Experimental Study Of Apoptosis Of Human Prostate Cancer Cells Induced By STRAIL Of Hyriopsis Schlegelii

Prostate cancer is one of the major cancers that threaten the health of men worldwide.In Europe and America,the incidence of prostate cancer has been ranked first among male malignant tumors for many years,and the mortality rate is the second.Although the incidence of prostate cancer in China is lower than that in Europe and the United States,but in recent years,with the aging of the population,changes in dietary structure,the improvement of medical diagnostic technology and the popularization of medical checkups,the morbidity and mortality of prostate cancer in China are increasing year by year.It is estimated that there were about 60300 new cases and 26600 deaths of prostate cancer in China in 2015.Unfortunately,after 12-18 months of median treatment,most patients end up with castration-resistant prostate cancer(CRPC).Once hormone resistance occurs,CRPC is insensitive to both chemotherapy and radiotherapy at current treatment levels,leading to a decline in the quality of life in patients with CRPC who face an ineffective endocrine therapy.A series of effects,such as shortening of survival time,etc.Therefore,the development of new and effective prostate cancer treatment drugs has become a key issue in the treatment of CRPC.In recent years,molecular targeted therapy based on the mechanisms of promoting tumor cell apoptosis and changing intracellular signal transduction pathway has attracted much attention.For example,scholars at home and abroad,such as Yang Gao-yi,found that down-regulation of Bcl-2 protein can induce apoptosis of prostate cancer cells.It was also found that Bcl-2 antisense oligodeoxynucleotide oblimersensodium combined with docetaxel in the treatment of steroid resistant prostate cancer showed good therapeutic effect.In addition,adenovirus carrying soluble human tumor necrosis factor-related apoptosis-inducing ligand(Strail)could induce apoptosis in human tumor cells.Among them,TRAIL has attracted people's attention.Studies on TRAIL and its mediated apoptotic pathway have shown that TRAIL or its extracellular domain degrades to form soluble cytokines sTRAIL(Soluble TRAIL),which can act on the death receptors DR4 and DR5 on the surface of target cells to form death inducible signal complexes.Caspase8 and its downstream caspase cascade were activated and then the death receptor pathway and mitochondrial dependent apoptotic pathway were activated.The death receptor pathway eventually induces apoptosis by activating effective enzymes such as caspase3 and acting on substrates such as protein kinase(PKC).Mitochondria dependent pathway releases cytC and other apoptosis-related factors through the formation of active tBid(truncated BID.Bcl2 is one of the inhibitory factors in this pathway.In addition,some tumor cells overexpress the deceptive receptor DcRl and DcR2,which can compete with DR4/DR5 to bind to TRAIL,inhibit apoptosis mediated by TRAIL,and make tumor cells exhibit tolerance to TRAIL.However,in recent years,although the anticancer effects of TRAIL proteins in vertebrates such as human beings have been studied,the apoptotic effect on PC3 cells of prostate cancer is rarely reported,and the detailed molecular mechanism of its action has not been reported.However,the antitumor function of TRAIL gene and its recombinant protein in invertebrates such as shellfish has not been reported internationally.During the previous cloning of immne-related genes of Hyriopsis schlegelii by RT-PCR,we obtained a partial sequence of the soluble tumor necrosis factor-related apoptosis-inducing ligand(TNF-related apoptosis-inducing ligand)of mussel Hyriopsis schlegelii.It was also found that the TRAIL had some anti-tumor properties.In order to investigate the antitumor effect of shellfish sTRAIL gene and its possible mechanism,we intend to further determine the role of sTRAIL in promoting apoptosis of prostate cancer cell line PC-3 through the study of this project.It also' reveals the possible mechanism of its action,such as signal transduction pathway of death receptor,so that protein is used as a new anticancer drug,and TRAIL and related signal molecules are used as effective targets in the treatment of prostate cancer and other cancers.Method:1.RT-PCR and Western Blotting techniques were used to clone the sTRAIL gene sequence of Hyriopsis schlegelii,and the sTRAIL gene was expressed in the prokaryotic cell,and the polyclonal antibody was obtained.2.Using different concentrations(10ng-10 g)from the recombinant sTRAIL protein was induced by stimulation of the cells were analyzed on sTRAIL induced PC-3 cell apoptosis after 0-96h stimulation by flow cytometry and DNA ladder assay.3.Using Real-time quantitative PCR and Western Blotting Technology to detect the expression of DR4,DR5,DcR1 and DcR2 at the mRNA and protein level of sTRAIL recombinant protein induced/uninduced tumor cells(PC-3).4 Respectively with caspase8 inhibitor-Z-AEVD-FMK and Caspase 3 inhibitor-Ac-DEVD-CHO induced by sTRAIL in PC-3 cells,using the above method to detect cell apoptosis,and the detection of inhibitors of caspase8 before and after treatment,Caspase3,expression of PKC molecules in mRNA and protein levels.5 In the above study of the death receptors,based on the study of caspase signaling pathway,PKC gene expression changes before and after PKC inhibitor Staurosporine induced by sTRAIL PC-3 cells by quantitative real-time PCR and Western blotting technology-6.The changes of the expression level of anti apoptotic factor bcl-2 gene in PC3 cells were detected after sTRAIL induction.The apoptosis rate was detected again after the treatment with Bcl-2 inhibitor(ABT-737,5nM).Results:1.The sequence of sTRAIL protein of Hyriopsis schlegelii was cloned,and the toxic effect of sTRAIL protein on PC3 cells was confirmed.2.sTRAIL has a strong cytotoxic effect on PC3 cells and can induce apoptosis of PC3 cells.With the increase of sTRAIL concentration and induction time,the apoptotic rate of PC3 cells increases gradually,showing a significant dose and time effect.In the 100ng sTRAIL induced group,the apoptotic rate was over 23.8%at 24 h and the formation of DNA ladder could be detected.3 After sTRAIL induction,the expression of DR4 and DR5 was found in both,but DR4 was higher than DR5.DcR1 and DcR2 were not expressed in PC3 cells induced by sTRAIL.4.sTRAIL induced caspase8 caspase3 gene expression was significantly up-regulated and accompanied by an increase in apoptotic rate,which could be down-regulated by caspase8 inhibitor Z-IETD-FMK or caspase3 inhibitor Ac-DEVD-CHO respectively.5.Induced by sTRAIL,the expression of PKC gene was up-regulated,but the apoptosis rate was significantly decreased after treatment with Saurosporine,but still significantly higher than that in the control group.6.The expression of bcl-2 was up-regulated within 24 hours after induction of sTRAIL,but not significantly,and the apoptotic rate was significantly increased after treated with bcl-2 inhibitor ABT-737.Conclusion:The effect of sTRAIL on the apoptosis of prostate cancer cell line PC3 was determined,and the effects of sTRAIL on the death receptor pathway related to the apoptosis of prostate cancer cell line(PC3),such as caspases,PKC and Bcl2,were analyzed.TRAIL,DR4 and bcl-2 were identified as new targets for targeting PC-3 cell.