β-咔啉类生物碱抑制SGC-7901细胞迁移和侵袭的机制研究
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摘要
该研究主要探索β-咔啉类生物碱抑制SGC-7901细胞迁移和侵袭的机制与黏着斑激酶(focal adhesion kinase,FAK)基因表达的相关性。采用CCK-8法测定不同浓度条件下β-咔啉类生物碱对胃癌SGC-7901细胞的增殖抑制率;并通过Transwell小室测定β-咔啉类生物碱对SGC-7901细胞迁移与侵袭能力的影响;qRT-PCR及Western blot技术检测FAK基因mRNA及蛋白的表达差异。然后将si-FAK-1051重组质粒转染到SGC-7901细胞中,再次采用qRT-PCR及Western blot技术鉴定FAK基因沉默效果,最后采用同样的方法检测FAK基因沉默对胃癌SGC-7901细胞增殖和迁移的影响。随着β-咔啉类生物碱浓度增加,人胃癌SGC-7901细胞增殖抑制率逐渐升高,IC50=13.364 mg·L-1;阳性对照组(5-FU,5 mg·L-1)和β-咔啉类生物碱组中Transwell小室内SGC-7901细胞均可见显著减少(P<0.01),且β-咔啉类生物碱组SGC-7901细胞数目较阳性对照组明显降低(P<0.01);阳性对照组、β-咔啉类生物碱实验组FAK基因mRNA及蛋白表达水平较空白对照组显著降低(P<0.05)。si-FAK-1051转染入胃癌SGC-7901细胞后,FAK基因的mRNA及蛋白表达被明显下调(P<0.05),细胞增殖和细胞迁移能力也显著降低(P<0.05)。β-咔啉类生物碱较5-FU具有更有效的抑制胃癌SGC-7901细胞迁移与侵袭能力,而该机制可能与β-咔啉类生物碱抑制FAK基因mRNA及蛋白表达水平有关。
To explore the mechanism of β-carboline alkaloids inhibiting the migration and invasion of SGC-7901 cells and its correlation with FAK gene expression,CCK-8 method was used to determine the inhibitory rate of β-carboline alkaloids on the proliferation of gastric cancer SGC-7901 cells under different concentrations.The effect of β-carboline alkaloids on the migration and invasion of SGC-7901 cells was used by Transwell compartment.Detection of mRNA and protein expression of FAK genes were used by qRT-PCR and Western blot.Then si-FAK-1051 recombinant plasmid was transfected into SGC-7901 cells.FAK gene silencing effect was identified by qRT-PCR and Western blot technique again.Finally,the effects of FAK gene silencing on proliferation and migration of gastric cancer SGC-7901 cells were detected by CCK-8 kit and Transwell chamber assay respectively.With the increase of the concentration ofβ-carboline alkaloids,the inhibitory rate of SGC-7901 cells in human gastric cancer cells increased gradually,with IC5013.364 mg·L-1.The number of SGC-7901 cells of Transwell compartment in the positive experimental group(5-FU,5 mg·L-1) and the β-carboline alkaloids group decreased significantly(P<0.01) and the number of SGC-7901 cells in the β-carboline alkaloids group was significantly lower than that in the positive experimental group(P<0.01).Compared with the blank control group,the mRNA and protein expression level of FAK genes in the positive experimental group was significantly lower than that in the experimental group of β-carboline alkaloids(P<0.05).After transfection of si-FAK-1051 into gastric cancer SGC-7901 cells,the expression of mRNA and protein of FAK gene was significantly down regulated(P<0.05).SGC-7901 cell proliferation and cell migration ability also decreased significantly(P<0.05).β-carboline alkaloids are more effective than 5-FU in inhibiting migration and invasion of gastric cancer SGC-7901 cells,and the mechanism may be related to the inhibition of mRNA and protein expression of FAK gene by β-carboline alkaloids.
To explore the mechanism of β-carboline alkaloids inhibiting the migration and invasion of SGC-7901 cells and its correlation with FAK gene expression,CCK-8 method was used to determine the inhibitory rate of β-carboline alkaloids on the proliferation of gastric cancer SGC-7901 cells under different concentrations.The effect of β-carboline alkaloids on the migration and invasion of SGC-7901 cells was used by Transwell compartment.Detection of mRNA and protein expression of FAK genes were used by qRT-PCR and Western blot.Then si-FAK-1051 recombinant plasmid was transfected into SGC-7901 cells.FAK gene silencing effect was identified by qRT-PCR and Western blot technique again.Finally,the effects of FAK gene silencing on proliferation and migration of gastric cancer SGC-7901 cells were detected by CCK-8 kit and Transwell chamber assay respectively.With the increase of the concentration ofβ-carboline alkaloids,the inhibitory rate of SGC-7901 cells in human gastric cancer cells increased gradually,with IC5013.364 mg·L-1.The number of SGC-7901 cells of Transwell compartment in the positive experimental group(5-FU,5 mg·L-1) and the β-carboline alkaloids group decreased significantly(P<0.01) and the number of SGC-7901 cells in the β-carboline alkaloids group was significantly lower than that in the positive experimental group(P<0.01).Compared with the blank control group,the mRNA and protein expression level of FAK genes in the positive experimental group was significantly lower than that in the experimental group of β-carboline alkaloids(P<0.05).After transfection of si-FAK-1051 into gastric cancer SGC-7901 cells,the expression of mRNA and protein of FAK gene was significantly down regulated(P<0.05).SGC-7901 cell proliferation and cell migration ability also decreased significantly(P<0.05).β-carboline alkaloids are more effective than 5-FU in inhibiting migration and invasion of gastric cancer SGC-7901 cells,and the mechanism may be related to the inhibition of mRNA and protein expression of FAK gene by β-carboline alkaloids.
