过表达PDCD4逆转胃癌顺铂耐药的初步机制
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摘要
目的程序性细胞死亡因子(PDCD4)是近年来新发现的一类促凋亡基因,文中主要研究PDCD4逆转胃癌顺铂耐药的分子机制,为胃癌的耐药机制研究和治疗提供新的基因靶点。方法通过在人胃癌细胞系SGC7901/DDP中稳定过表达PDCD4基因,实验分为对照组(仅转染p CMV质粒),高表达组(仅转染p CMV-PDCD4质粒),对照加药组(转染p CMV质粒并加入顺铂处理),高表达加药组(转染p CMV-PDCD4质粒并加入顺铂处理),进行细胞免疫荧光﹑流式细胞术、RT-PCR和Western blot实验。实验分为空载加药组(转染p CMV质粒并加入顺铂处理)﹑PDCD4加药组(转染p CMV-PDCD4质粒并加入顺铂处理)和PDCD4加激活剂组(转染p CMV-PDCD4质粒,先加入SC79再加入顺铂处理)进行p Akt回补实验。结果高表达组PDCD4蛋白和mRNA表达较对照组明显升高(P<0.05),高表达加药组PDCD4蛋白和mRNA表达较对照加药组明显升高(P<0.05)。流式细胞术检测细胞早期凋亡水平显示,高表达加药组顺铂诱导的细胞早期凋亡率(25.22%)较对照加药组(21.08%)显著增高(P<0.05)。通过Western blot法表明,相较于对照加药组,高表达加药组中p Akt、p GSK3β和BCL-2的表达量均有明显降低(0.73±0.06 vs 0.42±0.04、0.88±0.05 vs 0.53±0.08、1.15±0.13 vs 0.84±0.14),差异均有统计学意义(P<0.05);而相较于对照加药组,高表达加药组中促凋亡蛋白BAK的表达水平则明显升高(1.28±0.09 vs 1.48±0.09),差异有统计学意义(P<0.05)。PDCD4加药组p Akt、p GSK3β表达较空载加药组、PDCD4加激活剂组明显降低(P<0.05),PARP(C)表达较空载加药组、PDCD4加激活剂组明显升高(P<0.05)。结论过表达PDCD4能够通过p Akt/p GSK3β促进顺铂引起的胃癌细胞凋亡,有利于逆转细胞顺铂耐药的形成。
Objective Gastric cancer is the most common cancer in the world. In China,Patients with gastric cancer are mostly treated with platinum-based chemotherapy. Programmed cell death 4( PDCD4) was found as an important proapoptosis recently,the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell. The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer. Methods Stable transfection with p CMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP; the cells were divided into control group,over-expression group,control with cisplatin group,over-expression with cisplatin group for following experiments. Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro; Real-time PCR was used to detect the mRNA expression levels of PDCD4,and the protein levels of PDCD4,p AK,p GSK3β,BCL-2 and Bak were detected by Western blot.The cells were divided into vector group,PDCD4 group,PDCD4 with activator group for detect the level of PARP( C) by Western blot.Results Compared with control group,the results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group( also in the over-expression with cisplatin group),which was indicated stable transfection with PDCD4 was successful. Immunofluorescence( with hoechst dying) and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin. PDCD4 overexpression attenuated the protein levels of p Akt,p GSK3β and BCL-2,but increased the protein levels of BAK.Furthermore,incubation with SC-79( the activator of Akt) reversed cell apoptosis induced by PDCD4. Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through p AKT/p GSK3β pathway,which is favorable to reverse cisplatin resistance in gastric cancer.
Objective Gastric cancer is the most common cancer in the world. In China,Patients with gastric cancer are mostly treated with platinum-based chemotherapy. Programmed cell death 4( PDCD4) was found as an important proapoptosis recently,the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell. The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer. Methods Stable transfection with p CMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP; the cells were divided into control group,over-expression group,control with cisplatin group,over-expression with cisplatin group for following experiments. Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro; Real-time PCR was used to detect the mRNA expression levels of PDCD4,and the protein levels of PDCD4,p AK,p GSK3β,BCL-2 and Bak were detected by Western blot.The cells were divided into vector group,PDCD4 group,PDCD4 with activator group for detect the level of PARP( C) by Western blot.Results Compared with control group,the results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group( also in the over-expression with cisplatin group),which was indicated stable transfection with PDCD4 was successful. Immunofluorescence( with hoechst dying) and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin. PDCD4 overexpression attenuated the protein levels of p Akt,p GSK3β and BCL-2,but increased the protein levels of BAK.Furthermore,incubation with SC-79( the activator of Akt) reversed cell apoptosis induced by PDCD4. Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through p AKT/p GSK3β pathway,which is favorable to reverse cisplatin resistance in gastric cancer.
