副溶血弧菌耐热直接溶血毒素抑制人肺腺癌细胞活性的研究
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摘要
目的海洋细菌毒素抗肿瘤是近年来兴起的用于癌症治疗的新思路。本文旨在研究耐热直接溶血毒素(TDH)对人肺腺癌细胞SPC-A-1细胞的抑制作用。方法利用DEAE-纤维素、DEAE-Sephadex A-50、葡聚糖G-75层析的方法,从海洋细菌-副溶血弧菌ATCC33846的代谢物中分离纯化得到纯度较高的耐热直接溶血毒素(TDH)。通过MTT法、细胞划痕试验、凋亡试验、caspase-3活性试验比较TDH作用于不同细胞的半抑制剂浓度IC50,研究了TDH对人肺腺癌细胞SPC-A-1细胞的作用。结果人结肠上皮细胞NCM460、人肝成纤维细胞LO2、人肺腺癌细胞SPC-A-1在TDH处理24h之后,细胞的半抑制剂浓度IC50分别为151μg/mL、118μg/mL和6.7μg/mL,正常细胞的IC50高出肺腺癌细胞近20倍。在低于5μg/mL浓度下,TDH能剂量依赖性抑制SPC-A-1细胞的生长、增殖和迁移能力。流式细胞法和荧光试剂检测表明:5μg/mL的TDH能诱导33%的SPC-A-1细胞发生早期凋亡,并激活caspase-3。结论 TDH可能通过激活caspase-3途径诱导细胞凋亡,从而抑制SPC-A-1细胞的生长和增殖。
Anti-tumor of marine bacterial toxin is a new idea for the treatment of cancer in recent years.The aim of this study was to investigate the inhibitory effects of thermostable direct hemolysin(TDH)on human lung cancer cell line SPC-A-1.TDH was separated and purified from marine bacteria-Vibrio parahaemolyticus ATCC33846metabolites(TDH)by DEAEcellulose,DEAE-Sephadex A-50 and Sephadex G-75 chromatography.In this paper,the comparison of the concentration of semi inhibitor in different cells treated by TDH,the effect of TDH on human lung cancer cell line SPC-A-1was studied by MTT assay,cell scratch experiment,apoptosis experiment and caspase-3activity experiment for the first time.It showed that the IC50 of human colon epithelial cells NCM460,human liver fibroblast cells LO2 and human lung cancer cell SPC-A-1treated by TDH with 24 hwere 151μg/mL,118μg/mL and 6.7μg/mL,IC50 of the normal cells is higher than that of lung cancer cell about 20 times.The results of growth curve and wound test showed that TDH could inhibit the growth,proliferation and migration of SPC-A-1cells in a dose-dependent manner below 5μg/mL.Further flow cytometry revealed that5μg/mL TDH induced early apoptosis in 33% of SPC-A-1cells.These results suggest that TDH may inhibit the growth and proliferation of SPC-A-1cells by inducing apoptosis.
Anti-tumor of marine bacterial toxin is a new idea for the treatment of cancer in recent years.The aim of this study was to investigate the inhibitory effects of thermostable direct hemolysin(TDH)on human lung cancer cell line SPC-A-1.TDH was separated and purified from marine bacteria-Vibrio parahaemolyticus ATCC33846metabolites(TDH)by DEAEcellulose,DEAE-Sephadex A-50 and Sephadex G-75 chromatography.In this paper,the comparison of the concentration of semi inhibitor in different cells treated by TDH,the effect of TDH on human lung cancer cell line SPC-A-1was studied by MTT assay,cell scratch experiment,apoptosis experiment and caspase-3activity experiment for the first time.It showed that the IC50 of human colon epithelial cells NCM460,human liver fibroblast cells LO2 and human lung cancer cell SPC-A-1treated by TDH with 24 hwere 151μg/mL,118μg/mL and 6.7μg/mL,IC50 of the normal cells is higher than that of lung cancer cell about 20 times.The results of growth curve and wound test showed that TDH could inhibit the growth,proliferation and migration of SPC-A-1cells in a dose-dependent manner below 5μg/mL.Further flow cytometry revealed that5μg/mL TDH induced early apoptosis in 33% of SPC-A-1cells.These results suggest that TDH may inhibit the growth and proliferation of SPC-A-1cells by inducing apoptosis.
引文
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[2]Ceccarelli D,Hasan NA,Huq A,et al.Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors[J].Front Cell Infect Microbiol,2013,3(8):97.DOI:10.3389/fcimb.2013.00097
[3]Iida T,Honda T.Hemolysins produced by Vibrios[J].J Toxicol Toxin Rev,1997,16:215-227.
[4]Shimohata T,Takahashi A.Diarrhea induced by infection of Vibrio parahaemolyticus[J].J Med Invest,2010,57(3-4):179-182.DOI:10.2152/jmi.57.179
[5]Lin YR,Chen YL,Wang KB,et al.The thermostable direct hemolysin from grimontia hollisae causes acute hepatotoxicity in vitro and in vivo[J].PLoS ONE,2013,8(2):e56226.DOI:10.1371/journal.pone.0056226
[6]Raghunath P.Roles of thermostable direct hemolysin(TDH)and TDH-related hemolysin(TRH)in Vibrio parahaemolyticus[J].Front Microbiol,2015,5:805.DOI:10.3389/fmicb.2014.00805
[7]Naim R,Pasaribu FH.Thermostable direct hemolysin of Vibrio parahaemolyticus induces morphological changes and disrupts actin in cultured human epithelial cells[J].African Journal of Microbiology Research,2009,3(11):727-730.
[8]杨靖亚,陆晓帆,张建,等.副溶血弧菌TDH单克隆抗体的研制与性质鉴定[J].中国免疫学杂志,2012,28(3):235-239.
[9]杨靖亚,刘建文,刘承初,等.羧甲基壳聚糖对人高转移肺腺癌细胞—95-D增殖能力和体外转移能力的抑制作用[J].中国临床药理学与治疗学,2006,11(6):640-643.
[10]Liu CC,Yang H,Zhang LL,et al.Biotoxins for cancer therapy[J].Asian Pacific J Cancer Prevention Apjcp,2014,15:4753-6.DOI:10.7314/APJCP.2014.15.12.4753
[11]Yang X,Kessler E,Su LJ,et al.Diphtheria toxin-epidermal growth factor fusion protein DAB389EGF for the treatment of bladder cancer[J].Clin Cancer Res,2013,19(1):148-57.
[12]Piascik P.FDA approves fusion protein for treatment of lymphoma[J].J Am Pharm Assoc,1999,39:571-2.
[13]Chowdhury P,Pore D,Mahata N,et al.Thermostable direct hemolysin downregulates human colon carcinoma cell proliferation with the involvement of E-cadherin,andβ-catenin/Tcf-4Signaling[J].Plos One,2012,6(5):e20098.DOI:10.1371/journal.pone.0020098
[14]Price JE,Clonogenicity and experimental metastatic potential of spontaneous mouse mammary neoplasms[J].J Natl Cancer,Inst,1986,77:529-35.DOI:10.1093/jnci/77.2.529
[15]Kaufmann SH,Vaux DL.Alterations in the apoptotic machinery and their potential role in anticancer drug resistance[J].Oncogene,2003,22(47):7414.DOI:10.1038/sj.onc.1206945
[16]翟中和,王喜忠,丁明孝.细胞生物学[M].4版,北京:高等教育出版社,2011:12-28.
[17]赵彦超,顾耘.细胞凋亡通路研究进展[J].现代医学,2013,41(4):285-288.