芹菜素7-O-葡萄糖苷对抗TRAIL乳腺癌细胞凋亡的作用机制研究

Action mechanism of apigenin 7-O-glucoside on the apoptosis of anti-TRAIL breast cancer cells

关键词:  乳腺癌  芹菜素7-O-葡萄糖苷  肿瘤坏死因子相关的凋亡诱导配体  氧化应激  细胞凋亡

Key words:  breast cancer  apigenin 7-O-glucoside  tumor necrosis factor-related apoptosis-inducing ligand  oxidative stress  apoptosis

摘要:

[目的] 探讨芹菜素7-O-葡萄糖苷(AGL)对抗肿瘤坏死因子相关凋亡诱导配体(TRAIL)乳腺癌细胞凋亡的影响。[方法] 通过CCK-8法、蛋白免疫印迹、流式细胞法检测AGL、TRAIL、AGL+TRAIL对乳腺癌细胞活性、聚二磷酸腺苷核糖聚合酶-1(PARP-1)水平和细胞凋亡的影响,通过激光扫描共焦显微镜检测细胞ROS水平,采用ELISA法检测丙二醛(MDA)水平和超氧化物歧化酶(SOD)活性以及Western-blot检测PARP-1、p53、p53上调凋亡调制物(PUMA)水平。建立MCF7移植瘤模型,观察AGL+TRAIL处理下移植瘤生长和凋亡情况。[结果] TRAIL(50 ng/mL)+AGL(20、40 μmol/L)处理时,MCF7细胞存活率降低(P＜0.05),Cleaved PARP-1水平升高(P＜0.05);TRAIL(50 ng/mL)+AGL(20 μmol/L)处理时,MCF7细胞凋亡率、Cleaved-Cas-3、Cleaved-Cas-9水平均显著升高(P＜0.05)。AGL(10、20 μmol/L)处理时,MCF7细胞ROS(+)细胞数、MDA水平显著升高(P＜0.05),SOD活性显著降低(P＜0.05);TRAIL(50 ng/mL)+AGL(20 μmol/L)MCF7细胞ROS(+)细胞数目、p53、PUMA、Cleaved PARP-1相对蛋白水平显著高于TRAIL(50 ng/mL)处理(P＜0.05),TRAIL(50 ng/mL)+AGL(20 μmol/L)+NAC(4 mmol/L)MCF7细胞ROS(+)细胞数、p53、PUMA、Cleaved PARP-1相对蛋白水平显著低于TRAIL(50 ng/mL)+AGL(20 μmol/L)处理(P＜0.05)。AGL+TRAIL组小鼠移植瘤质量、体积和肿瘤组织p53阳性率、PUMA3阳性率均显著低于Control组(P＜0.05),细胞凋亡率显著高于Control组(P＜0.05)。[结论] AGL联合TRAIL可促抗TRAIL MCF7细胞凋亡,可能与增强细胞氧化应激损伤和激活Caspase级联反应有关。

Abstract:

[Objective] To explore the effects of apigenin 7-O-glucoside (AGL) on the apoptosis of anti- tumor necrosis factor-related apoptosis inducing ligand (TRAIL) breast cancer cells.[Methods] The effects of AGL,TRAIL and AGL+TRAIL on activity of breast cancer cells,level of poly (ADP-ribose) polymerase-1 (PARP-1) and apoptosis were detected by CCK-8,Western-blot and flow cytometry. ROS level was detected by laser scanning confocal microscope. The level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were detected by ELISA. The levels of PARP-1,p53 and p53up-regulated modulator of apoptosis (PUMA) were detected by Western-blot. MCF7 models of transplanted tumor were established to observe growth and apoptosis of transplanted tumor treated with AGL+TRAIL.[Results] With the treatment of TRAIL (50 ng/mL) +AGL (20,40 μmol/L),survival rate of MCF7 cells was decreased (P＜0.05),and Cleaved PARP-1 level was increased (P＜0.05). With the treatment of TRAIL (50 ng/mL) +AGL (20 μmol/L),apoptosis rate of MCF7 cells,levels of Cleaved-Cas-3 and Cleaved-Cas-9 were significantly increased (P＜0.05). With the treatment of AGL (10,20 μmol/L),number of ROS (+) cells and MDA level in MCF7 cells were significantly increased (P＜0.05),while SOD activity was significantly decreased (P＜0.05). The number of ROS (+) cells in MCF7 cells,related levels of p53,PUMA and Cleaved PARP-1 proteins with the treatment of TRAIL (50 ng/mL) +AGL (20 μmol/L) were significantly higher than those with the treatment of TRAIL (50 ng/mL) (P＜0.05),which were significantly lower with the treatment of TRAIL (50 ng/mL) +AGL (20 μmol/L) +NAC (4 mmol/L) than those with the treatment of TRAIL (50 ng/mL) +AGL (20 μmol/L) (P＜0.05). The mass and volume of transplanted tumors, positive rates of p53 and PUMA3 in AGL+TRAIL group were significantly lower than those in Control group (P＜0.05),while apoptosis rate was significantly higher than that in Control group (P＜0.05).[Conclusion] AGL combined with TRAIL can promote apoptosis of anti-TRAIL MCF7 cells,which may be related to enhancing oxidative stress damage and activating caspase cascade.