抑制Vav3基因表达对胃癌细胞凋亡抑制蛋白及肿瘤多药耐药性的影响
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摘要
目的探讨Vav3基因在胃癌多药耐药性(MDR)中的作用及可能机制。方法采用荧光定量反转录聚合酶链(QRT-PCR)及蛋白印迹技术检测Vav3在胃癌、癌旁组织及人胃癌细胞株SGC7901、胃上皮细胞GES-1中的表达;合成针对Vav3的siRNA,并转染SGC7901;MTT法检测氟尿嘧啶(5-FU)、奥沙利铂(L-OHP)对转染前后胃癌细胞的抑制率;荧光定量RT-PCR和蛋白质印迹法检测转染前后凋亡抑制蛋白家族成员xIAP、Survivin、Livin表达,并检测Caspase-3、Caspase-8表达及活性。结果 Vav3在胃癌组织及细胞株的表达高于癌旁组织及胃上皮细胞株(P<0.05);Vav3-siRNA转染SGC7901后Vav3表达明显受到抑制(P<0.01);Vav3-siRNA转染SGC7901 48h后5-FU、L-OHP对肿瘤细胞的抑制作用均明显增强(P<0.05);转染后SGC7901中xIAP、Survivin的表达均较转染前降低(P<0.05),Livin表达在转染前后无明显变化;转染后Caspase-3、Caspase-8表达及活性升高(P<0.05)。结论 Vav3可通过调控胃癌细胞凋亡抑制途径参与胃癌多药耐药,抑制Vav3表达可能有助于逆转胃癌细胞的化疗耐药。
Objective To explore the role of Vav3 gene in multidrug resistance(MDR)of gastric cancer and the related mechanism.Methods QRT-PCR was used to examine the expressions of Vav3 gene in gastric cancer tissues,tumor-adjacent tissues,human gastric cancer cell line SGC7901,and gastric epithelial cell line GES-1.Then Vav3-siRNA was synthesized and tansfected into SGC7901 cells. MTT assay was then used to determine the inhibition rates of tumor cells exposed to chemotherapeutic agents(5-FU,L-OHP)before and after Vav3-siRNA transfection.Real-time RT-PCR and Western blotting analysis were used to observe the expressions of inhibitor of apoptosis proteins(IAPs):xIAP,Survivin,and Livin;meanwhile,the expression and activity of Caspase-3and Caspase-8 were also determined.Results Vav3 was over-expressed in gastric cancer tissues and gastric cell line compared with those in tumor-adjacent tissues and gastric epithelial cell line GES-1(P<0.05).Expression of Vav3 was significantly inhibited by Vav3-siRNA(P<0.01).Inhibition rates of tumor cells exposed to 5-FU and L-OHP were significantly increased 48 hafter Vav3-siRNA tansfection(P<0.05).The expressions of xIAP and Survivin were significantly decreased in cancer cells after Vav3-siRNA tansfection(both P<0.05),and no notable change was found for Livin expression;also the expression and activity of Caspase-3and Caspase-8protein were significantly increased after Vav3-siRNA tansfection in SGC7901cells(all P<0.05).Conclusion Vav3 can participate in MDR of gastric cancer by regulating apoptotic pathways,and inhibition of Vav3 can help reverse MDR of gastric cancer cells by regulating some IAPs.
Objective To explore the role of Vav3 gene in multidrug resistance(MDR)of gastric cancer and the related mechanism.Methods QRT-PCR was used to examine the expressions of Vav3 gene in gastric cancer tissues,tumor-adjacent tissues,human gastric cancer cell line SGC7901,and gastric epithelial cell line GES-1.Then Vav3-siRNA was synthesized and tansfected into SGC7901 cells. MTT assay was then used to determine the inhibition rates of tumor cells exposed to chemotherapeutic agents(5-FU,L-OHP)before and after Vav3-siRNA transfection.Real-time RT-PCR and Western blotting analysis were used to observe the expressions of inhibitor of apoptosis proteins(IAPs):xIAP,Survivin,and Livin;meanwhile,the expression and activity of Caspase-3and Caspase-8 were also determined.Results Vav3 was over-expressed in gastric cancer tissues and gastric cell line compared with those in tumor-adjacent tissues and gastric epithelial cell line GES-1(P<0.05).Expression of Vav3 was significantly inhibited by Vav3-siRNA(P<0.01).Inhibition rates of tumor cells exposed to 5-FU and L-OHP were significantly increased 48 hafter Vav3-siRNA tansfection(P<0.05).The expressions of xIAP and Survivin were significantly decreased in cancer cells after Vav3-siRNA tansfection(both P<0.05),and no notable change was found for Livin expression;also the expression and activity of Caspase-3and Caspase-8protein were significantly increased after Vav3-siRNA tansfection in SGC7901cells(all P<0.05).Conclusion Vav3 can participate in MDR of gastric cancer by regulating apoptotic pathways,and inhibition of Vav3 can help reverse MDR of gastric cancer cells by regulating some IAPs.
