ACCREDITATION InnoVision Communications is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. CREDIT DESIGNATION STATEMENT InnoVision Communications designates this educational activity for a maximum of 1.5 AMA PRA Category 1 CreditsTM. Physicians should only claim credit commensurate with the extent of their participation in the activity. STATEMENT OF PURPOSE Natural products are being used as supplements by cancer patients, with or without the knowledge of their cancer treatment team. It is important to know which of these products show efficacy against diseases such as cancer, and which are ineffective. It is also essential to define the mechanism(s) of action of natural products, especially as relevant to cancer prevention or treatment. The purpose of this article is to review the use of one such natural product, a fermented wheat germ extract (FWGE ), in the treatment regimen of cancer patients. FWGE has shown efficacy in both animal cancer models and human clinical trials with cancer patients, but more well-controlled trials in humans are necessary to assess the full potential of FWGE in cancer treatment. 

FWGE exerts its anticancer effect via an array of mechanisms, likely because there are many undefined components in this product that modulate numerous biological systems in cancer patients. TARGET AUDIENCE This activity is designed to meet the educational needs of physicians and other healthcare professionals who diagnose, treat and manage patients who have or are at risk for cancer. OBJECTIVES Upon completion of this article, participants should be able to: 1. Define FWGE and discuss its safety profile. 2. Describe the mechanisms by which FWGE exerts its anticancer activity. 3. Review the efficacy of FWGE in cancer prevention and therapy, both in animal models and human clinical trials. (Altern Ther Health Med. 2007;13(2)56-63.) DISCLOSURE InnoVision Comunications assesses conflict of interest with its faculty, planners, and manager of CME activities. Conflicts of interest that are identified are thoroughly vetted by the CME committee for fair balance, scientific objectivity of studies utilized in this activity, and patient care recommendations. InnoVision is committed to providing its learners with highquality, unbiased, and state-of-the-art education. Gary L. Johanning, PhD, and Feng Wang-Johanning, MD, PhD, have no real or apparent conflicts of interest to report.

The American Cancer Society estimated in 2006 that more than 2.4 million new cancer cases, including basal and squamous cell skin cancers, would be diagnosed in the United States that year.1 Cancer patients in the United States generally are treated using conventional therapy, which includes surgery, chemotherapy, radiotherapy, and newer, more targeted therapies such as immunotherapy, gene therapy, angiogenesis inhibitors and targeted therapies.2 With improved diagnosis and treatment, the 5- year survival rate of cancer patients will likely increase, and these cancer survivors will try to find treatments to prevent cancer recurrence and to advance longevity after a diagnosis of cancer. A recent study found that a large percentage of breast and prostate cancer patients use some form of complementary therapy, with vitamins being used by 63% and 37% of breast and prostate cancer patients, respectively, and diet and nutrition therapy being used by 84% and 46% of these patients.3 This study suggests that cancer patients have a strong desire to seek out alternative diet and nutritional therapies to augment their conventional cancer therapy. The challenge to physicians and healthcare providers, then, is to provide information to cancer patients about which therapies are likely to be beneficial to them in preventing recurrence of cancer and promoting their well-being. This review will critically evaluate the efficacy of a plant extract that is currently being evaluated in clinical trials for treatment of cancer patients. The product is a fermented extract of wheat germ called FWGE . This article discusses how the extract is made, whether it is safe, its mode of action, and, finally, the use of FWGE in clinical cancer trials.

