异烟肼联合多柔比星选择性抑制肝癌细胞增殖的作用机制
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摘要
目的研究异烟肼联合多柔比星对肝癌细胞增殖的影响及机制,为开发新的化疗药物增效剂提供实验依据。方法体外培养人肝癌细胞系HepG2及人永生化肝细胞L-02,分为无药物干预的对照组、异烟肼作用组,多柔比星作用组、异烟肼+多柔比星联合组,分别进行相应处理。磺酰罗丹明B(SRB)法检测细胞增殖能力,利用荧光探针DCFH-DA检测胞内活性氧浓度变化,采用酶循环法检测胞内rGSH水平、rGSH/GSSG比值变化,Western blot法检测细胞中Nrf2蛋白表达。结果相较L-02细胞,异烟肼与多柔比星联合组对HepG2细胞增殖的抑制作用显著增强(P<0.05),诱导HepG2细胞内生成更多的活性氧(P<0.05)、降低胞内rGSH/GSSG比值(P<0.05);相较HepG2细胞其它组,联合组胞内Nrf2蛋白表达量减少,但核内Nrf2蛋白表达量升高。结论异烟肼联合多柔比星通过诱导活性氧的产生,降低了还原型谷胱甘肽水平,进而抑制了肝癌细胞的增殖。虽然HepG2细胞核内Nrf2蛋白表达量升高,但不能缓解高活性氧水平对肝癌细胞增殖的抑制。
Objective To study the effects of isoniazid combined with doxorubicin on the hepatoma cells proliferation and the mechanism,and to provide experimental evidence for the development of new synergists of chemotherapeutic drugs.MethodsThe HepG2 and L-02 were cultured in vitro.They were divided into the control group without drug intervention,isoniazid group,doxorubicin group,isoniazid + doxorubicin combination group.Cell proliferation was determined by Sulfonyl rhodamine B(SRB)assay.The changes of the intracellular reactive oxygen concentration,rGSH level and rGSH/GSSG ratio were detected,and the cell Nrf2 protein was determined by Western blotting.Results Compared with L-02 cells,the combination of isoniazid and doxorubicin inhibited the proliferation of HepG2 cells significantly(P<0.05);more reactive oxygen species were induced in HepG2 cells(P<0.05);the ratio of intracellular GSH/GSSG was significantly decreased(P<0.05).Compared with other groups of HepG2,the intracellular Nrf2 protein decreased,but the nuclear Nrf2 protein increased in combination group.Conclusion Isoniazid combined with doxorubicin can reduce the reduced glutathione level through inducing the production of ROS.Although the nuclear Nrf2 protein is increased,the inhibition of hepatoma cell proliferation by high ROS cannot be alleviated.
Objective To study the effects of isoniazid combined with doxorubicin on the hepatoma cells proliferation and the mechanism,and to provide experimental evidence for the development of new synergists of chemotherapeutic drugs.MethodsThe HepG2 and L-02 were cultured in vitro.They were divided into the control group without drug intervention,isoniazid group,doxorubicin group,isoniazid + doxorubicin combination group.Cell proliferation was determined by Sulfonyl rhodamine B(SRB)assay.The changes of the intracellular reactive oxygen concentration,rGSH level and rGSH/GSSG ratio were detected,and the cell Nrf2 protein was determined by Western blotting.Results Compared with L-02 cells,the combination of isoniazid and doxorubicin inhibited the proliferation of HepG2 cells significantly(P<0.05);more reactive oxygen species were induced in HepG2 cells(P<0.05);the ratio of intracellular GSH/GSSG was significantly decreased(P<0.05).Compared with other groups of HepG2,the intracellular Nrf2 protein decreased,but the nuclear Nrf2 protein increased in combination group.Conclusion Isoniazid combined with doxorubicin can reduce the reduced glutathione level through inducing the production of ROS.Although the nuclear Nrf2 protein is increased,the inhibition of hepatoma cell proliferation by high ROS cannot be alleviated.
引文
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[33]Khalil H S,Langdon S P,Goltsov A,et al.Quantitative analysis of NRF2pathway reveals key elements of the regulatory circuits underlying antioxidant response and proliferation of ovarian cancer cells[J].J Biotechnol,2015,202:12-30.
