胃癌细胞VEGF/NRP-1自分泌途径及临床作用的探讨
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摘要
目的:对胃癌细胞VEGF/NRP-1自分泌途径的分析,探明其启动及效应的细胞信号途径;分析阻断VEGF/NRP-1自分泌途径后胃癌细胞的生物学活性,探讨其临床治疗的价值。
方法:以人胃癌细胞株SCG7901为研究对象。MTT法检测12G5对细胞粘附Matrigel的抑制作用,筛选出刚出现抑制作用的最小浓度用于后续实验;检测12G5对SCG7901细胞的侵袭转移能力的影响,Transwell小室进行人工重组基底膜侵袭和运动实验;台盼蓝染色法检测12G5对细胞增殖的影响;流式细胞术检测12G5对细胞凋亡的影响;RT-PCR及Western blot检测SCG7901细胞VEGF自分泌途径因子的mRNA和蛋白的表达,12G5对CXCR4表达的影响。
结果:12G5可使人胃癌细胞株SCG7901对基底膜粘附能力下降,浓度为10ug/ml的12G5即可使胃癌细胞粘附能力抑制,与对照组相比具有显著意义(P<0.01),且随药物浓度的增大和时间的延长出现粘附抑制增强。胃癌细胞株对照组和实验组侵袭迁移实验穿膜细胞数分别为:121.7±5.51,54.3±9.07;140.3±6.03,62.0±11.53。胃癌细胞株SCG7901实验组的侵袭迁移能力明显低于对照组,有非常显著性差异(P<0.01)。
12G5对人胃癌细胞株SCG7901有细胞增殖抑制作用,经12G5处理SCG7901细胞后出现细胞生存率明显低于对照组(P<0.01),且随时间的延长与对照组比较抑制增强。流式细胞仪检测细胞的凋亡率,实验组与对照组比较,结果显示实验组的凋亡率明显高于对照组,差异具有显著性意义(P<0.01)。
检测结果显示胃癌细胞株SCG7901存在VEGF165mRNA、NRP-1mRNA、CXCR4mRNA及蛋白的表达,12G5处理细胞后,实验组的CXCR4mRNA及蛋白水平较对照组显著下降(P<0.01)。
结论:抗CXCR4单克隆抗体12G5一方面可能通过下调CXCR4的表达减少了与其配体SDF-1的结合;另一方面可能直接阻断了CXCR4与SDF-1的结合,对人胃癌细胞株SCG7901的粘附、侵袭转移能力有抑制作用,且可抑制胃癌细胞增殖,促使其凋亡,提示胃癌细胞存在VEGF/NRP-1自分泌途径,且胃癌可能存在SDF-1的自分泌,通过阻断此自分泌途径有望成为今后胃癌临床治疗的手段之一。
OBJECTIVE: To analyze the VEGF/NRP-1 autocrine pathway in the gastric cancer cells,and try to find out the starting and responding of cell signal pathway;Observe the biologic activity of gastric cancer cells after blocking the VEGF/NRP-1 autocrine pathway,to explore the value for clinical treatment.
METHODS: Human gastric cancer cell lines SCG7901 was used in this study.The effects of CXCR4 inhibitor 12G5 on the cells adhering Matrigel was measured with MTT assay,and the minimum concentration was sieved for next step; The effects of 12G5 on the invasive and metastatic abilities of SCG7901cells was explored by Transwell Chamber.The effects of 12G5 on the proliferation of SCG7901 cells was explored by trypanblau staining method;The effects of 12G5 on apoptosis of SCG7901 cells was explored by flow cytometry;The expression of VEGF autocrine pathway was detected by RT-PCR and Western blot in the untreated group and 12G5-treated group.
RESULTS: 12G5 showed a time/dose-dependent inhibition effect on SCG7901 cells adhering Matrigel,and the adhesion of gastric cancer cells was inhibited on the concentration of 10ug/ml of 12G5, and compared with untreated group,adhesion of SCG7901 cells was lower(P<0.01);The invaded cell numbers in invasive and metastatic experiments of SCG7901 cell lines untreated or treated with 12G5 were 121.7±5.51,54.3±9.07;140.3±6.03,62.0±11.53 respectively,the invasive and metastatic abilities of treated group were lower than untreated group significantly(P<0.01).
12G5 showed a time-dependent growth inhibition effect on SCG7901 cells,and the survival rate of SCG7901 cells in the 12G5-treated group was lower than untreated group significantly(P<0.01);The apoptosis rate of SCG7901 cells was explored by flow cytometry,and it showed the apoptosis rate in the 12G5-treated group was higher than untreated group significantly(P<0.01).
The mRNA and protein expression level of VEGF165、NRP-1 was detected in SCG7901 cell lines,and the mRNA and protein expression level of CXCR4 in the 12G5-treated group was lower than untreated group significantly(P<0.01).
CONCLUSIONS: The expression of CXCR4 inhibited by anti- CXCR4 monoclonal antibody 12G5 may reduce the binding of CXCR4 and SDF-1,and 12G5 may directly blocked the binding of them ,and play a inhibiting effect on the adhesive、invasive and metastatic abilities of SCG7901 cells,and the growth of cancer cells was inhibited,and the apoptosis was promoted.It showed SCG7901 cells have a VEGF/NRP-1 autocrine pathway,and SDF-1 may be autocrined by gastric cancer cells. It may become one of clinical treatment means by blocking VEGF autocrine pathway.
