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The Role of Folate, Vitamin B-12, and Vitamin B-6 in the Provision of Methyl Groups. John M. Scott. Department of Biochemistry, Trinity College, Dublin, Ireland.
All human cells have several dozen methyltransferases. The range of methylated substrates varies widely and such methylations serve a wide range of functions. The methylation of cytosine residues in preformed DNA controls gene expression. The methylation of phospholipids affects receptor function. Protein methylation can change stability. However, although the substrates vary widely, the form in which these methyl groups must be provided to the various methyltransferases is always the same, namely as S-adenosylmethionine (SAM). The immediate origin of SAM is always the same: methionine is activated to SAM by the enzymatic addition of ATP. The three sources of this methyl group on methionine are dietary methionine, methylation of homocysteine with betaine, and methylation of homocysteine with 5-methyltetrahydrofolate (5-MTHF). Where a methyl group originated depends on whether the methylation reaction took place in the hepatocytes in the liver or in some other cell type, because liver hepatocytes play an additional role in methionine and choline catabolism. They are responsible for the disposal of the 
60% of methionine that exists in the normal diet over and above that required for protein synthesis. Nature has decided to degrade this methionine via SAM, S-adenosylhomocysteine (SAH), and homocysteine, which can then be passed down the transsulfuration pathway via cystathionine synthase to cysteine. Cysteine is important in its own right, being used for protein and glutathionine biosynthesis. Excess over the requirement of cysteine is further catabolized to pyruvate and sulfate with the former being used for energy or gluconeogenesis. Hepatocytes can also remethylate homocysteine back to methionine as part of the methylation cycle, a feature shared with all other cells and requiring a methyl group to be supplied as 5-MTHF using the vitamin B-12–dependent enzyme methionine synthase. How much remethylation occurs in the liver depends on the need to maintain a constant level of SAM. The liver and consequently the hepatocytes also have a further catabolic function, namely the catabolism of excess choline over requirements that have arisen from biosynthesis or as part of the diet. Choline is degraded in the mitochondria to betaine, which is exported to the cytoplasm and used to methylate homocysteine by the enzyme betaine homocysteine methyltransferase, which is independent of vitamin B-12. Thus, nature has charged the liver with the catabolism of both methionine and choline.
As mentioned, this catabolic pathway involves the conversion to SAM, SAH, homocysteine, etc. It is obvious that this involves the donation of the methyl group of SAM via a methyltransferase to some appropriate methyl acceptor. Methyltransferases are involved in a variety of critical functions. It would thus be quite inappropriate for the liver to use one of these as a method of disposing of or dumping excess methyl groups. Consequently, the liver has 2 unusual methyl acceptors that can mop up large amounts of methyl groups supplied by methionine and choline as SAM. These are guanidinoacetate, which is methylated in the biosynthesis of creatine, and sarcosine, which is produced by the methylation of glycine. Creatine biosynthesis is constant, being related to body muscle mass, whereas creatine phosphate acts as an energy store. Its importance is that it is extensive and 
80% of the methyl groups in the body are used in this pathway. The methylation of glycine is elective and it is used to get rid of the variable excess of methyl groups over and above the large but constant amount removed for creatine biosynthesis. Interestingly, glycine methyltransferase is the only methyltransferase not inhibited by SAH and clearly it is expected to dispose of methyl groups even if SAH accumulates and other methyltransferases are inhibited. The liver has a range of other methyltransferases that it uses for specific methylations, and these will be subject to the level of SAM, the substrate, and SAH of the product. These, like in other cells, will be very much influenced by the relative levels of these two derivatives, sometimes called the SAM-SAH ratio. To maintain a favorable SAM-SAH ratio the hepatocytes, in common with other cells, will have the option of remethylating homocysteine back to methionine and then to SAM using the methionine synthase. For this to happen, the liver must have an adequate supply of vitamin B-12, a cofactor for this enzyme, and an adequate supply of 5-MTHF as the methyl donor. This in effect will mean that the methylation cycle occurs in the liver to a varying extent.
What about the provision of methyl groups for other cells in the body? Clearly other cells would have the option like the liver of using circulating methionine. However, this would mean that for each methylation reaction a SAH and consequently a homocysteine would be generated. It would thus fall to such cells to either export or catabolize this resultant homocysteine. Nature has elected not to attempt this and most cells either do not have any of the enzymes of the transsulfuration pathway or only have the first one or two. It clearly does not make sense for most cells to generate large amounts of cysteine. The catabolism of such cysteine by the liver to pyruvate involves transaminating the potentially toxic amino group, which the liver can get rid of in the urea cycle, an option not open to other cells. In addition, unlike most other cells, hepatocytes have a high demand for cysteine, protein, and glutathione biosynthesis. To maintain their SAM levels, other cells complete the methylation cycle (i.e., they remethylate the homocysteine being generated via SAH, after the actions of the different methyltransferases, back to methionine and thus provide a replacement of SAM). For this they need a source of methyl groups that come solely via 5-MTHF. Most cells are going to be very dependent on an adequate supply of these methyl groups via 5-MTHF and for an adequate amount of folate and vitamin B-12 for methionine synthase. If either is compromised, the methylation cycle in all cells will be compromised. The cells' response to such dropping SAM levels will be to increase the activity of the enzyme that provides 5-MTHF, namely 5,10-methyltetrahydrofolate reductase (MTHFR). This enzyme has an inhibitory allosteric binding site for SAM. As the level of SAM decreases, this enzyme becomes more and more active, channeling more and more 1-carbon groups away from 5,10-methylenetetrahydrofolate (5,10-methylene THF) and 10-formyltetrahydrofolate (10-formyl THF) used by the cell for pyrimidine and purine biosynthesis, respectively. This response will reinstate the SAM levels. However, if folate status is inadequate, the cell is faced with a choice of distributing its folate cofactors between the DNA biosynthesis cycle and the methylation cycle. If the cell is replicating, this may have metabolic consequences. It is interesting that there is evidence that being homozygous for the C
T 677 polymorphism, which lowers the activity of MTHFR, appears to protect against the development of colon cancer but only in subjects who have inadequate folate status. However, given adequate folate the cell appears to adjust to get the required amount of methyl groups for all of its methyltransferase by upregulating MTHFR. This consequently increases amounts of 5,10-methylene THF converted to 5-MTHF. Cells appearto have an unlimited supply of 5,10-methylene THF if folate status is adequate. This 5,10-methylene THF was traditionally thought to be synthesized by the action of the enzyme serine hydroxymethyltransferase, which attaches carbon 3 of serine to tetrahydrofolate to make 5,10-methylene THF. Serine is widely available in all cells, being synthesized in 3 enzymatic steps from 3-phosphoglycerate, an intermediate of the glycolytic pathway. The source of the amino group is glutamate, which is also readily available in all cells. The 5,10-methylene THF so synthesized can then be channeled into the methylation cycle by the enzyme MTHFR. Alternatively, it can be used directly by the enzyme thymidylate synthase to make the pyrimidine thymidylate. It can also be metabolized by a complex enzyme called MTHF dehydrogenase-1, through 5,10-methenyl THF to 10-formyl THF. This latter form of folate acts as the carbon 1 donor for 2 of the enzymes involved in purine biosynthesis. These make up the carbon 2 and the carbon 8 of the eventual purine ring.
