至真方抑制自噬改善大肠癌HCT-116/L-OHP细胞耐药性
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摘要
[目的]探讨至真方对大肠癌HCT116/L-OHP细胞耐药性的影响,并从自噬角度探讨至真方改善大肠癌HCT116/L-OHP耐药的可能作用机制。[方法]CCK-8及Western blot检测大肠癌HCT116/L-OHP细胞耐药性;mRFP-GFP-LC3双荧光自噬指示体系检测至真方对大肠癌HCT116/L-OHP细胞自噬的影响;流式细胞术检测至真方对大肠癌HCT116/L-OHP细胞凋亡诱导作用;Western blot检测至真方干预后耐药细胞自噬标志蛋白Beclin-1、SQSTM1/P62、LC3的表达。[结果]至真方可下调LC3、Beclin-1、上调SQSTM1/P62的表达(P<0.05),经低、中、高浓度干预后凋亡率分别为5.1%、7.7%、13.1%(P<0.05)。mRFP-GFP-LC3双荧光自噬指示体系检测发现至真方干预组随着浓度的降低,黄点(自噬体)和红点(自噬溶酶体)数量均减少(P<0.05)。[结论]至真方可通过抑制HCT-116/L-OHP细胞自噬活性,增加大肠癌HCT-116/L-OHP细胞凋亡率,恢复其部分敏感性。
[Objective]To investigate the effect of Zhi-zhen-fang(ZZF)on the multidrug resistance of colorectal cancer HCT116/L-OHP cells and try to explain the mechanism through autophagy.[Methods]CCK-8and western blot were used to detect the drug resistance of colorectal cancer HCT116/L-OHP cells;mRFP-GFP-LC3 double fluorescence autophagy indicator system was used to detect the effect of ZZF on autophagy of colorectal cancer HCT116/L-OHP cells;the expression of autophagy marker proteins Beclin-1,SQSTM1/P62 and LC3in drug-resistant cells was detected by Western blotting.[Results]HCT116/LOHP cells were resistant;ZZF could down-regulate LC3,Beclin-1,up-regulate the expression of SQSTM1/P62(P<0.05),and the rate of apoptosis was 5.1%,7.7%,13.1%(P<0.05).The mRFP-GFPLC3dual-fluorescence autophagy indicator system found that the number of yellow spots(autophagosomes)and red spots(autophagy lysosomes)decreased with the decrease of concentration in the ZZF intervention group(P<0.05).[Conclusion]ZZF can increase the apoptotic rate of colorectal cancer HCT-116/L-OHP cells by inhibiting the autophagy activity of HCT-116/L-OHP cells and reverse drug resistance partly.
[Objective]To investigate the effect of Zhi-zhen-fang(ZZF)on the multidrug resistance of colorectal cancer HCT116/L-OHP cells and try to explain the mechanism through autophagy.[Methods]CCK-8and western blot were used to detect the drug resistance of colorectal cancer HCT116/L-OHP cells;mRFP-GFP-LC3 double fluorescence autophagy indicator system was used to detect the effect of ZZF on autophagy of colorectal cancer HCT116/L-OHP cells;the expression of autophagy marker proteins Beclin-1,SQSTM1/P62 and LC3in drug-resistant cells was detected by Western blotting.[Results]HCT116/LOHP cells were resistant;ZZF could down-regulate LC3,Beclin-1,up-regulate the expression of SQSTM1/P62(P<0.05),and the rate of apoptosis was 5.1%,7.7%,13.1%(P<0.05).The mRFP-GFPLC3dual-fluorescence autophagy indicator system found that the number of yellow spots(autophagosomes)and red spots(autophagy lysosomes)decreased with the decrease of concentration in the ZZF intervention group(P<0.05).[Conclusion]ZZF can increase the apoptotic rate of colorectal cancer HCT-116/L-OHP cells by inhibiting the autophagy activity of HCT-116/L-OHP cells and reverse drug resistance partly.
引文
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[3] Klukovits A,Krajcsi P.Mechanisms and therapeutic potential of inhibiting drug efflux transporters[J].Expert Opin Drug Metab Toxicol,2015,11(6):907-920.
[4] Popeda M,Pluciennik E,Bednarek AK.Proteins in cancer multidrug resistance[J].Postepy Hig Med Dosw(Online),2014,68:616-632.
[5] Ding J,Miao ZH,Meng LH,et al.Emerging cancer therapeutic opportunities target DNA-repair systems[J].Trends Pharmacol Sci,2006,27(6):338-344.
[6]胡兵,安红梅,郑佳露,等.藤龙补中汤对大肠癌RKO细胞转移相关基因表达影响[J].中国中西医结合消化杂志,2018,26(5):416-419.
[7]唐阳,王松坡.丹参、黄芪、女贞子有效成分对HCT-8/VCR耐药性的影响及机制[J].中国中西医结合消化杂志,2018,26(4):340-343.
[8]孔令春,陈志霞,孙玉舫,等.至真方对人大肠癌多药耐药细胞株HCT-8/VCR的增殖抑制及凋亡诱导作用[J].世界中医药,2010,5(4):285-288.
[9]段佩雯,王浩,伏杰,等.至真方调控Hedgehog/ABCG2信号途径改善大肠癌细胞的多药耐药性[J].中国中西医结合消化杂志,2018,26(3):270-274.
[10]Saha S,Panigrahi DP,Patil S,et al.Autophagy in health and disease:A comprehensive review[J].Biomed Pharmacother,2018,104:485-495.
[11]Gutierrez-Casado E,Khraiwesh H,Lopez-Dominguez JA,et al.The impact of aging,calorie restriction and dietary fat on mitochondrial ultrastructure,dynamics and autophagy markers in mouse skeletal muscle[J].J Gerontol A Biol Sci Med Sci,2018.[Epub ahead of print].
[12]Giampieri F,Afrin S,Forbes-Hernandez TY,et al.Autophagy in Human Health and Disease:Novel Therapeutic Opportunities[J].Antioxid Redox Signal,2019:30(4):577-634.
[13]Pavlides S,Vera I,Gandara R,et al.Warburg meets autophagy:cancer-associated fibroblasts accelerate tumor growth and metastasis via oxidative stress,mitophagy,and aerobic glycolysis[J].Antioxid Redox Signal,2012,16(11):1264-1284.
[14]Grander D,Panaretakis T.Autophagy:cancer therapy's friend or foe?[J].Future Med Chem,2010,2(2):285-297.
[15]Vegliante R,Ciriolo MR.Autophagy and Autophagic Cell Death:Uncovering New Mechanisms Whereby Dehydroepiandrosterone Promotes Beneficial Effects on Human Health[J].Vitam Horm,2018,108:273-307.
[16]Li H,Jin X,Chen B,et al.Autophagy-regulating microRNAs:potential targets for improving radiotherapy[J].J Cancer Res Clin Oncol,2018:144(9):1623-1634.
[17]Basciani S,Vona R,Matarrese P,et al.Imatinib interferes with survival of multi drug resistant Kaposi's sarcoma cells[J].FEBS Lett,2007,581(30):5897-5903.
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[20]谢昆,李密杰,蒋成砚,等.自噬相关蛋白ATG5/BECLIN-1调控细胞自噬和凋亡的分子机理研究进展[J].中国人兽共患病学报,2018,34(3):272-275.
[21]陈易斯,宋福永.选择性自噬接头蛋白p62/sequestosome 1的研究进展[J].中国药理学与毒理学杂志,2016,30(3):258-265.