摘要
目的探究柚皮素对顺铂耐药宫颈癌细胞(HeLa/DDP)顺铂耐药性的影响,并探究其分子机制。方法体外培养人HeLa/DDP细胞、HeLa细胞,CCK-8法检测不同质量浓度顺铂对HeLa/DDP、He La细胞增殖的影响及不同质量浓度柚皮素对HeLa/DDP细胞增殖的影响。设置对照组、顺铂组(2 mg/L顺铂)、顺铂+柚皮素组(2 mg/L顺铂+60 mg/L柚皮素)和顺铂+柚皮素+AMPK通路抑制剂组(2mg/L顺铂+60mg/L柚皮素+50μmol/LCompoundC);CCK-8法检测各组HeLa/DDP细胞增殖情况;流式细胞仪检测各组HeLa/DDP细胞凋亡情况;蛋白免疫印迹(Westernblotting)法检测各组HeLa/DDP细胞中腺苷酸活化蛋白激酶(AMPK)、p-AMPK、Bcl-2相关X蛋白(Bax)、Beclin1、微管相关蛋白1轻链3(LC3)Ⅰ、LC3Ⅱ蛋白表达情况。结果顺铂对HeLa/DDP细胞、HeLa细胞的半数抑制浓度(IC50)分别在4~8、1~2 mg/L,柚皮素对HeLa/DDP细胞的IC50在30~60mg/L。与对照组相比,顺铂组He La/DDP细胞增殖抑制率、凋亡率及细胞中p-AMPK/AMPK、Bax、Beclin1、LC3Ⅱ蛋白表达水平显著升高(P<0.05),LC3Ⅰ蛋白表达水平显著降低(P<0.05);与顺铂组相比,顺铂+柚皮素组HeLa/DDP细胞增殖抑制率、凋亡率及细胞中p-AMPK/AMPK、Bax、Beclin1、LC3Ⅱ蛋白表达水平显著升高(P<0.05),LC3Ⅰ蛋白表达水平显著降低(P<0.05);与柚皮素组相比,AMPK通路抑制剂组He La/DDP细胞增殖抑制率、凋亡率及细胞中p-AMPK/AMPK、Bax、Beclin1、LC3Ⅱ蛋白表达水平显著降低(P<0.05),LC3Ⅰ蛋白表达水平显著升高(P<0.05)。结论柚皮素可能通过激活AMPK通路介导的自噬反应,与顺铂联用发挥对HeLa/DDP细胞增殖抑制作用及凋亡促进作用。
Objective To investigate the effect of naringenin on cisplatin resistance of cervical cancer He La/DDP cells and its molecular mechanism.Methods HeLa/DDP cells and HeLa cells were cultured in vitro,CCK-8 method was used to detect the effects of different concentrations of cisplatin on the proliferation of HeLa/DDP and He La cells,and the effects of naringenin on the proliferation of HeLa/DDP cells.HeLa/DDP cells were divided into control group,cisplatin group(2 mg/L cisplatin),cisplatin+naringenin group(2 mg/L cisplatin+60 mg/L naringenin)and cisplatin+naringenin+AMPK pathway inhibitor group(2 mg/L cisplatin+60 mg/L naringenin+50μmol/L Compound C).The proliferation of HeLa/DDP cells was detected by CCK-8 method,the apoptosis of HeLa/DDP cells was detected by flow cytometry.Western blotting(WB)method was used to detect the expression of adenosine monophosphate activated protein kinase(AMPK),p-AMPK,Bcl-2 associated X protein(Bax),Beclin1,microtubule-associated protein 1 light chain 3(LC3)I and LC3 II protein in HeLa/DDP cells.Results The IC50 value of cisplatin to HeLa/DDP cells and HeLa cells were 4—8 mg/L and 1—2 mg/L,respectively.IC50 value of naringenin to HeLa/DDP cells was in the range of 30—60 mg/L.Compared with those in the control group,the proliferation inhibition rate,apoptosis rate and the protein expression levels of p-AMPK/AMPK,Bax,Beclin1 and LC3Ⅱin HeLa/DDP cells were significantly higher in cisplatin group(P<0.05),and the expression level of LC3Ⅰprotein was significantly lower(P<0.05).Compared with those in cisplatin group,the proliferation inhibition rate and apoptosis rate of HeLa/DDP cells and the expression levels of p-AMPK/AMPK,Bax,Beclin1 and LC3Ⅱprotein in cisplatin+naringenin group were significantly higher,and the expression level of LC3Ⅰprotein was significantly lower(P<0.05).Compared with those in cisplatin+naringenin group,the proliferation inhibition rate and apoptosis rate of HeLa/DDP cells and the protein expression levels of p-AMPK/AMPK,Bax,Beclin1 and LC3Ⅱwere significantly lower in cisplatin+naringenin+AMPK pathway inhibitor group,the expression level of LC3Ⅰprotein was significantly higher(P<0.05).Conclusion Naringenin can inhibit proliferation and promote apoptosis of HeLa/DDP cells by activating autophagy mediated by AMPK pathway.
作者
贾艳利
李军芳
梁高峰
JIA Yan-li;LI Jun-fang;LIANG Gao-feng(Department of Obstetrics and Gynecology,Mengzhou People’s Hospital,Jiaozuo 454750,China;Department of Clinical Pharmacy,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471000,China)
出处
《现代药物与临床》
CAS
2021年第5期878-884,共7页
Drugs & Clinic
基金
国家自然科学基金资助项目(81741147)。
关键词
柚皮素
腺苷酸活化蛋白激酶通路
自噬
宫颈癌
HeLa/DDP细胞
顺铂耐药性
naringenin
adenosine monophosphate activated protein kinase pathway
autophagy
cervical cancer
Hela/DDP cells
cisplatin resistance
