Tumstatin185-191逆转肺腺癌化疗耐药及其对Akt、ERK活性的影响
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摘要
目的Tumstatin是胶原蛋白Ⅳ胶原a3链的C端包括非胶原区在内约28KD大小的一个片断,体外试验和动物模型已经发现Tumstatin对包括黑色素瘤、神经胶质瘤、前列腺癌、喉鳞癌等在内的多种肿瘤均有抑制作用,有望成为极具潜力的新型抗肿瘤药物。目前Tumstatin与肺癌的相关报道比较少见,Tumstatin对PI3K/Akt及MAPK/ERK信号通路的影响尚未完全明了,Tumstatin是否与常用化疗药物存在协同作用亦有待研究。本项目以体外培养的肺腺癌细胞株A549及耐顺铂的肺腺癌耐药细胞株A549/DDP为研究对象,观察Tumstatin185-191(Tum185-191)单药、顺铂单药及两药联合应用时对肺腺癌细胞增生与凋亡的影响,并检测干预前后细胞中Akt与ERK的活性变化,以探讨Tumstatin对肺腺癌细胞的作用及其可能作用机制。
方法MTT法测定Tum185-191、顺铂单药及两药联合干预48小时后对A549及A549/DDP细胞增殖的影响,并计算其不同药物干预下的IC_(50)、耐药指数和逆转指数;Hoechest 33258 DNA染色及流式检测分析Tum185-191及顺铂对肺癌细胞凋亡的影响;细胞免疫组织化学及Western blot检测Tum185-191及顺铂对p-Akt、p-ERK蛋白表达的影响。
结果Tum185-191对A549及A549/DDP细胞的增殖均有抑制作用,其IC_(50)分别为73.67μM、80.25μM;顺铂对A549细胞的IC_(50)为5.24μM,当其与20μM或40μM的Tum185-191联合应用时,顺铂对A549细胞的IC_(50)分别下降为3.48μM、1.39μM;A549/DDP细胞相对于A549细胞对顺铂的耐药指数为14.73;顺铂对A549/DDP细胞的IC_(50)为77.16μM;当顺铂与20μM的Tum185-191联合应用时,顺铂对A549/DDP细胞的IC_(50)下降为57.97μM,耐药逆转指数为1.33;当顺铂与40μM的Tum185-191联合时,顺铂对A549/DDP细胞的IC_(50)为26.40μM,耐药逆转指数为2.92。联合应用Tum185-191及顺铂对A549、A549/DDP细胞凋亡的诱导作用均较单药应用时显著增加。细胞免疫组织化学及Western blot检测发现p-Akt、p-ERK在未经干预的A549及A549/DDP细胞内均呈高表达状态,Tum185-191能显著抑制p-Akt、p-ERK的表达,顺铂对p-Akt、p-ERK的表达无明显影响,Tum185-191与顺铂联合使用并不增加Tum185-191对p-ERK、p-Akt的抑制效应。
结论Tum185-191单药或联合顺铂对于A549、A549/DDP细胞均有显著抑制作用。Tum185-191能够增加A549细胞对顺铂的敏感性并部分逆转A549/DDP细胞对顺铂的化疗耐药。Tum185-191可能通过下调A549及A549/DDP细胞内p-Akt、p-ERK水平发挥其抑制细胞生长、促进细胞凋亡的作用。Tum185-191增加细胞对顺铂的敏感性、部分逆转肺腺癌的化疗耐药可能与其对Akt、ERK活性的抑制作用有关。
Purpose Tumstatin and the peptides derived from tumstatin have previously been shown to be effective in vivo and in vitro against a wide range of human tumors.To data,however,few studies have investigated the combination antitumor activity of tumstatin with conventional anticancer drugs in cancinoma,and insufficient data are available on the antitumor effects by the tumstatin on non-small lung cancer(NSCLC) cells.In this report,we investigated the effects of tumstatin 185-191(Tum185-191) as a single agent or combination with cisplatin on NSCLC cell lines A549 and A549/DDP.In addition,we evaluated the changes in the Akt and ERK signaling in cultured NSCLC cells treated by tumstatin 185-191 and cisplatin.
