Effect of Wuzhishan Callicarpa nudiflora in Hainan Province on Cisplatin-induced Apoptosis of Nasopharyngeal Carcinoma Cells and Mechanism： Based on Nrf2/ARE Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo explore the effect of Wuzhishan Callicarpa nudiflora （LHZZ） on the sensitivity of nasopharyngeal carcinoma （NPC） cells to cisplatin （DDP） and the mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the survival rate of NPC HNE1 cells after treatment with different concentration of DDP （0，2，4，8，16，32 mg·L-1） and different concentration of LHZZ （0，25，50，75，100 mg·L-1）. The following groups were designed： control group （normal HNE1 cells），DDP group （8 mg·L-1 DDP，24 h），LHZZ group （50 mg·L-1 LHZZ，24 h），DDP + LHZZ group （8 mg·L-1 DDP + 50 mg·L-1 LHZZ，24 h），DDP + LHZZ + nuclear factor erythroid 2-related factor 2 （Nrf2） activator sulforaphane （SFN） group （8 mg·L-1 DDP + 50 mg·L-1 LHZZ，24 h， 10 μmol·L-1 SFN，24 h）. Then CCK-8 assay was employed to examine cell survival rate，colony formation test the colony-forming ability，flow cytometry and in situ terminal end-labeling（TUNEL） staining cell apoptosis，fluorescent probe 2'，7'-dichlorofluorescin diacetate （DCFH-DA） the level of reactive oxygen species （ROS） in cultured cells，Western blot the expression of apoptosis-related proteins in cells，such as B-cell lymphoma/leukemia-2 （Bcl-2），Bcl-2 associated X protein （Bax），and cysteinyl aspartate specific proteinase-3 （Caspase-3），and Real-time quantitative polymerase chain reaction （Real-time PCR） the expression of Nrf2 and antioxidant response element （ARE） mRNA in cells. ResultThe survival rates of cells treated with different concentration of DDP and different concentration of LHZZ decreased compared with that in the control group （P<0.05）. Compared with the DDP group and the LHZZ group，DDP + LHZZ group demonstrated decrease in cell survival rate，number of cell colonies，and Bcl-2 level，and increase in the apoptosis level and the expression of Bax and Caspase-3 （P<0.05）. However，after the addition of SFN，the Nrf2/ARE signaling pathway was activated and the above variation was inhibited （P<0.05）. In addition，the level of intracellular ROS in the LHZZ group was lower than that in the control group （P<0.05） and the level in the DDP + LHZZ group was lower than that in the DDP group （P<0.05）. Moreover，the ROS level in the DDP + LHZZ + SFN group was higher than that in the DDP+LHZZ group （P<0.05）. ConclusionLHZZ can enhance the sensitivity of DDP-induced NPC apoptosis，possibly by blocking the Nrf2/ARE signaling pathway and inhibiting the level of ROS.