抗菌肽对肝癌多药耐药细胞Bel-7402/ADM增殖和耐药性影响
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摘要
目的检测抗菌肽对耐药细胞Bel-7402/ADM增殖和耐药性的影响,为探讨抗菌肽在肝癌治疗方面的前景和应用价值提供参考依据。方法采用阿霉素浓度梯度递增法体外诱导建立人肝癌多药耐药细胞系Bel-7402/ADM,检测细胞对化疗药物的敏感性;提取家蝇幼虫抗菌肽,采用噻唑蓝比色法(MTT)检测抗菌肽对肝癌细胞的抑制作用,流式细胞仪检测抗菌肽对细胞周期分布和进入细胞内阿霉素浓度的影响,并采用荧光定量聚合酶链反应(PCR)检测抗菌肽对Bel-7402/ADM细胞多药耐药基因MDR1表达的影响。结果建立了对阿霉素以及多种化疗药物均耐药的肝癌细胞系Bel-7402/ADM,Bel-7402/ADM对5–氟尿嘧啶、长春新碱、紫杉醇和阿霉素的耐药指数(RI)分别为15.94、8.45、8.11和10.96。抗菌肽对Bel-7402/ADM细胞的半数抑制浓度(IC_(50))为(463.67±16.93)μg/mL,低于对Bel-7402细胞IC_(50)的(596.34±19.27)μg/mL(t=8.959,P <0.001)。抗菌肽作用后,Bel-7402和Bel-7402/ADM细胞均出现G_0/G_1期比例升高,S期比例下降;Bel-7402/ADM细胞对阿霉素的RI由10.96降至7.10,而Bel-7402细胞对阿霉素的敏感性无明显变化;Bel-7402/ADM细胞内阿霉素的平均荧光强度由(24.16±3.53)增高至(48.27±5.50)(t=6.390,P=0.003),而Bel-7402细胞荧光强度增高不明显;Bel-7402/ADM细胞多药耐药基因1(MDR1)的相对表达量由(1.749±0.020)降至(1.071±0.044)(t=24.297,P <0.001)。结论肝癌多药耐药细胞Bel-7402/ADM对抗菌肽不耐药,且抗菌肽可逆转Bel-7402/ADM细胞的耐药性。
Objective To evaluate the effect of antibacterial peptides(AMPs) on proliferation and drug resistance in human hepatocellular carcinoma(HC) cells(Bel-7402/ADM) with multidrug resistance for providing evidences to possible application of AMPs in HC treatment. Methods We established a MDR HC cell line with the administration of adriamycin in culture medium with gradient increment and then detected the cells′ sensitivity to chemotherapeutic drugs. The AMPs were extracted from Musca domestica larva. The inhibitive effect of the AMPs on HC was determined with methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay; the effect of AMPs on cell cycle and concentration of intracellular adriamycin was detected with flow cytometry; and the effect of AMPs on the expression of multidrug resistance gene 1(MDR1) in Bel-7402/ADM cells was determined with reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR). Results Bel-7402/ADM cell line resistant to adriamycin and other chemotherapeutic drugs was established successfully and the resistance index(RI) of the Bel-7402/ADM cells to 5-fluorouracil, vincristine, paclitaxe, and adriamycin were 15.94, 8.45, 8.11, and 10.96, respectively. The 50% inhibitory concentration(IC_(50)) of AMPs against Bel-7402/ADM cells was 463.67 ± 16.93 μg/mL and was lower than that of AMPs against Bel-7402 cells(596.34 ± 19.27μg/mL). After treatment with AMPs, the proportion of cells in G_0/G_1 phase was significantly increased but the proportion of cells in S phase declined in both Bel-7402 and Bel-7402/ADM cells; the RI of Bel-7402/ADM cells to adriamycin was decreased from 10.69 to 7.10, while the RI of Bel-7402 cells to adriamycin did not changed significantly; the average fluorescence intensity of adriamycin in Bel-7402/ADM cells was increased from 24.16 ± 3.53 to 48.27 ± 5.5, while that of adriamycin in Bel-7402 cells did not increase obviously; the relative expression of MDR1 in Bel-7402/ADM cells was decreased significantly from 1.749 ± 0.020 to 1.071 ± 0.044(t = 24.297,P < 0.001). Conclusion The multidrug resistant human hepatocellular carcinoma Bel-7402/ADM cells are not resistant to AMPs and AMPs could reverse drug resistance of Bel-7402/ADM cells.
