Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; Neuroscience Research Center, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; Iranian National Center for Addiction Studies (INCAS), Tehran University of Medical Sciences, Tehran, Iran.
Life Sci. 2017 Mar 15;173:11-19. doi: 10.1016/j.lfs.2017.02.005. Epub 2017 Feb 12.
Glioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGF) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGF blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway.
CD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system. The cell survival, apoptosis, and protein expression values were measured using MTT assay, TUNEL staining and western blot, successively. Intracellular cAMP and free secreted VEGF levels were assessed by cAMP enzyme immunoassay and ELISA, respectively.
Bevacizumab suppressed GCSCs survival with IC~6.5μg/ml and enhanced the levels of apoptosis, p53 and cleaved-caspase3 along with a decrease in free VEGF levels and ERKs activation. However, there was no significant modulation of AKT phosphorylation on serine 473, the intracellular PDE4A, VEGF and cAMP levels. More cytotoxicity in co-treated cells coupled with a more substantial decline in the free VEGF levels and a greater increase in the quantities of p53 and cleaved-caspase3 compared to those treated with bevacizumab alone. Co-treatment reduced phospho-AKT, endogenous VEGF and PDE4A values but elevated cAMP levels.
This study highlighted a booster cytotoxic effect of combined rolipram and bevacizumab treatment on the GCSCs primary culture, suggesting that this approach is warranted in treatment of GBMs overexpressing VEGF and PDE4A.
神经胶质瘤癌症干细胞样细胞（GCSCs）通过自分泌方式分泌血管内皮生长因子 A（VEGF）来促进自身增殖，这种方式受到磷酸二酯酶 4（PDE4）的正向调节。在本研究中，我们研究了贝伐单抗（一种 VEGF 阻断剂）单独使用以及与一种名为罗利普兰的 PDE4 特异性抑制剂联合使用对从人手术肿瘤标本中分离出的 GCSCs 的潜在细胞毒性作用，重点关注 PI3K/AKT 通路。
通过流式细胞术对 CD133+/CD15+ GCSCs 进行鉴定，并在无血清原代培养系统中进行扩增。依次使用 MTT 测定法、TUNEL 染色和 Western blot 测定细胞存活、凋亡和蛋白表达值。通过 cAMP 酶免疫测定法和 ELISA 分别评估细胞内 cAMP 和游离分泌的 VEGF 水平。
贝伐单抗以 IC6.5μg/ml 抑制 GCSCs 存活，并增强细胞凋亡、p53 和 cleaved-caspase3 的水平，同时降低游离 VEGF 水平和 ERKs 激活。然而，AKT 丝氨酸 473 磷酸化、细胞内 PDE4A、VEGF 和 cAMP 水平没有明显的调节作用。与单独使用贝伐单抗相比，联合治疗的细胞具有更高的细胞毒性，同时游离 VEGF 水平显著下降，p53 和 cleaved-caspase3 的数量显著增加。联合治疗降低了磷酸化 AKT、内源性 VEGF 和 PDE4A 的水平，但升高了 cAMP 水平。
本研究强调了罗利普兰和贝伐单抗联合治疗对 GCSCs 原代培养的增强细胞毒性作用，表明在治疗过表达 VEGF 和 PDE4A 的 GBM 时，这种方法是合理的。
