| BackgroundProstate cancer is one of the most commonly diagnosed malignant tumors of urogenital system in men. The incidence of this cancer is significantly different in different regions, different countries and different races. Prostate cancer is the most common malignant tumor in men of Europe and the United States, also the second leading cause of cancer-related death. The American Cancer Society estimated that there were approximately238,590men diagnosed with prostate cancer and that29,720would die from prostate cancer related causes in2013. With the improving of living standard, the incidence of prostate cancer in China increased year by year. Now the prostate cancer has became the number3of the male urogenital system tumor in men. Endocrine therapy is the first choice for the treatment of prostate cancer, but the application of endocrine therapy after a period of time, the patient would inevitably produce steroid resistance, or parts of the patients are not sensitive to endocrine therapy primary, known as Castration Therapy-Resistant prostate cancer. This kind of prostate cancer can be regard as the terminal period of the disease, mainly depends on chemotherapy, whose effects are still poor, posing a threat to survival rate and quality of life of patient’s. Understanding the pathogenesis and exploring new targets of Castration Therapy-Resistant prostate cancer may potentially provide significant clinical impact.At present, INPP4B is involved in a wide variety of tumors and emerges as a potential tumor suppressor by negative regulation of PI3K/AKT pathway and blocking the cell malignant transformation. AR induces expression of INPP4B at the level of transcription in human prostate cancer cell lines, some study speculated that castration therapy-resistant prostate cancer cells may have lost AR expression, other study showed significant decrease in INPP4B expression in human prostate cancers compared to benign prostate epithelium tissue by examination of radical prostatectomy specimens. All of these led to the hypothesis that re-expression of INPP4B may exert antitumor effect. Function loss of PTEN is frequently observed in Castration Therapy-Resistant prostate cancer, contributing to defective homologous recombination. PARP inhibitor can promote cell death by inhibiting the compensatory DNA repair pathway in PTEN-null tumor. However, evidence suggested that inhibition of PARP resulted in activation of the PI3K/AKT pathway.which may counteract the anti-tumor effect and associate with cytostatic resistance. So the idea of investigate the effects of combing of INPP4B gene transfection and PARP inhibitor on castration therapy-resistant prostate cancer cell line PC3was produced.ObjectiveThis study intends to investigate the effects of INPP4B gene transfection and PARP inhibitor separating and combining on castration therapy-resistant prostate cancer cell line PC3.Materials and methods PC3and LNcap cells were cultured in vitro and the expression of INPP4B at the mRNA level was detected by reverse transcription PCR. PC3cells transfected with recombinant lentivirus vector carrying the human INPP4B gene and the expression of INPP4B was confirmed by Real-time PCR and Western blot. We set up CON (control by untreated PC3cells), Lenti-GFP, Lenti-INPP4B, PARP inhibitor AGO14699, Lenti-GFP+PARP inhibitor and Lenti-INPP4B+PARP inhibitor six groups, the effects of INPP4B transfection and/or PARP inhibitor treatment on the proliferation, apoptosis and cell cycle, and the level of p-AKT of PC3cells were determined by CCK-8assay, flow cytometry and Western blot, respectively.ResultsINPP4B gene was lost in PC3cells. After transfection recombinant lentivirus, real-time PCR showed the relative levels of INPP4B mRNA in lenti-INPP4B group is87.84±7.62; Similar results were obtained by Western blot analysis, noticeable expression of INPP4B protein was shown in the Lenti-INPP4B group. lenti-INPP4B group:the CCK-8showed the vitality of cells were (66.18±2.32)%,(45.10±0.00)%and (29.35±3.19)%in Lenti-INPP4B group, It demonstrated transfecting INPP4B gene resulted in a significant reduction of cell growth (P<0.05); Apoptosis rate measured by flow cytometry did not increase significantly in the Lenti-INPP4B group compared to the Lenti-GFP group (P>0.05); The levels of p-AKT decreased upon treatment with transfection of INPP4B gene, but not in Lenti-GFP. PARP inhibitor groups:the CCK-8showed the cells vitality were (93.58±3.51)%,(64.63±2.18)%,(57.15±3.38)%,(43.88±3.02)%,(40.60±0.86)%,(99.76±0.06)%, respectively after treated with0.1,1,10,20,40μM five concentrations of AG014699and DMSO48hours, AGO14699has a significant reduction of cell growth compared with DMSO (P<0.05);10μM was selected as the fixed single concentration for use and in combination treatments; CCK-8showed the vitality of PC3cells were (88.53±1.73)%,(60.77±2.26)%and (54.12±1.96)%at24,36and48hours after treated with10μM AG014699(P<0.05); Effects of AG014699on the distribution of cell cycle phase showed the ratio of G2/M phase were (23.50±2.90)%,(35.10±5.11)%and (55.97±3.75)%when treated with1,10and20μM after48hours, which implied that the ratio of G2/M phase increased with the mounting concentration of AGO14699, a high concentration of PARP inhibitor can induce a G2/M accumulation; Annexin V-FITC/PI showed the apoptosis of PC3cells were6.54%、12.16%、23.53%after treated with1μM,1OμM,20μM of AGO1469948hours, demonstrated AGO14699could promote cell apoptosis in a dose dependent pattern; The levels of p-AKT increased upon treatment with PARP inhibitor AGO14699in a dose dependent mannet. Lenti-INPP4B+PARP inhibitor group:First, observed the number of PC3cells decreased significantly and the shape of PC3cells became irregular after treated transfecting of INPP4B gene combined with PARP inhibitor treatment compared with single treatment; CCK-8showed the vitality of cells were (49.85±0.30)%,(26.30±1.81)%and (9.72±3.10)%after24,36and48hours, significant growth inhibition could be observed upon combination than transfection of INPP4B gene and PARP inhibitor given separately (P<0.05); Transfection of INPP4B gene combined with PARP inhibitor treatment caused a G1arrest; Compared with PARP inhibitor alone, combined with transfection of INPP4B gene did not result in any further significant increase apoptosis rate (P>0.05);Compared with AGO14699used alone, the levels of p-AKT decreased when combined with transfection of INPP4B gene, which was not detected when combined with Lenti-GFP.Conclusions1. INPP4B gene was lost in PC3cells.2. Transfection of INPP4B gene had a antitumor effect in PC3cells when used alone.3. PARP inhibitor had a antitumor effect in PC3cells when used alone.4. The combination of INPP4B gene transfection and PARP inhibitor had a synergistic antitumor effect on PC3cells, may be through decreasing the levels of p-AKT of PC3cells, which was expected to shed new light on combined biological therapy in castration therapy-resistant prostate cancer. |
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