 WHAT IS FWGE , AND HOW IS IT PRODUCED? The wheat grain kernel consists of 3 parts. The endosperm is the embryo of the kernel. It makes up 83% of the kernel and is the source of energy for new wheat plants if the kernel is planted and sprouts. It is high in starch and gluten (the protein in wheat flour), but relatively low in vitamin and mineral content. The endosperm is used to make white flour. The bran (14%) consists of the thin outer layers of the wheat kernel and contains vitamins, minerals, and fiber. The germ makes up only 2%-3% of the wheat kernel and is the most nutritious part of the wheat kernel. Nutrients are concentrated in the germ, and it is rich in vitamins, minerals, proteins, and fats. Wheat germ contains high levels of tocopherol and B vitamins. It is separated from the other wheat components by the milling process. Whole-wheat flours are made by milling the whole kernel; that is, all 3 of the above parts of the wheat kernel. In addition to the nutrients listed above, wheat germ can be subjected to fermentation with Saccharomyces cerevisiae (yeast) to yield the benzoquinones 2,6-dimethoxy-benzoquinone (DMBQ ) and 2-methoxy-benzoquinone.4 These benzoquinones are present in unfermented wheat germ as glycosides; yeast glycosidase activity present during fermentation leads to release of the benzoquinones as aglycones. FWGE is an aqueous extract of wheat germ, fermented with Saccharomyces cerevisiae for 18 hours at 30˚C.5 After fermentation, water is decanted, and the product is spray-dried, homogenized, encapsulated, and formulated. 

The wheat germ fermentation end-product, which is suitable for human consumption, is a dried extract standardized to contain methoxy-substituted benzoquinones (2-methoxy-benzoquinone and 2,6-DMBQ ) at a concentration of 0.04%. Since FWGE is a complex mixture, additional, as yet poorly characterized molecules remain in the product. Nobel laureate and Hungarian scientist Dr Albert SzentGyörgyi initially proposed the use of methoxy-substituted benzoquinones like those present in FWGE as anticancer agents. He hypothesized that disorders of metabolism might play important roles in cancer development, and found that high redox potential quinones such as those discussed above could block cell replication6 and suggested that they might prove to be useful in reversing disorders of cellular metabolism. FWGE was developed by the Hungarian biochemist Máté Hidvégi and was registered in Hungary as medical nutriment no. 503 in 2002. It is approved there as a non-prescription medical nutriment for cancer patients. FWGE also has been registered as a special nutriment for cancer patients in the Czech Republic and Bulgaria and is on the Australian register of Therapeutic Goods. It is currently available in 10 countries. In the United States and a number of other countries, FWGE is classified as a dietary supplement. It is manufactured in a Good Manufacturing Practices (GMP) facility by Biromedicina First Hungarian Corporation for Cancer Research and Oncology in Budapest and is distributed in the United States as Avé, a dietary supplement instant-drink mix. IS FWGE  SAFE TO CONSUME?

 Several studies have been carried out to evaluate the safety of FWGE in doses used for treatment of cancer and autoimmune diseases. Boros et al discussed some of the studies in animals and humans that provide an indication of its safety;7 studies in these species to date suggest few adverse effects of FWGE . Acute and subacute toxicology tests carried out in a Good Laboratory Practice (GLP) setting revealed minimal side effects. Toxicity studies in the rat and mouse demonstrated an acute oral LD50 of FWGE in male and female mice and rats of greater than 2,000 mg/kg. The no-observable adverse effect level, which is the greatest concentration or amount of FWGE  that causes no detectable adverse alteration, was 2,000 mg/kg/day in rats, and in a subchronic study with mice and rats was found to be 3,000 mg/kg/day. There is a wide therapeutic window for FWGE . Doses toxic to normal cells are more than 50 times higher than the dosage recommended for therapy, which suggests that a wide range of therapeutic dosages can be tested before the product becomes toxic. The US Food and Drug Administration recently granted FWGE a status of Generally Recognized As Safe (GRAS), which allows it to be used in foods, drinks, and dietary supplements. Significant side effects have not been reported, but mild and transient diarrhea, nausea, flatulence, soft stool, constipation, dizziness, and increase in body weight can accompany the consumption of FWGE . 

