Background Aurora kinase A (AURKA) has been implicated in the regulation of cell cycle progression, mitosis and a key number of oncogenic signaling pathways in various malignancies including neuroblastoma. Small molecule inhibitors of AURKA have shown potential, but still not as good as expected effects in clinical trials. Little is known about this underlying mechanism. Here, we evaluated the inhibitory effects of AURKA inhibitor MLN8237 on neuroblastoma cells to understand the potential mechanisms responsible for tumor therapy. Methods MLN8237 treatment on neuroblastoma cell line IMR32 was done and in vivo inhibitory effects were investigated using tumor xenograft model. Cellular senescence was evaluated by senescence-associated β-gal Staining assay. Flow cytometry was used to tested cell cycle arrest and cell apoptosis. Senescence-associated signal pathways were detected by western blot. CD133 microbeads and microsphere formation were used to separate and enrich CD133+ cells. AURKA small interfering RNA transfection was carried to downregulate AURKA level. Finally, the combination of MLN8237 treatment with AURKA small interfering RNA transfection were adopted to evaluate the inhibitory effect on neuroblastoma cells. Results We demonstrate that MLN8237, an inhibitor of AURKA, induces the neuroblastoma cell line IMR32 into cellular senescence and G2/M cell phase arrest. Inactivation of AURKA results in MYCN destabilization and inhibits cell growth in vitro and in a mouse model. Although MLN8237 inhibits AURKA kinase activity, it has almost no inhibitory effect on the AURKA protein level. By contrast, MLN8237 treatment leads to abnormal high expression of AURKA in vitro and in vivo. Knockdown of AURKA reduces cell survival. The combination of MLN8237 with AURKA small interfering RNA results in more profound inhibitory effects on neuroblastoma cell growth. Moreover, MLN8237 treatment followed by AURKA siRNA forces senescent cells into apoptosis via suppression of the Akt/Stat3 pathway. Conclusions The effect of AURKA-targeted inhibition of tumor growth plays roles in both the inactivation of AURKA activity and the decrease in the AURKA protein expression level.

中文翻译：

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AURKA 的沉默增强了 AURKA 抑制剂 MLN8237 对神经母细胞瘤细胞的抗肿瘤功效。

背景 Aurora 激酶 A (AURKA) 与各种恶性肿瘤（包括神经母细胞瘤）中细胞周期进程、有丝分裂和关键数量的致癌信号通路的调节有关。AURKA 的小分子抑制剂已显示出潜力，但在临床试验中仍不如预期效果。人们对这种潜在机制知之甚少。在这里，我们评估了 AURKA 抑制剂 MLN8237 对神经母细胞瘤细胞的抑制作用，以了解负责肿瘤治疗的潜在机制。方法MLN8237对神经母细胞瘤细胞系IMR32进行处理，并使用肿瘤异种移植模型研究体内抑制作用。通过衰老相关的β-gal染色测定评估细胞衰老。流式细胞仪用于检测细胞周期停滞和细胞凋亡。通过蛋白质印迹检测衰老相关的信号通路。CD133 微珠和微球形成用于分离和富集 CD133+ 细胞。进行 AURKA 小干扰 RNA 转染以下调 AURKA 水平。最后采用MLN8237联合AURKA小干扰RNA转染的方法评价对神经母细胞瘤细胞的抑制作用。结果 我们证明 AURKA 的抑制剂 MLN8237 可诱导神经母细胞瘤细胞系 IMR32 进入细胞衰老和 G2/M 细胞期停滞。AURKA 的失活导致 MYCN 不稳定并在体外和小鼠模型中抑制细胞生长。MLN8237 虽然抑制 AURKA 激酶活性，但对 AURKA 蛋白水平几乎没有抑制作用。相比之下，MLN8237 治疗导致 AURKA 在体外和体内异常高表达。敲除 AURKA 会降低细胞存活率。MLN8237 与 AURKA 小干扰 RNA 的组合对神经母细胞瘤细胞生长产生更深远的抑制作用。此外，MLN8237 处理后跟 AURKA siRNA 通过抑制 Akt/Stat3 通路迫使衰老细胞凋亡。结论 AURKA 靶向抑制肿瘤生长的作用在 AURKA 活性失活和 AURKA 蛋白表达水平降低中均起作用。MLN8237 处理后跟 AURKA siRNA 通过抑制 Akt/Stat3 通路迫使衰老细胞凋亡。结论 AURKA 靶向抑制肿瘤生长的作用在 AURKA 活性失活和 AURKA 蛋白表达水平降低中均起作用。MLN8237 处理后跟 AURKA siRNA 通过抑制 Akt/Stat3 通路迫使衰老细胞凋亡。结论 AURKA 靶向抑制肿瘤生长的作用在 AURKA 活性失活和 AURKA 蛋白表达水平降低中均起作用。