杨哲豪,喻超,潘耀振,邓路,郑迪杰,孙诚谊
YANG Zhehao,YU Chao,PAN Yaozhen,DENG Lu,ZHENG Dijie,SUN Chengyi

• 1:贵州医科大学附属医院肝胆外科贵州医科大学肝胆胰脾重点实验室贵州省肝胆胰脾疾病研究所

摘要(Abstract)：

目的:构建乐伐替尼诱导的原发性肝细胞癌(HCC) SMMC-7721耐药细胞株,并探讨其生物学特性。方法:选取HCC细胞株SMMC-7721进行培养传代,采用药物逐步递增法建立体外乐伐替尼诱导的SMMC-7721耐药细胞株(耐药组),以未经处理的肝癌细胞为对照组;采用CCK-8法检测乐伐替尼、阿霉素、氟尿嘧啶和顺铂对两组细胞的半数抑制浓度(IC50),并计算乐伐替尼耐药指数(RI);采用Real time-PCR检测两组细胞耐药基因MDR1、LRP、MRP2、GST-pi、TopoⅡɑ和BCRP的表达,采用Western blot检测两组细胞P-gp、LRP、MRP2、GST-pi、TopoⅡɑ和BCRP蛋白的表达。结果:乐伐替尼对对照组和耐药组SMMC-7721细胞IC50分别为(0.15±0.03)μmol/L和(4.34±0.12)μmol/L,RI为28;阿霉素、氟尿嘧啶和顺铂对耐药组SMMC-7721细胞的IC50较对照组降低(P <0.05);耐药组SMMC-7721细胞中MDR1、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP耐药基因的相对表达量均较对照组下降(P <0.05或P <0.01),耐药组SMMC-7721细胞中P-gp、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP蛋白表达均较对照组降低(P <0.05)。结论:实验成功诱导获得耐乐伐替尼的体外肝癌SMMC-7721细胞株,该细胞株对阿霉素、氟尿嘧啶和顺铂的敏感性增高,其体制可能与耐药基因MDR1、LRP、MRP2、GST-pi、TopoⅡɑ及BCRP表达降低有关。
Objective: To construct levatinib-induced hepatocellular carcinoma( HCC) SMMC-7721 drug-resistant cell line and explore its biological characteristics. Methods: HCC cell line SMMC-7721 was selected to culture and subculture. The levatinib-resistant cell line was established by step-by-step drug-increasing method. The untreated HCC cells were selected as the control group and the levatinibresistant cells as the drug-resistant group. The median inhibition concentration( IC_(50)) of levatinib,doxorubicin,fluorouracil and cisplatin of both groups was detected by CCK-8 assay and the drug resistance index( RI) of levatinib was calculated. Real time-PCR was used to detect the expression of drug-resistance genes MDR1,LRP,MRP2,GST-pi,TopoⅡɑ and BCRP in two groups. Western blot was used to detect the expression of P-gp,LRP,MRP2,GST-pi,TopoⅡɑ and BCRP proteins of the cells. Results: The IC50 of levatinib on SMMC-7721 cells of two groups were( 0. 15 ± 0. 03) μmol/L and( 4. 34 ± 0. 12) μmol/L( P < 0. 05). The drug resistance index was 28,and the IC50 of doxorubicin,fluorouracil and cisplatin of the drug-resistant group was lower than that in the control group( P < 0. 05). The expression of genes MDR1,MRP2,LRP,GST-pi,TopoⅡɑ and BCRP was low in drug-resistant cells( P < 0. 05 or P < 0. 01) while the expression of protein P-gp,LRP,MRP2,GST-pi,Topo Ⅱ ɑ and BCRP decreased in drug-resistant cells. Conclusion: The experiment successfully established HCC cell line SMMC-7721 with levatinib-resistance and found that the increased sensitivity to doxorubicin,fluorouracil and cisplatin may be related to the low expression of genes MDR1,LRP,MRP2,GST-pi,TopⅡɑ and BCRP in levatinib-resistance HCC cells.

关键词(KeyWords)： 癌,肝细胞;分子靶向治疗;乐伐替尼;耐药细胞;化疗敏感性;多药耐药
carcinoma,hepatocellular;molecular yargeted therapy;levatinib;drug-resistant cells;chemosensitivity;multidrug resistance

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81860505,81860506);; 贵州省科学技术厅-贵州医科大学附属医院联合基金[黔科合LH字(2016)7229];贵州省肝胆外科临床医学研究中心[黔科合平台人才(2017)5404]

作者(Author): 杨哲豪,喻超,潘耀振,邓路,郑迪杰,孙诚谊
YANG Zhehao,YU Chao,PAN Yaozhen,DENG Lu,ZHENG Dijie,SUN Chengyi