More recently, it has become clear that in most cells the carbon 3 of serine is also channeled into purine and pyrimidine and ultimately also into the methylation cycle but by a more circuitous route. Cytoplasmic serine enters the mitochondria and via the mitochondrial version of serine hydroxymethyltransferase gets converted to 5,10-methylene THF. This is further metabolized to formate, which exits the mitochondria into the cytoplasm. There, by the action of the trifunctional enzyme (MTHF dehydrogenase-1) mentioned above, the formate is converted into 10-formyl THF and via 5,10-methenyl to 5,10-methylene THF, the first of the cofactors again to be used for purine biosynthesis and the last being used for pyrimidine biosynthesis. Again, MTHFR can convert 5,10-methylene THF into 5-methyl THF and thus into the methylation cycle. This latter pathway involves the intermitochondrial metabolism of serine and probably takes place for some reason related to balancing the redox state within the cell consequent on the need for reducing equivalents to convert ribose into the deoxyribose needed for DNA biosynthesis. Although most of the evidence of this use of cellular serine for the provision of carbon 1 units is from tissue culture or yeast systems, it does appear that this pathway is very extensive. It will obviously depend on the cell type and different needs for purines and pyrimidines or need for the provision of carbon 1 groups to the methylation cycle. In either event, the original source of these carbon groups is cytoplasmic serine.
Thus, under conditions of full folate and vitamin B-12 status, cells have a relatively unlimited supply of carbon 1 groups from serines for both DNA biosynthesis and the methylation cycle. In addition, the liver has 2 other sources of methyl groups, namely the uptake of dietary methionine from the circulation and the methyl groups of betaine, which has arisen solely from the oxidation of choline. It is interesting to consider when folate status begins to diminish. Depending on the cell type, folate cofactors can be segregated between the two cycles that use them, but the balance is not clear. The cells' needs for SAM seems to be ensured by the fact that the lower the SAM the higher the activity of MTHFR and the more that 5,10-methylene THF will be diverted into 5-MTHF and into the methylation cycle. At what point this demand for SAM compromises purine and pyrimidine biosynthesis is unclear. It is also unclear whether in such circumstances of reduced folate status the provision of methyl groups is protected to the detriment of the ability to synthesize DNA. As mentioned, some evidence shows that this may happen. The common CT 677 polymorphism of MTHFR is known to result in an unstable form of this enzyme, which has the effect of reducing its activity in cells. Being polymorphic for this variant has been found in a few studies to protect against the development of certain forms of cancer (e.g., colorectal cancer). It is suggested that when folates are low, their retention for DNA biosynthesis, as distinct from their use for methylation, protects against the transformation of normal cells into adenomatous cells. However, no direct evidence supports this hypothesis. It is equally possible that reduction in the methylation cycle might reduce tumor survival or, in the change from the types of cells seen in the precursor polyps of such tumors, produce full-blown colon cancer.
The reduction of folate status in the liver seems to produce a different pattern. Liver cells do not have a high requirement for purines and pyrimidines because under normal circumstances they do not have a high replication rate. Although they have a high demand for methyl groups principally for creatine biosynthesis, methyl groups are provided by dietary methionine and the catabolism of choline via betaine. The biggest effect of reduced folate status on the liver would probably be its inability to remethylate the homocysteine so generated. Such increased homocysteine usually arises from an excessive burden of methionine to catabolize because of, say, a high protein diet. However, in such circumstances the concentration of SAM would be excessively high. Such high SAM concentrations as well as decreasing the activity of MTHFR increases the activity of cystathionine synthase, which would be the first enzyme to catabolize the excess homocysteine. However, during low folate status, even with a normal uptake of dietary methionine the SAM level may not become high. This will tend to reduce the amount of homocysteine being catabolized by cystathionine synthase, resulting in export into the plasma. Much of the excess homocystine found in the plasma may consequently arise from the export from the liver. Cystathionine synthase also requires vitamin B-6 as a cofactor and its reduced status, which apparently infrequently also alters plasma homocysteine.
Reduction in vitamin B-12 status will progressively impair the provision of methyl groups using the methylation cycle. This will not have a severe effect on the liver because it can convert homocysteine into methionine using betaine-homocysteine methyltransferase, which is independent of vitamin B-12. It also can use dietary methionine to feed its methylation cycle. Thus even in severe vitamin B-12 deficiency there is no evidence that the major use of methyl groups to make creatine is reduced. The effect of reduced vitamin B-12 status on other cells however will be profound. They are not cushioned by betaine or plasma methionine, as in the liver. The reduction in the methylation cycle will directly reduce the level of all of the methyltransferases with potentially a wide range of consequences. This would be as varied as inability to control expression of certain genes, changes in receptor function, protein stability, etc.
Diet, DNA methylation, and cancer
DNA Methyltransferase Function and Regulation. Keith Robertson. Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.
