石蒜碱调节AKT/mTOR信号通路对乳腺癌细胞曲妥珠单抗耐药的影响

Effect of Lycorine on Trastuzumab Resistance in Breast Cancer Cells by Regulating AKT/mTOR Signaling Pathway

石蒜碱具有显著的抗肿瘤活性,可增强顺铂对肝癌细胞敏感性及细胞毒性.本研究采用体外培养人乳腺癌细胞SKBR3,以5μg/mL的TZB连续给药诱导建立TZB耐药细胞SKBR3/TR,以CCK-8法筛选石蒜碱最佳作用浓度后检测其对SKBR3/TR细胞TZB耐药指数的影响.构建SKBR3/TR移植瘤裸鼠模型,随机分为对照组、TZB组、TZB+石蒜碱组,药物干预后检测各组裸鼠肿瘤体积与质量.将体外培养的SKBR3/TR细胞随机分为对照组、TZB组、TZB+SC79组、TZB+石蒜碱组、TZB+石蒜碱+SC79(AKT激活剂)组,药物分组干预后以免疫印迹实验检测AKT/mTOR信号通路相关蛋白表达;以流式细胞实验、CCK-8法、结晶紫染色分别检测各组细胞凋亡和增殖;以免疫印迹实验检测各组细胞增殖(PCNA)、凋亡(Bax、Cleaved Caspase-3、Bcl-2)及耐药P-糖蛋白(P-gp)相关蛋白表达.探讨石蒜碱调节AKT/mTOR信号通路对乳腺癌细胞曲妥珠单抗(TZB)耐药的影响.结果显示:石蒜碱可明显降低SKBR3/TR细胞TZB耐药指数,并增强TZB对SKBR3/TR移植瘤裸鼠肿瘤生长的抑制作用.与对照组、TZB组相比,TZB+石蒜碱组细胞的凋亡率、Bax与Cleaved Caspase-3蛋白表达升高(P<0.05),p-AKT/AKT、p-mTOR/mTOR、细胞活力、集落形成率、PCNA与Bcl-2、P-gp蛋白表达降低(P<0.05);TZB+SC79组细胞各指标变化趋势与TZB+石蒜碱组相反(P<0.05).SC79可减弱石蒜碱对SKBR3/TR细胞的作用.上述结果表明,石蒜碱可抑制AKT/mTOR信号通路激活,从而减轻乳腺癌细胞的TZB耐药性.

Lycorine has significant anti-tumor activity and can enhance the sensitivity and cytotoxicity of cisplatin to liver cancer cells.In this study,SKBR3 cells were cultured in vitro and induced to establish trastuzumab(TZB)resistant cells SKBR3/TR by continuous administration of 5μg/mL TZB.The optimal concentration of lycorine was screened using CCK-8 method,and its effect on the TZB resistance index of SKBR3/TR cells was detected.A SKBR3/TR nude mouse model of transplanted tumors was constructed and randomly separated into a control group,TZB group,and TZB+lycorine group.After drug intervention,the tumor volume and mass of nude mice in each group were measured.SKBR3/TR cells cultured in vitro were randomly separated into control group,TZB group,TZB+SC79 group,TZB+lycorine group,and TZB+lycorine+SC79(AKT activator)group.After grouping and drug intervention,immunoblotting experiments were applied to detect the expression of AKT/mTOR signaling pathway related proteins.Flow cytometry,CCK-8 assay,and crystal violet staining were applied to detect apoptosis and proliferation of cells in each group.Immunoblotting experiments were applied to detect cell proliferation(PCNA),apoptosis(Bax,Cleaved Caspase-3,Bcl-2),and expression of resistance(P-glycoprotein(P-gp))related proteins in each group.The effect of lycorine on TZB resistance in breast cancer cells by regulating AKT/mTOR signaling pathway was investigated.The results show that lycorine could obviously reduce the TZB resistance index of SKBR3/TR cells and enhance the inhibitory effect of TZB on the growth of SKBR3/TR transplanted tumors in nude mice.Compared with the control group and TZB group,the apoptosis rate,expression of Bax and Cleaved Caspase-3 proteins in cells increased in the TZB+lycorine group(P<0.05),the p-AKT/AKT,p-mTOR/mTOR,the cell viability,colony formation rate,expression of PCNA and Bcl-2,P-gp proteins decreased(P<0.05);the trend of changes of various cellular indicators in the TZB+SC79 group was opposite to that in the TZB+Lycorianine group(P<0.05).SC79 can weaken the effect of lycorine on SKBR3/TR cell lines.The results showed that lycorine can inhibit the activation of AKT/mTOR signaling pathway,thereby reducing the TZB resistance of breast cancer cells.