiting tumor growth than using either compound alone. However, concurrent DADS treatment significantly (P < 0.05) inhibited the depression in leukocyte counts and spleen weight and prevented the elevated plasma urea caused by 5-FU treatment. These data suggest that DADS, a constituent of garlic oil, reduces the toxicity of 5-FU and is an effective antitumorigenic agent against xenografts resulting from an established human colon tumor cell line.

Takahashi, Y. and Y. Niitsu (1994). “[Glutathione S transferases-pi].” Gan To Kagaku Ryoho 21(7): 945-51.

It has been known that many drug resistant factors including p-glycoprotein related to anticancer drug resistance. It is assumed that Glutathione s-transferase (GST) is one of the resistant factors. In this present study, we examined the relationship between GST (especially GST-pi) and drug resistance, and also possibility of overcoming of drug resistance for GST-pi related drug resistance. We studied whether GST-pi directly related to anticancer drug resistance by transfection of GST-pi antisense cDNA into human colonic cancer cell line (M 7609). By transfection, cytosolic GST-pi concentrations decreased and sensitivity for adriamycin increased. It was confirmed that GST-pi directly related to some anticancer drug resistance including adriamycin. Moreover, we also have found that ketoprofen, which is an inhibitor of GST-pi activity, increased Adriamycin sensitivity. That is, partial overcoming of drug resistance was obtained. In future, it will be expected that GST-pi inhibitors etc are tried for overcoming of drug resistance.

Veeravagu, A., A. R. Hsu, et al. (2007). “Vascular endothelial growth factor and vascular endothelial growth factor receptor inhibitors as anti-angiogenic agents in cancer therapy.” Recent Patents Anticancer Drug Discov 2(1): 59-71.

New blood vessel formation (angiogenesis) is fundamental to the process of tumor growth, invasion, and metastatic dissemination. The vascular endothelial growth factor (VEGF) family of ligands and receptors are well established as key regulators of these processes. VEGF is a glycoprotein with mitogenic activity on vascular endothelial cells. Specifically, VEGF-receptor pathway activation results in signaling cascades that promote endothelial cell growth, migration, differentiation, and survival from pre-existing vasculature. Thus, the role of VEGF has been extensively studied in the pathogenesis and angiogenesis of human cancers. Recent identification of seven VEGF ligand variants (VEGF [A-F], PIGF) and three VEGF tyrosine kinase receptors (VEGFR- [1-3]) has led to the development of several novel inhibitory compounds. Clinical trials have shown inhibitors to this pathway (anti-VEGF therapies) are effective in reducing tumor size, metastasis and blood vessel formation. Clinically, this may result in increased progression free survival, overall patient survival rate and will expand the potential for combinatorial therapies. Having been first described in the 1980s, VEGF patenting activity since then has focused on anti-cancer therapeutics designed to inhibit tumoral vascular formation. This review will focus on patents which target VEGF-[A-F] and/or VEGFR-[1-3] for use in anti-cancer treatment.

Versantvoort, C. H., H. J. Broxterman, et al. (1995). “Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein.” Br J Cancer 72(1): 82-9.

Wang, C. Z., X. Luo, et al. (2007). “Notoginseng enhances anti-cancer effect of 5-fluorouracil on human colorectal cancer cells.” Cancer Chemother Pharmacol 60(1): 69-79.

PURPOSE: Panax notoginseng is a commonly used Chinese herb. Although a few studies have found that notoginseng shows anti-tumor effects, the effect of this herb on colorectal cancer cells has not been investigated. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of colorectal cancer that interferes with the growth of cancer cells. However, this compound has serious side effects at high doses. In this study, using HCT-116 human colorectal cancer cell line, we investigated the possible synergistic anti-cancer effects between notoginseng flower extract (NGF) and 5-FU on colon cancer cells. METHODS: The anti-proliferation activity of these modes of treatment was evaluated by MTS cell proliferation assay. Apoptotic effects were analyzed by using Hoechst 33258 staining and Annexin-V/PI staining assays. The anti-proliferation effects of four major single compounds from NGF, ginsenosides Rb1, Rb3, Rc and Rg3 were also analyzed. RESULTS: Both 5-FU and NGF inhibited proliferation of HCT-116 cells. With increasing doses of 5-FU, the anti-proliferation effect was slowly increased. The combined usage of 5-FU 5 microM and NGF 0.25 mg/ml, significantly increased the anti-proliferation effect (59.4 +/- 3.3%) compared with using the two medicines separately (5-FU 5 microM, 31.1 +/- 0.4%; NGF 0.25 mg/ml, 25.3 +/- 3.6%). Apoptotic analysis showed that at this concentration, 5-FU did not exert an apoptotic effect, while apoptotic cells induced by NGF were observed, suggesting that the anti-proliferation target(s) of NGF may be different from that of 5-FU, which is known to inhibit thymidilate synthase. CONCLUSIONS: This study demonstrates that NGF can enhance the anti-proliferation effect of 5-FU on HCT-116 human colorectal cancer cells and may decrease the dosage of 5-FU needed for colorectal cancer treatment.

Wierstra, I. and J. Alves (2008). “The c-myc promoter: still MysterY and challenge.” Adv Cancer Res 99: 113-333.

