[1]姜秀星,傅若秋,高宁.千金藤素协同多柔比星逆转K562/ADR耐药的分子机制研究[J].第三军医大学学报,2018,40(23):2154-2161.
 JIANG Xiuxing,FU Ruoqiu,GAO Ning.Molecular mechanism of cepharanthine synergistically acting with doxorubicin in reversing multidrug resistance of K562/ADR cells[J].J Third Mil Med Univ,2018,40(23):2154-2161.
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千金藤素协同多柔比星逆转K562/ADR耐药的分子机制研究(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第23期
页码:
2154-2161
栏目:
基础医学
出版日期:
2018-12-15

文章信息/Info

Title:
Molecular mechanism of cepharanthine synergistically acting with doxorubicin in reversing multidrug resistance of K562/ADR cells
作者:
姜秀星傅若秋高宁
陆军军医大学(第三军医大学)药学与检验医学系生药学教研室
Author(s):
JIANG Xiuxing FU Ruoqiu GAO Ning

Department of Pharmacognosy, Faculty of Pharmacy and Laboratory Medicine, Army Military Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
千金藤素K562/ADR细胞耐药线粒体分裂凋亡
Keywords:
cepharanthine K562/ADR cells drug resistance mitochondrial fission apoptosis
分类号:
R285.5; R733.7; R915
文献标志码:
A
摘要:

目的探讨千金藤素协同多柔比星逆转K562/ADR细胞耐药的分子机制。 方法MTT法测定K562、K562/ADR细胞存活率;Western blot检测多药耐药蛋白Mdr-1、凋亡相关蛋白以及线粒体分裂关键蛋白Drp1和pDrp1(Ser637)、线粒体外膜蛋白Tom20蛋白的表达:流式细胞仪检测K562/ADR细胞凋亡以及ROS生成量;免疫共沉淀技术检测Drp1、Tom20蛋白之间特异性结合。结果梯度浓度多柔比星均会降低K562、K562/ADR细胞存活率,两者的IC50值分别为(0.75±0.11)、(75.97±2.17)μmol/L,差异有统计学意义(P<0.01)。K562细胞中检测不到多药耐药蛋白,而在K562/ADR细胞中Mdr1高表达。千金藤素与多柔比星协同作用于K562/ADR细胞,导致K562/ADR存活率明显降低并呈较好的量效关系,协同系数均小于1,K562/ADR细胞凋亡率显著增加(P<0.01)。千金藤素与多柔比星协同作用于K562/ADR细胞导致ROS生成量显著升高,Drp1线粒体转位增加。抑制ROS则可阻断两种药物协同作用引起的Drp1线粒体转位,降低K562/ADR细胞凋亡率(P<0.01)。 结论千金藤素协同多柔比星诱导ROS生成促进Drp1线粒体转位,导致线粒体过度分裂和细胞凋亡,最终逆转K562/ADR耐药。

Abstract:

ObjectiveTo investigate the molecular mechanism of cepharanthine synergistically acting with doxorubicin in the reverse of multidrug resistance of K562/ADR cells. MethodsAfter K562 cells and K562/ADR cells were treated with doxorubicin at various concentration respectively, cell viability was assessed by MTT assay. Then cepharanthine (6 μmol/L), doxorubicin (4 μmol/L), NAC (ROC inhibitor, 4 μmol/L) alone, or in combination of the former 2 agents or 3 agents together were used to treat the cells respectively. Cell apoptosis and production of reactive oxygen species (ROS) were determined by flow cytometry with Annexin V-/PI staining. The expression of multidrug resistant protein Mdr-1, apoptosis and mitochondrial fission related proteins, Drp1 and p-Drp1 (Ser637), and translocase of the mitochondrial outer membrane Tom20 were detected by Western blot analysis. Co-immunoprecipitation was used detected the binding of Drp1 and Tom20. Results  The IC50 value of K562 cells to doxorubicin was 0.75±0.11 μmol/L, and that of K562/ADR cells was 75.97±2.17 μmol/L, with significantly difference (P<0.01). Mdr-1 was mildly expressed in K562 cells, but was highly expressed in K562/ADR cells. Combined treatment of cepharanthine and doxorubicin resulted in marked decrease in cell viability, which was in a dosedependent manner and the synergistic coefficient less than 1, and also resulted in increases in apoptotic rate (P<0.01) in K562/ADR cells. The synergistic action of the 2 agents also promoted the production of ROS and mitochondrial translocation of Drp1. What’s more, inhibiting ROS production by NAC treatment could block the mitochondrial translocation of Drp1 and decrease the apoptotic rate induced by the 2 agents (P<0.01). ConclusionCepharanthine synergistically acting with doxorubicin induces the production of ROS and promotes mitochondrial translocation of Drp1, thus results in mitochondrial fission and cell apoptosis, and finally reverses multidrug resistance of K562/ADR cells.