DNA methylation is an epigenetic modification of the genome catalyzed by a group of 3 DNA methyltransferases: DNMT1, 3A, and 3B. Methyl groups are not randomly distributed in mammalian cells but rather are compartmentalized in repetitive DNA, heterochromatic regions, and parasitic elements. Other regions of the genome, such as CpG island promoters, are almost always unmethylated. Given the minimal sequence requirements of the DNMTs (CpG), it is likely that they are directed to sequences that are to be methylated by interactions with other proteins, particularly chromatin-associated factors. This compartmentalization is essential because genetic knockouts of the DNMTs leads to embryonic lethality, and reversal of the normal DNA methylation patterns is a hallmark of most transformed cells. The de novo DNA methyltransferase Dnmt3a has been shown to play crucial roles in embryonic development, genomic imprinting, and transcriptional silencing. Despite its importance, very little is known about how the enzymatic activity and transcriptional repression functions of Dnmt3a are regulated. Using a yeast 2-hybrid screen, we found that Dnmt3a interacts with multiple components of the sumoylation machinery, namely the E2 sumo conjugating enzyme Ubc9 and the E3 sumo ligases PIAS1 and PIASx
, all of which are involved in conjugating the small ubiquitin-like modifier protein, SUMO-1, to its target proteins. Dnmt3a is modified by SUMO-1 in vivo and in vitro and the regions of Dnmt3a responsible for interaction and sumoylation map to the N-terminal regulatory domain. SUMO-1 modification of proteins can dramatically alter their properties. Functionally, sumoylation of Dnmt3a disrupts its ability to interact with histone deacetylases (HDAC1/2) but not with another interaction partner, Dnmt3b. Conditions that enhance the sumoylation of Dnmt3a in vivo, by either SUMO-1 or PIAS protein overexpression, abolished its capacity to repress transcription. These studies reveal a new level of regulation governing Dnmt3a whereby a posttranslational modification can dramatically regulate its interaction with specific protein partners and alter its ability to repress transcription. Alterations in the ability of Dnmt3a to interact with histone deacetylases may also affect its ability to access chromatin or be targeted to regions undergoing histone deacetylation as a precursor to permanent gene silencing.
Family History as a Tool for Evaluating Cancer Genetic Risk. Maren T. Scheuner. Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, and Office of Genomics and Disease Prevention, Centers for Disease Control and Prevention, Atlanta, GA.
Family history is a risk factor for many common cancers, including breast, ovarian, prostate, colon, and endometrial cancer. It reflects the interaction of genetic, environmental, cultural, and behavioral risk factors. Stratification of familial risk into different risk categories (e.g., average, moderate, high) is possible by considering: the number of relatives affected with cancer and their degree of relationship, ages of cancer onset, occurrence of associated cancer diagnoses or other conditions, and gender of affected relatives. Individuals with increased familial risk for cancer might benefit from personalized prevention recommendations specific to their familial risk, which could include assessment and modification of risk factors, lifestyle changes, early detection strategies, and chemopreventive therapies. Individuals with the highest familial risk might have Mendelian disorders associated with a large amount of risk for a spectrum of cancers usually with an early age of onset. In these cases, referral for genetic evaluation should be considered, including pedigree analysis, risk assessment, genetic counseling and education, discussion of available genetic testing, and recommendations for risk-appropriate screening and preventive interventions.
Breast Cancer and Family Genetics—Tackling the Ethical Issues. Marcia Van Riper. Carolina Center for Genome Sciences, School of Nursing, University of North Carolina at Chapel Hill, NC.
Genetic testing for BRCA1-2 mutations is inherently a family experience that can result in family members confronting difficult ethical, legal, and psychosocial issues. To date, few researchers in this area have used a family perspective. The purpose of this research, which was part of a larger study concerning the family experience of genetic testing for 4 different types of genetic testing (multiple marker screening for Down syndrome, carrier testing for cystic fibrosis, BRCA1-2 testing, and mutation analysis for Huntington disease), was to explore how families define and manage the ethical issues that emerge during the genetic testing experience. The guiding theoretical framework for this research was the Family Management Style Model developed by Knafl and colleagues. Family members from 15 families at increased risk for breast cancer were interviewed using a semistructured interview guide. Preliminary findings underscore the need to use a family perspective when exploring ethical issues that emerge during the genetic testing experience. Most of the family members who underwent genetic testing reported that the genetic testing experience had a profound effect on their life, the lives of other family members, and communication patterns within and outside the family. Ethical issues faced by families in this study include concerns about autonomy (the right to know and the right not to know), informed consent, responsibility to future directions, breaches of confidentiality, discrimination by health and life insurance providers, discrimination in the workplace, uncertainties inherent in cancer risks figures, the predictive value of the test results, decisions regarding cancer risk-reduction strategies, and the testing of children. The examination of individual and family responses to ethical dilemmas generated during genetic testing will inform practitioner and consumer decision making as well as policy makers. Furthermore, identification of the particular ethical dilemmas that arise in the context of family is necessary for the development of strategies or interventions that support the family experience.
Epidemiology of Polyphenols and Cancer. Stephanie A. Smith-Warner. Department of Nutrition, Harvard School of Public Health, Boston, MA.
Polyphenols consist of many types of compounds including phenolic acids, lignans, and flavonoids. Flavonoids have been hypothesized to be associated with a lower risk of cancer because of their antioxidant, antiproliferative, antiinflammatory, and estrogenic properties. Until recently, comprehensive food composition databases for specific flavonoids were not available. Consequently, few epidemiologic studies have estimated flavonoid intakes or examined their associations with the risk of cancer. Total flavonol and flavone intake generally has been estimated in epidemiologic studies as the sum of the intakes of the flavonols quercetin, kaempferol, and myricetin and of the flavones luteolin and apigenin. Tea, onions, and apples generally have been the main contributors to total flavonol and flavone intake. In a limited number of studies, frequently with small numbers of cases, total flavonol and flavone intake has not been associated with cancer mortality. Of the specific cancer sites examined, associations with lung cancer have been reported most frequently. All but one of the studies have reported a relative risk in the protective direction for total flavonol and flavone consumption; however, few associations were statistically significant. Limited evidence is available evaluating the association between specific flavonoids and cancer risk or between total flavonol and flavone intake and the risk of other types of cancer. With the recent formation of food composition databases of specific flavonoids, further investigation of the association between total and specific flavonoid intakes and cancer risk will be possible.
