CRBGP通过抑制FAK-AKT通路和白介素-6的分泌抑制胶质瘤U251细胞的活性

CRBGP Inhibited The Activity of Glioma U251 Cells Through Suppressing FAK-AKT Pathway and The Secretion of Interleukin-6

目的电压门控钠通道(voltage-gated sodium channels,VGSCs)表达于胶质瘤U251细胞,并影响U251细胞的增殖、侵袭和凋亡。富含半胱氨酸的颊腺蛋白(cysteine-rich buccal gland protein,CRBGP)是一种从日本七鳃鳗颊腺中分离出来的VGSCs阻断剂。本文旨在探究CRBGP是否因具有VGSCs阻断功能从而能够抑制U251细胞的活性。方法首先采用MTT法检测了CRBGP对U251细胞增殖的作用,并分别利用莱特-吉姆萨、FITC-phalloidin和Hoechst 33258染色法观察CRBGP处理后U251细胞的形态、细胞骨架和细胞核。细胞外基质蛋白如IV型胶原蛋白、纤连蛋白和层黏连蛋白用于检测CRBGP对U251细胞黏附的影响。此外,本文采用transwell法检测CRBGP处理后U251细胞的迁移和侵袭能力,并通过Western blot方法探讨CRBGP诱导U251细胞凋亡并抑制其运动的内在机理。最后,通过酶联免疫吸附测定法检测CRBGP对U251细胞的抑炎效果。结果CRBGP通过线粒体依赖途径诱导U251细胞凋亡,阻断促炎因子白介素-6的释放,从而抑制U251细胞增殖。同时,CRBGP对VGSCs的阻断作用和抗炎活性有助于其抑制U251细胞的黏附、迁移和侵袭。最后,CRBGP通过抑制FAK-AKT信号通路影响U251细胞的增殖和运动。结论CRBGP可能因具有VGSCs抑制特性而表现出抗肿瘤活性,为VGSCs在胶质瘤中的作用提供了依据。

Objective Voltage-gated sodium channels(VGSCs)are expressed in glioma U251 cells and affect the proliferation,invasion and apoptosis of U251 cells.It is reasonable to hypothesize that the cysteine-rich buccal gland protein(CRBGP),a VGSCs blocker isolated from the buccal glands of Lampetra japonica,may suppress the activity of U251 cells.Methods Firstly,the proliferation of U251 cells in the presence of CRBGP was detected by MTT assay.And the morphology,cytoskeleton and nucleus of U251 cells after treated with CRBGP were observed by Wright-Giemsa,FITC-phalloidin and Hoechst 33258 staining assays,respectively.Subsequently,extracellular matrix proteins such as collagen IV,fibronectin and laminin were used to detect the effect of CRBGP on U251 cells’adhesion.In addition,the migration and invasion of U251 cells treated with CRBGP were detected by transwell assays.And the internal mechanisms of CRBGP on U251 cells’apoptosis and mobility were explored by Western blot.Finally,the anti-inflammatory effect of CRBGP on U251 cells was detected by enzyme linked immunosorbent assay.Results CRBGP inhibited the proliferation of U251 cells by inducing apoptosis in a mitochondrial-dependent pathway and preventing the release of proinflammatory factor interleukin-6.Also,the VGSCs blocking and anti-inflammatory activities of CRBGP contributed to its inhibitory effects on the adhesion,migration and invasion of U251 cells.Finally,CRBGP affected the proliferation and mobility of U251 cells through the suppression of the FAK-AKT pathway.Conclusion Together,our data indicated that CRBGP could exhibit its anti-tumor activity probably by its VGSCs inhibitory property,providing a basis for the functional information of VGSCs to the gliomas.