背景:胶质母细胞瘤(GBM)是最常见的脑肿瘤,预后最差。替莫唑胺是治疗GBM的唯一一线药物。不幸的是,阻力问题是一个经典问题。因此,开发治疗GBM的新药至关重要。作为一种致癌基因,Skp2参与包括GBM在内的各种癌症的发病机理。在这项研究中,我们研究了AAA237对人胶质母细胞瘤细胞的抗癌作用及其潜在机制。
方法:进行CCK-8测定以评估AAA237在48和72小时的IC50值,分别。采用细胞热转移测定(CETSA)来确定Skp2作为细胞环境内AAA237的内在靶标的状态。EdU-DNA合成试验,进行软琼脂测定和基质胶测定以检查AAA237对细胞生长的抑制作用。鉴定GBM细胞的迁移和侵袭能力,进行了transwell测定。采用RT-qPCR和Western印迹来验证BNIP3的水平。mRFP-GFP-LC3指示系统用于评估自噬通量的变化,并研究AAA237对自噬体和溶酶体之间的动态融合过程的影响。探讨化合物AAA237对体内肿瘤生长的影响,在原位模型中将LN229细胞注射到小鼠的脑中。
结果:AAA237可以抑制体外培养的GBM细胞的生长。AAA237可以与Skp2结合并抑制Skp2的表达以及p21和p27的降解。以剂量依赖的方式,AAA237证明了抑制集落形成的能力,迁移,和GBM细胞的侵袭。AAA237处理可以上调BNIP3作为hub基因,因此通过mTOR途径诱导BNIP3依赖性自噬,而3-MA可以在某种程度上逆转这一过程。在体内,AAA237的给药有效地抑制了神经胶质瘤肿瘤的发展,没有副作用。
结论:化合物AAA237,一种新型Skp2抑制剂,抑制集落形成,通过上调BNIP3作为hub基因,以剂量依赖性方式和时间依赖性方式迁移和侵袭GBM细胞,并通过mTOR途径诱导BNIP3依赖性自噬,因此它可能是治疗GBM的可行药物。
BACKGROUND: Glioblastoma (GBM) is the most common brain tumor with the worst prognosis. Temozolomide is the only first-line drug for GBM. Unfortunately, the resistance issue is a classic problem. Therefore, it is essential to develop new drugs to treat GBM. As an oncogene, Skp2 is involved in the pathogenesis of various cancers including GBM. In this study, we investigated the anticancer effect of AAA237 on human glioblastoma cells and its underlying mechanism.
METHODS: CCK-8 assay was conducted to evaluate IC50 values of AAA237 at 48, and 72 h, respectively. The Cellular Thermal Shift Assay (CETSA) was employed to ascertain the status of Skp2 as an intrinsic target of AAA237 inside the cellular milieu. The EdU-DNA synthesis test, Soft-Agar assay and Matrigel assay were performed to check the suppressive effects of AAA237 on cell growth. To identify the migration and invasion ability of GBM cells, transwell assay was conducted. RT-qPCR and Western Blot were employed to verify the level of
BNIP3. The mRFP-GFP-LC3 indicator system was utilized to assess alterations in autophagy flux and investigate the impact of AAA237 on the dynamic fusion process between autophagosomes and lysosomes. To investigate the effect of compound AAA237 on tumor growth in vivo, LN229 cells were injected into the brains of mice in an orthotopic model.
RESULTS: AAA237 could inhibit the growth of GBM cells in vitro. AAA237 could bind to Skp2 and inhibit Skp2 expression and the degradation of p21 and p27. In a dose-dependent manner, AAA237 demonstrated the ability to inhibit colony formation, migration, and invasion of GBM cells. AAA237 treatment could upregulate
BNIP3 as the hub gene and therefore induce
BNIP3-dependent autophagy through the mTOR pathway whereas 3-MA can somewhat reverse this process. In vivo, the administration of AAA237 effectively suppressed the development of glioma tumors with no side effects.
CONCLUSIONS: Compound AAA237, a novel Skp2 inhibitor, inhibited colony formation, migration and invasion of GBM cells in a dose-dependent manner and time-dependent manner through upregulating
BNIP3 as the hub gene and induced
BNIP3-dependent autophagy through the mTOR pathway therefore it might be a viable therapeutic drug for the management of GBM.