Tea and Cancer Prevention. Chung S. Yang. Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ.
Consumption of tea (Camellia sinensis) has been suggested to prevent cancer and other diseases. Animal studies have shown that tea and tea constituents inhibit carcinogenesis of the skin, lung, oral cavity, esophagus, stomach, liver, prostate, and other organs. The mechanisms of this inhibitory activity, however, are not clear. In some studies, the inhibition correlated with an increase in tumor cell apoptosis and decrease in cell proliferation. Studies with human cancer cell lines demonstrated that epigallocatechin-3-gallate (EGCG), a major tea polyphenol that has been proposed to be the major active ingredient, inhibits mitogen-activated protein kinases, cyclin-dependent kinases, growth factor–related cell signaling, the activation of activator protein 1 and nuclear factor–
B, topoisomerase I, and matrix metalloproteinases as well as other enzyme activities. Most of these experiments were conducted with 10 µmol/L, 20 µmol/L, or higher concentrations of EGCG. The importance of these activities in the inhibition of carcinogenesis in animals and humans remains to be demonstrated. Other active constituents also need to be considered. For example, in the UV-induced skin tumorigenesis model in mice, caffeine has been demonstrated to be the key effective constituent. In humans, tea polyphenols undergo glucuronidation, sulfation, methylation, and ring fission. After tea ingestion, the peak plasma concentration of EGCG is 
1 µmol/L. The bioavailability of tea constituents as well as the quantity of tea ingestion determine the biological effects of tea consumption. This factor may explain the inconsistent results concerning the effect of tea consumption on human cancer in epidemiologic studies. More quantitative information or biomarkers for tea consumption and proper control of confounding factors are needed to provide a better understanding of this topic. Even though the results from epidemiologic studies are inconclusive, tea may still be explored for prevention of cancer at selected organ sites. [Supported by National Institutes of Health grants CA 56673 and CA 88961.]
Cranberry Constituents and Prostate Cancer Cell Growth. P. J. Ferguson*
E. Kurowska# D. Freeman
A. Chambers** D. J. Koropatnick***. *Department of Physiology & Pharmacology, Department of Medicine, **Department of Oncology, Department of Medical Biophysics, and Department of Microbiology & Immunology, University of Western Ontario, the London Regional Cancer Centre and #KGK Synergize, Inc., London, Ontario, Canada.
Prostate cancer is the second leading cause of cancer death among males in North America. Despite the many advances in cancer treatment, chemotherapy of solid tumors is still greatly limited by lack of selectivity to tumor cells and recurrence of drug-resistant tumors. Antiandrogenic therapies can control prostate tumor growth for up to 2–3 y, but the agents used have numerous unpleasant and toxic side effects. This tumor eventually undergoes changes and emerges with an androgen-independent population against which chemotherapy has very limited success. Alternative treatments are needed that are effective against tumors that have progressed to androgen-independence and are at least limited in their side effects. The significantly lower incidence of prostate cancer in Asia compared with North America, in light of the very different diets of these populations, suggests that dietary components may prevent this disease (1). Therefore, safely ingested foods may be a source of agents that can be used to treat prostate cancer without untoward side effects.
In a preliminary experiment, cranberry fed to human breast–tumor-bearing, immunodeficient mice reduced the growth of tumors and metastases (2). Cranberry extracts were also shown to have anticarcinogenic (3) and antiproliferative activities (4). Numerous phytochemicals, including flavonoids and phenolics, have been previously characterized for their anticancer properties. Although cranberries (Vaccinia macrocarpa) are a rich source of phytochemicals, the components of cranberry responsible for its anticancer activity are not known. Using fractionating techniques and in vitro assays against a variety of human tumor cell lines, we have identified groups of components of cranberry that inhibit proliferation of cancer cells. Extracts were obtained from cranberry presscake (the material remaining after the extraction of juice) and also homogenized whole berry (Ocean Spray, Inc., Lakeville-Middleboro, MA). The total berry extract (UTH), a crude fraction from presscake containing flavonoids of many different structures (fraction 6), and also a fraction from whole berry composed of proanthocyanidins (PACs; polymers of flavanols) have significant antiproliferative activity against tumor cell lines. The human cell lines that were tested included androgen receptor–positive prostate cancer LNCaP, the androgen receptor–negative prostate line DU145, and estrogen receptor–negative breast cancer MDA-MB-435 as well as others.
Against the androgen-dependent prostate cancer cell line LNCaP, these preparations strongly inhibited proliferation; they also inhibited proliferation of the androgen-independent prostate tumor line DU145. The IC50 values for inhibition of proliferation of LNCaP and DU145 cells by fraction 6 were, respectively, 10 and 234 µg/mL. These cell lines also differed in sensitivity to UTH by 15-fold. To distinguish cytotoxic activity that was independent of the androgen receptor, functional studies were first conducted using the breast cancer cell line MDA-MB-435, which exhibited sensitivity to fraction 6 equivalent to that of the DU145 line: Fraction 6 inhibited cell cycle progression by arresting cells in both G1 and G2/M, and cells treated with fraction 6 underwent apoptosis in a time- and concentration-dependent manner.
To help identify the components responsible for the antiproliferative activity, further fractionation of fraction 6 and PACs was done with HPLC. This procedure yielded additional purified fractions that exhibited significant inhibition of LNCaP, but their identity is as yet unknown. Therefore, several significant constituents of cranberry were chosen as possible active ingredients because of their known anticancer properties. These ingredients were characterized based on their cytotoxicity to the various cancer cell lines and their elution pattern on the HPLC: quercetin, myricetin, resveratrol, and the family of catechins and their gallic acid conjugates and polymers (3,5–8). Quercetin (9,10), resveratrol (9,11), and the catechin derivative epigallocatechin gallate (EGCG) (12) have all been shown to inhibit proliferation of prostate tumor cells in vitro, in vivo, or both. High myricetin consumption is correlated with reduced prostate cancer risk (13). EGCG, as a major component of green tea, has also been implicated in reducing prostate cancer risk in populations that commonly consume this drink (1).
