PFKFB3基因增强阿帕替尼对胃癌凋亡的影响及机制
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摘要
[目的]探讨沉默PFKFB3基因对阿帕替尼处理的胃癌细胞凋亡的影响及机制。[方法]通过Western blotting检测人胃黏膜上皮细胞株GES1及SGC-7901、BGC823和MKN45胃癌细胞PFKFB3蛋白表达。将PFKFB3特异性siRNA(si-PFKFB3)转染SGC-7901细胞,Western blotting检测转染效果。MTT法检测转染siPFKFB3或不同浓度(10、20、30、40μmol/L)阿帕替尼处理SGC-7901细胞48h的细胞活力。流式细胞术检测转染si-PFKFB3或/和40μmol/L阿帕替尼处理SGC-7901细胞48h的细胞凋亡率,Western blotting检测Bcl-2、Bax、PI3K、AKT和p-AKT蛋白表达。[结果]与对照GES1细胞相比,PFKFB3在SGC-7901、BGC823和MKN45胃癌细胞中的表达均明显升高(P<0.05)。si-PFKFB3转染SGC-7901细胞后,PFKFB3蛋白表达明显降低(P<0.05)。转染si-PFKFB3及不同浓度阿帕替尼均可抑制SGC-7901细胞活力,阿帕替尼对细胞活力抑制呈现浓度依赖性(P<0.05)。转染si-PFKFB3及阿帕替尼均可诱导SGC-7901细胞凋亡,下调Bcl-2、PI3K和p-AKT表达,上调Bax表达,二者合用对SGC-7901细胞凋亡诱导更明显(P<0.05)。[结论]沉默PFKFB3基因表达可明显增强阿帕替尼对胃癌细胞的凋亡诱导作用,机制可能与下调PI3K/AKT信号通路有关。
[Objective]To investigate the effect and mechanism of silencing PFKFB3 gene on apoptosis of gastric cancer cells treated with apatinib.[Methods]Western blotting was used to detect the expression levels of PFKFB3 in human gastric epithelial cell lines GES1 and gastric cancer cells SGC-7901,BGC823 and MKN45.PFKFB3 specific siRNA(si-PFKFB3)was transfected into SGC-7901 cells,and the transfection effect was detected by Western blotting.MTT assay was applied to determine the cell viability of SGC-7901 cells transfected with si-PFKFB3 or treated with apatinib at different concentrations(10,20,30,40μmol/L)for 48 hours.Flow cytometry was used to measure the apoptotic rate of SGC-7901 cells transfected with si-PFKFB3 or/and treated with 40μmol/L apatinib for 48 hours.Western blotting was used to detect the expression of Bcl-2,Bax,PI3 K,AKT and p-AKT proteins.[Results]Compared with the control GES1 cells,the expression levels of PFKFB3 in SGC-7901,BGC823 and MKN45 gastric cancer cells were significantly increased(P<0.05).After transfection with si-PFKFB3,the expression level of PFKFB3 protein in SGC-7901 cells remarkably decreased(P<0.05).Either transfection of si-PFKFB3 or treatment with different concentrations of apatinib inhibited the cell viability of SGC-7901 cells.And apatinib inhibited cell viability in a concentration-dependent manner(P <0.05).Knock-down PFKFB3 and treatment with apatinib both induced apoptosis of SGC-7901 cells,down-regulated the expression of Bcl-2,PI3 Kand p-AKT,and up-regulated the expression of Bax.The combination of the two methods was more significantly on inducing apoptosis of SGC-7901 cells(P<0.05).[Conclusion]Silencing the expression of PFKFB3 gene can significantly enhance the apoptotic induction effect of apatinib on gastric cancer,which may be related to down-regulation of PI3 K/AKT signaling pathway.
[Objective]To investigate the effect and mechanism of silencing PFKFB3 gene on apoptosis of gastric cancer cells treated with apatinib.[Methods]Western blotting was used to detect the expression levels of PFKFB3 in human gastric epithelial cell lines GES1 and gastric cancer cells SGC-7901,BGC823 and MKN45.PFKFB3 specific siRNA(si-PFKFB3)was transfected into SGC-7901 cells,and the transfection effect was detected by Western blotting.MTT assay was applied to determine the cell viability of SGC-7901 cells transfected with si-PFKFB3 or treated with apatinib at different concentrations(10,20,30,40μmol/L)for 48 hours.Flow cytometry was used to measure the apoptotic rate of SGC-7901 cells transfected with si-PFKFB3 or/and treated with 40μmol/L apatinib for 48 hours.Western blotting was used to detect the expression of Bcl-2,Bax,PI3 K,AKT and p-AKT proteins.[Results]Compared with the control GES1 cells,the expression levels of PFKFB3 in SGC-7901,BGC823 and MKN45 gastric cancer cells were significantly increased(P<0.05).After transfection with si-PFKFB3,the expression level of PFKFB3 protein in SGC-7901 cells remarkably decreased(P<0.05).Either transfection of si-PFKFB3 or treatment with different concentrations of apatinib inhibited the cell viability of SGC-7901 cells.And apatinib inhibited cell viability in a concentration-dependent manner(P <0.05).Knock-down PFKFB3 and treatment with apatinib both induced apoptosis of SGC-7901 cells,down-regulated the expression of Bcl-2,PI3 Kand p-AKT,and up-regulated the expression of Bax.The combination of the two methods was more significantly on inducing apoptosis of SGC-7901 cells(P<0.05).[Conclusion]Silencing the expression of PFKFB3 gene can significantly enhance the apoptotic induction effect of apatinib on gastric cancer,which may be related to down-regulation of PI3 K/AKT signaling pathway.
