左金丸逆转KRAS突变型大肠癌西妥昔单抗耐药的作用机制研究

Effect and mechanism of Zuojin Wan on reversing cetuximab resistance in KRAS mutant colorectal cancer

目的探讨左金丸(ZJW)对KRAS突变型大肠癌西妥昔单抗(CET)耐药的逆转作用及其机制。方法①常规培养KRAS突变人结肠癌细胞HCT116,通过细胞增殖率(CCK-8法检测)确定ZJW无毒剂量(即10 mg·L^(-1)),观察ZJW、CET的协同效应(ZJW 10 mg·L^(-1)与CET 100、200 mg·L^(-1)具有协同效应,本研究选择CET 100 mg·L^(-1)进行后续实验)。将处于对数生长期的HCT116细胞分为对照组、CET 100 mg·L^(-1)组(CET组)、ZJW 10 mg·L^(-1)组(ZJW组)、CET 100 mg·L^(-1)+ZJW 10 mg·L^(-1)组(ZJW+CET组),作用48 h;采用流式细胞仪分析细胞周期和凋亡情况,Western blot法检测细胞中核因子-κB p65(NF-κB p56)、B淋巴细胞瘤-2(Bcl-2)蛋白表达情况,免疫荧光法检测细胞中NF-κB p65、磷酸化核因子-κB p65(p-NF-κB p65)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)表达情况,实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测细胞中NF-κB p65、Bcl-2、Caspase-3 mRNA表达情况。②将HCT116细胞皮下注射于裸鼠腋下,建立大肠癌皮下移植瘤模型,然后将16只裸鼠随机分为对照组(PBS灌胃)、CET组(CET尾静脉注射,15 mg·kg^(-1))、ZJW+CET组(CET尾静脉注射,15 mg·kg^(-1);ZJW灌胃,2055 mg·kg^(-1))、ZJW组(ZJW灌胃,2055 mg·kg^(-1)),每组4只,连续干预28 d,绘制肿瘤生长图,28 d后剥离肿瘤组织,RT-qPCR法检测皮下移植瘤组织NF-κB p65、Bcl-2、Caspase-3 mRNA表达情况。结果①ZJW在HCT116细胞中的IC_(10)为10.71 mg·L^(-1);CET抑制HCT116增殖的IC_(50)为578.6 mg·L^(-1),与10 mg·L^(-1)左金丸联合使用时IC_(50)值降低为231.3 mg·L^(-1);CET 100、200 mg·L^(-1)与ZJW 5、10 mg·L^(-1)联合使用时,药物联合指数(CI)<1。②与对照组相比,ZJW组、ZJW+CET组细胞S期比例、早期凋亡率升高(P<0.05);与CET组相比,ZJW组细胞S期比例、早期凋亡率升高(P<0.05);与ZJW组相比,ZJW+CET组S期细胞比例、早期凋亡率升高(P<0.05)。③与对照组比较,CET组、ZJW组细胞中Bcl-2表达降低(P<0.05),ZJW组细胞中Caspase-3表达升高(P<0.05),ZJW+CET组细胞Bcl-2、NF-κB p65、p-NF-κB p65表达降低(P<0.05);与CET组相比,ZJW+CET组细胞中Bcl-2、NF-κB p65、p-NF-κB p65表达降低(P<0.05),Caspase-3表达升高(P<0.05);与ZJW组相比,ZJW+CET组细胞Bcl-2、NF-κB p65、p-NF-κB p65表达降低(P<0.05),Caspase-3表达升高(P<0.05)。④与对照组相比,ZJW组、ZJW+CET组细胞Bcl-2、NF-κB p65 mRNA表达降低(P<0.05);与CET组相比,ZJW组、ZJW+CET组细胞Bcl-2、NF-κB p65 mRNA表达降低(P<0.05);与ZJW组相比,ZJW+CET组细胞Bcl-2、NF-κB p65 mRNA表达降低(P<0.05)。⑤相较于对照组、CET组,ZJW组、ZJW+CET组裸鼠瘤体生长较为缓慢。给药28 d后剥离瘤体,与对照组相比,ZJW组、ZJW+CET组裸鼠皮下移植瘤体积缩小(P<0.05);与CET组相比,ZJW、ZJW+CET组裸鼠皮下移植瘤体积缩小(P<0.05);与ZJW组比较,ZJW+CET组裸鼠皮下移植瘤体积缩小(P<0.05)。⑥与对照组相比,ZJW组、ZJW+CET组瘤体组织中NF-κB p65、Bcl-2 mRNA表达降低,Caspase-3 mRNA表达升高(P<0.05);与CET组相比,ZJW组瘤体组织中Caspase-3 mRNA表达升高(P<0.05);与ZJW组相比,ZJW+CET组瘤体组织中Caspase-3 mRNA表达升高(P<0.05)。结论ZJW可通过抑制细胞增殖、阻滞细胞周期、促进细胞凋亡等途径逆转KRAS突变大肠癌细胞对CET的耐药,继而抑制大肠癌生长,其机制可能与调控NF-κB/Bcl-2/Caspase-3通路相关。

