Maternal vitamin B6 deficient or supplemented diets on expression of genes related to GABAergic, serotonergic, or glutamatergic pathways in hippocampus of rat dams and their offspring.

SCOPE: Vitamin B6 plays crucial roles on brain development and its maternal deficiency impacts the gamma-aminobutyric acid (GABA)ergic, serotonergic, glutamatergic, and dopaminergic systems in offspring. However, the molecular mechanisms underlying these neurological changes are not well understood. Thus, we aimed at evaluating which components of those neurotransmitter metabolism and signaling pathways can be modulated by maternal vitamin B6 -deficient or B6 -supplementated diets in the hippocampus of rat dams and their offspring. METHODS AND RESULTS: Female Wistar rats were fed three different diets: control (6 mg vitamin B6 /kg), supplemented (30 mg vitamin B6 /kg) or deficient diet (0 mg vitamin B6 /kg), from 4 weeks before pregnancy through lactation. Newborn pups (10 days old) from rat dams fed vitamin B6 -deficient diet presented hyperhomocysteinemia and had a significant increase in mRNA levels of glutamate decarboxylase 1 (Gad1), fibroblast growth factor 2 (Fgf2), and glutamate-ammonia ligase (Glul), while glutaminase (Gls) and tryptophan hydroxylase 1 (Tph1) mRNAs were downregulated. Vitamin B6 supplementation or deficiency did not change hippocampal global DNA methylation. CONCLUSION: A maternal vitamin B6 -deficient diet affects the expression of genes related to GABA, glutamate, and serotonin metabolisms in offspring by regulating Gad1, Glul, Gls, and Tph1 mRNA expression.

Suppression of Mammary Carcinogenesis Through Early Exposure to Dietary Lipotropes Occurs Primarily In Utero.

The study determined whether feeding during lactation affects the suppressive effect of maternal dietary lipotropes (i.e., methionine, choline, folate, and vitamin B12) on mammary carcinogenesis. Pregnant Sprague-Dawley rats were randomly allocated to the control diet during pregnancy and lactation (CC), lipotropes-fortified diet during pregnancy (LC), lipotropes-fortified diet during pregnancy plus lactation (LL), or lipotropes-fortified diet during lactation (CL). Randomly selected female offspring from each group were injected intraperitoneally with 50 mg/kg body weight of N-nitroso-N-methylurea at 50 days of age to induce mammary tumors. The LC and LL diets significantly increased tumor latency and survival (P < 0.05). Tumor volumes were significantly suppressed in LC and LL offspring as compared with the CC and CL pups (3759.1 ± 563.0 and 3603.7 ± 526.1 vs. 7465.0 ± 941.1 and 5219.3 ± 759.8 mm(3), respectively; P < 0.05). Both LC and LL lowered tumor multiplicity as compared with CC and CL (P < 0.05). The LC and LL diets repressed transcription of histone deacetylase (HDAC) 1 as well as total HDAC enzyme activity as compared with CC and CL diets (P < 0.05). Data suggest that the tumor suppressive effect of maternal dietary lipotropes is primarily in utero and may be linked to regulation of proteins involved in chromatin remodeling.

Pubertal supplementation of lipotropes in female rats reduces mammary cancer risk by suppressing histone deacetylase 1.

PURPOSE: The time from puberty to the first pregnancy is known to be important for a woman's life-time breast cancer risk. Recent studies suggest that epigenetic mechanisms may involve pubertal maturation processes, which can affect the risk of breast cancer in later life. Epigenetic alterations are related to lipotropes (methionine, choline, folate, and vitamin B12), which are methyl donors and cofactors. However, the effects of pubertal supplementation of lipotropes in breast cancer remain largely unknown. METHODS: Twenty female Sprague-Dawley rats, aged 6 weeks, were divided into two groups and fed a normal control diet or a lipotrope-fortified diet formulated to provide five times basal levels of lipotropes during puberty. All rats were injected intraperitoneally with N-nitroso-N-methylurea at 50 days of age to induce mammary tumors. RESULTS: Tumor multiplicity and tumor volume decreased significantly as a result of lipotrope supplementation. Interestingly, quantitative RT-PCR revealed significantly decreased expression of histone deacetylase 1 (Hdac1) and DNA methyltransferase 1 (Dnmt1) genes in tumor tissues of the rats supplemented with lipotrope-fortified diet, suggesting that reduced risk of breast cancer can be attributed, at least in part, to decreased expression of these two genes. CONCLUSIONS: This study demonstrates that supplementation of lipotrope-fortified diet during puberty suppresses tumor growth, potentially through down-regulating Hdac1 and Dnmt1 gene expression. Our findings suggest that pubertal methyl diet plays an important role in the etiology of breast cancer, and further studies are warranted to develop preventative strategies against breast cancer.

