Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

Nutrition exerts considerable effects on health, and dietary interventions are commonly used to treat diseases of metabolic aetiology. Although cancer has a substantial metabolic component1, the principles that define whether nutrition may be used to influence outcomes of cancer are unclear2. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. By contrast, whether specific dietary interventions can influence the metabolic pathways that are targeted in standard cancer therapies is not known. Here we show that dietary restriction of the essential amino acid methionine-the reduction of which has anti-ageing and anti-obesogenic properties-influences cancer outcome, through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and is the target of a variety of cancer interventions that involve chemotherapy and radiation. Methionine restriction produced therapeutic responses in two patient-derived xenograft models of chemotherapy-resistant RAS-driven colorectal cancer, and in a mouse model of autochthonous soft-tissue sarcoma driven by a G12D mutation in KRAS and knockout of p53 (KrasG12D/+;Trp53-/-) that is resistant to radiation. Metabolomics revealed that the therapeutic mechanisms operate via tumour-cell-autonomous effects on flux through one-carbon metabolism that affects redox and nucleotide metabolism-and thus interact with the antimetabolite or radiation intervention. In a controlled and tolerated feeding study in humans, methionine restriction resulted in effects on systemic metabolism that were similar to those obtained in mice. These findings provide evidence that a targeted dietary manipulation can specifically affect tumour-cell metabolism to mediate broad aspects of cancer outcome.

Effects of chronic alcohol consumption on DNA damage and immune regulation induced by the environmental pollutant dibenzo[a,l]pyrene in oral tissues of mice.

Previously, we showed that oral application of the environmental pollutant dibenzo[a,l]pyrene (DB[a,l]P) induces oral tumors in mice. Thus, in the present investigation we examined the effect of alcohol on DB[a,l]P-induced DNA damage and immune regulation; we showed that alcohol (6.4% v/v in the diet, 35% of Calories) significantly enhanced the levels of (-)-anti-trans-DB[a,l]P-dA while decreased the levels of GSH in the mouse oral tissues. Analysis of RNA expression revealed that DB[a,l]P alone upregulates inflammatory genes while alcohol suppresses several markers of immune surveillance. Collectively, these results suggest that alcohol may enhance oral carcinogenesis induced by DB[a,l]P.

Mushrooms: A rich source of the antioxidants ergothioneine and glutathione.

While mushrooms are the highest dietary source for the unique sulfur-containing antioxidant ergothioneine, little is known regarding levels of the major biological antioxidant glutathione. Thus, our objectives were to determine and compare levels of glutathione, as well as ergothioneine, in different species of mushrooms. Glutathione levels varied >20-fold (0.11-2.41mg/gdw) with some varieties having higher levels than reported for other foods. Ergothioneine levels also varied widely (0.15-7.27mg/gdw) and were highly correlated with those of glutathione (r=0.62, P<0.001). Both antioxidants were more concentrated in pileus than stipe tissues in selected mushrooms species. Agaricus bisporus harvested during the third cropping flush contained higher levels of ergothioneine and glutathione compared to the first flush, possibly as a response to increased oxidative stress. This study demonstrated that certain mushroom species are high in glutathione and ergothioneine and should be considered an excellent dietary source of these important antioxidants.

Influence of Obesity on Breast Density Reduction by Omega-3 Fatty Acids: Evidence from a Randomized Clinical Trial.

Preclinical data indicate that omega-3 fatty acids (n-3FA) potentiate the chemopreventive effect of the antiestrogen (AE) tamoxifen against mammary carcinogenesis. The role of n-3FA in breast cancer prevention in humans is controversial. Preclinical and epidemiologic data suggest that n-3FA may be preferentially protective in obese subjects. To directly test the protective effect of n-3FA against breast cancer, we conducted a 2-year, open-label randomized clinical trial in 266 healthy postmenopausal women (50% normal weight, 30% overweight, 20% obese) with high breast density (BD; ≥25%) detected on their routine screening mammograms. Eligible women were randomized to one of the following five groups (i) no treatment, control; (ii) raloxifene 60 mg; (iii) raloxifene 30 mg; (iv) n-3FA lovaza 4 g; and (v) lovaza 4 g plus raloxifene 30 mg. The 2-year change in BD, a validated biomarker of breast cancer risk, was the primary endpoint of the study. In subset analysis, we tested the prespecified hypothesis that body mass index (BMI) influences the relationship between plasma n-3FA on BD. While none of the interventions affected BD in the intention-to-treat analysis, increase in plasma DHA was associated with a decrease in absolute breast density but only in participants with BMI >29. Our results suggest that obese women may preferentially experience breast cancer risk reduction from n-3FA administration.

