Multifunctional therapeutic strategy for malignant gliomas targeting cathepsin D

我们研究了以天冬氨酸氨基转移酶D为靶点的恶性胶质瘤新的治疗方法的有效性。有报道称，组织蛋白酶D基因高表达的胶质母细胞瘤患者经常发生软脑膜播散，他们的生存期明显短于那些低表达的患者。用酶联免疫吸附试验(ELISA)检测血清组织蛋白酶D浓度，发现高级别胶质瘤患者血清组织蛋白酶D水平明显高于低级别胶质瘤患者，提示该酶可作为胶质瘤生物标志物。我们首先利用RNA干扰技术下调了神经胶质瘤细胞系(U87 MG、U138 MG、U251 MG、U373 MG；A172)中天冬氨酸蛋白酶D的基因表达。在原本高表达组织蛋白酶D的U87 MG和A172细胞中，组织蛋白酶D的表达得到了有效的下调，这些细胞株的侵袭能力相应降低。然后，我们使用9L胶质肉瘤细胞和序列MRI监测系统建立了大鼠脑胶质瘤模型。在该模型中，用照射灭活的全瘤细胞进行外周免疫和脑内注射IL-2基因转导载体可以诱导已建立的脑肿瘤的完全排斥反应。用组织蛋白酶D作为皮下疫苗，对大鼠脑胶质瘤模型的治疗效果不及灭活全细胞瘤苗。免疫组织化学分析显示，IL-2在脑肿瘤组织中大量表达，但CD8~+T细胞和CD_4~+T细胞大量浸润。随访3个月，未见不良反应。更多的胶质瘤特异性蛋白将是预防胶质瘤疫苗的理想选择。

We investigated efficacies of the novel therapeutic approaches for malignant gliomas targeting one of the aspartic protease, cathpsin D. We have reported that glioblastoma patients with high gene expression of cathepsin D frequently had leptomeningeal dissemination and they lived significantly shorter than those with low expression. Measurement of the serum cathepsin D concentrations by enzyme-linked immunosorbent assay (ELISA) demonstrated a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors, which suggests that this protease would be useful as a glioma biomarker. We first knocked down the gene expression of cathpsin D in glioma cell lines (U87MG, U138MG, U251MG, U373MG; A172) using RNA interference. Effective down-regulations of cathepsin D was achieved in U87MG and A172 which originally express high levels of cathepsin D, the invasive abilities of these cell lines were accordingly dcreased. Then we used a rat glioma model using 9L gliosarcoma cells and serial MRI monitoring system. For this model, the peripheral vaccination with whole tumor cells inactivated by irradiation and intracerebral administration of IL-2 gene transduced vectors could induce complete rejection of established brain tumors. When cathepsin D was used as subcutaneous vaccine, the treatment efficacies against a rat glioma model did not reach those obtained by the inactivated whole tumor cell vaccine. Immunohistochemical analysis revealed that IL-2 was sufficiently expressed in the brain tumors, but the CD4^+ T cells and CD8^+ T cells were abundantly infiltrated. During the 3-month follow-up period, no adverse effect was observed. More glioma-specific proteins would be desirable for vaccination against gliomas.