Role For E2F1in The Regulation Of Transcription Factor Dimerization Partner-3Expression And Apoptosis In Prostate Cancer PC3Cells

Prostate cancer (PCa) is one of the most common cancer in male’s genitourinarysystem around the world and the second leading cause of death due to cancer in men in theUnited State. Traditionally, androgen-ablation therapy, anatomical castration andchemical castration are mostly used to reduce androgen level for PCa treatment.However, almost all the patients will gradually progress to androgen-independent prostatecancer(AIPC), which accelerats the proliferation of cancer cell without the inhibition bythe low androgen levels and eventually leads to tumor progression or metastasis. Atpresent there is still no effective curing methold for AIPC. With the development ofmodern molecular biology, gene therapy is expected to become a new and effective meansfor AIPC. Therefore, there will be both scientific and clinical significance to clarify themolecular pathogenesis and transcriptional regulation mechanism of prostate cancer.TFDP3has been previously identified as an inhibitor of E2F molecules. In our formerstudies, the expression of TFDP3both in normal tissue and cancer tissue has been analyzed by microarray and in situ hybridization. It has been demonstrated that TFDP3gene is not a specific gene in tumor/testicular tissue. It is expressed widely in variousnormal tissues and particularly high in various cancer tissues, especially in liver andprostate cancer. We also demanstrated that TFDP3could not only inhibit E2F1-inducedproliferation and apoptosis of prostate cancer cell but also induce its autophagy, whichhelps to improve the survivability of the hormone-dependent prostate cancer cell lineLNCap. TFDP3-induced autophagy and anti-apoptosis may be one of the most importantself-protection mechanisms that drive prostate cancer cells from anandrogen-dependent toandrogen-independent.Through analyzing sequence of TFDP3promoter region, we found a transcriptionfactor E2F1binding site, which indicates that E2F1may enhance TFDP3promoter’sactivity. To confirm that, a luciferase reporter system (pGL3-TFDP3-promoter) containingTFDP3gene promoter was constructed in this study. Next, western blotting was employedto analyze the change of TFDP3expression under the treatment of E2F1. Finally, flowcytometry was used to detect apoptosis of prostase cancer cells by co-transfectingplasmids respectively containing TFDP3and E2F1gene.The main results:1. Total DNA was extracted from human prostate cancer cell line PC3and used toamplify the fragment containing TFDP3promoter by PCR. The amplified product ofupstream sequence of TFDP3gene (1064bp from ATG) was inserted into pGL3-Basicvector to constructpGL3-TFDP3-promoter vector. We transfected pGL3-TFDP3-promotervector into PC3cells, then we could detect the the luciferase activity to guarantee theTFDP3promoter was functional.The luciferase activity was significantly higher in PC3cells co-transfected withpGL3-TFDP3-promoter and pCMV-E2F1-HA than PC3cells transfected withpGL3-TFDP3-promoter alone (1.14±0.06vs0.61±0.05, P<0.05), which suggested thatE2F1could stimulate the TFDP3promoter.2. PC3cells were transfected with pCMV-E2F1-HA. Then TFDP3protein content inthe transfectants were determined by Western blotting analysis. It was showed that the TFDP3content in PC3cells transfected with pCMV-E2F1-HA was2.7times(0.089±0.02vs0.24±0.03,P<0.05) higher than that in non-transfected cells, whichsuggested that E2F1could upregulate the expression of TFDP3.3. PC3cells were transfected with pGL3-TFDP3and pCMV-E2F1-HA, alone ortogether. Apoptosis of the transfected cells were then analyzed by flow cytometry. Theproportion of apoptotic cells transfected with pCMV-E2F1-HA was significantly higherthan that both in non-transfected PC3cells ([2.66±0.001]%vs[7.1±0.003]%, P<0.05) andin PC3cells co-transfected with pGL3-TFDP3and pCMV-E2F1-HA ([4.92±0.002]%vs[7.1±0.003]%, P <0.05), which confirmed that TFDP3could inhibit the apoptosis ofPC3induced by E2F1.4. TFDP3and E2F1expression was assessed by immunohistochemistry in normalhuman tissues and prostate cancer tissues. The association between TFDP3and E2F1inprostate cancer developmentwas analyzed in various stages. The results show that TFDP3was always expressed in coordination with E2F1at equivalent expression levels inprostate cancer tissues, and was highly expressed particularly in samples of high stage.In summary, TFDP3promoter-luciferase reporter vector pGL3-TFDP3-promoter wasconstructed successfully. It has been initially proved that E2F1could bind to TFDP3promoter and has regulation function in transcription and expression level by dualluciferase assays and Western blotting. The immunohistochemistry results showed thatTFDP3was always expressed in coordination with E2F1at equivalent expression levels inprostate cancer tissues, and was highly expressed particularly in samples of high stage.Flow cytometry assay showed that TFDP3could effectively inhibite apoptosis of prostatecells, which may suggest that TFDP3plays an important role in the survival of prostatecancer cell.