引文
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[13] Hermida MA,Dinesh Kumar J,Leslie NR. GSK3 and its interactions with the PI3K/AKT/m TOR signalling network[J]. Adv Biol Regul,2017,65(8):5-15.
[14] Mc Cubrey JA,Steelman LS,Bertrand FE,et al. GSK-3 as potential target for therapeutic intervention in cancer[J]. Oncotarget,2014,5(10):2881-2911.
[15]林高阳,徐克.MicroRNA调控肿瘤耐药的研究进展[J].中国肺癌杂志,2014,17(10):741-749.
[16]徐冬,许利剑.凋亡抑制基因Survivin在胰腺癌中的研究进展[J].医学研究生学报,2013,26(3):319-322.
[17]姜艳,杨大群,王聪洋,等.抑癌基因PDCD4与肿瘤关系的研究进展[J].现代肿瘤医学,2015,23(22):3363-3366.
[18] Biyanee A,Singh P,Klempnauer KH. Translation,Pdcd4 and e IF4A[J]. Oncoscience,2015,2(9):731-732.
[19] Liu C,Yu J,Yu S,et al. MicroRNA-21 acts as an oncomir through multiple targets in human hepatocellular carcinoma[J].J Hepatol,2010,53(1):98-107.
[20]宋晓红. PI3K/AKT/GSK3β信号传导通路在化疗药物诱导卵巢癌细胞凋亡中的实验研究[D].华中科技大学,2009.
[21] Zhang X,Wang X,Song X,et al. Programmed cell death 4 enhances chemosensitivity of ovarian cancer cells by activating death receptor pathway in vitro and in vivo[J]. Cancer Sci,2010,101(10):2163-2170.
[22] Dan HC,Ebbs A,Pasparakis M,et al. Akt-dependent activation of m TORC1 complex involves phosphorylation of m TOR(mammalian target of rapamycin)by IκB kinaseα(IKKα)[J].J Biol Chem,2014,289(36):25227-25240.
[23]刘馨骏,宫剑滨,潘涛.红景天苷对大鼠缺血再灌注损伤心肌Akt/GSK-3β作用的研究[J].医学研究生学报,2015,28(2):146-148.
[24] Fu Y,Hu D,Qiu J,et al. Overexpression of glycogen synthase kinase-3 in ovarian carcinoma cells with acquired paclitaxel resistance[J]. Int J Gynecol Cancer,2011,21(3):439-444.
[25] Gao Y,Liu Z,Zhang X,et al. Inhibition of cytoplasmic GSK-3βincreases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells[J]. Cancer Lett,2013,336(1):231-239.
[26] Domoto T,Pyko IV,Furuta T,et al. Glycogen synthase kinase-3βis a pivotal mediator of cancer invasion and resistance to therapy[J]. Cancer Sci,2016,107(10):1363-1372.
[27] Wang WQ,Zhang H,Wang HB,et al. Programmed cell death 4(PDCD4)enhances the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by inhibiting the PI3K/Akt signaling pathway[J]. Mol Diagn Ther,2010,14(3):155-161.
[28] Feng MG,Liu CF,Chen L,et al. MiR-21 attenuates apoptosistriggered by amyloid-βvia modulating PDCD4/PI3K/AKT/GSK-3βpathway in SH-SY5Y cells[J]. Biomed Pharmacother,2018,101(5):1003-1007.
[2] Zhu X,Zhang K,Wang Q,et al. Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells[J]. Zhonghua Zhong Liu Za Zhi,2015,37(6):404-411.
[3]张国华,徐颖,邹晨,等.长链非编码RNA DGCR5在胃癌中的表达及临床意义[J].医学研究生学报,2018,31(1):33-38.
[4]余丽莉,鲍军,江盼,等.鞘氨醇-1-磷酸转运体在胃癌组织的表达及其对胃癌细胞增殖和迁移的影响[J].医学研究生学报,2018,31(10):1020-1025.
[5] Quintero-Aldana G,Jorge M,Grande C,et al. Phase II study of first-line biweekly docetaxel and cisplatin combination chemotherapy in advanced gastric cancer[J]. Cancer Chemother Pharmacol,2015,76(4):731-737.
[6]郑伟,王聪洋,姜艳,等.PDCD4在Hela细胞中过表达致抗凋亡蛋白Bcl-2表达下调机制探讨[J].现代肿瘤医学,2016,24(17):2690-2694.
