Beijing Tongren Eye Center, Beijing Ophthalmology & Visual Sciences Key Lab, Beijing Tongren Hospital, Capital Medical University, Beijing, PR China.
Curr Eye Res. 2011 Jan;36(1):71-7. doi: 10.3109/02713683.2010.510943.
The purpose of this study was to investigate the pro-apoptotic effect of camptothecin (CPT) on Y79 retinoblastoma cells and the role of Forkhead box, class O (FOXO1) in CPT-induced apoptosis.
CPT-induced apoptosis was determined by flow cytometry with annexin V-FITC positive cells and Western blot of PARP expression, respectively. The expressions of FOXO1 were detected by Western blot. The transcriptional activity of FOXO1 was determined by luciferase reporter assay. siRNAs specifically inhibiting FOXO1 were used, and flow cytometry and Western blot were executed to test the role of FOXO1 in CPT-induced apoptosis.
CPT was extremely effective in inducing apoptosis of Y79 retinoblastoma cells. FOXO1 was highly expressed in Y79 cells. CPT not only elevated the FOXO1 dephosphorylation level but also promoted its transcriptional activity, suggesting that the activation of FOXO1 was, at least in part, triggered by CPT. The decreased annexin V positive cells and less PARP cleavage demonstrated that siRNAs-mediated inhibition of FOXO1 significantly abrogated CPT-induced apoptosis, indicating that FOXO1 plays an important role in CPT-induced apoptosis. Moreover, the expression of Bim was also elevated with the treatment of CPT, which is in accordance with the activation of FOXO1.
Our study provides the evidence that a high level of endogenous FOXO1 expression in retinoblastoma cells contributes, at least in part, to CPT-induced apoptosis, which may help broad application of CPT in retinoblastoma therapy in the future.
本研究旨在探讨喜树碱（CPT）对 Y79 视网膜母细胞瘤细胞的促凋亡作用，以及叉头框蛋白 O（FOXO1）在 CPT 诱导细胞凋亡中的作用。
通过流式细胞术检测 annexin V-FITC 阳性细胞和 PARP 表达的 Western blot，分别确定 CPT 诱导的细胞凋亡。Western blot 检测 FOXO1 的表达。通过荧光素酶报告基因检测法测定 FOXO1 的转录活性。使用特异性抑制 FOXO1 的 siRNA，通过流式细胞术和 Western blot 检测 FOXO1 在 CPT 诱导细胞凋亡中的作用。
CPT 对 Y79 视网膜母细胞瘤细胞的凋亡作用非常有效。FOXO1 在 Y79 细胞中高表达。CPT 不仅提高了 FOXO1 的去磷酸化水平，而且促进了其转录活性，提示 FOXO1 的激活至少部分是由 CPT 触发的。 Annexin V 阳性细胞减少和 PARP 裂解减少表明，siRNA 介导的 FOXO1 抑制显著阻断了 CPT 诱导的细胞凋亡，表明 FOXO1 在 CPT 诱导的细胞凋亡中发挥重要作用。此外，CPT 处理还增加了 Bim 的表达，这与 FOXO1 的激活一致。
本研究提供的证据表明，视网膜母细胞瘤细胞中内源性 FOXO1 表达水平高，至少部分有助于 CPT 诱导的细胞凋亡，这可能有助于 CPT 在未来的视网膜母细胞瘤治疗中的广泛应用。