参考文献/References:

[1]WANG J, SEEBACHER N, SHI H, et al. Novel strategies to prevent the development of multidrug resistance (MDR) in cancer[J]. Oncotarget, 2017, 8(48):84559-84571. DOI:10.18632/oncotarget.19187.
[2]WANG Y J, ZHAO H D,ZHU C F,et al.Tuberostemonine reverses multidrug resistance in chronic myelogenous leukemia cells K562/ADR[J].J Cancer, 2017, 8(6): 1103-1112. DOI:10.7150/jca.17688.
[3]FOGARTY C E, BERGMANN A. Killers creating new life:caspases drive apoptosisinduced proliferation in tissue repair and disease[J]. Cell Death Differ, 2017, 24(8): 1390-1400. DOI:10.1038/cdd.2017.47.
[4]GIAMPIETRI C, PETRUNGARO S, PADULA F, et al. Autophagy modulators sensitize prostate epithelial cancer cell lines to TNFalphadependent apoptosis[J]. Apoptosis, 2012, 17(11): 1210-1222. DOI:10.1007/s104950120752z.
[5]LEE J, LEE D G. Melittin triggers apoptosis in Candida albicans through the reactive oxygen speciesmediated mitochondria/caspasedependent pathway[J]. FEMS Microbiol Lett, 2014, 355(1): 36-42. DOI:10.1111/15746968.12450.
[6]PAGLIUSO A, THAM T N, STEVENS J K, et al. A role for septin 2 in Drp1mediated mitochondrial fission[J]. EMBO Rep, 2016, 17(6): 858-873. DOI:10.15252/embr.201541612.
[7]DESGROUAS C, CHAPUS C, DESPLANS J, et al. In vitro antiplasmodial activity of cepharanthine[J]. Malar J, 2014, 13: 327. DOI:10.1186/1475287513327.
[8]WU J, SUZUKI H, ZHOU Y W, et al. Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines[J]. J Cell Biochem, 2001, 82(2): 200-214.
[9]IKEDA R, CHE X, YAMAGUCHI T,et al. Cepharanthine potently enhances the sensitivity of anticancer agents in K562 cells[J]. Cancer Sci,2005, 96(6): 372-376. DOI:10.1111/j.13497006.2005.00057.x.
[10]PUGAZHENDHI A, EDISON T N J I, VELMURUGAN B K, et al. Toxicity of Doxorubicin (Dox) to different experimental organ systems[J]. Life Sci, 2018, 200: 26-30. DOI:10.1016/j.lfs.2018.03.023.
[11]FAN Y F, ZHANG W, ZENG L, et al. Dacomitinib antagonizes multidrug resistance (MDR) in cancer cells by inhibiting the efflux activity of ABCB1 and ABCG2 transporters[J]. Cancer Lett, 2018, 421: 186-198. DOI:10.1016/j.canlet.2018.01.021.
[12]TSURUO T, IIDA H, TSUKAGOSHI S, et al. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil[J]. Cancer Res, 1981, 41(5): 1967-1972.
[13]CZARNECKA A M, KUKWA W, KRAWCZYK T, et al. Mitochondrial DNA mutations in cancerfrom bench to bedside[J]. Front Biosci (Landmark Ed), 2010, 15: 437-460.
[14]JAESCHKE H, MCGILL M R, RAMACHANDRAN A. Oxidant stress, mitochondria, and cell death mechanisms in druginduced liver injury: lessons learned from acetaminophen hepatotoxicity[J]. Drug Metab Rev, 2012, 44(1): 88-106. DOI:10.3109/03602532.2011.602688.
[15]NADANACIVA S, DYKENS J A, BERNAL A,et al. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration[J]. Toxicol Appl Pharmacol, 2007, 223(3): 277-287. DOI:10.1016/j.taap.2007.06.003.
[16]NEUSTADT J, PIECZENIK S R. Medicationinduced mitochondrial damage and disease[J]. Mol Nutr Food Res, 2008,52(7): 780-788. DOI:10.1002/mnfr.200700075.
[17]ZHANG Z, LIU L, WU S, et al. Drp1, Mff, Fis1, and MiD51 are coordinated to mediate mitochondrial fission during UV irradiationinduced apoptosis[J]. FASEB J, 2016, 30(1):  466-476. DOI:10.1096/fj.15274258.
[18]ESTAQUIER J, ARNOULT D. Inhibiting Drp1mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis[J]. Cell Death Differ, 2007, 14(6): 1086-1094. DOI:10.1038/sj.cdd.4402107.
[19]OTERA H, MIHARA K. Mitochondrial dynamics: functional link with apoptosis[J]. Int J Cell Biol, 2012, 2012: 821676. DOI:10.1155/2012/821676.
[20]CEREGHETTI G M, STANGHERLIN A, MARTINS DE BRITO O, et al. Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria[J]. Proc Natl Acad Sci U S A,2008,105(41):15803-15808.DOI:10.1073/pnas.0808249105.
[21]ZHOU J, LI G,ZHENG Y,et al. A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1Lmediated mitochondrial fission[J]. Autophagy, 2015, 11(8): 1259-1279. DOI:10.1080/15548627.2015.1056970.

更新日期/Last Update: 2018-12-19