Study Of Arsenic Trioxide On Human Estrogen Receptor-negative Breast Cancer Cell Lines

Women breast cancer is the world's highest incidence of malignant tumor,and is clinically the most common hormone-dependent tumor. According to phenotypic differences in ER breast cancer were divided into two types:ER-positive breast cancer and ER-negative breast cancer.About 2 / 3 were ER-positive breast cancer patients,tumor cell growth with estrogen-dependent.For such patients,clinicians often take estrogen removal of endocrine treatment.Approximately 1 / 3 for ER-negative breast cancer patients with ER-positive breast cancer compared to a higher grade, is not sensitive to endocrine therapy,there is no ideal method of treatment, poor prognosis.If the induction of apoptosis in ER-negative breast cancer will be ER-negative breast cancer patients to open up new therapeutic approaches,in order to bring hope to these patients.As₂O₃ which is inorganic compounds,is Chinese medicine extract arsenic, having Highly toxic.A long period of time,the medicinal value of As₂O₃ has been overshadowed by its highly toxic.Now,with advanced quality control technology and deep studies on experimental and clinical of As₂O₃,its treatment of Cancer Clinical application is more and more wide. Modern pharmacological studies indicated that,As₂O₃ has varying degrees of inhibition for leukemia and many solid tumors.Chinese medicine also has a lot of literature on the arsenic treatment of documented tumor. In the experiment,we study that arsenic trioxide roles in estrogen receptor-negative breast cancer cell lines MDA-MB-435S of cell growth, cell morphology,cell cycle,apoptosis and apoptosis-related gene effect.We investigate the effect of arsenic trioxide on the ER-negative breast cancer cell lines the role of mechanisms of Chinese medicine treatment in ER-negative breast cancer has opened up new ideas.ObjectiveTo investigate the influence of arsenic on the cell proliferation, mitotic cycle and apoptosis of MDA-MB-435S and discuss the mechanism.MethodThe experiment was divided into six parts:MTT experiment,Light microscope to observe cell morphology,PI-flow single-dye experiments to measure the changes of cell cycle and apoptosis,Streaming Annexinâ…¤/ PI double staining to measure cell apoptosis,Immunohistochemical detection of intracellular Bcl-2 and Bax expression,PI 33342 fluorescent staining of apoptosis.We adopt the role of different concentrations in vitro MDA-MB-435S.The influence of different factors on growth of the cells were observed by MTT colorimetry assay and corresponding densities of As₂O₃ were screened preliminary by the results.At 1.75-28μg/ml range of linear relationship by dose-response curves,we select corresponding densities of As₂O₃ at upper and lower of IC50 value.Located the control group and the As₂O₃ group(1,5,10,15,20μg/ml),we measure the changes of cell cycle and apoptosis by flow cytometry,according to flow test results,we selecte the control group and the As₂O₃ group(1,5,10,15,20μg/ml) for being detected BCL-2 and BAX changes by immunohistochemical method.Cell apoptosis were observed by PI 33342 fluorescence staining experiments.results1.Greater than 1.75μg/ml arsenic trioxide could significantly inhibit the growth of MDA-MB-435S cells,and arsenic trioxide have a dose effect in 1.75～28μg/ml concentration range(P<0.001).2.Arsenic Trioxide can make the percentage of G0-G1 stage cells and G2/M stage cells increase in the MDA-MB-435S cell cycle,and make S stage cell decrease.To contrast with control group,all of the results have significant differences(p<0.05).3.PI-flow single-dye experiments show that MDA-MB-435S cells do not have obvious apoptosis and MCF-7 cells have obvious apoptosis in 1,5,10,15,20μg/ml(p<0.05).4.Streaming Annexinâ…¤/ PI double staining experiments show that the early apoptosis rate of MDA-MB-435S cells have no significant difference by As₂O₃ group(1,5,10,15,20μg/ml)compared with control group,and the early apoptosis rate of MDA-MB-435S cells have significant difference by As₂O₃ group(0.25,0.5,1,1.5,2μg/ml) compared with control group.With the action dose heightening,the rate of Apoptosis will be induced to increase obviously.MDA-MB-435S cells apoptosis probably induced by low concentrations of arsenic trioxide.5.Immunohistochemical detection experiments show that the group of Arsenic Trioxide(0.25,0.5,1,5,10μg/ml) compared with control group can down-regulate the express of BCL-2 and BAX.6.PI 33342 fluorescent staining experiments show that,under the same conditions,the group of Arsenic Trioxide(0.25,0.5,1,5,10μg/ml )compared with control group,apoptotic cells morphology can not be seen obviously in MDA-MB-435S cells and can be seen in MCF-7 cells.conclusion1.arsenic trioxide can inhibit MDA-MB-435S cell proliferation.2.Arsenic trioxide can block MDA-MB-435S cell cycle at G0-G1 and G2-M phase,so that reduction into the S phase cells,probably induced by MDA-MB-435S cell apoptosis.3.Arsenic Trioxide can down-regulate the express of BCL-2 and BAX. Arsenic trioxide are possible through reduced expression of BCL-2 to attain the inhibition MDA-MB-435S cells.Inhibition of the way should be studied further.