DOI: 10.19367/j.cnki.2096-8388.2020.08.003

参考文献(References)：

• [1]刘秀红，赵一鸣，赵晓飞，等．肝细胞癌诊断与治疗研究进展[J]．中国肝脏病杂志(电子版)，2017,9(2):20-25．
• [2]SAFFO S,TADDEI T H.Systemic management for advanced hepatocellular carcinoma:a review of the molecular pathways of carcinogenesis,current and emerging therapies,and novel treatment strategies[J].Digestive Diseases and Sciences,2019,64(4):1016-1029．
• [3]TAMAI T,HAYATO S,HOJO S,et al.Dose finding of Lenvatinib in subjects with advanced hepatocellular carcinoma based on population pharmacokinetic and exposure-response analyses[J].The Journal of Clinical Pharmacology,2017,57(9):1138-1147．
• [4]RAI V,ABDO J,ALSUWAIDAN A N,et al.Cellular and molecular targets for the immunotherapy of hepatocellular carcinoma[J].Molecular and Cellular Biochemistry,2018,437(1-2):13-36．
• [5]高彦君，姚颐，宋启斌，等．原发性肝癌索拉非尼耐药机制的研究进展[J]．肿瘤学杂志，2019,25 (9):836-840．
• [6]KUDO M.Targeted and immune therapies for hepatocellular carcinoma:predictions for 2019 and beyond[J].World Journal of Gastroenterology,2019,25(7):789-807．
• [7]CABANILLAS M E,HABRA M A.Lenvatinib:role in thyroid cancer and other solid tumors[J].Cancer Treatment Reviews,2016,42:47-55．
• [8]TAHARA M,SCHLUMBERGER M,ELISEI R,et al.Exploratory analysis of biomarkers associated with clinical outcomes from the study of Lenvatinib in differentiated cancer of the thyroid[J].European Journal of Cancer,2017,75:213-221．
• [9]于悦，时静，刘玉国．乐伐替尼治疗恶性肿瘤的研究进展[J]．中国医院药学杂志，2018,38(9):1005-1009．
• [10]RIMASSA L,PRESSIANI T,MERLE P.Systemic treatment options in hepatocellular carcinoma[J].Liver Cancer,2019,8(6):427-446．
• [11]陈旸，曾川，张献全．肝细胞肝癌的靶向及免疫治疗研究现状与进展[J]．医学信息，2019,32(3):45-48．
• [12]IKEDA K,KUDO M,KAWAZOE S,et al.Phase 2 study of lenvatinib in patients with advanced hepatocellular carcinoma[J].Journal of Gastroenterology,2017,52 (4):512-519．
• [13]杜豆，周娟，邱英，等．非小细胞肺癌奥希替尼获得性耐药细胞株的建立及耐药后化疗敏感性[J]．实用医学杂志，2019,35(04):516-519;524．
• [14]汤嵩，江啸，王翔，等．原发性肝癌多药耐药相关机制的研究进展[J]．中国肿瘤临床与康复，2018,25(8):1020-1024．
• [15]姚怡，杨柳，吴凯．结直肠癌多药耐药机制的研究进展[J]．解放军医学杂志，2019,44(7):624-630．
• [16]STAVROVSKAYA A A,RYBALKINA E Y.Recent advances in the studies of molecular mechanisms regulating multidrug resistance in cancer cells[J].Biochemistry Biokhimiia,2018,83(7):779-786．
• [17]CEBALLOS M P,RIGALLI J P,CERE L I,et al.ABCTransporters:regulation and association with multidrug resistance in hepatocellular carcinoma and colorectal carcinoma[J].Current Medicinal Chemistry,2019,26 (7):1224-1250．
• [18]RAZI B,ANANI S G,OMIDKHODA A,et al.Multidrug resistance 1 (MDR1/ABCB1) gene polymorphism and susceptibility to multiple myeloma:a systematic review and meta-analysis[J].Hematology,2018,23 (8):456-462．
• [19]WU Y J,WANG C,WEI W.The effects of DMARDs on the expression and function of P-gp,MRPs,BCRP in the treatment of autoimmune diseases[J].Biomedicine&Pharmacotherapy,2018,105:870-878．
• [20]TONG X,ZHAO J,ZHANG Y,et al.Expression levels of MRP1,GST-pi,and GSK3beta in ovarian cancer and the relationship with drug resistance and prognosis of patients[J].Oncology Letters,2019,18(1):22-28．
• [21]WANG B,SHEN C,LI Y,et al.Oridonin overcomes the gemcitabine resistant PANC-1/Gem cells by regulating GST-pi and LRP/1 ERK/JNK signalling[J].Onco Targets and Therapy,2019,12:5751-5765．
• [22]ZHANG Y,YANG S H,GUO X L.New insights into Vinca alkaloids resistance mechanism and circumvention in lung cancer[J].Biomedicine&Pharmacotherapy,2017,96:659-666．
• [23]TANG L J,ZHOU L J,ZHANG W X,et al.Expression of multidrug-resistance associated proteins in human retinoblastoma treated by primary enucleation[J].International Journal of Ophthalmology,2018,11(9):1463-1466．
• [24]ZHAO M,YU S,ZHANG M.Differential expression of multidrug resistancerelated proteins in adriamycinresistant(pumc91/ADM) and parental (pumc91) human bladder cancer cell lines[J].Molecular Medicine Reports,2016,14(5):4741-4746．
• [25]QIAN J Q,SUN P,PAN Z Y,et al.Annonaceous acetogenins reverses drug resistance of human hepatocellular carcinoma BEL-7402/5-FU and Hep G2/ADM cell lines[J].International Journal of Clinical and Experimental Pathology,2015,8(9):34-44．
• [26]GANTNER M E,PERONI R N,MORALES J F,et al.Development and validation of a computational model Ensemble for the early detection of BCRP/ABCG2 substrates during the drug design stage[J].Journal of Chemical Information and Modeling,2017,57(8):1868-1880.

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