【摘要】 目的 JTC-801是孤啡肽受体NOP[Nociceptin/orphanin FQ(N/OFQ)peptide]受体拮抗剂,适用于与神经损伤相关的神经性疼痛和异常性疼痛,在肿瘤中的研究鲜见。本研究探讨JTC-801对结肠癌HCT116及HT-29细胞增殖、迁移、侵袭及凋亡的影响,并探讨相关机制。方法采用JTC-801加药处理HCT116及HT-29细胞,用CCK8法、Transwell实验和细胞流式凋亡检测实验分别检测HCT116细胞增殖、迁移、侵袭及凋亡;采用蛋白质印迹法检测细胞凋亡相关蛋白以及PI3K信号通路相关蛋白表达。结果 CCK8实验表明,JTC-801显著抑制了HCT116及HT-29细胞增殖能力,并呈一定的剂量依赖性和时间依赖性。Transwell实验结果表明,10μmol/L JTC-801明显抑制了HCT116细胞迁移能力,与对照组迁移细胞数分别为44±2和96±5,t=16.72,P=0.000 1;也抑制了HT-29细胞迁移能力,与对照组迁移细胞数分别为45±4和93±7,t=10.32,P=0.005。同样10μmol/L JTC-801显著抑制了HCT116细胞,与对照组穿膜细胞数分别为21±5和43±3,t=6.54,P=0.002 8;也抑制了HT-29细胞,与对照组穿膜细胞数分别为23±3和40±2,t=8.17,P=0.001 2。细胞流式凋亡检测实验结果表明,10μmol/L JTC-801促进HCT116细胞凋亡,凋亡率为(13.74±0.42)%和(4.73±0.33)%,t=29.23,P<0.001;也促进HT-29细胞凋亡,凋亡率为(18.59±0.39)%和(8.51±0.57)%,t=25.28,P<0.001。且抗凋亡相关蛋白Bcl-2明显下降,同时促凋亡蛋白Bax及Active Caspase3蛋白水平则显著上升,P<0.05;10μmol/L JTC-801还显著抑制了p-Akt、p-mTOR及下游分子p70s6k蛋白的表达水平,均P<0.01。结论 JTC-801可以抑制结肠癌细胞增殖、迁移和侵袭,促进细胞凋亡,对结肠癌的发生发展进程产生重要作用。其作用可能通过影响PI3K信号通路活化实现。更多还原

【Abstract】 OBJECTIVE JTC-801 is a selective antagonist for NOP[Nociceptin/orphanin FQ(N/OFQ)peptide]receptor,suitable for neuropathic pain and abnormal pain associated with nerve injury.There is none research about effect of JTC-801 on tumor.The present study aimed to investigate the effect of JTC-801 on the proliferation,migration,invasion and apoptosis of colon cancer HCT116 and HT-29 cells,and to explore the relevant mechanisms.METHODS CCK8 assay,transwell assay and flow cytometry were adopted to detect cell proliferation,migration,invasion and apoptosis,respectively.Western blot was used to detect the expression of apoptosis-related proteins and PI3 K-related proteins.RESULTS CCK8 assay showed that JTC-801 treatment significantly inhibited the proliferation of HCT116 and HT-29 cells with a dose-dependent and time-dependent manner.Transwell assay showed that 10μmol/L JTC-801 treatment significantly inhibited migration of HCT116 and HT-29 cells[(44±2)vs(96±5),t=16.72,P= 0.000 1;(45±4)vs(93±7),t=10.32,P=0.005].JTC-801 treatment also significantly inhibited invasion of HCT116 and HT-29 cells[(21±5)vs(43±3),N=3,t=6.54,P=0.002 8;(23±3)vs(40±2),t=8.17,P=0.001 2].Flow cytometry results showed that JTC-801 promoted apoptosis of HCT116 and HT-29 cells[(13.74±0.42)% vs(4.73±0.33)% ,t=29.23,P<0.001;(18.59±0.39)% vs(8.51±0.57)% ,t=25.28,P<0.001],and the anti-apoptotic protein Bcl-2 decreased significantly,while the levels of pro-apoptotic protein Bax and Active Caspase3 were significantly increased(P<0.05).JTC-801 treatment also significantly inhibited the expression of p-Akt,p-mTOR and p70 s6 kprotein(P<0.01).CONCLUSION JTC-801 can inhibit proliferation,migration,and invasion and promote apoptosis of colon cancer,which may be achieved by affecting the activation of PI3 Kpathway.更多还原