具核梭杆菌感染对食管癌KYSE150细胞紫杉醇化疗抵抗的影响  

Effect of Fusobacterium nucleatum infection on paclitaxel resistance of esophageal cancer KYSE150 cells

作　　者：张哲源 刘怡文[2] 付臻 杨海军[1,4] 代宁涛 李军扩[1,4] 孙蔚[2] 郭艺博 孔金玉 周福有[1,5] ZHANG Zheyuan;LIU Yiwen;FU Zhen;YANG Haijun;DAI Ningtao;LI Junkuo;SUN Wei;GUO Yibo;KONG Jinyu;ZHOU Fuyou(Anyang Tumor Hospital,the Affiliated Anyang Tumor Hospital,Henan University of Science and Technology,Henan Medical Key Laboratory of Precise Prevention and Treatment of Esophageal Cancer,Anyang,Henan 455000;College of Clinical Medicine,Henan University of Science and Technology,the First Affiliated Hospital,Henan University of Science and Technology,Henan Key Laboratory of Microbiome and Esophageal Cancer Prevention and Treatment,Henan Key Laboratory of Cancer Epigenetics,Luoyang,Henan 471003;Department of Imaging,Anyang Tumor Hospital,Anyang,Henan 455000;Department of Pathology,Anyang Tumor Hospital,Anyang,Henan 455000;Department of Thoracic Surgery,Anyang Tumor Hospital,Anyang,Henan 455000)

机构地区：[1]安阳市肿瘤医院,河南科技大学附属安阳市肿瘤医院,河南省食管癌精准防治医学重点实验室,河南安阳455000 [2]河南科技大学临床医学院,河南科技大学第一附属医院,河南省微生态与食管癌防治重点实验室,河南省肿瘤表观遗传重点实验室,河南洛阳471003 [3]安阳市肿瘤医院影像科,河南安阳455000 [4]安阳市肿瘤医院病理科,河南安阳455000 [5]安阳市肿瘤医院胸外科,河南安阳455000

出　　处：《郑州大学学报（医学版）》2023年第5期603-610,共8页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省医学科技攻关计划项目(LHGJ20200806,LHGJ20200811,LHGJ20210585,SBGJ202103099,SBGJ202103100);安阳市科技计划项目重大专项(ZDKJJH2020006,2022A02SF002)。

摘　　要：目的:探讨具核梭杆菌(Fn)感染对食管癌细胞紫杉醇(PTX)化疗的影响及其机制。方法:将食管癌细胞KYSE150分为Fn未感染组及Fn感染组(12、24、48和72 h),通过CCK-8及Western blot检测各组细胞的PTX 24 h药物半数抑制浓度(IC_(50))及炎症小体NLRP3蛋白表达量。构建NLRP3敲降的食管癌细胞KYSE150-N,然后分为KYSE150组、KYSE150-N组、KYSE150+Fn组和KYSE150-N+Fn组4组。通过Western blot及流式细胞术检测各组细胞系中NLRP3、乙醛脱氢酶1(ALDH1)蛋白表达量及肿瘤干细胞(CSCs)百分比;通过平板克隆、成球实验及CCK-8检测各组细胞的体外增殖能力、克隆成球能力及PTX 24 h IC_(50)。将裸鼠分为上述4组,按分组接种细胞。成瘤后每3 d尾静脉注射一次PTX(10 mg/kg),共6次。对比各组裸鼠的成瘤能力和瘤体组织中NLRP3、ALDH1蛋白表达量及CSCs百分比。结果:与Fn未感染组相比,Fn感染组(12、24、48和72 h)KYSE150细胞的PTX 24 h IC_(50)及NLRP3蛋白表达量均增高(P均<0.05)。与KYSE150组相比,KYSE150+Fn组细胞的NLRP3及ALDH1蛋白表达量、CSCs百分比、体外增殖能力、克隆成球能力、PTX 24 h IC_(50)、裸鼠瘤体对PTX的抗性、瘤体内NLRP3与ALDH1蛋白表达量及CSCs百分比均增高(P均<0.05);而KYSE150-N组细胞上述指标均降低(P均<0.05)。与KYSE150+Fn组细胞相比,KYSE150-N+Fn组细胞上述指标均降低(P均<0.05)。结论:Fn可通过激活NLRP3诱导ALDH1高表达、富集CSCs,降低食管癌细胞对PTX的敏感性,促进其恶性增殖。Aim:To investigate the effect and mechanism of Fusobacterium nucleatum(Fn)infection on paclitaxel(PTX)resistance in esophageal cancer cells.Methods:Esophageal cancer KYSE150 cells were divided into Fn non-infection group and Fn infection group(12,24,48 and 72 hours).CCK-8 and Western blot were used to detect the 50%inhibitory concentration(IC_(50))of PTX for 24 hours and the expression of NLRP3 protein in each group.NLRP3 knockdown(KYSE150-N)cells were constructed.The cells were divided into KYSE150 group,KYSE150-N group,KYSE150+Fn group and KYSE150-N+Fn group.Western blot and flow cytometry were used to detect the protein expressions of NLRP3 and aldehyde dehydrogenase 1(ALDH1)and the percentage of cancer stem cells(CSCs)in each group.Plate clone formation assay,sphere formation assay,and CCK-8 assay were used to detect the proliferation ability,clone formation ability and PTX 24-hours IC_(50).The nude mice were divided into the above 4 groups,and the cells were inoculated accordingly.After tumor formation,PTX(10 mg/kg)was injected into the tail vein every 3 days for a total of 6 times.The tumor formation ability in each group,the expressions of NLRP3 and ALDH1 protein and the percentage of CSCs in tumor tissue were compared.Results:Compared with Fn non-infection group,the 24-hour IC_(50) of PTX and the expression of NLRP3 protein in KYSE150 cells of Fn infection group(12,24,48 and 72 hours)were increased(P<0.05).Compared with the KYSE150 group,in KYSE150+Fn group,the protein expressions of NLRP3 and ALDH1,the percentage of CSCs,in vitro proliferation ability,clonogenic sphere formation ability,PTX 24-hour IC_(50),tumor resistance to PTX in nude mice,the protein expressions of NLRP3 and ALDH1 and the percentage of CSCs were all increased(P<0.05).However,the above indexes of cells in KYSE150-N group were decreased(P<0.05).Compared with KYSE150+Fn group,the above indexes of KYSE150-N+Fn group were decreased(P<0.05).Conclusion:Fn can induce high expression of ALDH1 by activating NLRP3 to enrich CSCs,reduce the sens

关 键 词：具核梭杆菌 食管癌 炎症小体NLRP3 肿瘤干细胞 紫杉醇 化疗抵抗 KYSE150细胞 

分 类 号：R735.1[医药卫生—肿瘤]