惡性神經膠質瘤是中樞神經系統中最常見並且死亡率極高的原發性腦腫瘤。目前研究指出癌症的再復發、抗放化療，儘管最近在手術技術和藥物開發方面取得了進展，仍然難以治療。 即使是在手術切除方面取得進展，患者的預後仍然很差，平均生存期大約在15個月左右。在本篇研究中，我們使用市面上可取得之酪氨酸激酶抑制劑與熱休克蛋白90抑制劑NVP-AUY922探討其用於治療惡性神經膠質瘤患者對帝盟多膠囊(TMZ)有抗藥性的病人的可行性，以及相關的作用機轉。我們發現NVP-AUY922以濃度依賴性方式降低U87MG和T98G的細胞存活率，IC50分別為30 nM和35 nM。藉由細胞存活分析法(WST-1)測量比較NVP-AUY922與替莫唑胺（TMZ）的IC50，NVP-AUY922比TMZ更有效。群落形成測定顯示NVP-AUY922顯著降低了群落數，結果類似於U87MG和T98G細胞中的細胞存活測定。末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記（TUNEL assay）分析方法顯示NVP-AUY922顯著增加U87MG和T98G細胞中的TUNEL陽性細胞。我們評估了NVP-AUY922誘導的細胞死亡是否與細胞凋亡有關。我們發現NVP-AUY922通過西方墨點法處理48小時後，活化態凋亡蛋白酶-3, 活化態凋亡蛋白酶-9和聚腺苷酸二磷酸核糖转移酶 (PARP)的表現水平增加。此外我們研究了NVP-AUY922在體內的抗腫瘤活性。我們的初步結果顯示接種1×106 濃度U87細胞的腫瘤大小比接種2×105和5×105 濃度的U87細胞的腫瘤大小更明顯。我們將進一步將腫瘤細胞植入腦中或用1×106 濃度的U87膠質瘤細胞皮下接種它們以驗證NVP-AUY922在裸鼠實驗中的作用。

Glioblastoma multiforme (GBM), the most lethal primary brain tumor, and despite recent advances in surgical techniques and drug development, it remains difficult to treat. Even with advances in surgical resection, the prognosis of patients with GBM is still poor, with a median survival of 15 months. In this study, we report that heat shock protein 90 (HSP90) inhibitor 5-(2,4-Dihydroxy-5-isopropyl-phenyl)-N-ethyl-4-[4-(morpholinomethyl) phenyl] isoxazole-3-carboxamide NVP-AUY922 decreased cell viability of U87MG and T98G cells in a concentration-dependent manner with IC50 of 30 nM and 35 nM respectively. Comparing the IC50 measured by WST-1 assay for NVP-AUY922 versus Temozolomide (TMZ), NVP-AUY922 is more potent than TMZ. Colony formation assay showed that NVP-AUY922 significantly reduced colony number, the results similar to cell viability assay in both U87MG and T98G cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed that NVP-AUY922 significantly increased TUNEL positive cells in U87MG and T98G cell lines. We evaluated whether the NVP-AUY922 induced cell death was associated with apoptosis. We found that NVP-AUY922 increased levels of cleaved caspase-3, cleaved caspase-9 and poly (ADP-ribose) polymerase (PARP) after 48 h treatment by western blot analysis. In addition, we investigated the anti-tumor activity of NVP-AUY922 in vivo. Our preliminary results showed that tumor size inoculated with 1 x 106 U87MG cells was more obvious than those inoculated with 2 x 105 and 5 x 105 U87MG cells. We will further implant tumor cells into the brain or inoculate them subcutaneously with 1 x 106 U87MG glioma cells to verify the effect of NVP-AUY922 in nude mice model.

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