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

作　　者：周晶 卫真真 浦匀舟 李玲[2] 朱惠蓉[2] 季青[2] 李红山[1,4] ZHOU Jing;WEI Zhenzhen;PU Yunzhou;LI Ling;ZHU Huirong;JI Qing;LI Hongshan(Hepatology Department of Integrated Chinese and Western Medicine,Ningbo No.2 Hospital of Zhejiang Province,Ningbo,Zhejiang 315010,China;Department of Oncology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Medical Experiment Center,Jiading Branch of Shanghai General Hospital,Shanghai 201803,China;Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of Zhejiang Province,Ningbo,Zhejiang 315010,China)

机构地区：[1]浙江省宁波市第二医院中西医结合肝病科,浙江宁波315010 [2]上海中医药大学附属曙光医院肿瘤科,上海201203 [3]上海市第一人民医院嘉定分院实验医学中心,上海201803 [4]浙江省消化系统肿瘤诊治及研究重点实验室,浙江宁波315010

出　　处：《上海中医药杂志》2023年第5期37-45,共9页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81874399);浙江省自然科学基金项目(LQ23H290003);浙江省宁波市卫生局宁波市医学重点学科建设项目(2022Z01);中国博士后基金面上项目(2022M722159)。

摘　　要：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),ZJObjective 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 57

关 键 词：大肠癌 左金丸 靶向治疗 耐药 中药研究 作用机制 

分 类 号：R73[医药卫生—肿瘤]