Hematologic evaluations of hospitalized cancer patients in Hungary found that the white blood cell count, lymphocyte count, neutrophil granulocyte count, monocyte count, eosinophil granulocyte count, hemoglobin level, red blood cell count, erythrocyte sedimentation rate, hematocrit, platelet count, and prothrombin level were normal after 1-5 years of FWGE treatment.7 MECHANISM OF ACTION OF FWGE Since FWGE is a plant extract, the exact chemical composition is not known, andFWGE the constituent(s) that is active against cancer has not yet been identified. The methoxy-substituted benzoquinones are good candidates for the active ingredients in FWGE , but studies have shown that these may not be the important compounds in FWGE possessing immunostimulatory activity. As discussed in more detail in the “Immunomodulation” section of this article, FWGE in mice shortened the survival time of skin grafts in comparison to controls. However, DMBQ given in a dose equivalent to the lower FWGE dose did not have any effect on skin grafts whereas a DMBQ dose equivalent to the higher dose of FWGE actually elongated the graft survival. In addition, the higher DMBQ dose was toxic and resulted in the death of 5 experimental animals during the study.8 FWGE has documented anticancer activities, which will be discussed in the next section. Many cancer patients are using FWGE as a cancer treatment, so it is important to understand its mode of action, both from the standpoint of providing an explanation for any untoward effects that might develop with its use, as well as to identify potential novel pathways that lead to beneficial effects in cancer patients. 

What are some of the potential mechanisms that modulate the anticancer effects of FWGE ? The molecular targets of FWGE , discussed below, include apoptosis induction via poly (ADP-ribose) polymerase (PARP) and other pathways, the immune system, major histocompatibility complex (MHC) class I, ribonucleotide reductase (RNR), cyclooxygenase (COX-1 and COX-2) enzyme activity, intracellular adhesion molecule (ICAM) 1, tumor necrosis factor alpha (TNF-α) production, and transketolase (TK). This is a relatively large number of molecular targets, which suggests that several as yet undefined components of FWGE may promote its antineoplastic action. The discovery of individual active compounds in FWGE should thus be pursued to find which components are responsible for each biological effect.Cell Cycle, Induction of Apoptosis and Poly Polymerase Cleavage FWGE influences apoptosis (programmed cell death) via several molecular pathways. Since apoptosis involves the killing of cancer cells, a major mechanism of FWGE action is apoptosis induction. Probably the most significant effect on apoptosis is cleavage of PARP. As discussed below, FWGE activates downstream caspase-3 proteases, resulting in cleavage of PARP and subsequent prevention of DNA repair in cancer cells. 

The cytotoxic effects of FWGE have been documented in several studies, and cell death generally occurred by apoptosis and in some cases, necrosis. FWGE treatment decreased the number of Jurkat T-cell leukemia cells that accumulated a formazan dye (MTT), and this decrease was greater at higher FWGE doses, indicating that FWGE decreases cancer cell viability.9 Cell cycle analysis by flow cytometry after propium iodide (PI) staining revealed that cells treated with 0.7 and 1 mg/mL FWGE had an increase in the sub-G1 region of the cell cycle, which is associated with apoptosis, and a significant decrease in the S phase, and these changes became prominent at 48 and 72 hours following FWGE treatment. The effective dose of FWGE for inhibiting tumor metastasis formation in cancer patients in clinical trials is 0.5 to 1 mg/mL,10 so this dosage is physiologically relevant. FWGE caused an increase in apoptosis, as measured by flow cytometry after PI and annexin V staining, in Jurkat cells, beginning at doses of 0.5 mg/mL. Doses of 0.5 and 1 mg/mL showed a greater apoptotic response at 72 hours than at 24 hours of treatment, and doses of 5 and 10 mg/mL FWGE showed a time-independent maximal effect, with approximately 90% of cancer cells undergoing apoptosis. Laser scanning cytometry experiments showed that FWGE -treated cells had undergone apoptosis, not cell death by necrosis. The authors of this study used a caspase inhibitor to see whether the phosphatidylserine externalization characteristic of caspase action is reversed in FWGE -treated cells. Movement of phosphatidylserine from the inner to the outer plasma membrane of the cell is a characteristic that distinguishes apoptosis from necrosis.

