基于甲羟戊酸途径的盐酸小檗碱体外抗肺癌细胞药效及机制

Effect and Mechanism of Berberine Hydrochloride Against Lung Cancer Cells in Vitro Based on Mevalonate Pathway

目的:探究盐酸小檗碱(BBH)通过甲羟戊酸(MVA)途径抗肺癌细胞的药效及机制。方法:以人源肺癌细胞A549与鼠源肺癌细胞LLC作为研究对象,细胞增殖与活性检测(CCK-8)法检测BBH(10、20、30、40、50μmol·L^(-1))对2种肺癌细胞增殖的抑制作用(48 h);然后采用细胞划痕实验检测BBH(40μmol·L^(-1))对A549与LLC细胞迁移能力的影响(24、48 h);集落形成实验比较BBH(10、20、40μmol·L^(-1))给药下2种细胞的集落形成能力;试剂盒检测BBH(40μmol·L^(-1))对A549与LLC细胞中乙酰辅酶A(A-CoA)和总胆固醇(TC)含量的影响;运用AutoDock Vina软件对接BBH与MVA途径调控蛋白固醇调节结合原件蛋白2(SREBP2);实时荧光定量聚合酶链式反应(Real-time PCR)检测BBH(40μmol·L^(-1))对A549与LLC细胞中MVA途径9个相关mRNA[羟甲基戊二酸单酰辅酶A合酶1(HMGCS1)、羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、甲羟戊酸激酶(MVK)、磷酸甲羟戊酸激酶(PMVK)、5-焦磷酸甲羟戊酸脱羧酶(MVD)、法尼基焦磷酸合酶(FDPS)、角鲨烯单加氧酶(SQLE)、法尼基二磷酸法尼基转移酶1(FDFT1)、异戊二烯基二磷酸合酶1(GGPS1)]表达的影响;蛋白免疫印迹法(Western blot)检测BBH(40μmol·L^(-1))对2种细胞中MVA途径3个基因(HMGCS1、HMGCR、FDFT1)的关键蛋白表达的影响;使用TCGA数据库分析HMGCS1、HMGCR、FDFT1与SREBP2的转录基因SREBF2在非小细胞肺癌(NSCLC)中的关系。结果:与空白组比较,BBH组A549与LLC细胞的增殖、迁移与集落形成能力均显著降低(P<0.01);细胞的凋亡率显著升高(P<0.01);分子对接表明BBH与SREBP2具有良好的对接活性;与空白组比较,BBH组MVA途径的代谢产物A-CoA与TC的含量均显著下降(P<0.01);给药BBH后,A549与LLC细胞中MVA途径基因HMGCS1、HMGCR、MVK、PMVK、MVD、FDPS、SQLE、FDFT1、GGPS1 mRNA的表达降低(P<0.01),HMGCS1、HMGCR、FDFT1蛋白水平明显下降(P<0.05,P<0.01)。在NSCLC患者中,HMGCS1、HMGCR、FDFT1与SREBF2呈高度相关(R=0.54、R=0.57、R=0.48)。结论:BBH可抑制A549与LLC细胞的增殖、迁移与集落形成能力并促进细胞的凋亡,其原因可能与BBH结合SREBP2调控MVA途径有关。

Objective:To investigate the efficacy and mechanism of berberine hydrochloride(BBH)against lung cancer cells through the mevalonate(MVA)pathway.Method:Human lung cancer A549 cells and mouse Lewis lung carcinoma(LLC)cells were used as research subjects.Cell proliferation and cell counting kit-8(CCK-8)assay were performed to detect the inhibitory effect of BBH(10,20,30,40,50μmol·L^(-1))on the proliferation of the two kinds of cells(48 h).Then cell scratch assay was used to explore the influence of BBH(40μmol·L^(-1))on the migration of A549 and LLC cells(24,48 h),and colony formation assay was conducted to compare the colony formation ability of the cells under different concentrations of BBH(10,20,40μmol·L-1).Moreover,the effects of BBH(40μmol·L^(-1))on the content of acetyl-coenzyme A(A-CoA)and total cholesterol(TC)in A549 and LLC cells were determined by kit assay.AutoDock Vina was used for the dock of BBH and MVA pathway regulatory protein,sterol regulatory element-binding protein 2(SREBP2).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to observe the effects of BBH(40μmol·L^(-1))on the mRNA expression of nine genes related to the MVA pathway in A549 and LLC cells:hydroxymethylglutaryl-CoA synthase 1(HMGCS1),hydroxymethylglutaryl-CoA Reductase(HMGCR),mevalonate kinase(MVK),phosphomevalonate kinase(PMVK),mevalonate 5-pyrophosphate decarboxylase(MVD),farnesyl diphosphate synthase(FDPS),squalene epoxidase(SQLE),farnesyl-diphosphate farnesyltransferase 1(FDFT1),and geranylgeranyl diphosphate synthase 1(GGPS1).Western blot was performed to clarify the effects of BBH(40μmol·L^(-1))on the expression of three key proteins of the MVA pathway:HMGCS1,HMGCR,and FDFT1.The Cancer Genome Atlas(TCGA)database was searched to analyze the relationship between HMGCS1,HMGCR,FDFT1 and transcription gene SREBF2 in non-small cell lung cancer(NSCLC).Result:Compared with the conditions in the control group,the proliferation,migration,and colony formation of A549 and LLC cells in the BBH group were decreased(P<0.01),while the cell apoptosis rate was increased(P<0.01).Molecular docking showed that BBH had good binding activity with SREBP2.In addition,the content of A-CoA and TC of the MVA pathway was reduced(P<0.01).BBH downregulated the mRNA expression of HMGCS1,HMGCR,MVK,PMVK,MVD,FDPS,SQLE,FDFT1,and GGPS1 in A549 and LLC cells(P<0.01),and lowered the levels of HMGCS1,HMGCR,and FDFT1 proteins(P<0.05,P<0.01).In NSCLC patients,HMGCS1,HMGCR,and FDFT1 were highly correlated with SREBF2(R=0.54,R=0.57,and R=0.48).Conclusion:BBH can inhibit the proliferation,migration,and colony formation of A549 and LLC cells and promote cell apoptosis,which may be related to the regulation of MVA pathway by BBH binding to SREBP2.