Preliminary Pharmaceutical Evaluation Of S-allylmercaptocysteine And Its Antitumor Mechanism In Breast Cancer

Malignant tumor has been the biggest threat for human health since the morbidity rate has been ever increasing in last few decades. It has been reported that there were more than200types of cancers has been found, and more than10million new cases were diagnosed each year. Over700,000people die from cancers each year, which is about13%of total morbidity. Breast cancer is the top cancer in women and the breast cancer patients will increase26%by2020in the developing country. The common treatment methods for breast cancer at present include surgery, chemotherapy and radiotherapy. All the existing treatment methods for breast cancer have their own limitations; for example, the toxic and side effects of chemotherapy are usually serious and often accompanied by drug resistance. Therefore, the searching for new antitumor drugs with better efficacy and less toxicity has been the focus for past decades. Plants, animals and microorganisms have always been the important sources of medicines for treatment tumors and other diseases. Additional structural modifications based on natural compounds have become an important strategy for development of novel drugs.Garlic (Allium sativum), a member of the lily family, has been used as the flavors as well as healthy supplements for thousands of years. Garlic’s bioactivities are thought to be attributed to its pharmacological characteristics of many sulfur-containing compounds; more than33organosulfur compounds in garlic are now discovered. A large amount of data indicates that garlic’s anticarcinogenic activities are partially due to cancer cell apoptosis induced by organosulfur compounds. Among the garlic organosulfur compounds, the water-soluble constituent S-allylmercaptocysteine (SAMC) is not commercially available and its physical and chemical properties as well as anti-breast cancer mechanism have not been well studied. In this work, the synthetic routes and purification procedures of SAMC were studied in order to establish a starting point for the industrial scale production; preliminary pharmaceutical evaluation of SAMC was performed for further pharmaceutical development; and finally, the antitumor mechanism of SAMC in breast cancer cell lines MCF-7(ER-positive) and MDA-MB-231(ER-negative) was investigated.SAMC was synthesized in a two-step reaction by using L-cysteine hydrochloride, methoxycarbonyl chloride and allyl mercaptan as raw materials. The newly synthesized SAMC was purified by a PR-18column and the purity of the purified SAMC was determined to be above99%. The melting point, the equilibrium solubility, oil-water partition coefficients and stability of the purified SAMC were determined and the results indicate that the purified SAMC was suitable for further pharmaceutical development. SRB analysis indicated that SAMC exhibited an effective cell growth inhibition of human breast cancer cell lines MCF-7and MDA-MB-231in a dose and time-dependent manner. The IC50values of SAMC were found to be148μM and207μM for MCF-7and MDA-MB-231cells at72h, respectively. DAPI staining and Annexin V/PI analysis suggested that SAMC induced apoptosis along with the release of cytochrome-c, the cleavage of PARP and the expression change of Bcl-2family such as the increase of the pro-apoptotic protein Bax and decrease of the anti-apoptotic protein Bcl-2. The cell cycle arrest effect of SAMC was analysed by flow cytometry using PI, the results indicated that the cell cycles of MCF-7and MDA-MB-231were arrested in the G0/G1phase by SAMC, and the expression of cell cycle proteins, such as cyclin D1, cyclin E1were increased; p53and p21that is the downstream gene of p53were activated and responsible for the arrest effect. The down-regulation of PCNA suggested that SAMC inhibited the proliferation of cells. The mitochondrial membrane potential was decreased with the treatment of SAMC by JC-1staining. Caspase activity analysis indicated that SAMC activated the caspase-3/7, caspase-8and caspase-9. The wound scratch assay found that SAMC inhibited the migration of breast cancer cells and the expression of E-cadherin was increased.All the results indicated that SAMC inhibited cell proliferation by delaying the cell cycle at G0/G1phase, and induced the cell apoptosis through mitochondrial (intrinsic) and death receptor (extrinsic) pathways.