Maternal dietary canola oil suppresses growth of mammary carcinogenesis in female rat offspring.

As suggested by rodent studies and studies using human breast cancer cells, dietary canola oil is linked with lower breast cancer risk. Here, we investigated the effect of maternal (pregnancy plus lactation) dietary canola oil on the susceptibility of female Sprague-Dawley rat offspring to mammary carcinogenesis. Although the control diet had 10% soybean oil, the treatment diet was formulated to contain 10% canola oil as a fat source. N-nitroso-N-methylurea was injected to induce mammary cancer in offspring. The offspring of canola-fed dams showed significantly decreased tumor multiplicity (1.0 ± 0.3 vs. 1.9 ± 0.3, respectively; P = 0.04) and tumor volume (1232.5 ± 771.0 mm(3) vs. 6,302.5 ± 1,747.4 mm(3), respectively; P = 0.01), along with increased survival rate (87% vs. 47%, respectively; P = 0.01). In addition, the mRNA expression of development-related gamma-glutamyltransferase 1 was significantly higher in the lactating mammary tissues of the canola group dams and mammary tumor tissues of the offspring [2.5 ± 0.6 vs. 0.5 ± 0.2, respectively (P = 0.01) and 0.98 ± 0.03 vs. 0.56 ± 0.15, respectively (P = 0.05)]. These results suggest a potential anticancer effect of maternal dietary canola oil and may be useful in devising prenatal nutritional strategies to reduce breast cancer risk in humans.

Lipotropes enhance the anti-proliferative effect of chemotherapeutic drugs in MCF-7 human breast cancer cells.

Increasing evidence indicates that dietary intake of methyl nutrients is associated with the risk of breast cancer. Lipotropes are methyl group-containing essential nutrients (methionine, choline, folate and vitamin B12) which play key roles in one-carbon metabolism; however, little is known about the implications of lipotropes in possible tumor-suppressive effects with chemotherapeutic drugs for breast cancer. In the present study, we investigated the in vitro effects of lipotropes on cell growth and apoptosis of MCF-7 human breast cancer cells. Cells were cultured and treated with lipotropes, and cell proliferation, apoptosis and gene expression were determined. Also, the possible synergistic effects of lipotropes with anticancer drugs, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and doxorubicin (DOX), were examined. Lipotropes significantly reduced the growth of MCF-7 cells and increased apoptosis as well as upregulation of caspase-3 and tumor protein 53 (p53) enzyme activities. Gene transcription, as measured by quantitative real-time PCR, revealed a significant increase of p53 mRNA in MCF-7 cells treated with lipotropes, but there were no differences in two drug-resistant related genes. Moreover, lipotropes showed significant additive effects with SAHA and DOX on cell growth inhibition. These results suggest that lipotropes induce apoptosis, inhibit cell growth, and display anti-proliferative effects with SAHA and DOX in MCF-7 cells. Owing to the tumor-suppressive effects observed, lipotropes in combination with chemotherapeutic drugs may be tested further in animal models as potential therapeutic agents for reducing breast cancer risk.

Maternal high-methyl diet suppresses mammary carcinogenesis in female rat offspring.

Maternal nutrition during pregnancy influences the development and metabolism of the fetus. Recent studies suggest that the cancer risk of offspring later in life is associated with maternal diet, but little is known about the effect of a maternal diet high in methyl nutrients on breast cancer risk. Lipotropes are methyl group-containing essential nutrients (methionine, choline, folate and vitamin B(12)) that play key roles in one-carbon metabolism. In this study, we investigated the long-term effects of maternal dietary high-dose lipotropes (five times higher than in the control diet) on the development and progression of mammary tumors in rat offspring using two separate experiments (in utero exposure with and without postnatal supplementation). In both experiments, the female offspring were injected intraperitoneally with a single dose (50 mg/kg body wt) of N-nitroso-N-methylurea during puberty to induce mammary tumors. Tumor growth and development were recorded, and at the end of the study, tissues were collected for analysis. For both experiments, the offspring from dams fed a high-dose lipotropes showed significantly decreased tumor incidence, tumor multiplicity and tumor volume, while also displaying a significant increase in survival rate and tumor latency. Gene transcription analysis, as measured by quantitative real-time PCR, revealed a significant decrease of histone deacetylase 1 (Hdac1) messenger RNA in mammary tumors in both experiments. Our findings provide evidence that maternal dietary high-dose lipotropes reduce mammary carcinogenesis in offspring in association with long-term alterations in gene expression and may be useful in developing maternal dietary strategies to prevent breast cancer.