引文
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[8]Lv L,Zhang Z,Lei X,et al.Case Report Apatinib combined with S-1in patients with advanced pancreatic cancer causing sudden massive hemorrhage:report of two cases[J].Int J Clin Exp Med,2018,11(6):6358-6363.
[9]Gu M,Li L,Zhang Z,et al.PFKFB3promotes proliferation,migration and angiogenesis in nasopharyngeal carcinoma[J].J Cancer,2017,8(18):3887-3896.
[10]O'Neal J,Clem A,Reynolds L,et al.Inhibition of 6-phosphofructo-2-kinase(PFKFB3)suppresses glucose metabolism and the growth of HER2+breast cancer[J].Breast Cancer Res Treat,2016,160(1):29-40.
[11]Chen X,Wang A,Yue X.miR-449cinhibits migration and invasion of gastric cancer cells by targeting PFKFB3[J].Oncology Letters,2018,16(1):417-424.
[12]Wei Z,Liang Y,Zhang J,et al.PFK15,a Small Molecule Inhibitor of PFKFB3,Induces Cell Cycle Arrest,Apoptosis and Inhibits Invasion in Gastric Cancer[J].Plos One,2016,11(9):e0163768.
[13]Chen L,Li Z,Xu S,et al.Upregulation of miR-107inhibits glioma angiogenesis and VEGF expression[J].Cellular and molecular neurobiology,2016,36(1):113-120.
[14]Lecaros RLG,Huang L,Lee TC,et al.Nanoparticle delivered VEGF-A siRNA enhances photodynamic therapy for head and neck cancer treatment[J].Molecular Therapy,2016,24(1):106-116.
[15]Liao W C,Sohn Y S,Riutin M,et al.The Application of Stimuli-Responsive VEGF-and ATP-Aptamer-Based Microcapsules for the Controlled Release of an Anticancer Drug,and the Selective Targeted Cytotoxicity toward Cancer Cells[J].Advanced Functional Materials,2016,26(24):4262-4273.
[16]Roviello G,Ravelli A,Fiaschi A I,et al.Apatinib for the treatment of gastric cancer[J].Expert review of gastroenterology&hepatology,2016,10(8):887-892.
[17]Xu J,Liu X,Yang S,et al.Clinical response to apatinib monotherapy in advanced non-small cell lung cancer[J].Asia-Pacific Journal of Clinical Oncology,2018,14(3):264-269.
[18]侯兵,郭志旺,周涛,等.PI3K/AKT信号通路调控MKP3表达促进胶质母细胞瘤细胞增殖的实验研究[J].临床肿瘤学杂志,2018,23(7):577-581.
[19]Bugide S,Gonugunta VK,Penugurti V,et al.HPIPpromotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation[J].Cellular Oncology,2017,40(2):133-144.
[20]Baek SH,Ko JH,Lee JH,et al.Ginkgolic Acid Inhibits Invasion and Migration and TGF-β-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation[J].Journal of cellular physiology,2017,232(2):346-354.
[21]金伟伟,白山岭,肖明中,等.牛黄参含药血清调控Bax,Bcl-2表达诱导人肝癌细胞HepG-2凋亡的实验研究[J].湖北中医药大学学报,2017,19(2):11-14.
[22]Zheng Q,Wang B,Gao J,et al.CD 155knockdown promotes apoptosis via AKT/Bcl-2/Bax in colon cancer cells[J].Journal of cellular and molecular medicine,2018,22(1):131-140.
[23]Du M,Wen G,Jin J,et al.Mangiferin prevents the growth of gastric carcinoma by blocking the PI3K-Akt signalling pathway[J].Anti-cancer drugs,2018,29(2):167-175.
[24]Zhu X,Jiang X,Li A,et al.S-allylmercaptocysteine suppresses the growth of human gastric cancer xenografts through induction of apoptosis and regulation of MAPK and PI3K/Akt signaling pathways[J].Biochemical and biophysical research communications,2017,491(3):821-826.
[2]Yang Q,Hou P.Targeting PFKFB3in the Endothelium for Cancer Therapy[J].Trends Mol Med,2017,23(3):197-200.
