Chang Hsin-Han, Cheng Yu-Chen, Tsai Wen-Chiuan, Tsao Min-Jen, Chen Ying
Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan.
Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan.
Cell Physiol Biochem. 2018;48(4):1694-1702. doi: 10.1159/000492293. Epub 2018 Aug 3.
BACKGROUND/AIMS: Glioblastoma, also known as glioblastoma multiforme (GBM), is a fast-growing type of tumor that is the most aggressive brain malignancy in adults. According to GEO profile analysis, patients with high transient receptor potential canonical 3 (TRPC3) expression have poor survival rates. The aim of this study is to evaluate the effects of Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a selective TRPC3 channel blocker, on the proliferation and migration of human glioblastoma cells.
We first analyzed the TRPC3 mRNA expression in Gene Expression Omnibus (GEO) database. Then, TRPC3 protein expression was analyzed by Western blotting in three human GBM cell lines. The survival rate was measured by sulforhodamine B. JC1 staining was used to analyze the mitochondria membrane potential by flow cytometric analysis. Besides, the migration and invasion were evaluated by wound healing and Transwell assays. Annexin V and 7-aminoactinomycin D staining was used to monitor the apoptosis by flow cytometric analysis. The expression of apoptotic-related and migration-related proteins after Pyr3 treatment was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of Pyr3 in the in vivo study.
Basis on the results of bioinformatics study, glioma patients with higher TRPC3 expression had a shorter survival time than those with lower TRPC3 expression. GBM cell proliferation was decreased by Pyr3 treatment. The migration and invasion abilities of glioma cells were also inhibited via focal adhesion kinase and myosin light chain dephosphorization after Pyr3 treatment. Moreover, Pyr3 induced caspase-dependent apoptosis and mitochondria membrane potential imbalance in the GBM cells. In a xenograft animal model, Pyr3 in combination with temozolomide (TMZ) inhibited GBM tumor growth.
Pyr3 inhibited GBM tumor growth in vitro and in vivo. Pyr3-TMZ combination therapy could be used to treat glioblastoma in the future.
背景/目的：胶质母细胞瘤，也称为多形性胶质母细胞瘤（GBM），是一种生长迅速的肿瘤类型，是成人中最具侵袭性的脑恶性肿瘤。根据基因表达综合数据库（GEO）分析，瞬时受体电位经典型3（TRPC3）高表达的患者生存率较低。本研究旨在评估选择性TRPC3通道阻滞剂1-（4-（2,3,3-三氯丙烯酰胺）苯基）-5-（三氟甲基）-1H-吡唑-4-羧酸乙酯（Pyr3）对人胶质母细胞瘤细胞增殖和迁移的影响。
我们首先在基因表达综合数据库（GEO）中分析TRPC3 mRNA表达。然后，通过蛋白质免疫印迹法分析三种人GBM细胞系中的TRPC3蛋白表达。用磺酰罗丹明B检测细胞存活率。采用JC1染色通过流式细胞术分析线粒体膜电位。此外，通过伤口愈合实验和Transwell实验评估细胞迁移和侵袭能力。采用膜联蛋白V和7-氨基放线菌素D染色通过流式细胞术监测细胞凋亡。通过蛋白质免疫印迹法检测Pyr3处理后凋亡相关蛋白和迁移相关蛋白的表达。此外，在体内研究中使用原位异种移植小鼠模型来检测Pyr3的作用。
基于生物信息学研究结果，TRPC3表达较高的胶质瘤患者生存时间比TRPC3表达较低的患者短。Pyr3处理可降低GBM细胞增殖。Pyr3处理后，胶质瘤细胞的迁移和侵袭能力也通过粘着斑激酶和肌球蛋白轻链去磷酸化受到抑制。此外,Pyr3诱导GBM细胞中半胱天冬酶依赖性凋亡和线粒体膜电位失衡。在异种移植动物模型中，Pyr3与替莫唑胺（TMZ）联合使用可抑制GBM肿瘤生长。
Pyr3在体外和体内均抑制GBM肿瘤生长。Pyr3-TMZ联合疗法未来可用于治疗胶质母细胞瘤。
