The Plant Estrogen Go To The A Dihydro Yuchuang The Inhibition Of Tumor Cell Growth And Preliminary Study Of The Mechanisms Of Epigenetic Modification Of The Wood Acid (ndga,)

Background and Objective:Endocrine-disrupting chemicals (EDCs) in the environment have been linked to human health and disease, especially that of environmental estrogens. Environmental estrogens are comprised of two main groups phytoestrogens and synthetic substances released into the environment. While environmental estrogens are considered negative in almost any context, phytoestrogens are said to have a positive effect on guard against some cancers and other diseases.It has been reported that various environmental and dietary agents and lifestyles are suspected to be implicated in the development of a wide range of human cancers by eliciting epigenetic changes, though the contribution of epigenetic mechanisms to a given human cancer type and the precise targets of epigenetic alterations during ancer development are largely unknown. Epigenetic mechanism results in the heritable silencing of genes without any changes in their nucleic sequences and it involved DNA methylation, RNA associated silencing and histone modification. All critical changes in cancer cells, such as silencing of tumour suppressor genes, activation of oncogenes and defects in DNA repair, are caused not only by genetic but also by epigenetic mechanisms. It has been widely accepted that hypermethylation of CpG islands in promoter is a key mechanism for the silencing of many tumor suppressor genes and cancer related genes, such as p16, E-cad and RARβ. In many cases DNA methylation inhibitor can demethylate the CpG islands in the promoters and reactivate genes silenced by aberrant methylation. Results of recent studies show that a number of isoflavones, such as genistein, myricetin may partically reverse DNA hypermethylation and reactivate genes silenced by aberrant methylation. In comparison, few studies have investigated the effects of lignans on epigenetic alteration. We reported here for the first time that NDGA would inhibit growth of tumor cell lines and demethylate the promoter CpG island of tumor suppressor gene.Methods: Human colorectal cancer cell line RKO, breast cancer cell line T47D, SKBR3 and MDA-MB-435 were treated in the presence of different concentrations NDGA(10, 20, 50, 100μmol/L) and DNA methyltransferase inhibitor, 5-azazcytidine (1μmol/L).The growth inhibition was assayed by MTT; the methylation status of promoter in p16 and E-cad gene was analysed by MSP (methylation- specific PCR) and DNA sequencing. We used RT-PCR and Western to determine whether the changes in methylation status after NDGA treatment were reflected in the expression of its gene products. Given the known functions of p16INK4a in controlling cell-cycle progression at the G1 phase and a strong candidate for mediating cellular senescence. Using flow cytometry, we investigated the cell-cycle distribution of cells treated with varying concentration of NDGA and performed SA-β-gal assays to evaluate the state of cellular senescence. In order to examine the molecular mechanisms underlying changes in cell cycle pattern, we also checked cyclin D1 protein levels by Western blotting before and after treatment with NDGA.10 nu/nu female mice were grafted with human breast cancer cell line MDA-MB-435 into the flank of each animal. The mice were injected subcutaneously with 5×10~6 cells on day 0. Treatment with NDGA began on day 9. The mice were divided into 2 groups randomly at 5 mice per group: control group and NDGA treatment group. Tumors were measured every one week using a caliper, and the body weights of the mice were monitored for toxicity. Tumor volumes were determined by measuring the length (1) and the width (w), and the volumes were calculated (V= 1w~2/2). Experiment was terminated 7 weeks after tumor cell inoculation. Part of tumor tissue was immediately frozen and the rest were fixed in 10% neutral-buffered formalin and were embedded in paraffin. The methylation status of promoter in E-cad gene in NDGA treated group was analysed by MSP (methylation- specific PCR) and DNA sequencing. RT-PCR and Western was performed to evaluate the expression level of E-cad.Results:1. The hypermethylation of p16 gene in T47D and RKO, E-cad gene in SKBR3 and MDA-MB-435 was found and may served as a choice option for pharmaceutical study on demethylation. In comparison to the control, all the four different concentrations of NDGA was able to inhibit the growth of malignancy cell lines using MTT assay. The hypermethylation of p16 and E-cad gene had been successfully reversed after 144h disposal of NDGA, the expression of p16 gene and E-cad gene was obviously up-regulated in a concentration and time-dependent manner.2. Cell cycle data indicate that NDGA blocks the entry of cycling cells into S phase. We observed a moderate reduction in the expression of Cyclin D1 and the hyperphosphorylated form of pRB decreased at all concentrations of NDGA tested compared to the untreated control group. The data of senescence-associatedβ-galactosidase shows senescence characteristics in cells following NDGA treatment.3. Nude mice inoculated with human breast cancer MDA-MB-435 cells and treated with NDGA were effective in delaying the tumor incidence as well as reducing the tumor burden when compared to the control group. NDGA treatment were also found to reverse the methylation status of E-cad gene and the re-expression of protein levels was detected by immunohistochemistry.Conclusion:All these data sustain our contention that NDGA has anti-tumor properties via epigenetic modification. Our results suggest that NDGA may serve as an effective DNMT inhibitor to demethylate the hypermethylation of tumor suppressor gene in vitro and in vivo.