FoxO1在KCa激动剂开放血肿瘤屏障中的调控机制研究

How to selectively open the blood-brain tumor barrier (BTB) is an emerging strategy in treating brain tumor efficiently with chemotherapy. Calcium-activated potassium channel (KCa channel) activator is a kind of valuable drug for selective BTB opening. The previous study demonstrated that KCa channel activator could regulate the expression of adhere junction protein VE-cadherin in transcription level, down-regulate the expression of tight junction proteins, and then induce the opening of paracellular pathway. However, the related mechanism is still under investigation. FoxO1 is one of transcription factors, which can combine with DNA sequences 5′-TTGTTTAC-3′of target promoters, then regulate the transcription of down-stream target genes. Our previous study demonstrated that the promoter of human VE-cadherin (Ensembl:ENSG00000341529) has the target sequences 5′-TTGTTTAC-3′as well and it has ability to bind to neucleoprotein, which indicated that FoxO1 might regulate the transcription of VE-cadherin. VE-cadherin is an adhere junction protein which is involved in the location and regulation of tight junction protein. We presume that FoxO1 might affect the formation of adhere junction and tight junction by regulating the transcription of VE-cadherin, and then induce the opening of paracellular pathway. FoxO1 is an important effector for PI3K/PKB signal pathway and can on-off many genes transcription by phosphorylation and dephosphorylation. ROS may induce the FoxO1 phosphorylation by activating PI3K/PKB signal pathway, and help to transfer it from nucleus to endochylema, and then reduce the transcription to target genes. Activation of Kca channels could depolarize the mitochondrial membrane, and induce ROS production rapidly. Recently, we demonstrated that ROS is one of the important signal molecules in the mechanism of Kca channel activator-induced BTB opening. According to the previous data, we hypothesize that Kca channel activator might induce the ROS production rapidly, then ROS specifically activated the RhoA/PI3K/PKB signaling pathway, which contributed the phosphorylation of FoxO1 and caused the diversion of FoxO1 from nucleus to endochylema. Therefore, the transcription of VE-cadherin was attenuated, which indirectly change the expression and distribution of tight junction protein, and opening of cell to cell junctions. The first aim of this study is to investigate the effects of FoxO1 on the transcription of VE-cadherin, and on the changes of endothelial cell to cell junction; further study will focus on the role of FoxO1 in KCa channel activitor regulated transcription of VE-cadherin; at last, the role of ROS/RhoA/PI3K/PKB signal pathway will be examined during the biological process of KCa channel activitor regulating FoxO1 phosphorylation, and changing the BTB permeability. This project will indicate the role of FoxO1 in the mechanism of opening BTB induced by KCa channel activitor, and create a new target for drug developemnt.

如何选择性开放血肿瘤屏障(BTB)是当今研究有效治疗脑肿瘤的焦点。FoxO1是一种转录因子，与靶定基因启动子的DNA序列5′-TTGTTTAC-3′结合，调控下游靶基因的转录。前期实验发现FoxO1可能对VE-cadherin具有转录调控作用；而VE-cadherin的表达又受Kca通道激动剂在转录水平上的调控，且这种作用依赖于ROS的存在。因此，我们推测FoxO1是Kca通道激动剂开放BTB的重要调控靶点。本项目首先研究FoxO1对VE-cadherin转录调控的效果及其在细胞间连接调节中的作用；进一步研究FoxO1在Kca通道激动剂转录调控VE-cadherin中的作用；最后研究ROS/RhoA/PI3K/PKB信号通路在Kca通道激动剂调节FoxO1磷酸化和BTB通透性中的作用。本项目首次揭示FoxO1在Kca通道激动剂开放BTB中的调控机制，为选择性开放BTB的药物研究开创新靶点。

脑胶质瘤是神经系统最常见的肿瘤，占颅内肿瘤50%左右，其发病率居各种肿瘤的第二位。化疗是常用的脑胶质瘤治疗方法之一，但血脑肿瘤屏障的存在严重限制了抗肿瘤药物向脑肿瘤组织内转运。Ningaraj 等人首先在大鼠脑胶质瘤模型中证实：与正常脑组织相比，肿瘤血管内皮细胞和肿瘤细胞上钙离子激活性钾通道表达水平显著增高，KCa 通道激动剂可以选择性开放血肿瘤屏障而不影响正常血脑屏障，经静脉注射KCa通道激动剂可以增加抗肿瘤药物向脑肿瘤组织内转运，增加脑胶质瘤小鼠的存活率。上述研究结果表明KCa通道激动剂是非常具有临床应用价值的选择性开放BTB 的药物。阐明KCa 通道激动剂作用的分子机制，无疑会为开放BTB 的研究提供新思路。本项目在初步证明KCa通道激动剂时间依赖性的诱导细胞旁途径开放的基础上，首次发现人类粘附连接相关蛋白VE-cadherin（Ensembl:ENSG00000341529）的启动子序列内含有FoxO的靶定序列5′-TTGTTTAC-3′；进一步钾激动剂作用15 分钟后，在人胶质瘤细胞和人肿瘤血管内皮细胞上FoxO1 的蛋白表达显著增强，在人脑肿瘤血管内皮细胞上FoxO1 的蛋白表达显著增强，p-FoxO1 未见增高，但是钾通道激动剂作用2 小时后，FoxO1 蛋白表达仍显著增强，但p-FoxO1 表达显著上调，此时VE-cadherin和caveolin-1 表达水平显著下降，表明一旦FoxO1 被磷酸化后即失去对VE-cadherin 和caveolin-1的转录调控作用。此外，课题组进一步证实ROS/PI3K/PKB的激活参与FoxO1的磷酸化，为了进一步明确抑制FoxO1 磷酸化的必要性，脑血管内皮细胞转入突变的FoxO1:AAA后，体外BTB 的通透性显著增加，表明有效抑制FoxO1 磷酸化是开放BTB 的关键。本研究能揭示FoxO1在KCa通道激动剂开放BTB中的作用机制，拓宽选择性开放BTB途径的研究思路，为脑胶质瘤等中枢神经系统疾病的治疗开创新靶点。