[3]Ge X,Lyu P,Cao Z,et al.Overexpression of miR-206suppresses glycolysis,proliferation and migration in breast cancer cells via PFKFB3targeting[J].Biochem Biophys Res Commun,2015,463(4):1115-1121.
[4]Lu L,Chen Y,Zhu Y.The molecular basis of targeting PFKFB3as a therapeutic strategy against cancer[J].Oncotarget,2017,8(37):62793-62802.
[5]Han J,Meng Q,Xi Q,et al.PFKFB3was overexpressed in gastric cancer patients and promoted the proliferation and migration of gastric cancer cells[J].Cancer Biomarkers,2016,18(3):1-8.
[6]Weina L I,Deng Y.Affects of Apatinib in the Invasion Ability of Non-small Cell Lung Cancer Cells[J].Herald of Medicine,2017,36(9):967-970.
[7]Huang L,Wei Y,Shen S,et al.Therapeutic effect of apatinib on overall survival is mediated by prolonged progression-free survival in advanced gastric cancer patients[J].Oncotarget,2017,8(17):29346-29354.
[8]Lv L,Zhang Z,Lei X,et al.Case Report Apatinib combined with S-1in patients with advanced pancreatic cancer causing sudden massive hemorrhage:report of two cases[J].Int J Clin Exp Med,2018,11(6):6358-6363.
[9]Gu M,Li L,Zhang Z,et al.PFKFB3promotes proliferation,migration and angiogenesis in nasopharyngeal carcinoma[J].J Cancer,2017,8(18):3887-3896.
[10]O'Neal J,Clem A,Reynolds L,et al.Inhibition of 6-phosphofructo-2-kinase(PFKFB3)suppresses glucose metabolism and the growth of HER2+breast cancer[J].Breast Cancer Res Treat,2016,160(1):29-40.
[11]Chen X,Wang A,Yue X.miR-449cinhibits migration and invasion of gastric cancer cells by targeting PFKFB3[J].Oncology Letters,2018,16(1):417-424.
[12]Wei Z,Liang Y,Zhang J,et al.PFK15,a Small Molecule Inhibitor of PFKFB3,Induces Cell Cycle Arrest,Apoptosis and Inhibits Invasion in Gastric Cancer[J].Plos One,2016,11(9):e0163768.
[13]Chen L,Li Z,Xu S,et al.Upregulation of miR-107inhibits glioma angiogenesis and VEGF expression[J].Cellular and molecular neurobiology,2016,36(1):113-120.
[14]Lecaros RLG,Huang L,Lee TC,et al.Nanoparticle delivered VEGF-A siRNA enhances photodynamic therapy for head and neck cancer treatment[J].Molecular Therapy,2016,24(1):106-116.
[15]Liao W C,Sohn Y S,Riutin M,et al.The Application of Stimuli-Responsive VEGF-and ATP-Aptamer-Based Microcapsules for the Controlled Release of an Anticancer Drug,and the Selective Targeted Cytotoxicity toward Cancer Cells[J].Advanced Functional Materials,2016,26(24):4262-4273.
[16]Roviello G,Ravelli A,Fiaschi A I,et al.Apatinib for the treatment of gastric cancer[J].Expert review of gastroenterology&hepatology,2016,10(8):887-892.
[17]Xu J,Liu X,Yang S,et al.Clinical response to apatinib monotherapy in advanced non-small cell lung cancer[J].Asia-Pacific Journal of Clinical Oncology,2018,14(3):264-269.
[18]侯兵,郭志旺,周涛,等.PI3K/AKT信号通路调控MKP3表达促进胶质母细胞瘤细胞增殖的实验研究[J].临床肿瘤学杂志,2018,23(7):577-581.
[19]Bugide S,Gonugunta VK,Penugurti V,et al.HPIPpromotes epithelial-mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation[J].Cellular Oncology,2017,40(2):133-144.
[20]Baek SH,Ko JH,Lee JH,et al.Ginkgolic Acid Inhibits Invasion and Migration and TGF-β-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation[J].Journal of cellular physiology,2017,232(2):346-354.
[21]金伟伟,白山岭,肖明中,等.牛黄参含药血清调控Bax,Bcl-2表达诱导人肝癌细胞HepG-2凋亡的实验研究[J].湖北中医药大学学报,2017,19(2):11-14.
[22]Zheng Q,Wang B,Gao J,et al.CD 155knockdown promotes apoptosis via AKT/Bcl-2/Bax in colon cancer cells[J].Journal of cellular and molecular medicine,2018,22(1):131-140.
[23]Du M,Wen G,Jin J,et al.Mangiferin prevents the growth of gastric carcinoma by blocking the PI3K-Akt signalling pathway[J].Anti-cancer drugs,2018,29(2):167-175.
[24]Zhu X,Jiang X,Li A,et al.S-allylmercaptocysteine suppresses the growth of human gastric cancer xenografts through induction of apoptosis and regulation of MAPK and PI3K/Akt signaling pathways[J].Biochemical and biophysical research communications,2017,491(3):821-826.