目的 探讨氟喹诺酮类化合物三甲氧基苯甲醛环丙沙星席夫碱诱导人肝癌SMMC-7721细胞凋亡作用。方法 用不同浓度的1-环丙基-6-氟-7-(哌嗪-1-基)-3-[5-苄硫基-4-(3,4,5-三甲氧苯甲叉基氨基)-1,2,4-三唑-3-基]-喹啉(1-H)-4-酮 (M27)与人肝癌SMMC-7721细胞、结肠癌HCT-116细胞株、白血病JURKET细胞株体外培养。MTT法检测M27及其前体化合物环丙沙星盐酸盐对肿瘤细胞的增殖抑制作用;Hoechst 33258荧光染色法、TUNEL法检测细胞凋亡变化;以pBR322 DNA为底物,琼脂糖电泳法检测M27对拓扑异构酶Ⅱ的活性的影响;高内涵活细胞成像系统测定线粒体跨膜电位(△ψm)变化;Western blotting法检测p53、Caspase-9、Caspase-3、Caspase-8、Bcl-2和Bax等凋亡相关蛋白的表达以及细胞色素C在线粒体内外分布的变化。结果 M27在10～60 μmol·L-1内抑制肿瘤细胞增殖,呈浓度、时间依赖性。作用于SMMC-7721细胞、HCT-116细胞、JURKET细胞24 h IC50值分别为37.97、46.07、44.33 μmol·L-1,而环丙沙星盐酸盐对SMMC-7721细胞增殖抑制作用不显著。各组M27作用于SMMC-7721细胞24 h,细胞凋亡率显著高于对照组(P<0.05),并伴有细胞线粒体膜电位(Δψm)降低。与对照组比较,M27能够抑制DNA拓扑异构酶Ⅱ的活性,使细胞p53、Bax、Caspase-8、Caspase-9和Caspase-3蛋白表达及活性片段增加, Bcl-2的表达量降低;线粒体细胞色素C减少,胞浆细胞色素C增多。结论 三甲氧基苯甲醛环丙沙星席夫碱能够抑制拓扑异构酶Ⅱ的活性,造成DNA损伤,诱导人肝癌SMMC-7721细胞凋亡。
Abstract
OBJECTIVE To investigate the effect of 1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)-3-[5-benzylsulfanyl-4-(3,4,5-trimethoxybenzylidene) amino-4H-1,2,4-triazol-3-yl]-quinolin-4(1H)-one (M27) on apoposis in hepatocarcinoma SMMC-7721 cells in vitro. METHODS SMMC-7721 cells, colon adenocarcinoma cells(HCT-116)and leukemia cell line JURKET were treated by M27 with different concentrations for different time in vitro, the inhibitory effect of M27 and its precursor ciprofloxacin on the cell proliferation were examined by MTT assay. Cell apoptosis was determined by Hoechst 33258 fluorescence staining and TUNEL assay. The effect of M27 on topoisomerase Ⅱ activity was measured using agarose gel electrophoresis by Plasmid pBR322 DNA as the substrate. Mitochondrial membrane potential(Δψm)was measured by high content screening imaging system. The p53,Caspase-9,Caspase-3,Caspase-8,Bcl-2,Bax and cytochrome C protein expressions were determined by Western blotting analysis. RESULTS The proliferation of the cancer cells was inhibited by M27 at 10-60 μmol·L-1 in time-and dose-dependent manner. Ciprofloxacin showed weak cytotoxicity against SMMC-7721 cell. SMMC-7721 cells treated by M27 with different concentrations for 24 h increased the percentage of apoptosis cells obviously (P<0.05) with a decrease in the mitochondrial membrane potential. Compared with control group, M27 influenced obviously DNA topoisomerase Ⅱ activity, stimulated DNA cleavage and inhibited DNA reunion mediated by topoisomerase Ⅱ. In addition, M27 increased protein expression of p53, Bax, Caspase-8, Caspase-9, Caspase-3, as well as the cleaved activated forms of Caspase-9, Caspase-8 and Caspase-3 significantly, whereas the expression of Bcl-2 decreased. There was a significant increase of cytochrome C in the cytosol after 24 h of treatment with M27 and a decrease in the mitochondrial compartment. CONCLUSION M27 as a fluoroquinolone derivative exerted potent anticancer activity through the mechanism of eukaryotic topoisomerase Ⅱ poisoning. The growth inhibition is mainly mediated via apoptosis-associated mitochondrial dysfunction and regulation of Bcl-2 signaling pathways.
关键词
氟喹诺酮衍生物 /
细胞凋亡 /
拓扑异构酶Ⅱ /
线粒体膜电位
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Key words
fluoroquinolone derivatives /
apoptosis /
topoisomerase Ⅱ /
mitochondrial membrane potential
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中图分类号：
R329.26
R965
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参考文献
[1] RAJABALIAN S, FOROUMADI A, SHAFIEE A, et al. Functionalized N(2-oxyiminoethyl) piperazinyl quinolones as new cytotoxic agents [J]. J Pharm Pharm Sci, 2007, 10(2):153-158.