Canola oil inhibits breast cancer cell growth in cultures and in vivo and acts synergistically with chemotherapeutic drugs.

Certain fatty acids in canola oil (CAN) have been associated with a reduced risk of breast cancer. This study assessed the effects of CAN on proliferation and death of human breast cancer cells in vitro and in vivo in chemically induced mammary carcinogenesis. We hypothesize that CAN reduces breast cancer cell growth by inducing cell death. In a series of in vitro experiments, human breast cancer T47D and MCF-7 cells were cultured and treated with CAN and two chemotherapeutic drugs, tamoxifen and cerulenin. Cell proliferation and caspase-3 and p53 activities were measured. Reduced cancer cell growth and increased expression of caspase-3 and p53 were seen in T47D and MCF-7 cells treated with CAN. Moreover, CAN showed synergistic cancer cell growth inhibition effects with tamoxifen and cerulenin. In a subsequent live animal experiment, 42 female Sprague-Dawley rats were randomly assigned to corn oil (CORN) or CAN diets, and mammary tumors were chemically induced by N-nitroso-N-methylurea. CAN-dieted rats had reduced tumor volumes and showed an increased survival rate as compared to CORN-dieted rats. We demonstrated that CAN has suppressive effects on cancer growth, and reduces tumor volumes. The results suggest that CAN may have inhibitory effects on breast cancer cell growth, and warrants further investigation of the synergistic effects of CAN with anti-cancer drugs.

Methyl-donor nutrients inhibit breast cancer cell growth.

Lipotropes (methyl group containing nutrients, including methionine, choline, folate, and vitamin B(12)) are dietary methyl donors and cofactors that are involved in one-carbon metabolism, which is important for genomic DNA methylation reactions and nucleic acid synthesis. One-carbon metabolism provides methyl groups for all biological methylation pathways and is highly dependent on dietary supplementation of methyl nutrients. Nutrition is an important determinant of breast cancer risk and tumor behavior, and dietary intervention may be an effective approach to prevent breast cancer. Apoptosis is important for the regulation of homeostasis and tumorigenesis. The anti-apoptotic protein Bcl-2 may be a regulatory target in cancer therapy; controlling or modulating its expression may be a therapeutic strategy against breast cancer. In this study, the effects of lipotrope supplementation on the growth and death of human breast cancer cell lines T47D and MCF-7 were examined and found to inhibit growth of both T47D and MCF-7 cells. Furthermore, the ratios of apoptotic cells to the total number of cells were approximately 44% and 34% higher in the lipotrope-supplemented treatments of T47D and MCF-7 cancer cells, respectively, compared with the control treatments. More importantly, Bcl-2 protein expression was decreased by approximately 25% from lipotrope supplementation in T47D cells, suggesting that lipotropes can induce breast cancer cell death by direct downregulation of Bcl-2 protein expression. Cancer treatment failure is often correlated with Bcl-2 protein upregulation. These data may be useful in the development of effective nutritional strategies to prevent and reduce breast cancer in humans.

Role of compensatory mammary growth in epigenetic control of gene expression.

To better understand the role of nutrition in regulating mammary gland development and lactation, we designed a novel stair-step compensatory nutrition regimen that is a unique combination of dietary energy restriction and realimentation (refeeding) phases; the basic concept of this regimen is to exploit the biological nature of the compensatory growth phenomenon in concert with one or more hormone-sensitive allometric phases of mammary development (i.e., peripuberty through gestation). Nutritionally induced compensatory growth during different developmental stages before first parturition positively affects mammary development and life-long lactation performance. This permanent enhancement of mammary gland growth and lactation potential strongly suggests a possible mechanistic link between nutritionally induced compensatory growth, epigenetic control of mammary gene expression, and metabolic imprinting. We hypothesize that compensatory-directed metabolic imprinting once set during late pregnancy prior to the first parturition persistently maintains and exerts its adaptive response on mammogenesis and galactopoiesis (i.e., maintenance and/or enhancement of milk secretion). The ability to influence heritable genes regulating milk synthesis may be used to improve the quality and quantity of milk (e.g., infant health, the secretion of certain immunoglobulins or growth factors) as well as the longevity of lactation.

