Kv1.5通道蛋白参与人脑胶质瘤细胞的自噬

脑胶质瘤是原发性颅内恶性肿瘤。患者的5年存活率不足1%。目前,除手术切除外,尚无有效的治疗手段。近年来发现,脑胶质瘤发病可能与多种钾离子通道的异常表达有关。自噬是膜包裹部分胞质和细胞内需降解的蛋白质、细胞器,并与溶酶体一起降解其所包裹内容物的生理过程。诱导胶质瘤细胞的自噬,促进其凋亡是肿瘤治疗的一种新策略。本室前期研究发现,电压依赖型钾通道1.5(Kv1.5)参与胞膜小窖标志蛋白质(caveolae,Cav-1)介导的多种肿瘤细胞的增殖和凋亡,但是否参与胶质瘤细胞的自噬并不清楚。本文首先利用不同浓度的K+通道阻断剂四乙胺(tetra-ethylammonium,TEA)、Kv通道阻断剂四氨基吡啶(4-amino-pyridine,4-AP)和Kv1.5通道特异性阻断剂DPO-1(diphenyl phosphine oxide-1)分别在不同时间,作用于人脑胶质瘤细胞U251,观察其对细胞存活的影响。发现DPO-1对U251细胞具有双向作用:低浓度促进存活,高浓度抑制存活。其中,1 mmol/L DPO-1处理6 h,可促进自噬相关蛋白质LC3的表达,而抑制mTOR信号蛋白质的磷酸化水平,表明Kv1.5通道可能参与胶质瘤细胞的自噬。然后,利用基因转染技术分别敲低和过表达Kv1.5通道的蛋白质水平,发现敲低Kv1.5通道蛋白,促进胶质瘤细胞的自噬,激活ERK信号通路,而过表达Kv1.5通道蛋白,则抑制胶质瘤细胞的自噬。进一步利用流式细胞技术观察细胞凋亡,发现改变Kv1.5通道蛋白的表达水平,可诱发细胞早期凋亡。提示Kv1.5通道参与人脑胶质瘤细胞的自噬过程。这为临床利用特异性Kv通道阻断剂靶向治疗胶质瘤提供了新的理论和实验依据。

Kv1.5 Channel Protein is Involved in the Autophagy of Human Glioma Cells

Glioma is a primary intracranial malignant tumor with a 5-year survival rate of less than 1%.At present,there is no effective treatment except for surgical resection.In recent years,it has been found that glioma may be related to the abnormal expression of various potassium ion channels.Autophagy is a physiological process that membranes encapsulate part of the cytoplasm,proteins and organelles which need to be degraded.Inducing autophagy and promoting apoptosis of glioma cell is a new strategy for tumor therapy.Our previous studies found that Kv1.5 is involved in Cav-1-mediated proliferation and apoptosis of multiple tumor cells.But it is not clear that whether Kv1.5 is involved in autophagy of glioma cells.In this paper,we first used different blockers(TEA,4-AP and DPO-1)to observe the effect on cell survival in human glioma cell line U251.And found that DPO-1 has two-way effect on cell survival rate:low concentration of DPO-1 promotes but high concentration inhibits survival of U251 glioma cells.Treatment of 1 mmol/L DPO-1 for 6 h can promote the expression of LC3 and inhibit the phosphorylation of mTOR,suggesting that Kv1.5 channel may be in autophagy of glioma cells.Secondly,we knocked down and overexpressed the protein of Kv1.5 channel using transient transfection,respectively.It is found that knock down of Kv1.5 channel protein promoted autophagy,activated ERK pathway while overexpression of Kv1.5 inhibited autophagy.Furthermore,flow cytometry was used to detect apoptosis rates,and data found that changing the expression level of Kv1.5 channel protein can induce early apoptosis.These results suggested that the Kv1.5 channel is involved in the autophagy of human glioma cells.This study provides theoretical and experimental evidence for targeted therapy of glioma with specific Kv channel blockers.