Objective To evaluate the effect of antibacterial peptides(AMPs) on proliferation and drug resistance in human hepatocellular carcinoma(HC) cells(Bel-7402/ADM) with multidrug resistance for providing evidences to possible application of AMPs in HC treatment. Methods We established a MDR HC cell line with the administration of adriamycin in culture medium with gradient increment and then detected the cells′ sensitivity to chemotherapeutic drugs. The AMPs were extracted from Musca domestica larva. The inhibitive effect of the AMPs on HC was determined with methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay; the effect of AMPs on cell cycle and concentration of intracellular adriamycin was detected with flow cytometry; and the effect of AMPs on the expression of multidrug resistance gene 1(MDR1) in Bel-7402/ADM cells was determined with reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR). Results Bel-7402/ADM cell line resistant to adriamycin and other chemotherapeutic drugs was established successfully and the resistance index(RI) of the Bel-7402/ADM cells to 5-fluorouracil, vincristine, paclitaxe, and adriamycin were 15.94, 8.45, 8.11, and 10.96, respectively. The 50% inhibitory concentration(IC_(50)) of AMPs against Bel-7402/ADM cells was 463.67 ± 16.93 μg/mL and was lower than that of AMPs against Bel-7402 cells(596.34 ± 19.27μg/mL). After treatment with AMPs, the proportion of cells in G_0/G_1 phase was significantly increased but the proportion of cells in S phase declined in both Bel-7402 and Bel-7402/ADM cells; the RI of Bel-7402/ADM cells to adriamycin was decreased from 10.69 to 7.10, while the RI of Bel-7402 cells to adriamycin did not changed significantly; the average fluorescence intensity of adriamycin in Bel-7402/ADM cells was increased from 24.16 ± 3.53 to 48.27 ± 5.5, while that of adriamycin in Bel-7402 cells did not increase obviously; the relative expression of MDR1 in Bel-7402/ADM cells was decreased significantly from 1.749 ± 0.020 to 1.071 ± 0.044(t = 24.297,P < 0.001). Conclusion The multidrug resistant human hepatocellular carcinoma Bel-7402/ADM cells are not resistant to AMPs and AMPs could reverse drug resistance of Bel-7402/ADM cells.
引文
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[19]郎蕾.抗菌肽Temporin-1CEa对乳腺癌耐药细胞MCF7/ADR的作用研究[D].沈阳:辽宁师范大学硕士学位论文,2014.
[20]郭姗,邢佳欣,赵瑞君,等.肝癌耐药细胞株Bel-7402/ADM的建立以及家蝇幼虫抗菌肽对其耐药性逆转作用的研究[J].寄生虫与昆虫学报,2017,24(3):161-168.
[21]Holohan C,Van Schaeybroeck S,Longley DB,et al.Cancer drug resistance:an evolving paradigm[J].Nat Rev Cancer,2013,13(10):714-726.
[22]黄宇星,刘二伟.联合用药的药物相互作用及研究方法[J].药物评价研究,2014,37(3):276-279.
[2]Vander BS,Komuta M,Libbrecht L,et al.Expression of multidrug resistance-associated protein 1 in hepatocellular carcinoma is associated with a more aggressive tumour phenotype and may reflect a progenitor cell origin[J].Liver Int,2008,28(10):1370-1380.
[3]国果,吴建伟,付萍.家蝇幼虫分泌型抗菌肽对大肠埃希菌抑制作用[J].中国公共卫生,2012,28(10):1318-1320.
