粉防己碱诱导人视网膜母细胞瘤细胞凋亡及其机制研究

背景与目的：粉防己碱(tetrandrine，Tet)是一种天然化合物，其抗视网膜母细胞瘤作用尚不清楚。该研究拟检测Tet对人视网膜母细胞瘤细胞的抗肿瘤作用，并进一步阐明其作用机制。方法：采用CCK-8法检测Tet对视网膜母细胞瘤细胞活力的抑制作用；应用Annexin V/PI法检测细胞凋亡情况；在2’,7’-二氯荧光素二乙酸酯(2’, 7’-dichlorofluorescin diacetate，DCFH-DA)染色后，采用流式细胞术检测细胞内反应性活性氧(reactive oxygen species，ROS)含量；采用蛋白［质］印迹法(Western blot)检测细胞Akt、p-Akt蛋白表达量。结果：Tet显著抑制视网膜母细胞瘤细胞活力，Tet浓度为4、8、10和20 μmol/L处理细胞24 h时，WERI-Rb-1细胞抑制率分别为5.7%、25.0%、55.1%和84.9%，Y79细胞抑制率分别为2.4%、2.9%、23.8%和54.2% (P<0.01)；10 μmol/L Tet处理细胞12、24和48 h时，WERI-Rb-1细胞抑制率分别为6.0%、45.5%和74.7%，Y79细胞抑制率分别为2.9%、19.4%和43.3%(P<0.01)。Tet诱导细胞凋亡，以10 μmol/L Tet处理细胞24和48 h时，WERI-Rb-1细胞凋亡率分别为(23.70±1.75)%和(34.83±3.15)%，Y79细胞凋亡率分别为(9.62±2.69)%和(14.97±1.50)%(P<0.01)，凋亡抑制剂Z-VAD-FMK能够显著抑制Tet对视网膜母细胞瘤细胞的凋亡诱导作用(P<0.05)。10 μmol/L Tet作用细胞6及12 h后，细胞的ROS产生量较对照组明显上升(P<0.01)，N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine，NAC)能够抑制Tet诱导产生的ROS(P<0.01)，NAC抑制ROS后，细胞的凋亡率较单独Tet作用组明显下降(P<0.01)。Tet能够抑制视网膜母细胞瘤细胞PI3K/Akt信号通路。结论：Tet诱导视网膜母细胞瘤细胞凋亡，该作用机制与细胞内ROS升高、PI3K/Akt信号通路抑制有关。

Tetrandrine induces retinoblastoma cells apoptosis via ROS induction and PI3K/Akt inhibition

Background and purpose: Tetrandrine is a natural compound whose role in retinoblastoma remains unclear. This study investigated the effects of tetrandrine (Tet) on human retinoblastoma cells. Methods: CCK-8 assays were performed to analyze the effects of Tet on viability of retinoblastoma cells. The apoptosis rate was determined by Annexin V/PI assays. After staining with 2′,7′-dichlorofluorescin diacetate (DCFH-DA), cellular reactive oxygen species (ROS) was measured by flow cytometry. Akt and p-Akt were detected by Western blot. Results: Tet inhibited cell viability of retinoblastoma cells. After treatment with Tet (4, 8, 10 and 20 μmol/L) for 24 h, cell viability inhibition rates of WERI-Rb-I were 5.7%, 25.0%, 55.1% and 84.9%, whereas inhibition rates of Y79 cells were 2.4%,2.9%, 23.8% and 54.2% (P<0.01). In cells treated with 10 μmol/L of Tet for 12, 24 and 48 h, cell viability inhibition rates of WERI-Rb-I were 6.0%, 45.5% and 74.7%, whereas inhibition rates of Y79 cells were 2.9%, 19.4% and 43.3% (P<0.01). Tet induced retinoblastoma cell apoptosis. After treatment with Tet (10 μmol/L) for 24 and 48 h, apoptosis rates of WERI-Rb-I were (23.70±1.75)% and (34.83±3.15)%, respectively, whereas apoptosis rates of Y79 cells were (9.62±2.69)% and (14.97±1.50)%, respectively (P<0.01). Apoptosis inhibitor Z-VAD-FMK attenuated Tet-induced cell death (P<0.05). ROS levels were indeed increased in cells treated with Tet (10 μmol/L) for 6 and 12 h (P<0.01), while N-Acetyl-L-cysteine (NAC) decreased Tet-induced ROS (P<0.01). After ROS was inhibited by NAC, apoptosis rate was decreased compared with the control (P<0.01). Further study indicated that Tet inhibited PI3K/Akt pathway in retinoblastoma cells. Conclusion: Tet induces cell apoptosis via increasing ROS synthesis and inhibiting PI3K/Akt pathway.