曲古抑菌素A和多西他赛联合应用治疗前列腺肿瘤的实验研究

Evaluating the cytotoxicity of combination treatment with Trichostatin A and Docetaxel in prostate cancer chemotherapy

目的研究组蛋白去乙酰化抑制药物与常用的紫杉类化疗药物联合应用对人类前列腺癌细胞的杀伤作用,探讨其可能的分子机制。方法用不同浓度的组蛋白去乙酰化抑制药物曲古抑菌素A(trichostatin A,TSA)和紫杉类化疗药物多西他赛(Docetaxel,DTX)分别处理DU145和22Rv1前列腺肿瘤细胞,体外观察二者对肿瘤细胞的细胞毒作用。采用水溶性四唑盐(water soluble tetrazolium,WST-1)法检测细胞的增殖抑制率;蛋白质免疫印迹(western blot,WB)检测细胞中DNA损伤标志物磷酸化H2A.X(p-H2A.X)和细胞凋亡标志蛋白聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)特异性裂解片段的表达水平;采用定量实时荧光聚合酶链反应(quantitative real-time fluorescence polymerase chain reaction,q RT-PCR)检测细胞中相关肿瘤抑制分子Maspin、p21^(CIP1/WAF1)和Bax的转录表达水平。结果TSA和DTX对于前列腺肿瘤细胞增殖的抑制作用随着浓度升高而增强。TSA对DU145细胞的细胞毒作用(IC50=0.16μM)较对22Rv1细胞(IC50=0.06μM)弱。而DTX对DU145细胞的细胞毒作用则较对22Rv1细胞表现更显著。低剂量的TSA(0.01μM,0.1μM)能协同DTX抑制肿瘤细胞的增殖。DTX能诱导肿瘤细胞中的PARP分子产生细胞凋亡的特异性裂解、使p-H2A.X表达水平升高,并促进肿瘤抑制分子MASPIN、p21^(CIP1/WAF1)和细胞凋亡分子Bax的转录表达。使用低剂量的TSA处理虽未见产生PARP的细胞凋亡特异性裂解片段和p-H2A.X表达升高,但促进DU145细胞表达maspin和Bax,也使22Rv1表达maspin和p21^(CIP1/WAF1)升高。同时TSA协同DTX显著诱导DU145细胞转录表达maspin、p21^(CIP1/WAF1)和Bax,协同诱导22Rv1细胞转录表达maspin和Bax分子。结论TSA和DTX均能抑制肿瘤细胞增殖。DTX处理能有效损伤肿瘤细胞的DNA,诱导肿瘤细胞凋亡。联合应用TSA和DTX可以显著提高抗肿瘤相关分子的表达。这种药物诱导的细胞毒作用可能是通过诱导肿瘤抑制基因的表达而发挥作用,并具有组织和细胞特异性。该结果为临床应用TSA和DTX药物干预和治疗前列腺肿瘤提供用药指导,同时也提醒临床医师用药时必须考虑患者的个体化差异。

Objective To investigate the therapeutic efficacy of trichostatin A（TSA）in combination with Docetaxel（DTX）in human prostate cancer chemointervention through studying the cytotoxic effect in vitro,and the possible molecular mechanism.Methods Prostate cancer cells DU145 and 22Rv1 were treated with different doses of TSA or DTX respectively.The cytotoxic effect was evaluated by WST-1 assay.The PARP cleavage and level of phosphorylated H2A.X（p-H2A.X）were assessed by Western Blot.The genes expression of tumor suppressive maspin、p21^CIP1/WAF1 and Bax were analyzed by reverse transcription followed by quantitative real-time PCR.Results The treatment with either TSA or DTX inhibited cell proliferation of prostate cancer DU145 and 22Rv1 cells with differential drug sensitivity.The cytotoxic effect of TSA on 22Rv1 cell（IC50=0.06μM）was more significant than on DU145 cell（IC50=0.16μM）.But the cytotoxic effect of DTX on DU145 was more significant than on 22Rv1 cell.Combination treatment of TSA with DTX showed synergistic inhibitory effect on cell proliferation of both cell lines.Both PARP cleavage and p-H2A.X level were increased in DTX-induced cytotoxicity companied with increased expression of maspin and Bax in DU145 cells,and of maspin and p21^CIP1/WAF1 in 22Rv1 cells.Treatment with sub-lethal concentration of TSA（0.1μM）was able to promote DTX-induced expression of tumor suppressive maspin,p21^CIP1/WAF1 and Bax in these two cell lines with a differential manner.Conclusion Treatment with TSA or DTX could inhibit the proliferation of prostate cancer DU145 and 22Rv1 cells.DTX treatment was able to induce apoptotic cell death.The combination treatment approach of TSA with DTX showed synergistic effects on increasing expression of tumor suppressive maspin,p21^CIP1/WAF1 and Bax,which may be differentially indispensible to their cytotoxic effect.