Methods A549 or A549/DDP cells were treated with Tum185-191 and cisplatin.Cell Viability was assessed using the modified 3-(4,5-dimethylthiazoh2-yl)- 2,5-diphenyltetrazolium bromide(MTT) assay. 50%inhibiting concentration(IC_(50))values and reversing drug-resistance index(RI) of the chemotherapeutic drug were analyzed by MTT assays. Cell apoptosis was measured by morphological observation and flow cytometry.The activation of Akt and Erk were evaluated by immunocytochemistry and Western blotting.
Results Here we showed that Tum185-191 could inhibit the proliferation of A549 and A549/DDP cells.IC_(50) values of Tum185-191 were 73.67μM in A549 cells and 80.25μM in A549/DDP cells.After treated with 20μM Tum185-191,IC_(50) values of cisplatin in A549 cells reduced from 5.24μM to 3.48μM,while 40μM Tum185-191 reduced from 5.24μM to 1.39μM.In A549/DDP cells,20μM Tum185-191 reduced IC_(50) values of cisplatin from 77.16μM to 57.97μM,and 40μM Tum185-191 reduced to 26.40μM.The RI was 1.33 in 20μM Tum185-191 treated cells and 2.92 in 20μM Tum185-191 treated cells. Cotreatment with tumstatin 185-191 and cisplatin exerted significantly greater effects in promoting apoptosis than either agent used alone.A lower levels of phospho-Akt(p-Akt) and phospho-ERK(p-ERK) were found in the A549 and A549/DDP cells treated with higher dosage of tumstatin 185-191,while tumstatin 185-191 treatment whether alone,or in combination,had the similar effects on the protein levels of p-Akt and p-ERK in A549 and A549/DDP cells.
Conclusions Our data suggest that Tum185-191 might enhance the sensitivity of A549 cells to cisplatin and has reversal effects on cisplatin resistant cell line A549/DDP.The effects of promoting apoptosis and downregulation of proliferation induced by Tum185-191 may be mediated through inactivation of the Akt and ERK pathways. Tum185-191 has promising antitumor activity either as single agent or in combination with cisplatin in A549 or A549/DDP cells.These findings indicate that combining conventional anticancer drugs such as cisplatin with agents that inhibit specific survival signals may provide a molecular basis for novel chemotherapeutic strategies in the treatment of NSCLC.
方法MTT法测定Tum185-191、顺铂单药及两药联合干预48小时后对A549及A549/DDP细胞增殖的影响,并计算其不同药物干预下的IC_(50)、耐药指数和逆转指数;Hoechest 33258 DNA染色及流式检测分析Tum185-191及顺铂对肺癌细胞凋亡的影响;细胞免疫组织化学及Western blot检测Tum185-191及顺铂对p-Akt、p-ERK蛋白表达的影响。
结果Tum185-191对A549及A549/DDP细胞的增殖均有抑制作用,其IC_(50)分别为73.67μM、80.25μM;顺铂对A549细胞的IC_(50)为5.24μM,当其与20μM或40μM的Tum185-191联合应用时,顺铂对A549细胞的IC_(50)分别下降为3.48μM、1.39μM;A549/DDP细胞相对于A549细胞对顺铂的耐药指数为14.73;顺铂对A549/DDP细胞的IC_(50)为77.16μM;当顺铂与20μM的Tum185-191联合应用时,顺铂对A549/DDP细胞的IC_(50)下降为57.97μM,耐药逆转指数为1.33;当顺铂与40μM的Tum185-191联合时,顺铂对A549/DDP细胞的IC_(50)为26.40μM,耐药逆转指数为2.92。联合应用Tum185-191及顺铂对A549、A549/DDP细胞凋亡的诱导作用均较单药应用时显著增加。细胞免疫组织化学及Western blot检测发现p-Akt、p-ERK在未经干预的A549及A549/DDP细胞内均呈高表达状态,Tum185-191能显著抑制p-Akt、p-ERK的表达,顺铂对p-Akt、p-ERK的表达无明显影响,Tum185-191与顺铂联合使用并不增加Tum185-191对p-ERK、p-Akt的抑制效应。
