摘要
目的:探讨PI3K抑制剂MK2206和GDC0941分别协同紫杉醇(Paclitaxel)对肝癌HepG2细胞增殖、诱导凋亡和对上皮间质转化(epithelial-mesenchymal transition,EMT)靶蛋白表达的分子机制。方法:CCK-8检测Paclitaxel、MK2206和GDC0941三个药物单独处理,或Paclitaxel分别协同MK2206和GDC0941对HepG2细胞增殖的影响;流式细胞凋亡法检测协同处理对细胞凋亡的影响;RT-PCR检测协同处理对抗凋亡蛋白(Bcl-2)、促凋亡蛋白(Bax)和Slug基因表达的影响;Western blot检测协同处理对β-catenin、Slug、人第10号染色体缺失的磷酸酶及张力蛋白(Pten)、抗凋亡蛋白(Bcl-2)、促凋亡蛋白(Bax)、半胱氨酸蛋白酶-3(Cleaved caspase-3)、E-钙连蛋白(E-cadherin)和粘连蛋白(Vimentin)蛋白表达的影响,免疫荧光法检测E-cadherin和Vimentin表达。结果:单独使用三种药物可随浓度升高抑制HepG2细胞活力(P<0.01),呈剂量相关性;Paclitaxel协同MK2206或GDC0941后,可在较低浓度抑制HepG2细胞活性。Paclitaxel协同MK2206或GDC0941后,细胞凋亡率显著增加(P<0.05);降低Bcl-2、Slug和β-catenin的mRNA表达水平,促进Bax和Pten的mRNA表达水平;与单一用药相比,协同用药显著下调Bcl-2、β-catenin和Vimentin的表达,上调Pten、Bax和E-cadherin的表达(P<0.05);协同药物反转β-catenin过表达诱导的Bax、E-cadherin、Pten、Cleaved caspase-3、Vimentin和β-catenin的表达(P<0.05)。结论:PI3K抑制剂MK2206/GDC0941能通过β-catenin/Pten信号轴增强Paclitaxel诱导肝癌HepG2细胞凋亡实现对HepG2细胞增殖抑制和EMT蛋白表达调控。
Objective:To explore the molecular mechanisms underlying the synergistic effects of PI3K inhibitors MK2206 and GDC0941 with paclitaxel on the proliferation,induction of apoptosis,and expression of epithelial-mesenchymal transition(EMT)target proteins in liver cancer HepG2 cells.Methods:CCK-8 was used to detect the effects of paclitaxel,MK2206,and GDC0941 alone on HepG2 cell proliferation,or paclitaxel in combination with MK2206 and GDC0941 on HepG2 cell proliferation,respectively.Flow cytometry was used to detect the effect of synergistic treatment on cell apoptosis.RT-PCR was used to detect the effects of synergistic treatment on anti-apoptotic protein(Bcl-2),pro-apoptotic protein(Bax),and Slug gene expression.Western blot was used to detect the effects of collaborative processing on the protein expression ofβ-catenin,Slug,the human chromosome 10 deficiency phosphatase and tensin(Pten),anti-apoptotic protein(Bcl-2),pro apoptotic protein(Bax),Cleaved caspase-3,E-cadherin,and Vimentin.Immunofluorescence was used to detect E-cadherin and Vimentin expression.Results:The use of three drugs alone inhibited HepG2 cell viability with increasing concentration(P<0.01),showing a dose-dependent relationship.After synergistic treatment with MK2206 or GDC0941,paclitaxel can inhibit HepG2 cell activity at lower concentrations and significantly increase cell apoptosis rate(P<0.05).Reducing the mRNA expression levels of Bcl-2,Slug andβ-catenin,and promoting the mRNA expression levels of Bax and Pten.Compared with monotherapy,synergistic therapy significantly downregulated the expression of Bcl-2,β-catenin,and Vimentin,and upregulated the expression of Pten,Bax,and E-cadherin(P<0.05).Synergistic therapy reversed the expression of Bax,E-cadherin,Pten,Cleaved caspase-3,Vimentin,andβ-catenin induced byβ-catenin overexpression(P<0.05).Conclusion:The PI3K inhibitor MK2206/GDC0941 can enhance paclitaxel induced apoptosis in HepG2 cells through theβ-catenin/Pten signaling axis,thereby inhibiting HepG2 cell proliferation and regulating EMT protein expression.
作者
祝珊珊
吴心怡
林乐颖
李杰
秦飞
付文涛
雷辰霞
蔡颖莲
陈川
ZHU Shanshan;WU Xinyi;LIN Leying;LI Jie;QIN Fei;FU Wentao;LEI Chenxia;CAI Yinglian;CHEN Chuan(School of Pharmacy,Xiamen Medical College,Fujian Xiamen 361026,China;Innovation Research Center for Biological Enzyme Catalysis and Drug Synthesis,Xiamen Medical College,Fujian Xiamen 361026,China;Department of Pharmacy,Xiamen Hospital of Traditional Chinese Medicine,Fujian Xiamen 361003,China)
出处
《现代肿瘤医学》
2025年第10期1658-1666,共9页
Journal of Modern Oncology
基金
福建省自然科学基金面上项目(编号:2024J011398)
福建省厦门市医疗卫生指导性项目(编号:3502Z20244ZD1139)
厦门医学院大学生创新创业计划项目(编号:202412631013,202412631073)。
