Multifunctional therapeutic strategy for malignant gliomas targeting cathepsin D

我们研究了针对天冬氨酸蛋白酶之一，组织蛋白酶D的恶性胶质瘤的新治疗方法的疗效。我们曾报道过组织蛋白酶D基因高表达的胶质母细胞瘤患者常发生软脑膜播散，其生存时间明显短于低表达者。通过酶联免疫吸附试验（ELISA）的血清组织蛋白酶D浓度的测量表明，与低级别肿瘤相比，高级别胶质瘤患者的血清组织蛋白酶D浓度显著增加，这表明这种蛋白酶将是有用的胶质瘤生物标志物。我们首先利用RNA干扰技术敲低了脑胶质瘤细胞系（U87 MG、U138 MG、U251 MG、U373 MG; A172）中组织蛋白酶D的基因表达。U87 MG和A172细胞表达的组织蛋白酶D水平明显降低，细胞侵袭能力也相应降低。然后，我们使用大鼠胶质瘤模型，使用9 L胶质瘤细胞和系列MRI监测系统。在此模型中，用经辐射灭活的全肿瘤细胞进行外周接种和脑内施用IL-2基因转导载体可诱导已建立的脑肿瘤的完全排斥。当组织蛋白酶D被用作皮下疫苗时，对大鼠胶质瘤模型的治疗功效没有达到通过灭活的全肿瘤细胞疫苗获得的那些。免疫组织化学分析显示IL-2在脑肿瘤中充分表达，但CD 4 ^+ T细胞和CD 8 ^+ T细胞大量浸润。在3个月的随访期间，未观察到不良反应。更多的神经胶质瘤特异性蛋白质对于针对神经胶质瘤的疫苗接种将是期望的。

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.