Department of Medical Bioscience, Faculty of Natural Sciences, University of the Western Cape, Bellville, Cape Town, 7530, South Africa.
Division of Cell Biology, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, 7925, South Africa.
Apoptosis. 2020 Apr;25(3-4):261-274. doi: 10.1007/s10495-020-01594-5.
Glioblastoma remains the most malignant of all primary adult brain tumours with poor patient survival and limited treatment options. This study adopts a drug repurposing approach by investigating the anti-cancer activity of a derivative of the antipsychotic drug phenothiazine (DS00329) in malignant U251 and U87 glioblastoma cells. Results from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays showed that DS00329 inhibited short-term glioblastoma cell viability and long-term survival while sparing non-cancerous cells. Western blot analysis with an antibody to γH2AX showed that DS00329 induced DNA damage and flow cytometry and western blotting confirmed that it triggered a G1 cell cycle arrest which correlated with decreased levels in Cyclin A, Cyclin B, Cyclin D1 and cyclin dependent kinase 2 and an increase in levels of the cyclin dependent kinase inhibitor p21. DS00329 treated glioblastoma cells exhibited morphological and molecular markers typical of apoptotic cells such as membrane blebbing and cell shrinkage and an increase in levels of cleaved PARP. Flow cytometry with annexin V-FITC/propidium iodide staining confirmed that DS00329 induced apoptotic cell death in glioblastoma cells. We also show that DS00329 treatment of glioblastoma cells led to an increase in the autophagosome marker LC3-II and autophagy inhibition studies using bafilomycin A1 and wortmannin, showed that DS00329-induced-autophagy was a pro-death mechanism. Furthermore, DS00329 treatment of glioblastoma cells inhibited the phosphatidylinositol 3'-kinase/Akt cell survival pathway. Our findings suggest that DS00329 may be an effective treatment for glioblastoma and provide a rationale for further exploration and validation of the use of phenothiazines and their derivatives in the treatment of glioblastoma.
胶质母细胞瘤仍然是所有成人原发性脑肿瘤中最恶性的一种，患者生存预后差，治疗选择有限。本研究采用药物再利用方法，研究了抗精神病药吩噻嗪（DS00329）的衍生物在恶性 U251 和 U87 胶质母细胞瘤细胞中的抗癌活性。MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）和集落形成试验的结果表明，DS00329 抑制短期胶质母细胞瘤细胞活力和长期存活，同时保留非癌细胞。用抗 γH2AX 抗体进行的 Western blot 分析表明，DS00329 诱导 DNA 损伤，流式细胞术和 Western blot 证实它触发了 G1 细胞周期停滞，这与细胞周期蛋白 A、细胞周期蛋白 B、细胞周期蛋白 D1 和细胞周期蛋白依赖性激酶 2 的水平降低以及细胞周期蛋白依赖性激酶抑制剂 p21 的水平升高有关。DS00329 处理的胶质母细胞瘤细胞表现出典型的凋亡细胞形态和分子标志物，如细胞膜起泡和细胞收缩以及 cleaved PARP 水平升高。用 Annexin V-FITC/碘化丙啶染色的流式细胞术证实，DS00329 诱导了胶质母细胞瘤细胞的凋亡性细胞死亡。我们还表明，DS00329 处理胶质母细胞瘤细胞导致自噬体标志物 LC3-II 的增加，并且使用巴弗洛霉素 A1 和渥曼青霉素进行的自噬抑制研究表明，DS00329 诱导的自噬是一种促死亡机制。此外，DS00329 处理胶质母细胞瘤细胞抑制了磷脂酰肌醇 3'-激酶/Akt 细胞存活途径。我们的研究结果表明，DS00329 可能是胶质母细胞瘤的有效治疗方法，并为进一步探索和验证吩噻嗪及其衍生物在胶质母细胞瘤治疗中的应用提供了依据。