The transcription factor c-Myc is a key regulator of cell proliferation, cell growth, differentiation, and apoptosis. Deregulated c-myc expression possesses a high transformation potential and the proto-oncogene c-myc represents a promising target in anticancer therapy. This review on the c-myc promoter describes its organization, the different levels of its normal regulation (including initiation and elongation of transcription, the dual P1/P2 promoters, chromatin structure, c-Myc autosuppression) as well as its deregulation in Burkitt’s lymphoma. Furthermore, it summarizes the many different transcription factors, signal transduction pathways, and feedback loops that activate or repress c-myc transcription. Finally, a concept for regulation of the c-myc promoter in different biological settings, for example, immediate-early induction, constant expression throughout the cell cycle in continuously cycling cells, repression during terminal differentiation and deregulation in cancer, is formulated.

Woerner, S. M., M. Kloor, et al. (2005). “Microsatellite instability of selective target genes in HNPCC-associated colon adenomas.” Oncogene 24(15): 2525-35.

Microsatellite instability (MSI) occurs in most hereditary nonpolyposis colorectal cancers (HNPCC) and less frequently in sporadic tumors as the result of DNA mismatch repair (MMR) deficiency. Instability at coding microsatellites (cMS) in specific target genes causes frameshift mutations and functional inactivation of affected proteins, thereby providing a selective growth advantage to MMR deficient cells. At present, little is known about Selective Target Gene frameshift mutations in preneoplastic lesions. In this study, we examined 30 HNPCC-associated MSI-H colorectal adenomas of different grades of dysplasia for frameshift mutations in 26 cMS-bearing genes, which, according to our previous model, represent Selective Target genes of MSI. About 30% (8/26) of these genes showed a high mutation frequency (> or =50%) in colorectal adenomas, similar to the frequencies reported for colorectal carcinomas. Mutations in one gene (PTHL3) occurred significantly less frequently in MSI adenomas compared to published mutation rates in MSI carcinomas (36.0 vs 85.7%, P=0.023). Biallelic inactivation was observed in nine genes, thus emphasizing the functional impact of cMS instability on MSI tumorigenesis. Some genes showed a high frequency of frameshift mutations already at early stages of MSI colorectal tumorigenesis that increased with grade of dysplasia and transition to carcinoma. These include known Target Genes like BAX and TGFBR2, as well as three novel candidates, MACS, NDUFC2, and TAF1B. Overall, we have identified genes of potential relevance for the initiation and progression of MSI tumorigenesis, thus representing promising candidates for novel diagnostic and therapeutic approaches directed towards MMR-deficient tumors.

Wrzesinski, S. H., M. L. McGurk, et al. (2007). “Successful desensitization to oxaliplatin with incorporation of calcium gluconate and magnesium sulfate.” Anticancer Drugs 18(6): 721-4.

Since the results of the MOSAIC trial demonstrated an improved disease-free survival in stage III colorectal patients treated with oxaliplatin combined with 5-fluorouracil and folinic acid when they were compared with those treated with 5-fluorouracil and folinic acid alone, the addition of this organoplatin to 5-fluorouracil and folinic acid has become first-line adjuvant treatment for stage III colorectal cancer. Unfortunately, there is a small population of patients who develop grade III/IV hypersensitivity reactions to oxaliplatin which, until recently, have interfered with further treatment with oxaliplatin-containing regimens. Successful oxaliplatin desensitization protocols for patients having severe oxaliplatin hypersensitivity reactions have been reported. However, none of these protocols, have incorporated magnesium and calcium salts. Retrospective data has suggested that pretreating colorectal cancer patients with magnesium sulfate and calcium gluconate before the administration of oxaliplatin may reduce the incidence of neurotoxicities induced by this drug. Therefore, we modified a previously published oxaliplatin-desensitization protocol by incorporating intravenous calcium gluconate and magnesium sulfate, and report a patient with stage IIIc colorectal cancer and prior severe hypersensitivity reactions to oxaliplatin who underwent successful oxaliplatin desensitization using this protocol.

Wynter, M. P., S. T. Russell, et al. (2004). “Effect of n-3 fatty acids on the antitumour effects of cytotoxic drugs.” In Vivo 18(5): 543-7.

BACKGROUND: n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cachexia, and it is thus important to know of any potential interactions with ongoing cytotoxic drug therapy. MATERIALS AND METHODS: For this reason eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were administered to mice bearing the cachexia-inducing MAC16 colon adenocarcinoma, and the effect of epothilone, gemcitabine, 5-fluorouracil and cyclophosphamide on tumour growth and body weight determined. RESULTS: Epothilone alone had a minimal effect on tumour growth rate, but this was potentiated by DH4, while for 5-fluorouracil and cyclophosphamide tumour growth inhibition was enhanced by EPA. The antitumour effect of gemcitabine was not altered by either fatty acid. EPA arrested the development of cachexia, while DHA had no effect and the same was true for their effect on tumour growth rate. The anticachectic effect of EPA was only seen in combination with 5-fluorouracil. CONCLUSION: These results suggest that n-3 fatty acids do not interfere with the action of chemotherapy and may potentiate the effect of certain agents.

Zaman, G. J., J. Lankelma, et al. (1995). “Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.” Proc Natl Acad Sci U S A 92(17): 7690-4.