[34]Chang J,Zhang Y,Li Y,et al.NrF2/ARE and NF-κB pathway regulation may be the mechanism for lutein inhibition of human breast cancer cell[J].Future Oncology,2018,4(8):719-726.
[35]Keum Y S,Choi B Y.Molecular and chemical regulation of the Keap1-Nrf2signaling pathway[J].Molecules,2014,19(7):10074-10089.
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[2]Ferlay J,Colombet M,Soerjomataram I,et al.Estimating the global cancer incidence and mortality in 2018:globocan sources and methods[J].Int J Cancer,2019,144(8):1941-1953.
[3]World Health Organization.Global Health Observatory[DB/OL].Geneva:World Health Organization,2018.(2019-03-21).http://who.int/gho/database/en/.
[4]Hsu W L,Chien Y C,Chiang C J,et al.Lifetime risk of distinct upper aerodigestive tract cancers and consumption of alcohol,betel and cigarette[J].Int J Cancer,2014,135(6):1480-1486.
[5]Zahand W E,McLafferty S L.Contextual effects and cancer outcomes in the United States:a systematic review of characteristics in multilevel analyses[J].Ann Epidemiol,2017,27(11):739-748,e3.
[6]吴申田.静态调强技术用于原发性肝癌放射治疗中的效果评价[J].现代诊断与治疗,2017,28(15):2770-2771.
[7]吴垒,孟弘婧,董胜利.射频消融在原发性肝癌治疗中的应用进展[J].中国现代医生,2017,55(6):160-163.
[8]万芝清,段学章.原发性肝癌体部立体定向放射治疗的疗效及安全性[J].肝癌电子杂志,2016,3(3):7-12.
[9]Schlachterman A,Craft W W Jr,Hilgenfeldt E,et al.Current and future treatments for hepatocellular carcinoma[J].World J Gastroenterol,2015,21(28):8478-8491.
[10]邵亮,孔欣,施艳新,等.伽玛射线立体定向放射治疗早期原发性肝癌临床研究[J].创伤与急危重病医学,2017,5(1):36-37.
[11]Mokhtari R B,Homayouni T S,Baluch N,et al.Combination therapy in combating cancer[J].Oncotarget,2017,8(23):38022-38043.
[12]Kruse R L,Vanijcharoenkarn K.Drug repurposing to treat asthma and allergic disorders:Progress and prospects[J].Allergy,2018,73(2):313-322.
[13]Delavan B,Roberts R,Huang R,et al.Computational drug repositioning for rare diseases in the era of precision medicine[J].Drug Discov Today,2018,23(2):382-394.
[14]吴文博,董晓琴,吴志刚.药理剂量抗坏血酸联合二甲双胍选择性抑制肝癌细胞增殖的作用及机制[J].华中科技大学学报:医学版,2018,47(4):400-404.
[15]文海棠.异烟肼联用常见药物的相互作用与合理性[J].中国药业,2015,24(8):79-80.
[16]陈廷玉,王东伟,张金波,等.异烟肼通过ROS/Caspase-3信号通路诱导L-02细胞凋亡及槲皮素的保护作用[J].癌变·畸变·突变,2019,31(1):53-57,68.
[17]钟瑾怡,成丛丛,徐金媛,等.多柔比星通过Stat3-Oct-4/CD44途径诱导三阴性乳腺癌4T1细胞干性的产生[J].中国病理生理杂志,2019,35(2):360-364.
[18]王峰.盐酸多柔比星联合环磷酰胺治疗乳腺癌患者的临床疗效探讨[J].中国社区医师,2018,34(30):49-51.
[19]石丹,马旖旎.汉防己甲素体外辅助多柔比星杀伤胃癌细胞及机制研究[J].浙江中西医结合杂志,2018,28(11):916-919,927.
[20]张迪,李健莹,汪晓婵,等.聚(2-乙基-2-噁唑啉)-胆固醇氯甲酸酯修饰盐酸阿霉素脂质体的制备及评价[J].药学学报,2015,50(9):1174-1179.
