粉防己碱增敏长春新碱通过MAPK信号通路诱导SGC-7901/VCR细胞凋亡的分子机制研究

Tetrandrine sensitizes vincristine to induce apoptosis in SGC-7901/VCR cells through MAPK signaling pathway

目的阐明粉防己碱(tetrandrine)增敏长春新碱(vincristine)诱导胃癌SGC-7901/VCR细胞凋亡的分子机制。方法用不同浓度(0、2、4、6、8μmol/L)粉防己碱及不同浓度(0、50、100、150、200 nmol/L)长春新碱单用或联用处理SGC-7901/VCR细胞48 h,MTT实验检测细胞存活率,流式细胞术检测细胞凋亡。Western blot检测细胞凋亡相关蛋白PARP-1、cleaved-Caspase 9、cleaved-Caspase 3、Bcl-2及MAPK信号通路相关蛋白P38、p-P38、JNK、p-JNK、ERK、p-ERK的表达。应用p38-MAPK抑制剂SB203580、JNK-MAPK抑制剂SP600125或ERK-MAPK抑制剂PD98059预处理SGC-7901/VCR细胞2 h,再以粉防己碱联用长春新碱处理48 h,测定细胞凋亡及相关蛋白表达。粉防己碱及长春新碱单用或联用处理SGC-7901/VCR细胞24 h,流式细胞术检测细胞周期,Western blot检测细胞周期相关蛋白CDC2、p-CDC2的表达。结果粉防己碱联用长春新碱导致SGC-7901/VCR细胞增殖活力以剂量依赖的方式下降,联合作用效果为协同。联合用药导致细胞凋亡显著增加(P<0.05),与PARP剪切激活及Caspase 9、Caspase 3裂解激活有关。联合用药增强细胞MAPK信号通路p-P38和p-JNK表达,减弱p-ERK表达,应用p38-MAPK或JNK-MAPK通路抑制剂预处理SGC-7901/VCR细胞后减弱联合用药诱导凋亡的作用(P<0.05),而应用ERK-MAPK通路抑制剂预处理后增强联合用药诱导凋亡的作用(P<0.05)。联合用药导致SGC-7901/VCR细胞S/G_(2)周期阻滞(P<0.05)。结论粉防己碱增敏长春新碱抑制细胞增殖并诱导SGC-7901/VCR细胞凋亡,MAPK信号通路可能在诱导细胞凋亡中起关键作用。

Objective To explore the molecular mechanism of tetrandrine sensitizing vincristine to induce apoptosis of gastric cancer SGC-7901/VCR cells.Methods SGC-7901/VCR cells were treated with tetrandrine(0,2,4,6 and 8μmol/L)and vincristine(0,50,100,150 and 200 nmol/L)alone or in combination for 48 h.MTT assay was used to detect cell viability,and flow cytometry was adopted to observe apoptosis.The expression of apoptosis-related proteins PARP-1,cleaved-Casepase 9,cleaved-Casepase 3,Bcl2,and MAPK signal transduction pathway related proteins P38,p-P38,JNK,p-JNK,ERK and p-ERK were detected by Western blotting.After SGC-7901/VCR cells were pretreated with p38-MAPK inhibitor SB203580,JNK-MAPK inhibitor SP600125,or ERK-MAPK inhibitor PD98059 for 2 h and followed by the treatment of tetrandrine combined with vincristine for 48 h,the apoptosis and the expression levels of related proteins were observed once again.SGC-7901/VCR cells were treated with tetrandrine and vincristine alone or combined for 24 h,cell cycle was detected by flow cytometry,and the expression of CDC2 and p-CDC2 were detected by Western blotting.Results Co-administration of tetrandrine and vincristine resulted in decreases in cell viability in a dose-dependent manner in SGC-7901/VCR cells,with a synergistic effect,and led to increases in cell apoptosis(P<0.05),which were associated with degradation of PARP,cleavage/activation of Caspase 3 and Caspase 9.Furthermore,combination of tetrandrine and vincristine markedly increased the levels of phospho-p38 and phospho-JNK and decreased the level of phospho-ERK.Pretreatment of p38 inhibitor SB203580 or JNK inhibitor SP600125 attenuated the combination-induced apoptosis(P<0.05).In contrast,pretreatment of ERK inhibitor PD98059 potentiated the combination-induced apoptosis(P<0.05).Combination treatment caused S/G_(2)cycle block of SGC-7901/VCR cells(P<0.05).Conclusion Tetrandrine sensitizes vincristine to inhibit cell proliferation and induces apoptosis in SGC-7901/VCR cells.MAPK signaling pathway may play a critical role in the combination-induced apoptosis in the cells.