The Research Of Zinc Effects The Chemosensitivity And Exogenous P53 Stability Of Prostate Cancer Cell Line PC-3

Prostate cancer patients are more likely to be older than 60 years of age,its rate is high and found to be advanced stages,chemotherapy is the main method for the treatment of prostate cancer.About more than half of the patients with prostate cancer occured p53 mutation or inactivation,p53 status determines the sensitivity of chemotherapy drugs for prostate cancer;prostate cancer patients have decreased zinc concentration,and with the degree of malignancy of prostate cancer aggravated,the zinc concentration may also be further reduced.The lack of zinc can be the main reason for the loss of p53 function and resistance to chemotherapy of prostate cancer PC-3 cells.In this study,we investigated whether zinc can enhance the sensitivity of Paclitaxel to prostate cancer cell line PC-3 and further explore its mechanism.Also we transfected a co-expression plasmid Pmp53 expressing wild-type p53 and inhibit the expression of MDM2 into PC-3 cells,to investigate the effect of zinc on the stability of p53.[Objective] 1.We supplemented zinc in the prostate cancer cell line PC-3 to detect the effect of zinc on the chemosensitivity to Paclitaxel;2.We transfected Pmp53 into the cells to investigate the effect of zinc on the stability of exogenous p53.[Methods]1.The effect of zinc on chemosensitivity of PC-3 cells to PaclitaxelExperimental groups: control group,zinc group,Paclitaxel group,zinc + Paclitaxel group.We used MTT method to determine the optimal concentration of zinc combined with Paclitaxel in PC-3 cells.Morphological analysis assay to detect the effect of zinc combined with Paclitaxel on cell morphology.The colony formation assay and KI-67 staining assay to analyse cell proliferation ability.Cell apoptosis and mitochondrial membrane potential were detected by flow cytometry and Hochest staining assay.The level of apoptosis related genes and proteins levels of PCR and Western-blot.2.The effect of zinc on the stability of exogenous p53A co-expression plasmid Pmp53 was introduced into PC-3 cells.The experimental groups was divided into seven groups.PCR and Western-blot were used to detect the changes of p53 and MDM2 gene and protein levels in each group.The combination ability of p53 and MDM2 was detected by co-immunoprecipitation assay.The half-life of MDM2 and p53 was detected by CHX.[Results] 1.Zinc enhanced the chemosensitivity of paclitaxel to PC-3 cells1)Firstly,with different concentrations of zinc and Paclitaxel on PC-3 cells 48 h,the results showed that 10 nmol/L Paclitaxel combined with 250 ?mol/L zinc can significantly inhibit the proliferation of PC-3 cells(P<0.05).The clone formation ability assay observed that compared with control group and zinc group,Paclitaxel group and zinc combined with Paclitaxel group could significantly inhibit the cell clone formation ability(P<0.05);Annexin V-FITC flow cytometry detected that compared with control group and zinc group,Paclitaxel group and Paclitaxel combined with zinc group could significantly promote cell apoptosis(P<0.05);JC-1 staining assay found that compared with the control group and zinc group,Paclitaxel group and zinc combined with Paclitaxel group can significantly reduce the mitochondria membrane potential(P<0.05).2)RT-PCR study found that compared with the control group and zinc group,Paclitaxel group and zinc combined with Paclitaxel group could significantly reduce Bcl-2 and MDM2 gene expression level,enhance Bax,Caspase3 and caspase9 gene expression level(P<0.05).The Western-blot study found that compared with control group and zinc group,Paclitaxel group and zinc combined with Paclitaxel group could significantly reduce the expression level of Bcl-2 and MDM2 oncogene protein,enhance the expression level of Bax,Cleaved-caspase3 and Cleaved-caspase9 protein.(P<0.05).2.Zinc increased the stability of exogenous p53The co-immunoprecipitation experiment found that compared with the Pmp53 Group,the protein expression of p53 which combined with MDM2 in Pmp53+Zn group was significantly decreased;compared with Pmp53+Zn group,the protein expression of p53 which combined with MDM2 in Zn+Pmp53+TPEN group was significantly increased.The CHX capture experiment showed that the half-life of p53 in the Pmp53+Zn group was significantly prolonged compared with that in the single Pmp53 group.[Conclusions]1.Zinc combined with Paclitaxel could significantly inhibit prostate cancer PC-3 cell proliferation and promote PC-3 cell apoptosis.The results suggested that zinc could enhance the chemosensitivity of Paclitaxel.2.Zinc reduced the ubiquitination degradation of exogenous p53 which induced by MDM2 and prolonged the half-life of p53.It was suggested that zinc could increase the stability of exogenous p53.