Study On The Mechanisms Of MCF-7 Cells Apoptosis Induced By Ganodema Lucidum Polysaccharide Containing Selenium SeGLP-2B-1

As an organic selenium compound, selenium-polysaccharide displays both the activities of polysaccharide and selenium, and is easier for absorption and utilization by organisms. In this present study, inorganic selenium compounds Na2SeO3 were integrated into the Ganodema Lucidum mycelium, and the novel organic selenium compound, Ganodema Lucidum polysaccharide containing selenium(SeGLP), were synthesized by biotransformation. SeGLPs display a variety of physiological activities, such as anti-oxidation activity resulted from its rivalry to heavy metal. It can inhibit DNA synthesis and growth of tumor cells in cell cycle, and induce tumor cells apoptosis by oxidative stress, ROS, regulating of oncogene and tumor suppressor gene expression, adjusting of cell cycle related proteins, and activating of a series of caspase enzymes.In this study, Ganodema lucidum polysaccharides containing selenium SeGLP-2B-1 were isolated and purified from the rich-selenium Ganodema lucidum mycelium in the deeply fermenting. SeGLP-2B-1 could induce the apoptosis of MCF-7 cells. Effects of SeGLP-2B-1 on the proliferation of MCF-7 cells were measured by MTT assay after MCF-7 cells were incubated with SeGLP-2B-1 in vitro for 24 hours, 48 hours and 72 hours, respectively. The results showed that the apoptosis began to occurred after MCF-7 cells incubated with SeGLP-2B-1 for 24 hours, and the apoptosis rate was 25.39%. Subsequently, the highest degree of apoptosis was exhibited when MCF-7 cells incubated for 72 hours, the apoptosis rate was 80.53% and the IC50 was 0.158μM. The degradation of Chromosomal DNA was observed by the agarose gel electrophoresis. The changes of MCF-7 cell cycle were detected by PI color staining and flow cytometry. The AP peak was obviously observed after 24 hours. All the above results proposed that SeGLP-2B-1 could inhibit the growth of MCF-7 cells by inducing the MCF-7 cells apoptosis.We studied the molecular mechanism of SeGLP-2B-1 on inducing tumor cell MCF-7 to apoptosis. The changes of electric potential of intracellular mitochondria membrane was detected by Rhodamine123 fluorescence dyeing and flow cytometry. ,The results that the electric potential of mitochondria membrane declined after MCF-7 cells were incubated with SeGLP-2B-1 for 24 hours, and the electric potential declined more than 50% at 72 hours, indicated that the collapse of the electric potential(ΔΨm)of mitochondria membrane resulted in the opening of mitochondrial membrane permeability transferring pore. The releasing of cytochrome C from mitochondria to cytoplasm activated a series of caspase proteins, the apoptosis signals protein which lead to the cascade waterfall effect in apoptosis and the death of cells. In this study, caspase-9 and caspase-3 were activated and PARP was degradated into 83kD molecule after MCF-7 were treated with SeGLP-2B-1 for 48h. As a key enzyme in apoptosis pathway related with mitochondrial, caspase-9 activated by SeGLP-2B-1 activated caspase-3, the apoptosis effect signal protein and induced cell apoptosis.We found that caspase-8 was also activated after MCF-7 cells were incubated with SeGLP-2B-1 for 48h. As a death receptor signal protein in apoptosis pathway, caspase-8 was suggested to be concerned with the apoptosis induced by SeGLP-2B-1.After MCF-7 cells were incubated with SeGLP-2B-1 for 24h, 48h and 72h, the intracellular ROS level decreased with the increasing of incubated time, which showed that low concentration of SeGLP-2B-1 could enhance the antioxidation activity in cancer cells by decresing the ROS level.