The 4 candidate compounds were evaluated for antiproliferative activity against the previous panel of cell lines and were also eluted on the same HPLC system as above to determine whether they might be responsible for the antiproliferative activity of the cranberry extracts. Preliminary results indicate that 1) LNCaP, DU145, and MDA-MB-435 exhibit very similar sensitivity (within 2-fold) to quercetin and resveratrol; 2) LNCaP and DU145 are equally sensitive to myricetin and about 4 times less sensitive than MDA-MB-435; and 3) LNCaP and DU145 are similarly sensitive to EGCG and MDA-MB-435 is about twice as sensitive. HPLC analysis revealed that quercetin, myricetin, and resveratrol did not coelute with the fractions that contained most of the activity, but EGCG coeluted with the antiproliferative fraction of PACs. Because EGCG, quercetin, myriceti, and resveratrol did not exhibit the same pattern of cell line sensitivities as that shown by fraction 6 or PAC, these agents are unlikely to comprise the active component of these cranberry preparations. Therefore, an ingredient of cranberry yet to be identified may be responsible for this inhibition of prostate cancer cell growth. It is also possible that a combination of ingredients in the more crude fraction has greater activity than individual ingredients after separation by HPLC.
It is the intent of this research program to isolate and identify the component of cranberry that inhibits proliferation of cancer cells and is especially effective against the androgen-dependent prostate carcinoma line LNCaP. The above results indicate that a proanthocyanidin and perhaps one or more polymeric forms of this flavanol derivative may possess this activity. The active ingredient may also be a coeluting flavonol. Given that this anticancer activity is isolated from a safely ingested food, it is expected that the phytochemical responsible for the anticancer activity will have important clinical usefulness.
1. Agarwal, R. (2000) Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem. Pharmacol. 60: 1051–1059.[Medline]
2. Guthrie, N. (2000) Effect of cranberry juice and products on human breast cancer cell growth. FASEB J. 14: 531.
3. Kandil, F. E., Smith, M. A., Rogers, R. B., Pepin, M. F., Song, L. L., Pezzuto, J. M. & Seigler, D. S. (2002) Composition of a chemopreventive proanthocyanidin_rich fraction from cranberry fruits responsible for the inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)_induced ornithine decarboxylase (ODC) activity. J. Agric. Food Chem. 50: 1063–1069.[Medline]
4. Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. & Neto, C. C. (2002) Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J. Agric. Food Chem. 50: 5844–5849.[Medline]
5. Kähkönen, M. P., Hopia, A. I. & Heinonen, M. (2001) Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 49: 4076–4082.[Medline]
6. Chen, H., Zuo, Y. & Deng, Y. (2001) Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J. Chromatogr. 913: 387–395.
7. Bhagwat, S., Beecher, G. R., Haytowitz, D. B., Holden, J., Gebhardt, S., Dwyer, J., Peterson, J. & Eldridge, A. (2002) Development of a database for flavonoids in foods. J. Nutr. 132, 3548S.
8. Wang, Y., Catana, F., Yang, Y., Roderick, R. & Van Breemen, R. B. (2002) An LC-MS method for analyzing total resveratrol in grape juice, cranberry juice, and in wine. J. Agric. Food Chem. 50: 431–435.[Medline]
9. Kampa, M., Hatzoglou, A., Notas, G., Damianaki, A., Bakogeorgou, E., Gemetzi, C., Kouroumalis, E., Martin, P. M. & Castanas, E. (2000) Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines. Nutr. Cancer 37: 223–233.[Medline]
10. Xing, N., Chen, Y., Mitchell, S. H. & Young, C. Y. F. (2001) Quercetin inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells. Carcinogenesis 22: 409–414.
11. Rosenberg Zand, R. S., Jenkins, D. J. A., Brown, T. J. & Diamandis, E. P. (2002) Flavonoids can block PSA production by breast and prostate cancer cell lines. Clin. Chim. Acta 317: 17–26.[Medline]
12. Chung, L. Y., Cheung, T. C., Kong, S. K., Fung, K. P., Choy, Y. M., Chan, Z. Y. & Kwok, T. T. (2001) Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells. Life Sci. 68: 1207–1214.[Medline]
13. Knekt, P., Kumpulainen, J., Jarvinen, R., Rissanen, H., Heliovaara, M., Reunanen, A., Hakulinen, T. & Aromaa, A. (2002) Flavonoid intake and risk of chronic diseases. Am. J. Clin. Nutr. 76: 560–568.
Late Effects in Childhood Cancer Survivors: Approaches to Prevention. Anna T. Meadows. University of Pennsylvania School of Medicine and Follow-up Clinic in Oncology, Children's Hospital of Philadelphia.
Approximately 3 of every 4 children diagnosed with cancer can be expected to reach adulthood. There are now 250,000 childhood cancer survivors. Second cancers, near-lethal effects on vital organs such as the heart, lungs and kidneys, cognitive dysfunction, and issues surrounding sexual maturation and fertility are among the most serious known late effects of therapy for childhood cancer now being recognized. Preventing and ameliorating these late effects can take many forms: 1) surveillance for second cancers in survivors at greatest risk after therapy with alkylating agents, epipodophyllotoxins, and radiation; 2) providing educational support for children whose therapy has affected cognitive function; 3) initiating therapy with angiotensin converting enzyme inhibitors to survivors whose treatment with anthracyclines led to reduced cardiac function; 4) administering hormone replacement for survivors deficient in growth hormone, thyroid hormone, estrogen, or testosterone; and 5) using family history and lipid profiles to provide appropriate nutritional counseling and therapy to mitigate cardiac effects. Another method of preventing late effects involves modifying therapy based on our knowledge of the risk factors that determine long-term effects. Examples of such modifications that have already been accomplished include the elimination or reduction in dose of radiation for acute lymphoblastic leukemia, Wilms' tumor, and Hodgkin's disease, and, in the latter, the substitution of cyclophosphamide for nitrogen mustard, resulting in less gonadal toxicity and secondary leukemia. All of these modifications have already been shown to reduce long-term toxic effects in children and adolescents with cancer. Based on an understanding of the possible effects of treatment, pediatric oncologists have developed recommendations for surveillance and counseling of long-term survivors to provide instruction and intervention for the prevention of subsequent disease and the maintenance of health. Recommendations include understanding the elements of a sensible diet, engaging in appropriate exercise, and avoiding risk-taking behaviors such as smoking and excess sun exposure. If individuals exposed to therapy for cancer are followed for the remainder of their lives, the factors that play a role in the total lifetime incidence of late effects and that compromise quality of life will be appreciated. Such survivors are excellent subjects for the testing of intervention strategies for the prevention of cancer, heart disease, and other chronic conditions. The knowledge gained from the study of survivors will also enable us to provide the most rational follow-up care.
