帕博西尼逆转人食管鳞癌奥沙利铂耐药细胞株耐药性的研究  

Effect of palbociclib on oxaliplatin-resistant esophageal squamous cell carcinoma cells

作　　者：刘祥辰 董慧玲 孔鹏洲[2] 毕炀辉 贾军梅[3] 宋彬[3] Xiangchen Liu;Huiling Dong;Pengzhou Kong;Yanghui Bi;Junmei Jia;Bin Song(Shanxi Medical University,Taiyuan 030001,China;Translational Medicine Research Center,Shanxi Medical University,Taiyuan 030001,China;Department of Oncology,The First Hospital,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学,太原市030001 [2]山西医科大学医学转化中心,太原市030001 [3]山西医科大学第一附属医院肿瘤科,太原市030001

出　　处：《中国肿瘤临床》2019年第23期1193-1198,共6页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金青年基金项目(编号:817030161003251);山西省研究生教育创新计划项目(编号:2019SY243)资助~~.

摘　　要：目的:建立人食管鳞癌(esophageal squamous cell carcinoma,ESCC)奥沙利铂(oxaliplatin,L-OHP)耐药细胞株KYSE150-OXA,研究细胞周期蛋白依赖性激酶4/6(cyclin-dependent kinase 4/6,CDK4/6)抑制剂帕博西尼(palbociclib)对耐药细胞株耐药性的逆转及其机制。方法:CCK-8检测耐药细胞对奥沙利铂、紫杉醇(paclitaxel,PTX)、5-氟尿嘧啶(5-fluorouracil,5-FU)以及CDK4/6抑制剂palbociclib的耐药性;流式细胞术检测化疗药物对耐药细胞凋亡和周期的影响;Western blot检测细胞中多药耐药基因1(multidrug resistance gene 1,MDR1)及细胞周期相关蛋白的表达量;CalcuSyn软件计算palbociclib与L-OHP联合用药对细胞增殖的作用。结果:成功建立人体外ESCC L-OHP耐药细胞株KYSE150-OXA,耐药指数(resistance index,RI)为11.23±0.59,耐药细胞KYSE150-OXA对PTX和5-FU也表现出耐药性,Western blot结果显示耐药细胞中MDR1蛋白表达升高(4.46±0.22)倍;与亲本细胞KYSE150相比,耐药细胞株G1期细胞显著增多,且CDK4、CDK6及Cyclin D1表达显著升高;palbociclib增加KYSE150-OXA对L-OHP的敏感性;低浓度palbociclib与L-OHP表现出拮抗作用,高浓度时表现为协同作用;palbociclib处理组细胞中磷酸化视网膜母细胞瘤蛋白(phospho-retinoblastoma,pRb)降低(91±2)%及细胞周期蛋白A(Cyclin A)降低(89±6)%。结论:成功建立人体外ESCC L-OHP耐药细胞株KYSE150-OXA;palbociclib可能通过抑制异常激活的Cyclin D-CDK4/6-pRb信号通路逆转耐药细胞对L-OHP的耐药性。Objective:To establish oxaliplatin-resistant esophageal squamous cell carcinoma(ESCC)cell line and study the reversal effect and mechanism of palbociclib(an inhibitor of cyclin-dependent kinase 4/6)on this cell line.Methods:High-dose shock and low-dose induction were used to establish the oxaliplatin-resistant ESCC cell line,which was named KYSE150-OXA.CCK-8 assay was used to detect and calculate death rate of the cells treated with oxaliplatin(L-OHP),paclitaxel(PTX),5-fluorouracil(5-FU)and pabociclib(a CDK4/6 inhibitor),respectively.Flow-cytometry was performed to measure the apoptosis and cell cycle of the cells treated with the above chemotherapeutics.Western blot assay was used to determine the expression of multidrug resistance gene 1(MDR1)and related cell-cycle genes.CalcuSyn software was used to evaluate the combinative effects of palbociclib and L-OHP on proliferation of ESCC cells.Results:We established a L-OHP-resistant cell line KYSE150-OXA,which showed resistant capacity to both PTX and 5-FU,and the highest drug resistance index was 11.23±0.59.The results showed that MDR1 protein was significantly upregulated by(4.46±0.22)fold in drug-resistant cells.Compared with the control,the number of KYSE150-OXA cells in G1 phase was significantly increased.Expression of CDK4,CDK6 and Cyclin D1 was significantly increased,while Cyclin E expression was significantly decreased.Palboiclib was able to increase the sensitivity of KYSE150-OXA to L-OHP.In low concentration,palbociclib and L-OHP showed antagonism effect,but the synergistic effect was gradually increased with the increasing concentration of palbociclib.In the palbociclib treated group,expression of phospho-retinoblastoma protein decreased by(91±2)%and cyclin A decreased by(89±6)%.Conclusions:Oxaliplatin-resistant cell line which was named KYSE150-OXA,is successfully established.The results suggest that palbociclib can reverse oxaliplatin-resistance by inhibiting the abnormal activation of CyclinD-CDK4/6-pRb signaling pathway.

关 键 词：食管鳞癌 多药耐药 奥沙利铂 帕博西尼 

分 类 号：R735.1[医药卫生—肿瘤]