 The caspase inhibitor Z-VAD.fmk did indeed block the FWGE ar-induced increase in apoptosis in cells treated with 1 mg/mL FWGE for 72 hours, thus demonstrating that the apoptosis resulting from FWGE treatment is due to caspase activation. To further investigate the involvement of caspases in AvemFWGE r action, the effect of FWGE at doses of 0.3, 0.5, and 0.7 mg/mL on cleavage of PARP was determined, and cleavage of PARP was observed at FWGE doses above 0.5 mg/mL and was especially evident at a 0.7-mg/mL FWGE concentration. PARP plays an important role in DNA repair, and its cleavage leads to DNA fragmentation, resulting in the apoptosis that accompanies FWGE treatment. Breast cancer cells also respond to FWGE by inducing apoptosis. Marcsek et al reported that viability of the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) began to decrease when the cells were treated with levels of FWGE between 0.625 and 1.25 mg/mL,11 levels roughly the same as those cytotoxic to Jurkat cells in the studies described above. Cell cycle S phase and apoptosis were determined by flow cytometry based on PI and anti-5- bromo-2’-deoxyuridine (BrdU) fluorescence. FWGE strongly enhanced apoptosis of MCF-7 cells 24 and 48 hours after treatment, and this effect on apoptosis was even greater in cells treated with a combination of FWGE and the estrogen receptor modulator tamoxifen. In contrast to what was observed with Jurkat cells, the percentage of MCF-7 cells in the S phase of the cell cycle increased after 24 hours of FWGE treatment, followed by a decrease to control levels in cells treated for 48 hours.

 Colon cancer HT-29 cells showed decreased colony formation in clonogenic assays, with an IC50 FWGE for FWGE (concentration of FWGE that results in 50% of the colony formation observed in controls) of 0.118 mg/mL,12 which is considerably lower than the levels of 0.5 to 1 mg/mL that show clinical efficacy. When vitamin C was co-administered with FWGE , the IC50 was lowered still further, with a value of 0.075 mg/mL when 100 µM vitamin C was added. Vitamin C was used here because it was previously demonstrated that vitamin C influenced the effects of FWGE when these 2 compounds were co-administered. Similar to what was found in the studies with Jurkat cells, FWGE increased the percentage of cells in the G0-G1 phase of the cell cycle and led to an arrest of the cell cycle in the G1 phase, with a subsequent depletion of cells in the S and G2-M phases. In contrast to the effect of FWGE almost exclusively involving apoptosis in Jurkat cells, in HT-29 colon cancer cells, FWGE in concentrations of 0.8 to 3.2 mg/mL induced predominantly necrosis rather than apoptosis, although apoptosis did begin to increase at high FWGE concentrations. One important aspect of FWGE as it relates to cell death is that it does not induce apoptosis in normal cells such as peripheral blood mononuclear cells.13 Immunmodulation and Inhibition of MHC-I FWGE has a stimulatory effect on cellular immune response, an effect first observed in mice.8 In this study, FWGE increased the amount of 3H thymidine incorporated into mouse spleen lymphocytes in response to Concanavalin A, which means that it stimulated lymphoblastic transformation. 