Randomized controlled trial of oral glutathione supplementation on body stores of glutathione.

PURPOSE: Glutathione (GSH), the most abundant endogenous antioxidant, is a critical regulator of oxidative stress and immune function. While oral GSH has been shown to be bioavailable in laboratory animal models, its efficacy in humans has not been established. Our objective was to determine the long-term effectiveness of oral GSH supplementation on body stores of GSH in healthy adults. METHODS: A 6-month randomized, double-blinded, placebo-controlled trial of oral GSH (250 or 1,000 mg/day) on GSH levels in blood, erythrocytes, plasma, lymphocytes and exfoliated buccal mucosal cells was conducted in 54 non-smoking adults. Secondary outcomes on a subset of subjects included a battery of immune markers. RESULTS: GSH levels in blood increased after 1, 3 and 6 months versus baseline at both doses. At 6 months, mean GSH levels increased 30-35 % in erythrocytes, plasma and lymphocytes and 260 % in buccal cells in the high-dose group (P < 0.05). GSH levels increased 17 and 29 % in blood and erythrocytes, respectively, in the low-dose group (P < 0.05). In most cases, the increases were dose and time dependent, and levels returned to baseline after a 1-month washout period. A reduction in oxidative stress in both GSH dose groups was indicated by decreases in the oxidized to reduced glutathione ratio in whole blood after 6 months. Natural killer cytotoxicity increased >twofold in the high-dose group versus placebo (P < 0.05) at 3 months. CONCLUSIONS: These findings show, for the first time, that daily consumption of GSH supplements was effective at increasing body compartment stores of GSH.

Dietary methionine restriction inhibits prostatic intraepithelial neoplasia in TRAMP mice.

BACKGROUND: Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. METHODS: Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. RESULTS: As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P < 0.02). The reduction in PIN severity was associated with 46-64% reductions in cell proliferation rates (P < 0.02) and plasma IGF-1 levels (P < 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice. CONCLUSIONS: Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy.

Comparative effects of two different forms of selenium on oxidative stress biomarkers in healthy men: a randomized clinical trial.

Epidemiologic and laboratory studies indicate that dietary selenium protects against prostate cancer. Results from clinical trials suggest that selenium-enriched yeast (SY) but not selenomethionine (SeMet) may be effective at reducing prostate cancer risk. Our objectives were to directly compare for the first time the effects of SeMet and SY on prostate cancer relevant biomarkers in men. We performed a randomized double blind, placebo-controlled trial of SY (200 or 285 µg/day) and SeMet (200 µg/day) administered for 9 months in 69 healthy men. Primary endpoints included blood levels of selenium-containing compounds and oxidative stress biomarkers [urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin-F2α (8-iso-PGF2α) and blood glutathione (GSH)]. Secondary endpoints included plasma glucose and PSA levels. Compliance was high in all groups (>95%). Plasma selenium levels were increased 93%, 54%, and 86% after 9 months in SeMet and low- and high-dose SY groups, respectively, and returned to baseline levels after a 3-month washout (P < 0.05). Levels of 8-OHdG and 8-iso-PGF2α were decreased 34% and 28%, respectively, after 9 months in the high-dose SY group (P < 0.05). These decreases were greatest in individuals with low baseline plasma levels of selenium (<127 ng/mL). No changes in serum PSA or blood glucose and GSH were observed. Overall, we showed for the first time, reductions in biomarkers of oxidative stress following supplementation with SY but not SeMet in healthy men. These findings suggest that selenium-containing compounds other than SeMet may account for the decrease in oxidative stress.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis.

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

Age related changes in selenium and glutathione levels in different lobes of the rat prostate.

Aging represents a major risk factor for prostate cancer; however, mechanisms responsible for this relationship remain unclear. Preclinical and some clinical investigations support the protective role of selenium against prostate cancer possibly through the reduction of oxidative stress. While increased levels of oxidative stress together with decreases in selenium and the major cellular antioxidant glutathione (GSH) are common in tissues of old animals, there is little data available on these parameters in the prostate. In the present study we have compared the levels of selenium, GSH and protein-bound GSH (GSSP) in blood and prostate tissues in young (4-month), mature (12-month), old (18 month), and very old (24 month) male F344 rats. Each prostate lobe (dorsolateral, DL; anterior, AL; ventral, VL) was analyzed separately based upon their differing potential for prostate cancer development. At all ages, selenium levels were lowest in DL

Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.

