Mechanism Of Action Of Proliferating Cell Nuclear Antigen Antisense Oligodeoxynucleotide Induced Apoptosis In Androgen-Refractory Prostate Cancer Cell

It has been shown that the formation and development of malignant tumors are not noly associated with abnormal proliferation and differentiation, but also involved inhibition of apoptosis. Tumor cells, succeed in escaping from apoptosis by various mechanisms, are an important cause to gain immortalization, formation and progression, and the formation of cell strains, obtaining resistance to chemical therapy or radiation therapy, are also associated with its high activity against apoptosis. Therefore, it is important to demonstrate the pathogenesis of tumor by studying the relations among apoptosis-related genes, tumor formation and progression. And how to re-sensitize, activate or raise apoptosis has significant value and bright prospect, which will be benefit to promote healing rate and develop novel therapies of tumors.Antisense technique, one of the gene therapy, is based on the principle of Watson-Crick base pair, when antisense oligodeoxynucleotide (ASODN) incubated with tumor cells, sequence specific complementary to the target sequence would block the transcription and the expression of relevant gene. Because of itseffectiveness, safety, simplicity and no vector, antisense technique is a promising approach in the treatment of androgen-refractory prostate cancer. Previous studies have shown that proliferating cell nuclear antigen (PCNA) plays important role in prostate cancer's occurrance and development. PCNA is an auxiliary protein of DNA polymerase 8 and is found only in the nuclei of proliferating cells in the late Gl, and S phase, plays an essential role in DNA replication. It is one of the intracellar factors that is common to all pathways DNA synthesis. PCNA positivity is correlated with the histological grade and invasiveness of prostate cancer. Bcl-2 family has been known in numerous apoptosis-related genes, bcl-2 gene is an important factor to influence apoptosis, and overexpression of bcl-2 family can be observed in most human cancers. In recent years, a new important family of anti-apoptosis genes was discovered, namely the inhibitor of apoptosis proteins (IAPs). IAPs can directly inhibit Caspase and possess far stronger anti-apoptosis function than bcl-2 family, so it has become a hot point. The gene of survivin is a latest member of IAPs. Up to now, relevant studies referring to the relation of survivin, PCNA, bcl-2 and bax to the prostate cancer after PC-3 cells were treated with PCNA-ASODN are still no much, and no data is availiable concerning the gene of PCNA as a target for therapy in prostate cancer, too.In this study, we choose PCNA as a target gene, after PC-3 cells were treated with PCNA-ASODN, we investigated the mRNA expression of PCNA and survivin in PC-3 cell using guanidinium isothiocyanate reverse transcription polymerase chain reaction(RT-PCR) analysis, and also investigated the expression of bcl-2 , bax and PCNA using SABC immunohistochemical stain , to explore their relations to prostate cancer. And it is the first time that we study the influence of antisense PCNA oligodeoxynucleotide (PCNA-ASODN) on humanandrogen-independent PC-3 prostate cancer cells, such as cell growth, proliferation, and apoptosis, in order to search for a novel gene therapy for prostate cancer, particularly for androgen-independent prostate cancer.PART IThe effect of PCNA-ASODN on the cell growth and apoptosis of PC-3 cell lineObjective: To investigate the effect of PCNA-ASODN on the cell growth and apoptosis in androgen-independent prostate cancer PC-3 cell line.Methods: PC-3 cell was treated with different concentration of PCNA-ASODN with different time. The situation of cell proliferation was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltertrazolium bromide (MTT) method, cell cycle progression was assessed by flow cytometry (FCM), cell morphology was investigated by light microscope and transmission electron microscope.Results: The cell proliferation was inhibited effectively by PCNA-ASODN, The cell growth rate was reduced over 50% after 48 hours treatment. The inhibition effect showed in a dose-time dependent manner, and decreased after 72 hours. After PC-3 cell incubated with 30 u mol/L ASODN for 24, 48 and 72 hours , the cell apoptotic rate were 15.3%, 25.2% and 30.4% respectively(p<0.01). The change of apoptosis was seen in PC-3 cells examined by transmission electron microscope after it was treated by 30 u mol/L ASODN through the phase.Conclusion: 1. PCNA-ASODN could inhibit the proliferation of androgen-independent prostate cancer cell PC-3 cell line by dose and time-depend manner.2. PCNA-ASODN could induce apoptosis of androgen-independent prostate cancer cell PC-3 cell line.PART IIEffect of PCNA-ASODN on Survivin, PCNA, bcl-2 andbax expression in androgen-independentprostate cancer cell PC-3 cell lineObjectiverTo study the expression of survivin, PCNA, bcl-2 and bax genes during the apoptosis progress in androgen-independent prostate cancer PC-3 cell line.Methods: PC-3 cell was treated by different concentration of PCNA-ASDON for 48 hours. The expression levels of survivin and PCNA mRNA were analyzed using RT-PCR method. The expression levels of PCNA, bcl-2 and bax protein were analyzed by immunohistochemical SABC assay.Results: PC-3 cell was treated to PCNA-ASDON for 48 hours, the expression levels of survivin and PCNA mRNA decreased obviously than contrast group and PCNA-SODN group ( P<0.01 ) . The expression of bcl-2 and PCNA decreased obviously and the expression of bax increased obviously than contrast group and PCNA-SODN group (/70.05) .Conclusion: PCNA-ASODN can change the expression of multiple genes in PC-3 cell. PCNA-ASODN induce apoptosis of PC-3 cell through down-regulated the expression of mRNA and protein of PCNA, survivin and bcl-2, and then increased expression of bax.In summary, we draw following conclusion from the result of this study:1. Proliferating Cell Nuclear Antigen Antisense Oligodeoxynucleotide could inhibit the proliferation of androgen-independent prostate cancer cell PC-3 cell line.2. Proliferating Cell Nuclear Antigen Antisense Oligodeoxynucleotide could induce apoptosis of androgen-independent prostate cancer cell PC-3 cell line.3. PCNA-ASODN can change the expression of multiple genes in PC-3 cell. PCNA-ASODN induce apoptosis of PC-3 cell through down-regulated the expression of mRNA and protein of PCNA, survivin and bcl-2, and then increased expression of bax.