环磷酸腺苷拟似物联合硼替佐米诱导弥漫大B细胞淋巴瘤细胞凋亡作用及其机制研究

Studies on the apoptosis of diffuse large B cell lymphoma cells induced by cAMP analogue combined with bortezomib

目的探讨环磷酸腺苷(cAMP)拟似物8-对氯苯硫基环磷酸腺苷(8-CPT-cAMP)联合硼替佐米对弥漫大B细胞淋巴瘤(DLBCL)细胞的生物学效应及其作用机制。方法以不同浓度8-CPT-cAMP及硼替佐米单独或联合处理DLBCL细胞系OCI-Ly1和U2932,采用细胞计数试剂盒-8(CCK-8)检测细胞增殖抑制率,应用药物联合指数(CI)分析两药是否存在协同效应,同时应用流式细胞仪检测细胞凋亡率以及线粒体跨膜电位(ΔΨm)的变化,进一步利用蛋白质印迹法检测细胞凋亡相关蛋白包括含半胱氨酸的天冬氨酸蛋白酶(caspase)-3,聚腺苷二磷酸-核糖聚合酶(PARP),信号通路蛋白c-myc,蛋白激酶B(AKT)和磷酸化AKT(p-AKT)以及肌动蛋白(actin)的表达。结果 OCI-Ly1细胞和U2932细胞经8-CPT-cAMP及硼替佐米单用或联用处理24 h后,联合用药组细胞增殖抑制率分别为(55.25±0.30)%和(26.42±0.80)%,明显高于单药组,差异具有统计学意义(P<0.05);两药联合处理OCI-Ly1细胞和U2932细胞的CI值均小于1;联合用药组OCI-Ly1细胞和U2932细胞凋亡率分别为(38.48±0.30)%和(36.70±0.60)%,显著高于单药组,差异具有统计学意义(P<0.05);联合用药组OCI-Ly1细胞和U2932细胞内caspase-3和PARP蛋白均发生明显剪切活化;进一步研究还发现8-CPT-cAMP可协同硼替佐米促使OCI-Ly1细胞和U2932细胞内线粒体ΔΨm下降并下调细胞内c-myc和p-AKT蛋白的表达。结论 8-CPT-cAMP联合硼替佐米可协同诱导DLBCL细胞凋亡,并抑制其增殖,其机制可能与两药协同介导细胞内线粒体ΔΨm下降,下调c-myc表达,抑制PI3K/AKT信号通路有关。

Objective To investigate possible effect of 3', 5'-cyclic adenosine monophosphate(cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate(8-CPT-cAMP) combined with bortezomib in diffuse large B cell lymphoma(DLBCL) cells. Methods The DLBCL cell lines OCI-Ly1 cells and U2932 cells were treated with different concentrations of 8-CPT-cAMP or bortezomib alone and combination. The proliferation inhibition rates of OCI-Ly1 cells and U2932 cells were evaluated through cell counting kit(CCK-8) assay. The synergistic manners of 8-CPT-cAMP and bortezomib were determined by combination index(CI). Meanwhile, the flow cytometry was used to analyze the changes of cell apoptosis rate and mitochondrial transmembrane potential(ΔΨm). Furthermore, Western blot assay was used to detect expression levels of apoptosis related proteins including caspase-3 and PARP as well as signal transduction activators c-myc, AKT/p-AKT and actin. Results After treated with 8-CPT-cAMP and/or bortezomib, the proliferation inhibition rates of OCILy1 cells and U2932 cells in combination groups reached to(55.25±0.30)% and(26.42±0.80)%, which were significantly higher than those in 8-CPT-cAMP or bortezomib group, the differences were statistically significant(P<0.05). The CI values of OCI-Ly1 cells and U2932 cells in combination group were lower than 1. The apoptosis rates of OCI-Ly1 cells and U2932 cells in combination group were(38.48±0.30)% and(36.70±0.60)%, which were significantly higher than those in 8-CPTcAMP or bortezomib group, the differences were statistically significant(P<0.05). 8-CPT-cAMP combined with bortezomib could induce activation of caspase-3 and PARP in OCI-Ly1 cells and U2932 cells. Furthermore, 8-CPT-cAMP combined with bortezomib could trigger mitochondrial ΔΨm collapse and down-regulate the expression of c-myc and p-AKT in OCILy1 cells and U2932 cells. Conclusion 8-CPT-cAMP can synergize with bortezomib to induce the apoptosis of DLBCL cells, which may be mediated by mitochondrial ΔΨm collapse as well as suppression of c-myc expression and PI3K/AKT signaling pathway.