Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
PLoS One. 2010 Oct 29;5(10):e13731. doi: 10.1371/journal.pone.0013731.
Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.
METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.
CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
神经胶质瘤是最常见的原发性脑肿瘤，其特征是侵袭性和浸润性。uPAR 和组织蛋白酶 B 在高级别神经胶质瘤中过度表达，并与侵袭性癌症表型强烈相关。
方法/主要发现：在本研究中，我们观察到同时下调 uPAR 和组织蛋白酶 B 会诱导人神经胶质瘤细胞中一些促凋亡基因的上调和抗凋亡基因的下调。uPAR 和组织蛋白酶 B（pCU）下调的细胞表现出 Bcl-2/Bax 比例降低，并导致线粒体膜电位崩溃。我们还观察到广谱半胱氨酸蛋白酶抑制剂 Z-Asp-2,6-二氯苯甲酰基甲基酮（Z-Asp-2,6-DCB）挽救了 pCU 在 U251 细胞中诱导的凋亡，但不能挽救 5310 细胞。用 caspase-9 免疫沉淀分析 Apaf-1 发现，uPAR 和组织蛋白酶 B 下调激活了 U251 细胞中的凋亡体复合物形成。下调 uPAR 和组织蛋白酶 B 还会延迟 U251 和 5310 细胞中 CREB 的核转位和干扰 DNA 结合活性。进一步的 Western blot 分析表明，下调 uPAR 和组织蛋白酶 B 显著降低了信号分子 p-PDGFR-β、p-PI3K 和 p-Akt 的表达。裸鼠脑肿瘤切片和脑组织裂解物中 TUNEL 阳性细胞数量增加、Bax 表达增加和 Bcl-2 表达减少，证实了我们的体外结果。
总之，RNAi 介导的 uPAR 和组织蛋白酶 B 下调在 U251 细胞中引发 caspase 依赖性线粒体凋亡，在 5310 细胞中引发 caspase 非依赖性线粒体凋亡。因此，使用 siRNA 靶向 uPAR 和组织蛋白酶 B 介导的信号通路可能成为治疗神经胶质瘤的新治疗策略。