A compensatory nutrition regimen during gestation stimulates mammary development and lactation potential in rats.

The proper nutritional status during the hormone-sensitive growth phases preceding first parturition can affect mammary development and subsequent lactation performance. We developed a compensatory nutrition regimen (CNR), which is designed to stimulate mammary growth by exploiting the biological characteristics of the energy restriction and compensatory growth phenomenon. In the present study, we examined the effect of compensatory growth induced only once during late gestation upon mammary development and subsequent lactation potential over 2 lactation cycles. Female rats were mated and randomly assigned to either the control or the CNR group. Control rats were offered the control diet (AIN-93G) throughout the experiment. CNR rats were subjected to 40% energy restriction during the first 10 d of gestation followed by free access to the control diet for the remainder of the experiment. Dams on the CNR produced 14% more milk than control dams (P = 0.12). Mammary cell proliferation rates were approximately 46% (P < 0.05) and 27% (P = 0.07) higher in the CNR group than in the control during late gestation and early lactation of the first lactation cycle, respectively. Caspase-3 enzyme activity was decreased 15% (P < 0.05) and 22% (P = 0.11) in mammary tissues from the CNR group compared with that from the controls during the first and second lactation cycles, respectively. These results indicate that compensatory growth induced only once during late gestation increases mammary cell proliferation and differentiation and decreases regression of mammary cells throughout consecutive lactation cycles.

Methionine cytotoxicity in the human breast cancer cell line MCF-7.

Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.

Inhibition of excitotoxicity in cultured rat cortical neurons by a mixture of conjugated linoleic acid isomers.

Glutamate excitotoxicity, which is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors, directly contributes to the neuronal cell loss associated with both acute insults and chronic neurodegenerative disorders. Conjugated linoleic acid (CLA) is a group of dienoic derivatives of linoleic acid shown to have anticarcinogenic and antioxidative activities. To evaluate the effect of a mixture of CLA isomers (cis-9, trans-11 and cis-10, trans-12 octadecadienoic acids) on glutamate- and NMDA-induced excitotoxicity, primary cultures of rat cortical neurons were treated for 15 min with 300 microM glutamate or NMDA in the presence of various concentrations of CLA. After the exposure, cell cultures were maintained at 37 degrees C for 18 h in minimum essential medium supplemented with glucose. Neuronal injury was measured by a colorimetric cell proliferation assay, and a qualitative assessment was made by phase-contrast microscopy. CLA inhibited glutamate- and NMDA-induced neuronal cell death in a concentration-dependent fashion with the most effective dose for neuroprotection being 500 microM. These results demonstrate that a mixture of CLA isomers exhibits protective action against glutamate- and NMDA-induced excitotoxicity.

Lipotropes regulate bcl-2 gene expression in the human breast cancer cell line, MCF-7.

Lipotropes, a methyl group containing nutrients, including choline, methionine, folic acid, and vitamin B(12), are essential nutrients for humans. They are important methyl donors that interact in the metabolism of one-carbon units and are essential for the synthesis and methylation of deoxyribonucleic acid. The purpose of this study was to examine the effects of excess lipotropes on the growth of a human breast cancer cell line, MCF-7, and normal mammary cells, MCF-10A, in culture. Both cell lines were grown in basal culture medium for 24 h and then switched to medium supplemented with 50 times the amount of each lipotrope as basal culture medium (control). Although there were no significant differences in growth between treatments in either cell line, gene array and Northern analysis revealed that expression of bcl-2 was decreased in lipotrope-treated MCF-7 cells. The ability to induce tumor cell death could have many uses in the prevention and treatment of cancer. Bcl-2 regulates apoptosis and has been shown to directly affect the sensitivity of cancer cells to chemotherapy agents, and it is suggested that strategies designed to block Bcl-2 might prove useful in sensitizing tumor cells to chemotherapy-induced apoptosis. This study shows that although excess lipotropes do not inhibit the growth of breast cancer cells, they can down-regulate the bcl-2 gene, suggesting that lipotropes may increase the susceptibility of breast cancer cells to anticancer drugs.