Study On The Apoptosis Of Breast Cancer Cells Induced By Exopolysaccharide From Pseudoaltermonas Sp.S-5

Breast cancer is a malignant tumor that happened in the mammary gland epithelial tissue. Human breast cancer cells have lost the characteristics of the normal cells. The connections between cells have loosed, cells are easy to fall off from the tissue. Then cells could spread around the body with blood and lymph, and lead to death. The incidence of breast carcinoma is a growing trend in China in recent years. Mammmary cancer has become a threat to women’s physical and mental health. The therapeutic methods are surgery, chemotherapy, radiation and so on, these methods are harmful, to health. It is a new thinking to explore a new drag with little or without side effect and could inhibit or kill tumor cells.Our laboratory has completed the preliminary work that extracted and separated extracellular polysaccharide from Pseudoaltermonas sp.S-5,.Our research group found that PEP had good immune activity.This paper studys PEP could inhibit the proliferation of cancer cells or not, and explore the mechanism of apoptosis. We explore effects of PEP on MDA-MB-231.1. To evaluate the growth inhibitory by PEP, MDA-MB-231cells were treated with increasing concentrations of PEP for24and48h, and cell viability was measured bu MTT assay. The results show that PEP couls inhibit the growth of these cells. The inhibition rate is from2.06%to42.5%for24h, approximately to44.5%with1.0mg/ml PEP for48h.2. Hoechse33342stainging method is used to observe the morphological changes of breast cancer cells treated by increasing concentration of PEP for24h. In the control group, cells’nuclei have similarly sized, regularly shaped with control, and evenly stained (lightly stained). In the PEP group, cells expressed cell condensation of cytoplasm and nuclear fragmentation. This indicated that PEP inhibit the growth of cancer cells induced through the apoptosis. We confirm PEP induced apoptosis by using flow cytometry with Annexin V-FITC/PI staining. The results demonatrated that the populationg of pro-apopptosis elevated with increasing concentration PEP. The pro-apoptosis rate is15.82%with1.0mg/mL.This illustrated the phosphatidylserine lining flip to the outer membrane.3. To explore the apoptosis mechanism of breast cancer cells induced by PEP, we used DCFH-A was used to observe the variation of ROS, JC-1as a probe to detect the change of mitochondrial membrane potential, and the concentration of Ca2+was detected by Fluo-3/AM. These data suggested that apoptosis induced by PEP is through the mitochondrial way.4. To obtain furter imformation on the apoptosis induced by PEP, the expression of p53, Bcl-2, Bax, FasL and Fas mRNAs were examined in breast cancer cells with RT-PCR. Meanwhile, the expression of CHOP、GRP78、Calenexi、ATF4、 ATF6、Caspase3、Caspasel2were detected by QRT-PCR. The electrophoresis results show that the expression of Bcl-2was down-regulated, but the level of p53and Bax mRNA were up-regulated, compared to the control. The data demonstrated once again mitochondria involved the apoptosis. Then, QRT-PCR indicated that the expression of CHOP, Caspase-12and other related gene. The expression of CHOP, Caspase-12, Caspase-3, ATF4, ATF6were up-regluated, GRP78, Calenexin were down-regluated. This implied that endoplasmic reticulum also involved in the apoptosis indeced by PEP.The study suggested that the mechanism of apoptosis induced by PEP was mediated by intrinsic mitochondrial apoptotic pathway and death-receptor apoptosis signaling pathway, and endoplasmic reticulum pathway.This paper provided theoretical support and basis for breast cancer treatment.