Health Status of Adults Who Are Long-Term Childhood Cancer Survivors: A Report from the Childhood Cancer Survivor Study. M. M. Hudson A. C. Mertens Y. Yasui W. Hobbie H. Chen J. G. Gurney M. Yeazel C. J. Recklitis N. Marina L. R. Robison K. C. Oeffinger. St. Jude Children's Research Hospital, Memphis, TN.
BACKGROUND: The success in developing curative therapy for pediatric malignancies permits evaluation of cancer-related sequelae and their effect on the health status of long-term survivors. Numerous studies have demonstrated that cancer and its treatment predispose to a variety of medical and psychosocial sequelae that may adversely affect health status.
OBJECTIVE: We evaluated clinical and treatment factors predisposing to poor health outcomes of adults participating in the Childhood Cancer Survivor Study (CCSS).
METHODS: Health status was assessed in 9535 adult participants of the CCSS. The CCSS is a retrospective cohort study of individuals diagnosed with childhood cancer between 1970 and 1986, under age 21, and surviving 5 y. A randomly selected cohort of the survivors' siblings (N = 2916) served as a comparison group. Six health status domains were assessed: general health, mental health, functional status, activity limitations, cancer-related pain, and cancer-related anxiety and fears. The first 4 domains were assessed in the control group. Multivariate analyses were used to determine the odds ratio (OR) and 95% confidence intervals (CI) associated with health outcomes reported by self-administered questionnaire.
RESULTS: The mean age at interview for survivors was 26.8 y (range 18–48). The mean age at cancer diagnosis was 10.0 y (range 0.1–21.0), with a mean interval from diagnosis to completion of questionnaire of 17.4 y (range 6–29). The cohort included 53% male and 87.4% white, non-Hispanic survivors. Primary cancer diagnoses comprised leukemia, Hodgkin's disease, central nervous system tumor, bone tumor, soft tissue sarcoma, non-Hodgkin's lymphoma, Wilms' tumor, and neuroblastoma. Survivors were significantly more likely to report adverse general (OR = 2.5; 95% CI, 2.1–3.0) and mental health (OR = 1.8; 95% CI, 1.6–2.1), activity limitations (OR = 2.7; 95% CI, 2.3–3.3), and functional impairment (OR = 5.2; 95% CI, 4.1–6.6) compared with siblings. Although only 10.9% of survivors reported fair or poor general health, 44% reported at least one adversely affected health status domain. Sociodemographic factors associated with reporting at least one adverse health status domain included: female gender (OR = 1.4; 95% CI, 1.3–1.6), lower level of educational attainment (OR 2.0; 95% CI, 1.8–2.2), and annual income <$20,000 (OR = 1.8; 95% CI, 1.6–2.1). Relative to those with childhood leukemia, risk for at least one adverse health status domain was elevated among those with bone tumors (OR = 2.1; 95% CI, 1.8–2.5), central nervous system tumors (OR = 1.7; 95% CI, 1.5–2.0), and sarcomas (OR = 1.2; 95% CI, 1.1–1.5).
CONCLUSION: Clinicians caring for adult survivors of childhood cancer should be aware of the substantial risk for adverse health status, especially among females, those with low educational attainment, and those with low household incomes. Primary care providers should anticipate health problems in these clinical and sociodemographic groups when evaluating adults with a history of childhood cancer and be prepared to address physical and psychosocial sequelae adversely affecting health status.
Cardiovascular Effects of Therapy for Testicular Cancer. Jourik A. Gietema. Department of Medical Oncology, University Hospital Groningen, Groningen, The Netherlands.
Testicular cancer (TC) has become a very important oncologic disease. It is the most common malignancy in young men and thus has the potential to greatly shorten productive years of life. The primary age group is 15–35 y for nonseminomatous tumors and a decade older for seminomatous tumors. The goal of chemotherapy in TC is cure, never prolongation of survival. The success of cisplatin-containing chemotherapy for metastatic testicular cancer has resulted in an increasing number of cured TC patients (overall 10-y disease-free survival rate is 85–90%) and made this malignancy a model for a curable disease. In the early 1990s at the Department of Medical Oncology of the University Hospital Groningen, we observed that patients whose disseminated TC was cured with cisplatin-bleomycin chemotherapy several years later develop a high number of cardiovascular risk factors (CRF) such as hypercholesterolemia, hypertension, and overweight (1). These late toxic effects may affect the ultimate prognosis for these patients.
