鬼臼毒素衍生物LN-13诱导多药耐药细胞K562/A02凋亡及其机制
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摘要
目的研究新型鬼臼毒素衍生物LN-13对多药耐药肿瘤细胞株K562/A02产生的凋亡作用及潜在机制。方法MTT法测定LN-13和阳性对照药VP-16抑制K562/A02细胞48 h后的生长情况及其IC50值,Hoechst 33342、PI双染色观察LN-13作用K562/A02细胞48 h后的形态变化,流式细胞术测定LN-13作用K562/A02细胞48 h的凋亡情况,RTPCR检测LN-13作用K562/A02细胞后Bcl-2、Bax、Caspase-3、mdr-1基因表达的影响,Western blot检测LN-13作用K562/A02细胞后P-gp的表达情况。结果 LN-13对K562/A02细胞生长有显著地抑制,IC50值3.32μmol·L~(-1),Hoechst 33342、PI双染色观察到LN-13作用后,K562/A02细胞发生明显凋亡形态。流式细胞术检测LN-13(2、4、8μmol·L~(-1))作用K562/A02细胞48 h后,出现剂量递增趋势的凋亡比例,分别达到15.0%、48.0%、68.96%。另外,随着LN-13剂量增加,K562/A02细胞的Bax、Caspase-3基因表达增加,mdr-1基因表达减少,另外也下调了P-gp表达,差异有统计学意义。结论 LN-13可诱导多药耐药肿瘤细胞K562/A02产生凋亡作用,其机制可能是通过抑制P-gp蛋白表达及凋亡相关基因表达。
Aim To study the mechanism of action of the new derivative of podophyllotoxin( LN-13) in inducing the apoptosis of K562 / A02 cells. Methods The MTT method was taken to detect the inhibition of LN-13 and VP-16 on K562 / A02 proliferation and inhibition rate and IC_(50) values were obtained 48 hours later.The K562 / A02 cell morphological change induced by LN-13 were observed through Hochest33342 and PI staining after 48 hours later. Flow cytometry was taken to detect the apoptosis of K562 / A02 cells induced by LN-13. The reverse transcription-polymerase chain reaction was taken to detect the Bcl-2,Bax,caspase-3and mdr-1 mRNA expression. The expression of P-gp was detected by Western blot. Results The growth of K562 / A02 cells was obviously inhibited by LN-13 when IC_(50)value was 3. 32 μmol · L~(-1). LN-13 could obviously induced cell apoptosis observed by Hochest33342 and PI staining. Flow cytometry detection showed that LN-13( 2,4,8 μmol ·L- 1) could induce cell apoptosis and apoptosis ratio reached 15. 0%,48. 0%,68. 96%,respectively. The reverse transcription-polymerase chain reaction showed that LN-13 increased the Bax and Caspase-3 mRNA expression,and meanwhile the expression of mdr-1 mRNA decreased.Western blot showed that P-gp expression was decreased as the LN-13 dose increased. The data were significantly different from those of control group. Conclusion Podophyllotoxin derivative LN-13 can induce the apoptosis of K562 / A02 cells,which may be closely-related to regulating P-gp expression and apoptosis related gene mRNA expression.
Aim To study the mechanism of action of the new derivative of podophyllotoxin( LN-13) in inducing the apoptosis of K562 / A02 cells. Methods The MTT method was taken to detect the inhibition of LN-13 and VP-16 on K562 / A02 proliferation and inhibition rate and IC_(50) values were obtained 48 hours later.The K562 / A02 cell morphological change induced by LN-13 were observed through Hochest33342 and PI staining after 48 hours later. Flow cytometry was taken to detect the apoptosis of K562 / A02 cells induced by LN-13. The reverse transcription-polymerase chain reaction was taken to detect the Bcl-2,Bax,caspase-3and mdr-1 mRNA expression. The expression of P-gp was detected by Western blot. Results The growth of K562 / A02 cells was obviously inhibited by LN-13 when IC_(50)value was 3. 32 μmol · L~(-1). LN-13 could obviously induced cell apoptosis observed by Hochest33342 and PI staining. Flow cytometry detection showed that LN-13( 2,4,8 μmol ·L- 1) could induce cell apoptosis and apoptosis ratio reached 15. 0%,48. 0%,68. 96%,respectively. The reverse transcription-polymerase chain reaction showed that LN-13 increased the Bax and Caspase-3 mRNA expression,and meanwhile the expression of mdr-1 mRNA decreased.Western blot showed that P-gp expression was decreased as the LN-13 dose increased. The data were significantly different from those of control group. Conclusion Podophyllotoxin derivative LN-13 can induce the apoptosis of K562 / A02 cells,which may be closely-related to regulating P-gp expression and apoptosis related gene mRNA expression.
引文
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[4]陈昌明,刘慧芳,曹雪姣,等.姜黄素对人宫颈癌Hela细胞增殖的抑制作用[J].中国药房,2014,25(11):1006-8.[4]Chen C M,Liu H F,Cao X J,et al.The inhibitory effect of curcumin on the proliferation of human cervical cancer Hela cells[J].China Pharmacy,2014,25(11):1006-8.
[5]张元,王火,牛聪,等.多靶点鬼臼毒素新衍生物CCP-1抗肿瘤多药耐药作用机制研究[J].中国药理学通报,2016,32(1):144-5.[5]Zhang Y,Wang H,Niu C,et al.Anti-MDR tumor activities of CCP-1:a new multi-target podophyllotoxin derivative and its molecular mechanism[J].Chin Pharmacol Bull,2016,32(1):144-5.
[6]Shen Z T,Wu X H,Wang L,et al.Effects of gemcitabine on radiosensitization,apoptosis and Bcl-2 and Bax protein expression in human pancreatic cancer xenografts in nude mice[J].Genet Mol Res,2015,14(4):15587-96.
[7]Zeng J,Yang J,Chen D B,Lao K.The mechanisms by which Bax Induces the apoptosis of human ovarian cancer cells[J].Sichuan Da Xue Xue Bao Yi Xue Ban,2015,46(5):697-701.
[8]Chetsawang J,Suwanjang W,Pirompul N,et al.Calpastatin reduces Meth-amphetaine induced induction in c-Jun phosphorylation,Bax and cell death in neuroblastoma SH-SY5Y cells[J].Neurosci Lett,2012,506(1):7-11.
[9]Walters J,Pop C,Scott F L,et al.A constitutively active and uninhabitable Caspase-3 zymogen efficiently induces apoptosis[J].Biochem J,2009,424(3):335-45.
[10]Breier A,Gibalova L,Seres M,et al.New insight into p-glycoprotein as a drug target.[J].Anticancer Agents Med Chem,2013,13(1):159-70.
[11]Johnstone R W,Cretney E,Smyth M J.P-glycoprotein protects leukemia cells against caspase-dependent,but not caspase-independent,cell death[J].Blood,1999,93(3):1075-85.