引文
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[25]Rajani K,Batra S K,Lennon F E,et al.FAK and paxillin,two potential targets in pancreatic cancer[J].Oncotarget,2016,7(21):31586.
[2]Shaikh M S,Karpoormath R,Thapliyal N,et al.Current perspective of natural alkaloid carbazole and its derivatives as antitumor agents[J].Anticancer Agents Med Chem,2015,15(8):1049.
[3]许旭东,胡晓茹,杨峻山.抗肿瘤药用植物有效成分研究概况[J].中国中药杂志,2008,33(17):2073.
[4]Chatterjee P,Seal S,Mukherjee S,et al.A carbazole alkaloid deactivates m TOR through the suppression of rictor and that induces apoptosis in lung cancer cells[J].Mol Cell Biochem,2015,405(1/2):149.
[5]李强,曹明溶,刘志龙,等.去氢骆驼蓬碱诱导HepG2细胞凋亡并增强其对5-氟尿嘧啶和顺铂的敏感性[J].中国病理生理杂志,2013,29(2):284.
[6]Fan Y,Patima A,Chen Y,et al.Cytotoxic effects ofβ-carboline alkaloids on human gastric cancer SGC-7901 cells[J].Int J Clin Exp Med,2015,8(8):12977.
[7]曾凡业,樊玉祥,张洪亮.β-咔啉类生物碱对人胃癌SGC-7901细胞增殖,凋亡及PTEN/Akt表达的影响[J].临床肿瘤学杂志,2016,21(2):126.
[8]张立雯,季涛,宿树兰,等.桑叶黄酮类和生物碱类成分在正常和糖尿病大鼠体内的药代动力学研究[J].中国中药杂志,2017,42(21):4218.
[9]Sripisut T,Laphookhieo S.Carbazole alkaloids from the stems of Clausena excavata[J].Asian Nat Prod Res,2015,17(11):1048.
[10]Nagappan T,Ramasamy P,Wahid M E,et al.Biological activity of carbazole alkaloids and essential oil of Murraya koenigii against antibiotic resistant microbes and cancer cell lines[J].Molecules,2011,16(11):9651.
[11]Ma Y G,Wink M.The beta-carboline alkaloid harmine inhibits BCRP and can reverse resistance to the anticancer drugs mitoxantrone and camptothecin in breast cancer cells[J].Phytother Res,2010,24(1):146.
[12]Zhang H,Sun K,Ding J,et al.Harmine induces apoptosis and inhibits tumor cell proliferation,migration and invasion through down-regulation of cyclooxygenase-2 expression in gastric cancer[J].Phytomedicine,2014,21(3):348.
[13]Yu X J,Sun K,Tang X H,et al.Harmine combined with paclitaxel inhibits tumor proliferation and induces apoptosis through down-regulation of cyclooxygenase-2 expression in gastric cancer[J].Oncol Lett,2016,12(2):983.
[14]Sieg D J,Hauck C R,Ilic D,et al.FAK integrates growth-factor and integrin signals to promote cell migration[J].Nature Cell Biol,2000,2(5):249.
[15]Horton E R,Humphries J D,Stutchbury B,et al.Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition[J].J Cell Biol,2016,212(3):349.
[16]Alanko J,Ivaska J.Endosomes:emerging platforms for integrinmediated FAK signalling[J].Trends Cell Biol,2016,26(6):391.
[17]Dai F,Chen Y,Song Y,et al.A natural small molecule harmine inhibits angiogenesis and suppresses tumor growth through activation of p53 in endothelial cells[J].PLoS ONE,2012,7(12):e52162.
[18]陈瑛,王丹丹,朱虹,等.抗肿瘤新靶点黏着斑激酶FAK及其抑制剂研究进展[J].中国现代应用药学,2016,33(2):255.
[19]Gao H,Zhong F,Xie J,et al.PTTG promotes invasion in human breast cancer cell line by upregulating EMMPRIN via FAK/Akt/m TOR signaling[J].Am J Cancer Res,2016,6(2):425.
[20]Webb D J,Donais K,Whitmore L A,et al.FAK-Src signalling through paxillin,ERK and MLCK regulates adhesion disassembly[J].Nature Cell Biol,2004,6(2):154.
[21]Lim S T,Chen X L,Lim Y,et al.Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation[J].Mol Cell,2008,29(1):9.
[22]Palazzo A F,Eng C H,Schlaepfer D D,et al.Localized stabilization of microtubules by integrin-and FAK-facilitated Rho signaling[J].Science,2004,303(5659):836.
[23]Weiner T M,Liu E T,Craven R J,et al.Expression of focal adhesion kinase gene and invasive cancer[J].Lancet,1993,342(8878):1024.
[24]Miyazaki T,Kato H,Nakajima M,et al.FAK overexpression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma[J].Br J Cancer,2003,89(1):140.
[25]Rajani K,Batra S K,Lennon F E,et al.FAK and paxillin,two potential targets in pancreatic cancer[J].Oncotarget,2016,7(21):31586.