Further, an attempt was made to determine whether FWGE could influence the rejection period of skin grafts. A skin allograft from one mouse species was implanted into another, thymectomized mouse species. Thymectomized mice treated with FWGE at doses ranging from 0.03 g/kg to 3.0 g/kg had significantly shorter graft survival times than thymectomized mice not treated with FWGE . This indicates that FWGE stimulated cellular immune response of the recipient mice so that they rejected the skin grafts from donors more quickly than recipients not treated with FWGE . Tumor cells can avoid the adaptive immune response of cytotoxic T lymphocytes by downregulating major histocompatibility complex class I (MHC class I) expression on the cell surface. FajkaBoja et al found that FWGE treatment decreased major histocompatibility complex class I (MHC class I) antigen expression on the cell surface of lymphoid tumor cells. The cells included Jurkat leukemic T cells and 2 mutant phenotypes of this cell line (one of which was CD45 deficient), the Burkitt lymphoma B cell lines BL41 and Raji, and the myelo-monocytic cell line U937.13 They found that 4-hour treatment of Jurkat cells with 2 mg/mL FWGE caused a 90% decrease in cell surface MHC class I molecules and that the DMBQ component of FWGE caused a 70% decrease in MHC class I expression. In Raji cells treated with FWGE and DMBQ under the same conditions, MHC class I was downregulated by 69% and 30%, respectively.

 The wheat germ agglutinin present in FWGE was not responsible for the downregulation of cell surface MHC class I. When tyrosine phosphatase activity was inhibited with vanadate, MHC class I inhibition was greater than with FWGE alone, whereas blockage of the FWGE -induced Ca2+ influx with the Ca2+ chelator EGTA led to less downregulation than was observed with FWGE alone. Vanadate also increased FWGE apoptosis, while EGTA decreased apoptosis due to FWGE treatment. These changes in apoptosis mirror the effects of FWGE on MHC class I downregulation. In this same study, various other aspects of T and B cell metabolism were considered to help explain how protein phosphorylation and Ca2+ transport influence MHC class I downregulation in response to FWGE treatment. The authors first found that treatment of either B or T cell lines with FWGE at 5 mg/mL for 10 minutes led to reproducible tyrosine phosphorylation of specific proteins. The pattern of tyrosine phosphorylation in response to FWGE was different from the pattern when an antibody to the T cell receptor was used to stimulate the Jurkat T cells. Proteins with molecular weights of 76, 63, and 38 kDa were uniquely expressed in Jurkat cells treated with FWGE . This suggests that FWGE uses a mode of stimulation that is independent of the T cell receptor in Jurkat cells. Likewise, when the B cell line BL-41 was stimulated with FWGE at the same concentration and time used for Jurkat cells, the protein phosphorylation pattern was different from when these cells were B cell receptor-stimulated.

 The 63-kDa protein was again expressed in BL-41 cells but not in BL-41 cells stimulated with the B cell receptor, which indicates that the 63-kDa protein plays a specific role in response to FWGE that is independent of the B or T cell receptors. CD45 is a cell surface receptor expressed on leukocytes that plays a key role in leukocyte signaling.14 FWGE at 5 mg/mL inhibited the tyrosine phosphatase activity of CD45, but this inhibition was found to be due to the wheat germ agglutinin present in FWGE . Finally, FWGE treatment at a concentration of 5 mg/mL caused a transient (25 to 125 seconds after treatment) 3-fold increase in the intracellular Ca2+ concentration. This increase was blocked by the extracellular Ca2+ chelator EGTA, showing that the Ca2+ influx was from the intracellular space. MHC class I downregulation by FWGE in T and B cell lines may make them susceptible to natural killer (NK) cell activity. NK cells are a key component of anticancer immune defense, and NK killing is blocked by MHC class I proteins expressed on the cell surface.15 The overexpression of MHC class I molecules on the cell surface is one means by which cancer cells evade eradication by the immune system by avoiding the attack of NK cells. If FWGE decreases the expression of MHC class I proteins on the cancer cell surface, this should make the cancer cells more susceptible to NK cell killing.Pentose Phosphate Pathway, Glucose, and Nucleic Acid Metabolism The expression of genes that promote and suppress cancer, such as oncogenes and tumor suppressor genes, is modified in tumors; these genes play critical roles in cancer cell proliferation, differentiation, and death. Another difference between normal and tumor cells is the modification of biochemical pathways in tumors.