The effects of Tamoxifen and fish oil on mammary carcinogenesis in polyoma middle T transgenic mice.

In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER- tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.

Association of selenium status and blood glutathione concentrations in blacks and whites.

Selenium deficiency has been linked with increased cancer risk and, in some studies, selenium supplementation was protective against certain cancers. Previous studies have suggested that selenium chemoprevention may involve reduced oxidative stress through enhanced glutathione (GSH). Our objectives were to examine the relationships between selenium and GSH in the blood and the modifying effects of race and sex in free-living adults and individuals supplemented with selenium. Plasma selenium concentrations and free and bound GSH concentrations and γ-glutamyl cysteine ligase (GCL) activity in the blood were measured in 336 healthy adults (161 Blacks, 175 Whites). Plasma selenium and blood GSH were also measured in 36 healthy men from our previously conducted placebo-controlled trial of selenium-enriched yeast (247 µg/day for 9 mo). In free-living adults, selenium concentrations were associated with increased blood GSH concentration and GCL activity (P < 0.05). Further, selenium was significantly higher in Whites than in Blacks (P < 0.01). After 9 mo of supplementation, plasma selenium increased 114% in Whites and 50% in Blacks (P < 0.05), and blood GSH increased 35% in Whites (P < 0.05) but was unchanged in Blacks. These results indicate a direct association between selenium and GSH in the blood of both free-living and selenium-supplemented individuals, with race being an important modifying factor.

The impact of fish oil on the chemopreventive efficacy of tamoxifen against development of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate.

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

Functional significance of the GAG trinucleotide-repeat polymorphism in the gene for the catalytic subunit of gamma-glutamylcysteine ligase.

Gamma-glutamylcysteine ligase (GCL) is the rate-limiting enzyme in glutathione (GSH) synthesis. A GAG-repeat polymorphism in the 5' UTR of the gene coding for the catalytic subunit of GCL (GCLC) has been associated with altered GSH levels in vitro. Thus, we hypothesized that this polymorphism is associated with altered GCL activity and blood GSH levels in vivo. A total of 256 healthy United States black and white adults were genotyped for the GAG polymorphism and blood GSH levels were measured. In a subset of 107 individuals, blood GCL activity was determined. Five alleles with 4, 7, 8, 9, and 10 GAG repeats were observed. The most prevalent genotype was 7/9 (40%) followed by 7/7 (32%) and 9/9 (11%). GSH levels were 15% lower in 9/9 individuals than 7/9 individuals (P=0.05). GCL activity was 21% lower in 9/9 individuals than 7/7 individuals (P=0.04). A decreasing trend of GCL activity was observed in the order of 7/7>7/9>9/9 (P=0.04). These findings show that 9/9 individuals have lower blood GSH levels, which is likely due to a decrease in GCL activity. Such individuals might be more susceptible to oxidative stress-related diseases than individuals with other genotypes.

Enhanced levels of glutathione and protein glutathiolation in rat tongue epithelium during 4-NQO-induced carcinogenesis.

High glutathione (GSH) levels are commonly found in oral tumors and are thought to play an important role in tumorigenesis. While posttranslational binding of GSH to cellular proteins (protein glutathiolation) has recently been recognized as an important redox-sensitive regulatory mechanism, no data currently exist on this process during carcinogenesis. Our goal was to determine the effects of 4-nitroquinoline-N-oxide (4-NQO)-induced carcinogenesis on tongue levels of protein-bound and free GSH and related thiols in the rat. Male F-344 rats (6 weeks of age) were administered either 4-NQO (20 ppm) in drinking water or tap water alone (controls) for 8 weeks. Twenty-four weeks after cessation of 4-NQO, squamous cell carcinomas of the tongue were observed in all rats. The levels of both free and bound GSH in tumors, as well as in adjacent tissues, were 2- to 3-fold greater than in tongue epithelium from control rats (p < 0.05). Prior to tumor formation, at 8 weeks after cessation of 4-NQO, hyperplasia, dysplasia and carcinoma in situ were observed in 100%, 25% and 12.5% of 4-NQO-treated rats, respectively. At this early stage of carcinogenesis, levels of free and bound GSH were increased 50% compared with tongue tissues from control rats (p<0.05). Glutathione disulfide (GSSG) levels were also 2-fold greater in tongue tissues from 4-NQO treated vs. control rats (p<0.05). Altogether, these results suggest that protein glutathiolation, together with GSH and GSSG levels, are induced during oral carcinogenesis in the rat possibly as a result of enhanced levels of oxidative stress.