Mechanism of N-Acylthiourea-mediated activation of human histone deacetylase 8 (HDAC8) at molecular and cellular levels.

We reported previously that an N-acylthiourea derivative (TM-2-51) serves as a potent and isozyme-selective activator for human histone deacetylase 8 (HDAC8). To probe the molecular mechanism of the enzyme activation, we performed a detailed account of the steady-state kinetics, thermodynamics, molecular modeling, and cell biology studies. The steady-state kinetic data revealed that TM-2-51 binds to HDAC8 at two sites in a positive cooperative manner. Isothermal titration calorimetric and molecular modeling data conformed to the two-site binding model of the enzyme-activator complex. We evaluated the efficacy of TM-2-51 on SH-SY5Y and BE(2)-C neuroblastoma cells, wherein the HDAC8 expression has been correlated with cellular malignancy. Whereas TM-2-51 selectively induced cell growth inhibition and apoptosis in SH-SY5Y cells, it showed no such effects in BE(2)-C cells, and this discriminatory feature appears to be encoded in the p53 genotype of the above cells. Our mechanistic and cellular studies on HDAC8 activation have the potential to provide insight into the development of novel anticancer drugs.

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.

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.

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.

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.

Flaxseed sprouts induce apoptosis and inhibit growth in MCF-7 and MDA-MB-231 human breast cancer cells.

Flaxseeds have been shown to play a role in the prevention of cancer and heart disease, and it is believed that their more favorable fatty acid composition is responsible. Sprouting is a natural method to modify nutritional components and to decrease cyanide poisoning of raw flaxseeds. Here, we investigated the in vitro effects of flaxseed sprouts on cell growth and apoptosis of human breast cancer cells. In a series of in vitro experiments, estrogen-receptor-positive (MCF-7) and estrogen-receptor-negative (MDA-MB-231) cells were cultured and treated with flaxseed sprouts, and then cell proliferation, apoptosis, and gene expression were measured. Flaxseed sprouts significantly reduced the growth of both of MCF-7 and MDA-MB-231 cells and also increased apoptosis. However, flaxseed sprouts did not affect the growth of MCF-10A mammary epithelial cells. In gene transcription analysis using quantitative real-time polymerase chain reaction, flaxseed sprout treatment significantly upregulated p53 mRNA in both cell cancer lines. These results suggest that flaxseed sprouts induce apoptosis and inhibit cancer cell growth, thereby demonstrating their anti-proliferative effects in breast cancer cells. This study may provide important information for devising dietary strategies to reduce breast cancer risk.

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.