方法:以人胃癌细胞株SCG7901为研究对象。MTT法检测12G5对细胞粘附Matrigel的抑制作用,筛选出刚出现抑制作用的最小浓度用于后续实验;检测12G5对SCG7901细胞的侵袭转移能力的影响,Transwell小室进行人工重组基底膜侵袭和运动实验;台盼蓝染色法检测12G5对细胞增殖的影响;流式细胞术检测12G5对细胞凋亡的影响;RT-PCR及Western blot检测SCG7901细胞VEGF自分泌途径因子的mRNA和蛋白的表达,12G5对CXCR4表达的影响。
结果:12G5可使人胃癌细胞株SCG7901对基底膜粘附能力下降,浓度为10ug/ml的12G5即可使胃癌细胞粘附能力抑制,与对照组相比具有显著意义(P<0.01),且随药物浓度的增大和时间的延长出现粘附抑制增强。胃癌细胞株对照组和实验组侵袭迁移实验穿膜细胞数分别为:121.7±5.51,54.3±9.07;140.3±6.03,62.0±11.53。胃癌细胞株SCG7901实验组的侵袭迁移能力明显低于对照组,有非常显著性差异(P<0.01)。
12G5对人胃癌细胞株SCG7901有细胞增殖抑制作用,经12G5处理SCG7901细胞后出现细胞生存率明显低于对照组(P<0.01),且随时间的延长与对照组比较抑制增强。流式细胞仪检测细胞的凋亡率,实验组与对照组比较,结果显示实验组的凋亡率明显高于对照组,差异具有显著性意义(P<0.01)。
检测结果显示胃癌细胞株SCG7901存在VEGF165mRNA、NRP-1mRNA、CXCR4mRNA及蛋白的表达,12G5处理细胞后,实验组的CXCR4mRNA及蛋白水平较对照组显著下降(P<0.01)。
结论:抗CXCR4单克隆抗体12G5一方面可能通过下调CXCR4的表达减少了与其配体SDF-1的结合;另一方面可能直接阻断了CXCR4与SDF-1的结合,对人胃癌细胞株SCG7901的粘附、侵袭转移能力有抑制作用,且可抑制胃癌细胞增殖,促使其凋亡,提示胃癌细胞存在VEGF/NRP-1自分泌途径,且胃癌可能存在SDF-1的自分泌,通过阻断此自分泌途径有望成为今后胃癌临床治疗的手段之一。
OBJECTIVE: To analyze the VEGF/NRP-1 autocrine pathway in the gastric cancer cells,and try to find out the starting and responding of cell signal pathway;Observe the biologic activity of gastric cancer cells after blocking the VEGF/NRP-1 autocrine pathway,to explore the value for clinical treatment.
METHODS: Human gastric cancer cell lines SCG7901 was used in this study.The effects of CXCR4 inhibitor 12G5 on the cells adhering Matrigel was measured with MTT assay,and the minimum concentration was sieved for next step; The effects of 12G5 on the invasive and metastatic abilities of SCG7901cells was explored by Transwell Chamber.The effects of 12G5 on the proliferation of SCG7901 cells was explored by trypanblau staining method;The effects of 12G5 on apoptosis of SCG7901 cells was explored by flow cytometry;The expression of VEGF autocrine pathway was detected by RT-PCR and Western blot in the untreated group and 12G5-treated group.
RESULTS: 12G5 showed a time/dose-dependent inhibition effect on SCG7901 cells adhering Matrigel,and the adhesion of gastric cancer cells was inhibited on the concentration of 10ug/ml of 12G5, and compared with untreated group,adhesion of SCG7901 cells was lower(P<0.01);The invaded cell numbers in invasive and metastatic experiments of SCG7901 cell lines untreated or treated with 12G5 were 121.7±5.51,54.3±9.07;140.3±6.03,62.0±11.53 respectively,the invasive and metastatic abilities of treated group were lower than untreated group significantly(P<0.01).
12G5 showed a time-dependent growth inhibition effect on SCG7901 cells,and the survival rate of SCG7901 cells in the 12G5-treated group was lower than untreated group significantly(P<0.01);The apoptosis rate of SCG7901 cells was explored by flow cytometry,and it showed the apoptosis rate in the 12G5-treated group was higher than untreated group significantly(P<0.01).
The mRNA and protein expression level of VEGF165、NRP-1 was detected in SCG7901 cell lines,and the mRNA and protein expression level of CXCR4 in the 12G5-treated group was lower than untreated group significantly(P<0.01).
CONCLUSIONS: The expression of CXCR4 inhibited by anti- CXCR4 monoclonal antibody 12G5 may reduce the binding of CXCR4 and SDF-1,and 12G5 may directly blocked the binding of them ,and play a inhibiting effect on the adhesive、invasive and metastatic abilities of SCG7901 cells,and the growth of cancer cells was inhibited,and the apoptosis was promoted.It showed SCG7901 cells have a VEGF/NRP-1 autocrine pathway,and SDF-1 may be autocrined by gastric cancer cells. It may become one of clinical treatment means by blocking VEGF autocrine pathway.
引文
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[25]Endres MJ,Clapham PR,Marsh M,et al.CD4-independent infection by HIV-2 is mediated by fusin/CXCR4[J].Cell,1996,87(4):745-756.
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[3] Fakhari M,Pullirsch D,Abraham D,et al.Selective upregulation of vascular endo- thelial growth factor receptors neuropilin-1 and –2 in human neuroblastoma [J]. Cancer 2002;94(1):258-263
[4] Neuropilin-1 and neuropilin–2 co-expression is significantly correlated with increased vascularity and poor prognosis in nonsmall cell lung carcinoma[J]. Cancer 2002;95(10):2196-2201
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