Objective To investigate the effect and mechanism of Zuojin Wan(ZJW)on reversing the resistance to cetuximab(CET)in KRAS mutation colorectal cancer.Methods①KRAS mutant human colon cancer cells HCT116 were cultured and the non-toxic dose(10 mg·L^(-1))of ZJW was determined by cell proliferation rate(CCK-8 assay).The synergistic effect of ZJW and CET was observed(Due to the synergistic effect between ZJW 10 mg·L^(-1) and CET 100 and 200 mg·L^(-1),CET 100 mg·L^(-1) was selected for follow-up experiment in this study).HCT116 cells in logarithmic growth phase were divided into control group,CET 100 mg·L^(-1)(CET group),ZJW 10 mg·L^(-1)(ZJW group),CET 100 mg·L^(-1)+ZJW 10 mg·L^(-1)(ZJW+CET group),and were treated for 48 h.The cell cycle and apoptosis were analyzed by flow cytometry,and the expressions of nuclear factor-κB p65(NF-κB p65)and B lymphocytoma-2(Bcl-2)were detected by Western blot.The expressions of NF-κB p65,phosphorylated nuclear factor-κB p65(p-NF-κB p65)and cysteine aspartic protease-3(Caspase-3)were detected by immunofluorescence,and the mRNA expressions of NF-κB p65,Bcl-2 and Caspase-3 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR).②HCT116 cells were injected subcutaneously into the axilla of nude mice to establish a subcutaneously transplanted tumor model of colorectal cancer,and then 16 nude mice were randomly divided into control group(PBS gavage,n=4),CET group(CET caudal vein injection,15 mg·kg^(-1),n=4),ZJW+CET group(CET caudal vein injection,15 mg·kg^(-1);ZJW gavage,2055 mg·kg^(-1),n=4),and ZJW group(ZJW gavage,2055 mg·kg^(-1),n=4).Intervention were given continuously for 28 d,and tumor growth curves were plotted.The tumor tissue was stripped after 28 d,and the mRNA expressions of NF-κB p65,Bcl-2,Caspase-3 in subcutaneous transplanted tumor tissues were detected by RT-qPCR.Results①The IC_(10) value of ZJW in HCT116 cells was 10.71 mg·L^(-1);The IC_(50) value of CET inhibiting the proliferation of HCT116 was 578.6 mg·L^(-1),and the IC_(50) value decreased to 231.3 mg·L^(-1) with the combined use of 10 mg·L^(-1) ZJW.The drug combination index(CI)value was less than 1 when CET 100 and 200 mg·L^(-1) was used together with ZJW 5 and 10 mg·L^(-1).②Compared with those in control group,the S-phase proportion and early apoptosis rate of cells in ZJW and ZJW+CET groups increased(P<0.05).Compared with those in CET group,S-phase proportion and early apoptosis rate of cells in ZJW group increased(P<0.05).Compared with those in ZJW group,S-phase cell proportion and early apoptosis rate in ZJW+CET group increased(P<0.05).③Compared with those in control group,the expression of Bcl-2 in CET and ZJW groups decreased(P<0.05),the expression of Caspase-3 in ZJW group increased(P<0.05),and the expressions of Bcl-2,NF-κB p65 and p-NF-κB p65 in ZJW+CET group decreased(P<0.05).Compared with those in CET group,the expressions of Bcl-2,NF-κB p65 and p-NF-κB p65 in ZJW+CET group decreased(P<0.05),while the expression of Caspase-3 increased(P<0.05).Compared with those in ZJW group,the expressions of Bcl-2,NF-κB p65 and p-NF-κB p65 in ZJW+CET group decreased(P<0.05),while the expression of Caspase-3 increased(P<0.05).④Compared with those in control group,the mRNA expressions of Bcl-2 and NF-κB p65 in ZJW and ZJW+CET groups decreased(P<0.05).Compared with those in CET group,the mRNA expressions of Bcl-2 and NF-κB p65 in ZJW and ZJW+CET groups decreased(P<0.05).Compared with those in ZJW group,mRNA expressions of Bcl-2 and NF-κB p65 in ZJW+CET group decreased(P<0.05).⑤The tumor growth of nude mice in CET gtroup,ZJW group and ZJW+CET group was slower than that in control group.The tumors were stripped after 28 d of administration,and the volume of subcutaneous transplanted tumors in nude mice in ZJW group and ZJW+CET group was smaller than that in control group(P<0.05).The volume of subcutaneous transplanted tumors in nude mice in ZJW and ZJW+CET groups was smaller than that in CET group(P<0.05).The volume of subcutaneous transplanted tumors in nude mice in ZJW+CET group was smaller than that in ZJW group(P<0.05).⑥Compared with those in control group,the mRNA expressions of NF-κB p65 and Bcl-2 were lower and the mRNA expression of Caspase-3 was higher than that in CET Group in ZJW and ZJW+CET groups(P<0.05).The mRNA expression of Caspase-3 in ZJW group was higher(P<0.05).Compared with that in ZJW group,the mRNA expression of Caspase-3 in tumor tissues in ZJW+CET group was higher than that in ZJW group(P<0.05).Conclusions ZJW can reverse the resistance to CET in KRAS mutant colorectal cancer by inhibiting cell proliferation,blocking cell cycle and promoting cell apoptosis,and subsequently inhibiting the growth of colorectal cancer.The mechanism may be related to the regulation of NF-κB/Bcl-2/Caspase-3 pathway.