Recently, we found an increased incidence of cardiac events in our young TC survivors 10–20 y after chemotherapy, possibly as a result of increased occurrence of CRF (2). In 87 patients treated with cisplatin-bleomycin chemotherapy, major cardiac events were found in 5 (6%; age at time of event 30–42 y; 9–16 y after chemotherapy): 2 had a myocardial infarction (1 fatal) and 3 had angina pectoris with proven myocardial ischemia. An increased observed-to-expected ratio of 7.1 (95% CI 1.9–18.3) for coronary artery disease, as compared with the Dutch population, was found. Of the 87 patients, 62 were additionally evaluated for cardiac damage and CRF. Their cardiovascular risk profile was compared with that of 40 patients with comparable age and follow-up duration treated with orchidectomy only for stage I disease. Additional cardiovascular damage after chemotherapy was observed: subclinical dysfunction of the left ventricle, microalbuminuria, and a raise in markers of endothelial damage (2). Furthermore, we observed an unfavorable profile of CRF resembling syndrome-X (or metabolic syndrome) with insulin resistance, dyslipidemia, hypertension, and endothelial damage in 30% of the cured TC patients (3). It remains unresolved whether the increased number of cardiac events and the observed cardiovascular damage are a direct toxic effect of the chemotherapy or whether they are a result of the increased incidence of CRF. One can hypothesize that endothelial damage caused by the bleomycin-cisplatin chemotherapy together with the consequences of subclinical hypogonadism are the main causes of the observed increase in CRF and cardiovascular disease (CVD). Microalbuminuria, present in about one-fourth of the patients who received this chemotherapy, has been identified as an independent risk factor for CVD in large trials and thus might contribute to the high cardiac event rate, because endothelial activation is an early event in atherogenesis. We recently found that in these TC survivors, circulating platinum levels are still detectable more than 20 y after cisplatin combination chemotherapy (4). Chronic exposition of endothelium to platinum may therefore play an additional role in the development of the CVD.
Long-term survivors of metastatic TC appear to have an increased risk for CVD that is accompanied by an unfavorable CRF profile and signs of persisting endothelial damage. When compared with the threat for secondary malignancies after chemotherapy for TC, the increased risk for CVD may perhaps represent even a bigger problem. Unraveling of the pathogenesis of CVD after chemotherapy for TC cancer is of great importance and is the focus of several cross-sectional and prospective studies in TC patients currently underway at our institution. These investigations will provide opportunities for tailoring potential toxic treatment and guiding primary and secondary prevention strategies for serious side effects of chemotherapy treatment.
1. Gietema, J. A., Sleijfer, D. T., Willemse, P. H. B., Schraffordt Koops, H., Van Ittersum, E., Verschuren, W. M. M., Kromhout, D., Sluiter, W. J., Mulder, N. H. & De Vries, E. G. E. (1992) Long-term follow-up of cardiovascular risk factors in patients given chemotherapy for disseminated nonseminomatous testicular cancer. Ann. Intern. Med. 116: 709–715.
2. Meinardi, M. T., Gietema, J. A., Van der Graaf, W. T. A., Van Veldhuisen, D. J., Runne, M. A., Sluiter, W. J., De Vries, E. G. E., Willemse, P. H. B., Mulder, N. H., Van den Berg, M. P., Schraffordt Koops, H. & Sleijfer, D. T. (2000) Cardiovascular morbidity in long-term survivors of metastatic testicular cancer. J. Clin. Oncol. 18: 1725–1732.
3. Gietema, J. A., Meinardi, M. T., Van der Graaf, W. T. A. & Sleijfer, D. T. (2001) Syndrome X in testicular-cancer survivors. Lancet 357: 228–229.
4. Gietema, J. A., Meinardi, M. T., Messerschmidt, J., Gelevert, T., Alt, F., Uges, D. R. A. & Sleijfer, D. T. (2000) Circulating plasma platinum more than ten years after cisplatin treatment for testicular cancer. Lancet 355: 1075–1076.[Medline]
Second Primary Cancers: An Overview. Lois B. Travis. Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD.
As survival after a diagnosis of cancer improves, identification and quantification of the late effects of therapy become critical. The occurrence of second malignant neoplasms constitutes one of the most serious side effects of successful radiotherapy and chemotherapy. Although acute myeloid leukemia was the first observed carcinogenic effect of cancer treatment, solid tumors now represent the largest second cancer burden in some populations of survivors. Second primary cancers, however, do not necessarily represent an adverse effect of therapy but may reflect the operation of host determinants, shared etiologic influences, gene-environment interactions, and other factors. Critical to any assessment of second cancers is an evaluation of whether their occurrence exceeds the expectation and the size of the risk. Both cohort and case-control study designs have been used to quantify risk and will be described in the presentation. The characterization and estimation of second cancer risk is important in terms of patient management, enabling physicians to make informed decisions with regard to optimal therapy of the initial cancer, balancing efficacy against early and late toxicity. These investigations also provide a singular opportunity to study carcinogenesis because patients are exposed to measured amounts of potentially cancer-inducing agents, and dose-response relationships with radiation and chemotherapy can frequently be defined. The presentation will provide an overview of second primary cancers, focusing on selected highlights, with an emphasis on radiotherapy and chemotherapy in adults; areas for future research will also be summarized. Although cancer treatment is a double-edged sword, it should always be kept in mind that advances in therapy are largely responsible for the enormous improvements that have been observed in patient survival. Thus, the benefits of most cancer treatments far exceed any risk of developing a second cancer.
Molecular Epidemiology of Cancer. Fred F. Kadlubar. National Center for Toxicological Research. Jefferson, AR.
Molecular epidemiology not only provides us with the ability to predict interindividual differences in susceptibility to environmental exposures that lead to clinical disease, but also indicates effective prevention strategies. Biomarkers of susceptibility include polymorphisms in carcinogen and drug metabolism, DNA repair, and genes that control cell growth. Wide variations in carcinogen and drug metabolism are important determinants of individual cancer susceptibility. Such polymorphisms in carcinogen- and drug-metabolizing enzymes may be due to heritable or environmental factors, and the application of metabolic phenotyping and genotyping methods to epidemiologic studies has provided new insights into such gene-environment interactions. Polymorphisms in DNA repair or processing became evident from rare hereditary disorders involving defective repair or chromosomal stability. About 130 different genes are involved in DNA repair, and lower DNA repair proficiency or polymorphisms have recently been associated with increased susceptibility to cancers of the skin, brain, lung, stomach, breast, bladder, head and neck, and colon. Although over 100 genes have been identified that serve as positive or negative regulators of cell growth as well as of the cell cycle and apoptosis, these have been largely associated with rare hereditary disorders involving greatly increased human cancer susceptibility. The common polymorphisms in these genes have not yet received much attention, but studies indicate that these may be associated with breast, endometrial, ovarian, bladder, colon, lung, thyroid, gastric, nasopharyngeal, esophageal, multiple myeloma, and head and neck cancer. It should be emphasized that although these common genetic polymorphisms do not alone confer high individual cancer risk (low penetrance), they involve a large proportion of the population (high prevalence). Thus, their attributable risk can be high because it can affect a larger number of people in comparison with those rare defects (low prevalence) that greatly increase disease risk (high penetrance) but in much fewer individuals (low attributable risk). The combination of several high risk alleles in a single individual (gene-gene interactions) can result in further increases in relative risk. When increased relative risk is combined with carcinogen exposure, the probability of developing cancer becomes quite high. Examples of such multigene-environment interactions from our ongoing molecular epidemiologic studies of colon, breast, and prostate cancer will be presented.