[7] Allen KE,Weiss GJ. Resistance may not be futile:microRNA biomarkers for chemoresistance and potential therapeutics[J]. Mol Cancer Ther,2010,9(12):3126-3136.
[8] Bourguignon LY,Spevak CC,Wong G,et al. Hyaluronan-CD44interaction with protein kinase C(epsilon)promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21,leading to down-regulation of the tumor suppressor protein PDCD4,anti-apoptosis,and chemotherapy resistance in breast tumor cells[J]. J Biol Chem,2009,284(39):26533-26546.
[9] Keane NA,Glavey SV,Krawczyk J,et al. AKT as a therapeutic target in multiple myeloma[J]. Expert Opin Ther Targets,2014,18(8):897-915.
[10] Martini M,De Santis MC,Braccini L,et al. PI3K/AKT signaling pathway and cancer:an updated review[J]. Ann Med,2014,46(6):372-383.
[11] Faes S,Dormond O. PI3K and AKT:Unfaithful Partners in Cancer[J]. Int J Mol Sci,2015,16(9):21138-21152.
[12] Keane NA,Glavey SV,Krawczyk J,et al. AKT as a therapeutic target in multiple myeloma[J]. Expert Opin Ther Targets,2014,18(8):897-915.
[13] Hermida MA,Dinesh Kumar J,Leslie NR. GSK3 and its interactions with the PI3K/AKT/m TOR signalling network[J]. Adv Biol Regul,2017,65(8):5-15.
[14] Mc Cubrey JA,Steelman LS,Bertrand FE,et al. GSK-3 as potential target for therapeutic intervention in cancer[J]. Oncotarget,2014,5(10):2881-2911.
[15]林高阳,徐克.MicroRNA调控肿瘤耐药的研究进展[J].中国肺癌杂志,2014,17(10):741-749.
[16]徐冬,许利剑.凋亡抑制基因Survivin在胰腺癌中的研究进展[J].医学研究生学报,2013,26(3):319-322.
[17]姜艳,杨大群,王聪洋,等.抑癌基因PDCD4与肿瘤关系的研究进展[J].现代肿瘤医学,2015,23(22):3363-3366.
[18] Biyanee A,Singh P,Klempnauer KH. Translation,Pdcd4 and e IF4A[J]. Oncoscience,2015,2(9):731-732.
[19] Liu C,Yu J,Yu S,et al. MicroRNA-21 acts as an oncomir through multiple targets in human hepatocellular carcinoma[J].J Hepatol,2010,53(1):98-107.
[20]宋晓红. PI3K/AKT/GSK3β信号传导通路在化疗药物诱导卵巢癌细胞凋亡中的实验研究[D].华中科技大学,2009.
[21] Zhang X,Wang X,Song X,et al. Programmed cell death 4 enhances chemosensitivity of ovarian cancer cells by activating death receptor pathway in vitro and in vivo[J]. Cancer Sci,2010,101(10):2163-2170.
[22] Dan HC,Ebbs A,Pasparakis M,et al. Akt-dependent activation of m TORC1 complex involves phosphorylation of m TOR(mammalian target of rapamycin)by IκB kinaseα(IKKα)[J].J Biol Chem,2014,289(36):25227-25240.
[23]刘馨骏,宫剑滨,潘涛.红景天苷对大鼠缺血再灌注损伤心肌Akt/GSK-3β作用的研究[J].医学研究生学报,2015,28(2):146-148.
[24] Fu Y,Hu D,Qiu J,et al. Overexpression of glycogen synthase kinase-3 in ovarian carcinoma cells with acquired paclitaxel resistance[J]. Int J Gynecol Cancer,2011,21(3):439-444.
[25] Gao Y,Liu Z,Zhang X,et al. Inhibition of cytoplasmic GSK-3βincreases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells[J]. Cancer Lett,2013,336(1):231-239.
[26] Domoto T,Pyko IV,Furuta T,et al. Glycogen synthase kinase-3βis a pivotal mediator of cancer invasion and resistance to therapy[J]. Cancer Sci,2016,107(10):1363-1372.
[27] Wang WQ,Zhang H,Wang HB,et al. Programmed cell death 4(PDCD4)enhances the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by inhibiting the PI3K/Akt signaling pathway[J]. Mol Diagn Ther,2010,14(3):155-161.
[28] Feng MG,Liu CF,Chen L,et al. MiR-21 attenuates apoptosistriggered by amyloid-βvia modulating PDCD4/PI3K/AKT/GSK-3βpathway in SH-SY5Y cells[J]. Biomed Pharmacother,2018,101(5):1003-1007.