[4]国果,吴建伟,付萍,等.家蝇幼虫分泌型抗菌肽对小鼠免疫功能影响[J].中国公共卫生,2012,28(5):619-620.
[5]张庆华,赵瑞君,罗景星,等.家蝇幼虫抗菌肽对肿瘤细胞的作用效果观察[J].中华卫生杀虫药械,2007,13(3):202-204.
[6]原发家.家蝇幼虫抗菌肽defensin对食管癌细胞增殖、侵袭迁移的抑制作用研究[D].太原:山西医科大学硕士学位论文,2015.
[7]金小宝,龚水明,蒲俏虹,等.家蝇抗菌肽Cecropin对人源性肿瘤细胞增殖与凋亡的影响[J].中国热带医学,2012,12(3):293-295.
[8]Sun HX,Chen LQ,Zhang J,et al.Anti-tumor and immunomodulatory activity of peptide fraction from the larvae of Muscadomestica[J].J Ethnopharmacol,2014,153(3):831-839.
[9]Wang KR,Yan JX,Zhang BZ,et al.Novel mode of action of polybia-MPI,a novel antimicrobial peptide,in multi-drug resistant leukemic cells[J].Cancer Lett,2009,278(1):65-72.
[10]Bankovi?J,Andr?J,Todorovi?N,et al.The elimination of P-glycoprotein over-expressing cancer cells by antimicrobial cationic peptide NK-2:the unique way of multi-drug resistance modula-tion[J].Exp Cell Res,2013,319(7):1013-1027.
[11]聂守民,邢佳欣,赵瑞君,等.家蝇幼虫抗菌肽粗提物对原代FBL-3细胞增殖周期的影响[J].寄生虫与医学昆虫学报,2015,22(4):244-249.
[12]赵瑞君,于娟,崔晓东,等.家蝇幼虫抗弓形虫抗菌肽分离纯化[J].中国公共卫生,2008,24(10):1212-1213.
[13]Snow K,Judd W.Characterisation of adriamycin-and amsacrineresistant human leukaemic T cell lines[J].Br J Cancer,1991,63(1):17-28.
[14]张帆,黄鹤光.吉西他滨诱导胰腺癌PANC-1细胞耐药与C-IAP2表达关系的研究[J].中国临床药理学杂志,2016,32(6):540-542.
[15]王瑶,李婷,彭芳,等.美洲大蠊提取物逆转BEL-7402/5-Fu多药耐药性的作用及机制研究[J].药学研究,2017,36(6):315-318.
[16]黄静.抗菌肽Temporin-1CEa降低MCF-7/Adr细胞耐药性的作用研究[D].沈阳:辽宁师范大学硕士学位论文,2016.
[17]邱婷婷,项晓军,雷婉,等.耐5-氟尿嘧啶的人结肠癌HT-29/5-FU细胞株的建立及其生物学特性[J].细胞与分子免疫学杂志,2015,31(3):328-332.
[18]李明远,徐艳敏,钱程.索拉非尼诱导的耐药性肝癌细胞的生物学特征分析[J].第三军医大学学报,2016,38(6):580-584.
[19]郎蕾.抗菌肽Temporin-1CEa对乳腺癌耐药细胞MCF7/ADR的作用研究[D].沈阳:辽宁师范大学硕士学位论文,2014.
[20]郭姗,邢佳欣,赵瑞君,等.肝癌耐药细胞株Bel-7402/ADM的建立以及家蝇幼虫抗菌肽对其耐药性逆转作用的研究[J].寄生虫与昆虫学报,2017,24(3):161-168.
[21]Holohan C,Van Schaeybroeck S,Longley DB,et al.Cancer drug resistance:an evolving paradigm[J].Nat Rev Cancer,2013,13(10):714-726.
[22]黄宇星,刘二伟.联合用药的药物相互作用及研究方法[J].药物评价研究,2014,37(3):276-279.