Intraepithelial Neoplasia: Target for Prevention. Bernard Levin. Division of Cancer Prevention, University of Texas M.D. Anderson Cancer Center, Houston, TX.
Carcinogenesis is characterized by progressive disorganization at the molecular, cellular, and tissue levels. This process may be lengthy—sometimes up to several decades. Our understanding of carcinogenesis has been advanced by the concept of intraepithelial neoplasia (IEN). IEN usually occurs in most epithelial tissues as "moderate to severe dysplasia, is on the causal pathway leading from normal tissue to cancer and is close in stage of progression to cancer (invasive neoplasia)" (1). It is often multifocal and multiclonal. Subjects with IEN, particularly if severe, are at increased risk for developing invasive cancer in affected tissues. IEN may be detectable by familiar methods (e.g., use of endoscopy to locate adenomas in familial adenomatous polyposis) or discernible at a very early stage of development by using novel genomic, imaging, or proteomic techniques. The management of intraepithelial neoplasia requires an understanding of the natural history of specific lesions. Interventions may depend on the site or severity of the condition and the potential effect on quality of life. Interventions may include surgical removal of the IEN (e.g., severely dysplastic Barrett's esophagus), colonoscopic polypectomy (sporadic adenomas), or medical approaches (chemoprevention). Chemoprevention is the use of specific chemical compounds to prevent, inhibit, or reverse carcinogenesis before the development of invasive disease (2,3). Trials of chemopreventive agents are often large, lengthy, and expensive. The design and successful execution of chemopreventive trials often involves a complex set of partners including individuals at risk, the pharmaceutical industry, and federal agencies such as the National Cancer Institute and the Food and Drug Administration. To date, only 5 compounds have been approved for the treatment of IEN: topical 5-FU and topical diclofenac for multiple actinic-keratoses; intravesical bacillus Calmette-Guerin for bladder carcinoma in situ; tamoxifen for diffuse carcinoma in situ after lumpectomy and breast radiotherapy; celecoxib for adenomatous polyps in patients with familial adenomatous polyposis. In addition to defining molecular targets for chemopreventive intervention, genomic and proteomic techniques may help to refine entry criteria for trials aimed at eradication or control of IEN as well as enhance correlations with the outcome of such interventions.
1. O'Shaughnessy, J. A., Kelloff, G. J., Gordo, G. B., Dannenberg, A. J., Hong, W. K., Fabian, C. J., Sigman, C. C., Bertagnolli, M. M., Stratton, S. P., Lam, S., Nelson, W. G., Meyskens, F. L., Alberts, D. S., Follen, M., Rustgi, A. K., Papadimitrakopoulou, V., Scardino, P. T., Gazdar, A. F., Wattenberg, L. W., Sporn, M. B., Sakr, W. A., Lippman, S. M. & Von Hoff, D. D. (2002) Treatment and prevention of intra-epithelial neoplasia; an important target for accelerated new agent development. Clin. Cancer Res. 8: 314–346.
2. Kelloff, G. J. (2000) Perspectives on cancer chemoprevention research and drug development. Adv. Cancer Res. 78: 199–334.[Medline]
3. Umar, A., Viner, J. L., Richmond, E., Anderson, W. F. & Hawk, E. T. (2002) Chemoprevention of colorectal carcinogenesis. Int. J. Clin. Oncol. 7: 2–26.[Medline]
The Influence of Prenatal and Childhood Nutrition on the Development of Cancer. David Gunnell* George Davey Smith* Jeff Holly. *Department of Social Medicine and Division of Surgery, University of Bristol, Bristol, UK.
An individual's risk of developing cancer is determined by genetic predisposition together with the accumulation of adverse and protective exposures occurring from conception through to adulthood. Migrant studies, secular trends in incidence, and international variations point to the importance of environmental exposures in cancer etiology. There is growing recognition that patterns of nutrition in utero and throughout childhood influence risk of breast, prostate, colorectal, hematopoietic, and some other cancers. Indirect evidence for this comes from studies showing that anthropometric markers of prenatal and childhood growth—such as birthweight (growth in utero), leg length (prepubertal growth), and height (growth throughout childhood)—are positively associated with risk of a number of common malignancies. Intriguingly, in some studies low birthweight infants (<2500 g) also appear to be at increased risk. More direct evidence for the role of specific aspects of childhood diet comes from the relatively few studies with measures of diet recorded in childhood that are of sufficient size and duration of follow-up to investigate diet-cancer associations. Analysis of the Boyd Orr cohort—a 60-y follow-up of 5000 subjects with detailed contemporaneously recorded childhood (family) diet—suggests that low energy and high fruit intake in childhood are associated with lower cancer risk. Recent epidemiologic research suggests that nutritional influences on circulating levels of insulin-like growth factor-I (IGF-I) provide a plausible explanation for patterns of cancer risk in relation to diet, low and high birthweight, childhood height, and leg length. High levels of IGF-I promote cell turnover and reduce apoptosis; high levels are associated with raised birthweight and greater childhood height. High levels of energy, milk, dairy products and animal protein intake are associated with raised IGF-I. There is some evidence that vegetable intake is associated with low IGF-I levels but these aspects of diet may operate through other pathways, such as by reducing oxidative damage to DNA. Although raised IGF-I levels are associated with increased risk of prostate, colorectal, and premenopausal breast cancer, they may protect against other chronic diseases such as heart disease and neurodegeneration. Public health policies aimed at modifying circulating levels of IGFs should therefore be carefully tailored to optimize overall population health.
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