[21]郭瑶雪,邓晔,李春,等.异烟肼致线粒体损伤引起药物性肝损伤研究进展[J].中国临床药理学与治疗学,2015,20(3):356-360.
[22]何柳,徐江丽,郭丽梅,等.Nrf2/ARE信号通路保护奥沙利铂所致小鼠肝毒性[J].中国药学(英文版),2017,26(10):709-718.
[23]魏彩冰,周莲娣,张育珍,等.槲皮素通过核因子E2相关因子/抗氧化应答元件(Nrf2/ARE)信号通路发挥对免疫性肝损伤的保护作用[J].细胞与分子免疫学杂志,2017,33(3):300-304.
[24]Kaviyarasi R,Abilash V G,Tirupathi Pichiah P B,et al.Molecular mechanism of doxorubicin-induced cardiomyopathy-an update[J].Eur J Pharmacol,2018,818:241-253.
[25]Agudelo D,BérubéG,Tajmir-Riahi H A.An overview on the delivery of antitumor drug doxorubicin by carrier proteins[J].Int J Biol Macromol,2016,88:354-360.
[26]Li Y,Yang D,Wang Y,et al.Co-delivery doxorubicin and silybin for anti-hepatoma via enhanced oral hepatic-targeted efficiency[J].Int J Nanomedicine,2018,14:301-315.
[27]Schoenfeld J D,Sibenaller Z A,Mapuskar K A,et al.O2-and H2O2-mediated disruption of Fe metabolism cause the differential susceptibility of NSCLC and GBM cancer cells to pharmacological ascorbate[J].Cancer Cell,2017,32(2):268.
[28]Panieri E,Santoro M M.ROS homeostasis and metabolism:a dangerous liason in cancer cells[J].Cell Death Dis,2016,7(6):e2253.
[29]Cai N,Zhou W,Ye L L,et al.The STAT3inhibitor pimozide impedes cell proliferation and induces ROS generation in human osteosarcoma by suppressing catalase expression[J].Am J Transl Res,2017,9(8):3853-3866.
[30]Zhang X,Wang X,Wu T,et al.Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38MAPK/JNK activation[J].Sci Rep,2015,5:12579.
[31]Buldak R J,Buldak L,Kukla M,et al.Significance of selected antioxidant enzymes in cancer cell progression[J].Polish JPathol,2014,65(3):167-175.
[32]Singh D,Cho W C,Upadhyay G.Drug-induced liver toxicity and prevention by herbal antioxidants:an overview[J].Front Physiol,2016,6:363.
[33]Khalil H S,Langdon S P,Goltsov A,et al.Quantitative analysis of NRF2pathway reveals key elements of the regulatory circuits underlying antioxidant response and proliferation of ovarian cancer cells[J].J Biotechnol,2015,202:12-30.
[34]Chang J,Zhang Y,Li Y,et al.NrF2/ARE and NF-κB pathway regulation may be the mechanism for lutein inhibition of human breast cancer cell[J].Future Oncology,2018,4(8):719-726.
[35]Keum Y S,Choi B Y.Molecular and chemical regulation of the Keap1-Nrf2signaling pathway[J].Molecules,2014,19(7):10074-10089.
[36]Niture S K,Khatri R,Jaiswal A K.Regulation of Nrf2-an update[J].Free Radic Biol Med,2014,66:36-44.
[37]Ji A X,Chu A,Nielsen T K,et al.Structural insights into KCTD protein assembly and Cullin3recognition[J].J Mol Biol,2016,428(1):92-107.
[38]de SáJunior P L,CmaraD A D,Porcacchia A S,et al.The roles of ROS in cancer heterogeneity and therapy[J].Oxid Med Cell Longev,2017,2017(1):2467940.
[39]Tang J Y,Huang H W,Wang H R,et al.4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells[J].Environ Toxicol,2018,33(3):295-304.
[40]Raj L,Ide T,Gurkar A U,et al.Corrigendum:Selective killing of cancer cells by a small molecule targeting the stress response to ROS[J].Nature,2015,526(7574):596.
[41]Kovac S,Angelova P R,Holmstr9m K M,et al.Nrf2regulates ROS production by mitochondria and NADPH oxidase[J].Biochim Biophys Acta,2015,1850(4):794-801.