引文
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[12]Wang Y,Liu L,Liu X,Zhang H,Liu J,Feng B,et al.Shugoshin1enhances multidrug resistance of gastric cancer cells by regulating MRP1,Bcl-2,and Baxgenes[J].Tumour Biol,2013,34:2205-2214.
[13]Liu Y,Wu X,Dong Z,Lu S.The molecular mechanism of Vav3oncogene on upregulation of androgen receptor activity in prostate cancer cells[J].Int J Oncol,2010,36:623-633.
[14]Travert M,Huang Y,de Leval L,Martin-Garcia N,Delfau-Larue M H,Berger F,et al.Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets[J].Blood,2012,119:5795-5806.
[15]Chen X,Wang T,Yang D,Wang J,Li X,He Z,et al.Expression of the IAP protein family acts cooperatively to predict prognosis in human bladder cancer patients[J].Oncol Lett,2013,5:1278-1284.
[16]Wittkopf N,Günther C,Martini E,He G,Amann K,He Y W,et al.Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8[J].Gastroenterology,2013,145:1369-1379.
[17]Sikdar S,Mukherjee A,Ghosh S,Khuda-Bukhsh A R.Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer,in vitro and in vivo[J].Environ Toxicol Pharmacol,2013,37:300-314.
[2]Yan L H,Wang X T,Yang J,Kong F B,Lian C,Wei W Y,et al.Reversal of multidrug resistance in gastric cancer cells by E2F-1downregulation in vitro and in vivo[J].J Cell Biochem,2014,115:34-41.
[3]Xie X,Tang B,Zhou J,Gao Q,Zhang P.Inhibition of the PI3K/Akt pathway increases the chemosensitivity of gastric cancer to vincristine[J].Oncol Rep,2013,30:773-782.
[4]檀碧波,李勇,范立侨,赵群,刘羽,赵雪峰.胃癌凋亡相关蛋白Survivin、B淋巴细胞/白血病-2、bax表达与肿瘤细胞体外化疗药敏性的关系[J].中华实验外科杂志,2012,29:1315-1317.
[5]Li Y,Tan B B,Zhao Q,Fan L Q,Liu Y,Hao Y J,et al.Tumor chemosensitivity is correlated with expression of multidrug resistance associated factors in variously differentiated gastric carcinoma tissues[J].Hepatogastroenterology,2013,60:213-216.
[6]Gazzaniga P,Gradilone A,Petracca A,Nicolazzo C,Raimondi C,Iacovelli R,et al.Molecular markers in circulating tumour cells from metastatic colorectal cancer patients[J].J Cell Mol Med,2010,14:2073-2077.
[7]Fernandez-Salguero P M.A remarkable new target gene for the dioxin receptor:the Vav3proto-oncogene links AhR to adhesion and migration[J].Cell Adh Migr,2010,4:172-175.
[8]Chang K H,Sanchez-Aguilera A,Shen S,Sengupta A,Madhu M N,Ficker A M,et al.Vav3collaborates with p190-BCR-ABL in lymphoid progenitor leukemogenesis,proliferation,and survival[J].Blood,2012,120:800-811.
[9]Nomura T,Yamasaki M,Hirai K,Inoue T,Sato R,Matsuura K,et al.Targeting the Vav3oncogene enhances docetaxel-induced apoptosis through the inhibition of androgen receptor phosphorylation in LNCaP prostate cancer cells under chronic hypoxia[J].Mol Cancer,2013,12:27.
[10]Lee K,Liu Y,Mo J Q,Zhang J,Dong Z,Lu S.Vav3oncogene activates estrogen receptor and its overexpression may be involved in human breast cancer[J].BMC Cancer,2008,8:158.
[11]Kang J,Zhao G,Lin T,Tang S,Xu G,Hu S,et al.A peptide derived from phage display library exhibits antitumor activity by targeting GRP78in gastric cancer multidrug resistance cells[J].Cancer Lett,2013,339:247-259.
[12]Wang Y,Liu L,Liu X,Zhang H,Liu J,Feng B,et al.Shugoshin1enhances multidrug resistance of gastric cancer cells by regulating MRP1,Bcl-2,and Baxgenes[J].Tumour Biol,2013,34:2205-2214.
[13]Liu Y,Wu X,Dong Z,Lu S.The molecular mechanism of Vav3oncogene on upregulation of androgen receptor activity in prostate cancer cells[J].Int J Oncol,2010,36:623-633.
[14]Travert M,Huang Y,de Leval L,Martin-Garcia N,Delfau-Larue M H,Berger F,et al.Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets[J].Blood,2012,119:5795-5806.
[15]Chen X,Wang T,Yang D,Wang J,Li X,He Z,et al.Expression of the IAP protein family acts cooperatively to predict prognosis in human bladder cancer patients[J].Oncol Lett,2013,5:1278-1284.
[16]Wittkopf N,Günther C,Martini E,He G,Amann K,He Y W,et al.Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8[J].Gastroenterology,2013,145:1369-1379.
[17]Sikdar S,Mukherjee A,Ghosh S,Khuda-Bukhsh A R.Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer,in vitro and in vivo[J].Environ Toxicol Pharmacol,2013,37:300-314.