Brain Tumour North West (BTNW), Faculty of Science and Technology, University of Central Lancashire (UCLan), Preston PR1 2HE, UK.
FEBS Open Bio. 2013 Jun 20;3:271-8. doi: 10.1016/j.fob.2013.06.002. Print 2013.
Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.
热休克蛋白 90（Hsp90）促进肿瘤的进展和存活，并已成为一个重要的治疗靶点。以前我们报道过，17-AAG/siHsp90α 的联合治疗可显著下调多形性胶质母细胞瘤（GBM）中的 Hsp90α mRNA 和蛋白水平。在这里，我们研究了细胞穿透肽（Tat48-60 CPP）介导的 siRNA 诱导的 hsp90α 敲低作为单一药物以及与 17- 烯丙基-17-脱甲氧基格尔德霉素（17-AAG）联合使用，以抑制 GBM 肿瘤生长的能力，以及它是否具有治疗意义。用 siRNA 和/或 17-AAG 处理 GBM 和非致瘤细胞后，通过 qRT-PCR、免疫荧光、FACS 分析、定量 Akt、LDH 渗漏和细胞活力测定进行评估。进行 PAGE 以评估血清稳定性。siRNA/17-AAG 联合治疗可分别显著诱导 Hsp90α 基因和蛋白敲低 95%和 98%，同时 Akt 激酶活性降低 84%，诱导细胞周期停滞和肿瘤特异性细胞毒性 88%。CPP 和 siRNA 之间的有效复合物形成显示出提高了 siRNA 的血清稳定性，同时具有最小的内在体外毒性。初步的体内结果表明，联合治疗可诱导颅内胶质母细胞瘤小鼠模型中的 hsp90α 敲低和 Akt 激酶活性减弱。结果表明，RNAi 介导的 hsp90α 敲低可增加 GBM 中 17-AAG 治疗的疗效。此外，观察到的细胞毒性反应是 hsp90α 基因表达下调、Akt 激酶活性降低和 S-G2/M 细胞周期停滞的结果。这些结果是新颖的，突出了 Tat 有效递送至 GBM 中的 siRNA 的能力，并表明 Hsp90 的双重抑制具有治疗潜力。
