Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Texas College of Osteopathic Medicine, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Gene. 2019 Jul 15;705:67-76. doi: 10.1016/j.gene.2019.04.037. Epub 2019 Apr 13.
Medulloblastoma (MB) is characterized by highly invasive embryonal neuro-epithelial tumors that metastasize via cerebrospinal fluid. MB is difficult to treat and the chemotherapy is associated with significant toxicities and potential long-term disabilities. Previously, we showed that small molecule, clotam (tolfenamic acid: TA) inhibited MB cell proliferation and tumor growth in mice by targeting, survivin. Overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers, including MB. The aim of this study was to test combination treatment involving Vincristine® (VCR), a standard chemotherapeutic drug for MB and TA against MB cells. DAOY and D283 MB cells were treated with 10 μg/mL TA or VCR (DAOY: 2 ng/mL; D283: 1 ng/mL) or combination (TA + VCR). These optimized doses were lower than individual IC values. The effect of single or combination treatment on cell viability (CellTiterGlo kit), Combination Index (Chou-Talalay method based on median-drug effect analysis), activation of apoptosis and cell cycle modulation (by flow cytometry using Annexin V and propidium iodide respectively) and the expression of associated markers including survivin (Western immunoblot) were determined. Combination Index showed moderate synergistic cytotoxic effect in both cells. When compared to individual agents, the combination of TA and VCR increased MB cell growth inhibition, induced apoptosis and caused cell cycle (G/M phase) arrest. Survivin expression was also decreased by the combination treatment. TA is effective for inducing the anti-proliferative response of VCR in MB cells. MB has four distinct genetic/molecular subgroups. Experiments were conducted with MB cells representing two subgroups (DAOY: SHH group; D283: group 4/3). TA-induced inhibition of survivin expression potentially destabilizes mitotic microtubule assembly, sensitizing MB cells and enhancing the efficacy of VCR.
髓母细胞瘤 (MB) 是一种具有高度侵袭性的胚胎神经上皮肿瘤，通过脑脊液转移。MB 难以治疗，化疗伴随着明显的毒性和潜在的长期残疾。此前，我们发现小分子 Clotam（甲苯酰氨基乙酸：TA）通过靶向生存素抑制 MB 细胞增殖和小鼠肿瘤生长。生存素的过表达与包括 MB 在内的几种癌症的侵袭性和不良预后相关。本研究旨在测试包含长春新碱®（VCR）的联合治疗，VCR 是 MB 的标准化疗药物，以及 TA 对 MB 细胞的作用。用 10μg/mL TA 或 VCR（DAOY：2ng/mL；D283：1ng/mL）或联合（TA+VCR）处理 DAOY 和 D283 MB 细胞。这些优化剂量低于单独的 IC 值。单独或联合处理对细胞活力（CellTiterGlo 试剂盒）、组合指数（基于中值药物效应分析的 Chou-Talalay 方法）、凋亡激活和细胞周期调节（分别用 Annexin V 和碘化丙啶进行流式细胞术）以及相关标记物的表达（Western 免疫印迹）的影响。组合指数在两种细胞中均显示出适度的协同细胞毒性作用。与单独药物相比，TA 和 VCR 的联合使用增加了 MB 细胞生长抑制、诱导凋亡并导致细胞周期（G2/M 期）停滞。联合治疗还降低了生存素的表达。TA 对诱导 MB 细胞中 VCR 的抗增殖反应有效。MB 有四个不同的遗传/分子亚组。实验用代表两个亚组的 MB 细胞进行（DAOY：SHH 组；D283：第 4/3 组）。TA 诱导的生存素表达抑制可能使有丝分裂微管组装不稳定，使 MB 细胞敏感，并增强 VCR 的疗效。
