Downregulation of ROCK2 through Nanocomplex Sensitizes the Cytotoxic Effect of Temozolomide in U251 Glioma Cells

ObjectiveRho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells.MethodsGlycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration.ResultsOur results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone.ConclusionsIn conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.Rho相关卷曲螺旋激酶2（Rho-associated coiled-coil kinase 2, ROCK2）是极具吸引力的治疗靶点，因其在包括胶质瘤在内的多种恶性肿瘤中呈高表达。为此本研究旨在探讨ROCK2下调是否可增强替莫唑胺（temozolomide, TMZ）对U251细胞的细胞毒性作用。 方法：本研究采用乙二醇-聚乙烯亚胺（glycol-polyethyleneimine, PEG-PEI）作为载体，将siROCK2递送至U251细胞，并对PEG-PEI/siROCK2复合物（以下简称siROCK2复合物）的理化特性进行分析。分别通过流式细胞术（flow cytometry, FCM）与共聚焦激光扫描显微镜（confocal laser microscopy, CLSM）检测转染效率与细胞摄取情况。随后将U251细胞分别以100 μM替莫唑胺、siROCK2复合物以及二者联合处理。采用流式细胞术与划痕愈合实验分别检测细胞凋亡率与细胞迁移能力。通过检测Bax、Bcl-2、剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved caspase-3）、基质金属蛋白酶2（matrix metalloproteinase 2, MMP-2）以及基质金属蛋白酶9（matrix metalloproteinase 9, MMP-9）的蛋白水平，分析细胞凋亡与迁移程度。 结果：本研究结果显示，siROCK2复合物的理化特性与N/P比密切相关。PEG-PEI可作为递送siROCK2的优良载体，且对U251细胞毒性较低。共聚焦激光扫描显微镜实验证实，siROCK2复合物可被细胞成功摄取，且ROCK2的蛋白与mRNA水平均得到显著抑制。此外，联合处理组的细胞凋亡率更高，且划痕愈合实验中U251细胞的划痕间隙显著增大。与单一处理组相比，联合处理组的促凋亡蛋白Bax与剪切型半胱氨酸天冬氨酸蛋白酶-3水平显著升高，而抗凋亡蛋白Bcl-2以及迁移相关蛋白MMP-2、MMP-9的水平则显著降低。 结论：综上，本研究结果表明，替莫唑胺与siROCK2复合物联合可有效诱导U251细胞凋亡并抑制其迁移。因此，替莫唑胺与siROCK2复合物联合疗法有望成为人胶质瘤的潜在治疗策略。