苦蘵内酯J降解STAT3蛋白诱导H1975细胞凋亡的作用机制

Physagulin J Induces Cell Apoptosis by STAT3 Degradation in H1975 Cells

[目的]探索信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)新型抑制剂苦蘵内酯J诱导肺腺癌细胞凋亡的分子机制。[方法]以5、10、20、25、30、40μmol·L^(-1)苦蘵内酯J处理表皮细胞生长因子受体(epidermal growth factor receptor,EGFR)T790M继发突变的肺腺癌细胞株H1975细胞24h或48h后,CCK-8法检测细胞存活率;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞24h后,Annexin V-PI染色及流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白的表达;5、10、15、20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h或20μmol·L^(-1)苦蘵内酯J处理H1975细胞0.5、1、2、4h后,Western blot检测STAT3蛋白表达及其磷酸化水平;以20μmol·L^(-1)蛋白酶体抑制剂MG132及10μmol·L^(-1)苦蘵内酯J联合处理或单独处理H1975细胞,阐明苦蘵内酯J对STAT3蛋白的降解作用;20μmol·L^(-1)苦蘵内酯J处理H1975细胞4h后,实时荧光定量PCR检测STAT3 m RNA表达情况。[结果]H1975细胞存活率随苦蘵内酯J作用时间延长和作用浓度增加而不断降低,20μmol·L^(-1)苦蘵内酯J处理24h和48h,H1975细胞存活率分别为65.9%和44.2%;20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞凋亡率为51.1%;Western blot检测显示20μmol·L^(-1)苦蘵内酯J处理24h后,H1975细胞内cleaved-caspase 9、cleaved-caspase 3和cleaved-PARP蛋白表达水平显著高于未处理组(P<0.05,P<0.01,P<0.001);20μmol·L^(-1)苦蘵内酯J处理4h后,H1975细胞内STAT3总蛋白和STAT3-Tyr表达水平显著低于与未处理组(P<0.001);蛋白酶体抑制剂MG132可恢复STAT3的总蛋白水平;实时荧光定量PCR结果显示,苦蘵内酯J对STAT3 m RNA表达无影响(P>0.05)。[结论]苦蘵内酯J能够抑制H1975细胞增殖并诱导细胞凋亡,其可能的机制是引起STAT3泛素化降解并抑制STAT3磷酸化。

[Objeetive] To investigate the effect of Physagulin J, a novel inhibitor of signal transducer and activator of transcription 3（STAT3）, on lung adenocareinoma, and to explore the underlying mechanism. [Methods] H1975 cells were treated with 5, 10, 20, 25, 30, 40μmol·L^-1 Physagulin J and CCK-8 analysis was applied to detect cell viability; Annexin V-PI analysis was performed to verify cell apoptosis rate using 5, 10, 15, 20μmol·L^-1 Physagulin J treated for 24h, apoptosis related markers were also analyzed by Western blot; 5,10, 15, 20μmol·L^-1 Physagulin J treated for 4h, or 20μmol·L^-1 Physagulin J treated for 0.5, 1, 2, 4h, STAT3 and its phosphorylation level were detected by Western blot; Combinational treatment of 10μmol·L^-1 Physagulin J and 20μmol·L^-1 MG132 was performed to identify the STAT3 degradation function of Physagulin J; STAT3 mRNA level was deteeted by QRT-PCR after 0 or 20μmol·L^-1 Physagulin J treatment. [Results] CCK-8 assay showed that When 20μmol·L^-1 Physagulin J treated H1975 cells for 24h or 48h, cell survival rates were 65.9% and 44.2% respectively. FACS analysis showed that 20μmol·L^-1 Physagulin J treated H1975 cells for 24h, the apoptosis cell rate was 51.1%. Western blot assay indicated that the expression levels of cleaved-caspase 9（P〈0.05）, cleaved-caspase 3（P〈0.01） and cleaved- PARP（P〈0.001） were highly upregnlated in 20μmol·L^-1 Physagulin J treated group. Western blot analysis showed that total and phosphorylation protein level of STAT3 significantly reduced in 20μmol·L^-1 Physagulin J treated group（P〈0.001）. After treating with proteasome inhibitor MG132, STAT3 protein level could restore. [Conclusion] Physagulin J induced STAT3 ubiquitination and inhibited STAT3 phosphorylation, which resulted in cell apoptosis induction and proliferation inhibition of H1975.