[2] FORTERRE P, GRIBALDO S, GADELLE D, et al. Origin and evolution of DNA topoisomerases[J]. Biochimie, 2007, 89(4):427-446.
[3] KLOSKOWSKI T, GURTOWSKA N, NOWAK M, et al. The influence of ciprofloxacin on viability of A549, HepG2, A375. S2, B16 and C6 cell lines in vitro [J]. Acta Pol Pharm, 2011, 68(6):859-865.
[4] WALSBY E J, COLES S J, KNAPPER S, et al. The topoisomerase Ⅱ inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine [J]. Haematologica, 2011, 96(3):393-399.
[5] SEO K W, HOLT R, JUNG Y S, et al. Fluoroquinolone-mediated inhibition of cell growth, S-G2/M cell cycle arrest, and apoptosis in canine osteosarcoma cell lines [J]. PLoS One, 2012, 7(8):e42960.
[6] AL-TRAWNEH S A, ZAHRA J A, KAMAL M R, et al. Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents[J]. Bioorg Med Chem, 2010, 18(16):5873-5884.
[7] ROYCHOUDHURY S, MAKIN K M, TWINEM T L, et al. Development and use of a high-throughput bacterial DNA gyrase assay to identify mammalian topoisomerase Ⅱ inhibitors with whole-cell anticancer activity[J]. J Biomol Screen, 2003, 8(2):157-163.
[8] POMMIER Y, LEO E, ZHANG H, et al. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs[J]. Chem Biol, 2010, 17(5):421-433.
[9] CHEN Y S,WANG G Q,DUAN N N, et al. Synthesis and antitumor of fluoroquinolone C3-isostere derivatives:oxadiazole mannich base derivatives [J]. Chin J Appl Chem (应用化学), 2012, 29 (11):1246-1250.
[10] YOU Q D, LI Z Y, HUANG C H, et al. Discovery of a novel series of quinolone and naphthyridine derivatives as potential topoisomerase I inhibitors by scaffold modification [J]. J Med Chem, 2009, 52(18):5649-5661.
[11] GAO L Z,LI T,XIE Y S, et al. Synthesis and antitumor activity of fluoroquinolon-3-yl s-triazole sulfanylacetylhydrazones and s-triazole hydrazone derivatives (V) [J]. Chin Pharm J(中国药学杂志),2015, 50 (6):545-549 .
[12] XIE S Q, HU G Q, ZHANG Z Q, et al. Anti-tumour effects of HL-37, a novel anthracene derivative, in vivo and in vitro [J]. J Pharm Pharmacol, 2008, 60(2):213-219.
[13] MA Y C, HUANGFU C S, KANG Y H, et al. Effects of anisaldehyde amino-S-triazole schiff base on the differentiation and apoptosis of human hepatocarcinoma cells [J]. Chin Pharm J(中国药学杂志),2009, 44(20):1547-1551.
[14] REN Z, KANG Y H, SHI Z Y, et al. Cinnamaldehyde ofloxacin-3-ylhydrazone induces apoptosis of human hepatocarcinoma SMMC-7721 cells[J]. Acta Pharm Sin(药学学报), 2010, 45(9):1109-1115.
[15] SUN J P, SHI Z Y, LIU S M, et al. Trimethoxy-benzaldehyde levofloxacin hydrazone inducing the growth arrest and apoptosis of human hepatocarcinoma cells [J]. Cancer Cell Int, 2013, 13(1):67.
[16] SHI Z Y, LI Y Q, KANG Y H, et al. Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells [J]. Acta Pharmacol Sin(中国药理学报), 2012, 33(2):271-278.
[17] KLOSKOWSKI T, GURTOWSKA N, OLKOWSKA J, et al. Ciprofloxacin is a potential topoisomerase II inhibitor for the treatment of NSCLC[J]. Int J Oncol, 2012, 41(6):1943-1949.
[18] FU Y, YANG Y, ZHOU S, et al. Ciprofloxacin containing Mannich base and its copper complex induce antitumor activity via different mechanism of action[J]. Int J Oncol, 2014, 45(5):2092-2100.
[19] HAWTIN R E, STOCKETT D E, BYL J A, et al. Voreloxin is an anticancer quinolone derivative that intercalates DNA and poisons topoisomerase Ⅱ [J]. PLoS One, 2010, 5(4):e10186.
[20] HSU S C, YU C C, YANG J S, et al. A novel synthetic 2-(3-methoxyphenyl)-6, 7-methylenedioxoquinolin-4-one arrests the G2/M phase arrest via Cdc25c and induces apoptosis through caspase-and mitochondria-dependent pathways in TSGH8301 human bladder cancer cells[J]. Int J Oncol, 2012, 40(3):731-738.
[21] TSAI A C, PAN S L, SUN H L, et al. A new vascular targeting agent, induces apoptosis of human umbilical vein endothelial cells via p53-mediated death receptor 5 up-regulation[J]. J Biol Chem, 2010, 285(8):5497-5506.
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脚注
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基金
国家自然科学基金资助项目(20872028,21072045);河南省教育厅科学技术研究重点项目(13B320914);开封市社会发展科技攻关计划项目(1503029);河南大学科研基金项目(2013YBZR043)
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