磷酸酶PHLPP2抑制GPX4活性增强非小细胞肺癌铁死亡诱导剂RSL3敏感性的作用分析  

Phosphatase PHLPP2 Enhances Ferroptosis Inducer RSL3 Sensitivity by Inhibiting GPX4 Activity in Non-small Cell Lung Cancer

作　　者：王微 夏鑫杭[2] 杨海华 宋正波[1,3] WANG Wei;XIA Xin-hang;YANG Hai-hua;SONG Zheng-bo(Zhejiang Chinese Medical University,Hangzhou 310053,China;Taizhou Hospital of Zhe­jiang Province Affiliated to Wenzhou Medical University,Taizhou 317000,China;The Cancer Hospital of the University of Chinese Academy of Sciences(Zhejiang Cancer Hospital),Institute of Basic Medicine and Cancer(IBMC),Chinese Academy of Sciences,Hangzhou 310022,China)

机构地区：[1]浙江中医药大学,浙江杭州310053 [2]温州医科大学附属浙江省台州医院,浙江台州317000 [3]中国科学院大学附属肿瘤医院(浙江省肿瘤医院),中国科学院基础医学与肿瘤研究所,浙江杭州310022

出　　处：《肿瘤学杂志》2022年第6期459-464,共6页Journal of Chinese Oncology

基　　金：浙江省基础公益技术研究计划(LGF21H160027);台州市科技计划项目(20ywa09)。

摘　　要：[目的]探讨磷酸酶PHLPP2在铁死亡诱导剂RSL3诱导非小细胞肺癌细胞铁死亡发生中的作用及机制。[方法]体外培养人肺癌细胞株,GPX4抑制剂RSL3处理后MTT法检测RSL3抑制非小细胞肺癌细胞增殖作用,实时荧光定量PCR检测mRNA水平,Western blot法检测蛋白表达。流式细胞术检测脂质ROS水平,分别采用逆转录病毒/腺病毒感染上调/下调PHLPP2表达,两组间比较采用独立样本t检验。[结果]RSL3有效抑制非小细胞肺癌细胞的活力,RSL3处理后HCC827、H1975、H1650、A549和H1299细胞的增殖率分别为15.22%±2.35%、25.05%±5.92%、4.40%±1.61%、41.48%±10.01%和13.17%±2.54%。PHLPP2表达水平最高的H1650细胞对RSL3抑制增殖作用最显著,RSL3抑制增殖作用与PHLPP2表达水平呈正比。上调PHLPP2表达可以增加RSL3对A549细胞增殖抑制作用,并增加铁死亡关键标志脂质ROS的累积,A549-vector和A549-PHLPP2组的脂质ROS水平分别为4.34%±0.39%和15.36%±0.80%(P<0.001);相反,下调PHLPP2表达H1650细胞中,RSL3对H1650细胞增殖抑制作用减弱,减少了其诱导的脂质ROS的累积,H1650-shControl和H1650-shPHLPP2脂质ROS水平分别为14.76%±1.22%和4.89%±1.81%(P=0.0015)。公共数据库挖掘分析PHLPP2与铁死亡关键蛋白GPX4、SLC7A11和ASCL4的相关性,发现PHLPP2与GPX4表达呈负相关(r=-0.336,P<0.001)。Western blot进一步验证了上调PHLPP2表达可增强RSL3对GPX4蛋白的抑制作用。[结论]PHLPP2促进GPX4抑制剂RLS3诱导铁死亡发生,其机制主要通过协同抑制GPX4活性。[Objective]To explore the effect and mechanism of phosphatase PHLPP2 on ferroptosis inducers(FINs)RSL3 sensitivity in non-small cell lung cancer(NSCLC)cells.[Methods]Human NSCLC cell lines A549,H1650,HCC827,H1975 and H1299 were treated with GPX4 inhibitor RSL3,the cell proliferation was assessed by MTT assay in NSCLC cell lines.Real-time fluorescence quantitative PCR(qRT-PCR)and Western blot were used to analyze PHLPP2 expression.Lipid reactive oxygen species(ROS)was assessed by flow cytometry using fluorescent probes C11-BODIPY.Retrovirus/adenovirus transfection was used to up-regulate and down-regulate the expression of PHLPP2 in A549 and H1650 cells,respectively.Two-tailed unpaired Student’s t-test was used for comparison between the two groups.[Results]RSL3 effectively inhibited the viability of NSCLC cells.After RSL3 treatment,the proliferation rates of HCC827,H1975,H1650,A549and H1299 cells were 15.22%±2.35%,25.05%±5.92%,4.40%±1.61%,41.48%±10.01%and 13.17%±2.54%,respectively.Overexpression of PHLPP2 promoted cell death and increased lipid ROS production induced by RSL3 in A549 cells,the lipid ROS production in the A549-vector and A549-PHLPP2 groups were 4.34%±0.39%and 15.36%±0.80%,respectively(P<0.001).On the contrary,in H1650 cells with down-regulated PHLPP2 expression,RSL3 attenuated the proliferation inhibition of H1650 cells and reduced the accumulation of lipid ROS induced by RSL3.The levels of lipid ROS of H1650-shControl and H1650-shPHLPP2 were 14.76%±1.22%and 4.89%±1.81%,respectively(P=0.0015).Public database mining analysis showed the correlation between PHLPP2 and iron death key proteins GPX4,SLC7A11 and ASCL4.It was found that PHLPP2 was negatively correlated with the expression of GPX4(r=-0.336,P<0.001).Western blot further verified that up-regulating the expression of PHLPP2 enhanced the inhibitory effect of RSL3 on GPX4protein.[Conclusion]PHLPP2 may promote ferroptosis induced by GPX4 inhibitor RLS3 through synergistic inhibition of GPX4 activity.

关 键 词：非小细胞肺癌 铁死亡 PHLPP2 

分 类 号：R734.2[医药卫生—肿瘤]