Apoptosis And Molecular Mechanisms Of Prostate Cell Induced By Celecoxib

Background and Objective:With entering in the aging society and change of people' diet structure, the morbidity of prostate cancer in China is increasing greatly. Most patients of prostate cancer will transfer to hormone independent prostate cancer, HIPC or hormone refractory prostate carcinoma, HRPC. The two types of prostate cancer, especially HRPC, are insensitive to conventional therapy. Therefore it is needed the new way to conquer the obstacles. In recent years, many epidemic data show that non-steroidal anti-inflammatory drug, NSAIDs, which could suppress cyclooxygenase-2 and lipoxygenase in tumors cells, can take certain extent effect on cancer prevention, such as breast cancer, respiratory tract tumors, gastrointestinal tumors and urinary tumors, et al. Scientific experiments show that selective cyclooxygenase-2 inhibitor can effect on neovascularization of tumors and the growth, transformation, proliferation and apoptosis of tumors. These experiments offer theoretical and experimental basis for the new methods with prevention and treatment of tumors. However, there is little report on prostate cancer treated by selective cyclooxygenase-2 inhibitor at home and abroad. This study is aim to investigate the changes of apoptosis abilities and invasion abilities of prostate cancer cell line, DU-145 after being treated by selective cyclooxygenase-2 inhibitor, celecoxib. Meanwhile, we try to investigate the possible mechanism to find a new way to prevent and treat HIPC and HRPC.Content:The research is performed in hormone independent prostate cancer cell line, DU-145. Observe and screen cancer cell apoptosis after being treated by celecoxib. Then, detect the changes of mRNA of apoptosis and invasion relevant after reacted with the drug to investigate celecoxib'role in the cell apoptosis.Methods:1,Culture DU-145 with the medium DMEM contained 10% FBS and renew medium and passage the cell timely. The cell for experiment is under well condition at logarithmic phase.2,The apoptosis morphology of DU-145 cell was detected by Hoechst 33342/PI staining after 24 hours treated with celecoxib (50μmol/L). We record the picture using the Fluorescence microscope and compare with the control group.3,We divided the groups according the concentration of celecoxib(0,25,50,100,200μmol/L). Annexin V/PI dual-color flow cytometry was used to determine the apoptosis rate of DU-145 cell treated with these concentrations of celecoxib. Repeat the experiment three times and then record the data to statistics.4,RT-PCR was used to test the expression of mRNA levels of Bcl-2, E-cadherin, ICE and COX-2 after the cell being treated by celecoxib(50μmol/L) 24 hours later. PCR products electrophoresis in the volume fraction of 2% agaose gel 30 min, and observed and photographed under ultraviolet light. We analysis the optical density of PCR products by the software of Gel-proAnalyzer 4.0 and record the data to statistics.Results:1,Compare with the control group, DU-145 cell treated with celecoxib resulted in clear nuclear condensation with apoptotic bodies observed by Hoechst 33342/PI staining.2,Flow Cytometry revealed that the apoptosis of DU-145 induced by celecoxib was in a dose dependent and apoptosis rate in group (0,25,50,100,300μmol/L) were (1.10±0.15)%,(3.87±0.79)%,(10.59±1.58)%,(22.50±3.30)%,(33.85±2.71)%, respectively (P<0.05).3,RT-PCR demonstrated the mRNA level of Bcl-2 was down-regulated and E-cadherin up-regulated. There was no significant change in the mRNA level of ICE and no expression of COX-2.Conclusion:1,We can observe clearly cell apoptosis with Hoechst 33342/PI staining and we inferred that celecoxib as a selective COX-2 inhibitor can induce DU-145 apoptosis effectively.2,We get a quantitative measurement of apoptosis rate using Annexin V/PI dual-color flow cytometry. We confirmed that celecoxib induced apoptosis of DU-145 cells which is concentration dependent, that is, apoptosis rate and drug concentration has a positive correlation.3,We examined four kinds of mRNA expression of apoptosis related genes and tumor invasion related gene by RT-PCR. We don't detect any expression of COX-2 and we infer that the apoptosis is"COX-2 independent"way which is different to the traditional theory way of"COX-2 dependent"way. We get a new understanding of celecoxib as a COX-2 selective inhibitor in cell apoptosis. We detected the decrease of the cell expression of Bcl-2 in experiment after being treated by celecoxib. The family of Bcl-2 is an important regulatory factor in programmed cell death. They can inhibit cell apoptosis and involved in the apoptosis of mitochondria-mediated pathway. Our results suggest that celecoxib may be involved in mitochondrial apoptosis pathway. At the same time, we choose another apoptosis-related gene, ICE which is also important regulator of apoptosis. It is the core of Fas apoptosis pathway and a regulatory factor in the central of apoptosis. In our experiment, the expression of ICE gene in the experimental group and control group is not significantly different. Therefore, we conclude that celecoxib induced apoptosis in DU-145 cells may be independent of the Fas apoptosis pathway. E-cadherin plays an important role in cell-cell and cell-matrix adhesion reaction which can maintain cell and tissue morphology. During the growth of many tumor cells, the ability of tumor invasion is related with the function of E-cadherin. In our experiment we detected the increase of the expression of E-cadherin and we infer that celecoxib can enhance the adhesion between prostate cancer cells so it can reduce the ability of invasion and metastasis of DU-145 cells.In conclusion, celecoxib as a COX-2 selective inhibitor can induce apoptosis of hormone independent prostate cancer cell line, DU-145. Meanwhile, for the first time we discussed the invasive ability of tumor and confirmed celecoxib can reduce the invasive ability of tumor in our experiment, which can enhance the effect of the drug in anti-tumor. Our experiment provides an experimental basis for the treatment of hormone independent prostate cancer and gives a potential perspective for chemical prevention and treatment of prostate cancer.