Study On Apoptosis Of Human Prostate Cancer Cells Induced By 2-Methoxyestradiol

Objective:Prostate cancer?PCa?in the male urogenital tumor is one of the most common malignant tumor,its main characteristic is morbidity and high mortality rate,and harm to male health after the onset of the consumption and the medical resources of the society can not be ignored,which is more and more global attention.In our country,living habits,people's diet structure change,and the aging of the population has become increasingly serious,and people pay more attention to the disease degree and improve the level of diagnosis and treatment of modern medicine in recent years,the incidence of prostate cancer and mortality in malignancies in male urinary system respectively in sixth and ninth,and ranked in the incidence of male urinary tract tumors at an annual rate of 10%in the first rate.However,due to the atypical symptoms of early prostate cancer and the relative lag of disease screening,about 5%of the patients are still found to have distant metastases.For the treatment of the disease,the traditional treatment is castration?Androgen Deprivation Therapy,ADT?,also known as androgen deprivation therapy,including medical or surgical castration,mainly by blocking the androgen and androgen receptor?Androgen,Receptor,AR?to inhibit the growth of prostate tumors with this mechanism,reduce the prostate specific antigen?Protate Specific Antigen,PSA?,to improve the quality of life of patients and prolong the survival time.Most patients can benefit from castration,but after 18-30 months of treatment,most patients will be transferred to castration resistant prostate cancer?Castration Resistant Prostate Cancer,CRPC?,leading to the current existing treatment effect is poor,the median survival time is only about 18-24months.In recent years,with the continuous recognition and in-depth study of castrated resistant prostate cancer,a lot of clinical workers and researchers have made great progress in the pathogenesis and clinical treatment of this disease.2-methoxyestradiol?2-ME₂?began to be found in the urine of women in pregnancy.After the research has found that the anti angiogenesis,cell cycle arrest and differentiation induction of apoptosis,and reported its antitumor spectrum is more and more widely,such as breast cancer,liver cancer,nasopharyngeal carcinoma,hematopoietic malignancies,and because of its adverse reactions is small,so the research value is very high.In this experiment,non hormone dependent human prostate cancer cell PC-3 cell line as the research object,through the drug 2-methoxyestradiol?2-ME₂?and Huang Baisu?Nexrutine?on PC-3 cell to observe the changes of apoptosis of water products,in order to study 2-methoxyestradiol?2-ME₂?and Huang Baisu?Nexrutine?on apoptosis of prostate cancer cells.Methods:Human prostate cancer cell line PC-3 was cultured in a standard RPMI 1640basal medium containing 10%fetal bovine serum,and the culture bottle was incubated in a cell incubator with a set humidity of 5%CO₂ and 37 degrees.Cell growth to a certain density inoculated into 6-well plates in the incubation,meets the requirement of the experiment after drug intervention before and after the intervention using inverted microscope to observe the changes in cell morphology.1.With different concentrations?0.5um/l,1.0um/l,5.0um/l and 10.0um/l?2-methoxyestradiol and?2.5ug/ml,10.0ug/ml and 5.0ug/ml,and 20.0ug/ml?Huang Baisu?Nexrutine?expression were intervention cells 24 hours and 48 hours after collection of specimens is measured by RT-PCR to observe the c-FLIP mRNA change,with the expression of c-FLIP protein in Western-blot measurement the concentration of 2.5ug/ml;2.2.5ug/ml concentrations of Huang Baisu?Nexrutine?combined with different concentrations?0.5um/l,1.0um/l,5.0um/l and 10.0um/l?of 2-methoxyestradiol were used to intervene the cells.Then the above two methods were used to measure the change of target gene and target protein,and each group was set blank control.The data used SPSS 18.0 statistical analysis,Variance analysis method using factorial design,test the level of?=0.05,P<0.05 difference was statistically significant.Result:1.The morphological changes of cells were observed under inverted microscope.Normal cultured prostate cancer PC-3 cells were monolayer adherent growth cells,and the cells could be spindle shaped,triangular or irregular.The cytoplasm is full,the cytoplasm is well translucent,the nucleus can be mononuclear,and it can also be multicore.The use of 2-ME₂ in the two and Huang Baisu?Nexrutine?drugintervention growth rates of PC-3 cells compared with normal cells decreased,and the morphology of cells can be turned round,part of the visible cells under microscope appear membrane shrinkage and cytoplasm turbid,few nuclei can also appear concentrated phenomenon,refraction poor,adherent ability.Moreover,with the prolongation of the intervention time,the cell gap increased significantly.The volume of some cells was smaller than normal,and the chromatin and cytoplasm of nuclei were significantly concentrated,and the number of dead cells increased.After the combination of the two drugs,the number of dead cells increased significantly,the total cell number was also significantly reduced,and the time dependence of drug concentration and drug action was more obvious.2.RT-PCR determination of the relative expression of c-FLIP relative expression of mRNA not only single 2-methoxyestradiol and Huang Baisu?Nexrutine?can inhibit c-FLIP mRNA of the PC-3 cells,after 2-methoxyestradiol combined with Huang Baisu?Nexrutine?,PC-3 intracellular c-FLIP mRNA expression significantly decreased,which confirmed the effect of two kinds of drugs can induce apoptosis of PC-3 cells but,in Huang Baisu?Nexrutine?can enhance 2-methoxyestradiol to induce apoptosis of tumor cells,but there are time and concentration dependent manner.3.Using Western Blot to determing the relative expression of c-FLIP protein results also showed that the relative expression not only by 2-methoxyestradiol and Huang Baisu?Nexrutine?can obviously inhibit c-FLIP protein in PC-3 cells.After the combination 2-methoxyestradiolwith Huang Baisu?Nexrutine?,the c-FLIP protein in PC-3 cells relative expression significantly decreased,apoptosis can also be confirmed two drugs can induce PC-3 cells,but also in Huang Baisu?Nexrutine?can enhance 2-methoxyestradiol to induce apoptosis of tumor cells,but there are time and concentration dependent manner.Conclusion:2-methoxyestradiol and Huang Baisu?Nexrutine?significantly induce the apoptosis of prostate cancer PC-3 cells,and Huang Baisu?Nexrutine?can enhance the apoptosis of prostate cancer cells induced by 2-methoxyestradiol.