结论Tum185-191单药或联合顺铂对于A549、A549/DDP细胞均有显著抑制作用。Tum185-191能够增加A549细胞对顺铂的敏感性并部分逆转A549/DDP细胞对顺铂的化疗耐药。Tum185-191可能通过下调A549及A549/DDP细胞内p-Akt、p-ERK水平发挥其抑制细胞生长、促进细胞凋亡的作用。Tum185-191增加细胞对顺铂的敏感性、部分逆转肺腺癌的化疗耐药可能与其对Akt、ERK活性的抑制作用有关。
Purpose Tumstatin and the peptides derived from tumstatin have previously been shown to be effective in vivo and in vitro against a wide range of human tumors.To data,however,few studies have investigated the combination antitumor activity of tumstatin with conventional anticancer drugs in cancinoma,and insufficient data are available on the antitumor effects by the tumstatin on non-small lung cancer(NSCLC) cells.In this report,we investigated the effects of tumstatin 185-191(Tum185-191) as a single agent or combination with cisplatin on NSCLC cell lines A549 and A549/DDP.In addition,we evaluated the changes in the Akt and ERK signaling in cultured NSCLC cells treated by tumstatin 185-191 and cisplatin.
Methods A549 or A549/DDP cells were treated with Tum185-191 and cisplatin.Cell Viability was assessed using the modified 3-(4,5-dimethylthiazoh2-yl)- 2,5-diphenyltetrazolium bromide(MTT) assay. 50%inhibiting concentration(IC_(50))values and reversing drug-resistance index(RI) of the chemotherapeutic drug were analyzed by MTT assays. Cell apoptosis was measured by morphological observation and flow cytometry.The activation of Akt and Erk were evaluated by immunocytochemistry and Western blotting.
Results Here we showed that Tum185-191 could inhibit the proliferation of A549 and A549/DDP cells.IC_(50) values of Tum185-191 were 73.67μM in A549 cells and 80.25μM in A549/DDP cells.After treated with 20μM Tum185-191,IC_(50) values of cisplatin in A549 cells reduced from 5.24μM to 3.48μM,while 40μM Tum185-191 reduced from 5.24μM to 1.39μM.In A549/DDP cells,20μM Tum185-191 reduced IC_(50) values of cisplatin from 77.16μM to 57.97μM,and 40μM Tum185-191 reduced to 26.40μM.The RI was 1.33 in 20μM Tum185-191 treated cells and 2.92 in 20μM Tum185-191 treated cells. Cotreatment with tumstatin 185-191 and cisplatin exerted significantly greater effects in promoting apoptosis than either agent used alone.A lower levels of phospho-Akt(p-Akt) and phospho-ERK(p-ERK) were found in the A549 and A549/DDP cells treated with higher dosage of tumstatin 185-191,while tumstatin 185-191 treatment whether alone,or in combination,had the similar effects on the protein levels of p-Akt and p-ERK in A549 and A549/DDP cells.
Conclusions Our data suggest that Tum185-191 might enhance the sensitivity of A549 cells to cisplatin and has reversal effects on cisplatin resistant cell line A549/DDP.The effects of promoting apoptosis and downregulation of proliferation induced by Tum185-191 may be mediated through inactivation of the Akt and ERK pathways. Tum185-191 has promising antitumor activity either as single agent or in combination with cisplatin in A549 or A549/DDP cells.These findings indicate that combining conventional anticancer drugs such as cisplatin with agents that inhibit specific survival signals may provide a molecular basis for novel chemotherapeutic strategies in the treatment of NSCLC.
引文
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