环腺苷酸拟似物8-CPT-cAMP诱导骨髓瘤细胞凋亡的实验研究

Experimental study on cAMP analogue 8-CPT-cAMP inducing apoptosis in multiple myeloma cells

背景与目的:多发性骨髓瘤(multiple myeloma,MM)患者经标准方案治疗后缓解率虽高,但其缓解后复发及对化疗药物耐药性的产生较普遍。通过调变细胞内环腺苷酸浓度可以诱导多种肿瘤细胞增殖阻滞和凋亡,成为肿瘤治疗新途径。本研究观察环腺苷酸拟似物8-对氯苯硫基环腺苷酸[8-(4-chlorophenylthio)adenosine 3’,5’-cyclic monophosphate,8-CPT-cAMP]对MM细胞生物学行为的影响,探讨其诱导MM细胞凋亡的可能机制,为开发临床MM治疗新药提供研究方向。方法:以不同浓度8-CPT-cAMP处理MM细胞系U266,用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测其增殖,流式细胞仪检测细胞周期、凋亡率和线粒体跨膜电位的变化,实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-PCR)、蛋白质印迹法(Western blot)分别检测凋亡相关基因caspase-8、caspase-9、Bcl-2和Bax的转录及Bcl-2、Bax蛋白的表达。结果:U266细胞经不同浓度8-CPT-cAMP处理5 d后,生长受到明显抑制,呈浓度和时间依赖性;伴随作用时间延长,8-CPT-cAMP的半数有效抑制浓度(IC50)明显减低,第5天可达58.52μmol/L。U266细胞周期在8-CPT-cAMP浓度增加的状态下逐渐停滞在G0/G1期;各浓度组细胞增殖抑制率、凋亡率与对照组相比差异均有统计学意义(P<0.05)。同时8-CPT-cAMP能够诱导U266细胞线粒体跨膜电位下降;此外,经8-CPT-cAMP处理48和72 h后,不同浓度U266细胞Bcl-2 mRNA及蛋白表达较对照组明显下降(P<0.05),caspase-9、Bax mRNA及Bax蛋白表达明显升高(P<0.05),而caspase-8 mRNA表达与对照组比较无明显差异。结论:8-CPT-cAMP能够抑制骨髓瘤细胞增殖并诱导其凋亡,呈时间和浓度依赖性;该效应可能通过caspase依赖的线粒体诱导细胞凋亡途径来实现。

Background and purpose：Despite the high remission rate in patients with multiple myeloma （MM） after the standard regimen, but often relapsed and resistant. It has been shown that modulation of cAMP can induce cell cycle arrest and apoptosis in a variety of tumor cells, which has become an interesting approach to cancer therapy. This study aimed to investigate possible effects of cyclic adenosine monophosphate （cAMP） analogue 8-（4-chlorophenylthio） adenosine 3’, 5’-cyclic monophosphate （8-CPT-cAMP） on multiple myeloma cells, provide direction to develop new drugs for the treatment of MM. Methods：The myeloma cell line U266 cells were treated with 8-CPT-cAMP of different concentrations. The proliferation of U266 cells was evaluated through cell counting kit （CCK-8） assay, lfow cytometry was used to analyze the changes of cell were distribution, apoptosis rate as well as mitochondrial transmem-brane potential （ΔΨm） in U266 cells before and after the treatment. Meanwhile, real-time quantitative polymerase chain reaction （RT-PCR） and Western blot assay were used to detect expression levels of apoptosis regulators including caspase-8, caspase-9, Bcl-2 and Bax genes in U266 cells before and after the treatment. Results：The U266 cells were treated 5 days with 8-CPT-cAMP of different concentration, it was shown that 8-CPT-cAMP could signiifcantly inhibit cell growth of U266 cells in a concentration and time dependent manner, the IC50 of 8-CPT-cAMP was reduced obvious prolonged reaction time, and reached to 58.52μmol/L in the iffth day. The cell cycle of U266 cells was stopped in G0/G1 stage as the progress of concentration. It was showed statistical signiifcant difference associated with the cellular proliferation inhibition rate and apoptosis rate in different concentration and control （P〈0.05）. Meanwhile, 8-CPT-cAMP could induce mitochondrial transmembrane potential collapse in U266 cells. Compared with control groups, the levels of Bcl-2 mRNA transcripts and protein in U266 cells were reduced in 8-CPT-cAMP treated groups （P〈0.05）, while the levels of caspase-9, Bax mRNA transcription and the expression of Bax protein were increased in treatment groups, but the caspase-8 mRNA had no statistical signiifcant difference with controls. Conclusion：8-CPT-cAMP can inhibit the proliferation and promote apoptosis of myeloma cells, which might be mediated by caspase via mitochondrial pathway.