胡椒碱通过Keap1/Nrf2/ARE通路促进皮肤鳞状细胞癌细胞凋亡

Piperine promotes the apoptosis of skin squamous cell cancer cells through the Keap1/Nrf2/ARE pathway

目的探讨胡椒碱对人皮肤鳞状细胞癌细胞的功能影响及相关作用机制。方法用不同浓度梯度的胡椒碱处理人皮肤鳞状细胞癌细胞(A431和SCL-1)24 h或48 h后,检测对细胞存活率的影响;A431和SCL-1细胞单独用50μmol·L^(-1)和100μmol·L^(-1)胡椒碱处理,或先转染红系衍生的核因子2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)干扰RNA或Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)过表达载体pcDNA-Keap1后再用100μM胡椒碱进行诱导处理。Transwell和流式细胞仪分别检测细胞侵袭和凋亡;qPCR检测Keap1/Nrf2/抗氧化反应元件(antioxidant response elements,ARE)信号通路中Keap1、Nrf2和醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)的mRNA水平;Western blot检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、Keap1、Nrf2和NQO1蛋白表达水平。结果在0μmol·L^(-1)到300μmol·L^(-1)胡椒碱浓度梯度内,随着胡椒碱浓度和处理时间的增加,A431和SCL-1细胞存活率下降;与control组相比,50μmol·L^(-1)和100μmol·L^(-1)的胡椒碱处理细胞后,细胞活力和侵袭力减弱、凋亡率增加、Caspase-3的活性增加、Bax/Bcl-2比值升高、Keap1的mRNA和蛋白表达被抑制、Nrf2和NQO1的mRNA水平升高,N-Nrf2和NQO1的蛋白表达增加;转染si-Nrf2或pcDNA-Keap1后,逆转胡椒碱对细胞活力和侵袭能力的抑制、凋亡的促进及Keap1/Nrf2/ARE通路的激活作用。结论胡椒碱激活Keap1/Nrf2/ARE通路促进人皮肤鳞状细胞癌细胞的凋亡。

Objective To investigate the effect and mechanism of piperine on the function of human squamous cell carcinoma cells.Methods Human skin squamous cell carcinoma cells(A431 and SCL-1)were treated with different concentration gradients of piperine for 24 h or 48 h,followed by detection of cell survival.A431 and SCL-1 cells were treated with 50μmol·L^(-1) and 100μmol·L^(-1) piperine respectively,or transfected with nuclear factor erythroid 2-related factor 2(Nrf2)siRNA or Kelch-like ECH-associated protein 1(Keap1)overexpression vector(pcDNA-Keap1)first and then treated with 100μM piperine.Transwell and flow cytometry were used to detect cell invasion and apoptosis;qPCR was used to detect the mRNA levels of Keap1,Nrf2 and NAD(P)H:quinone oxidoreductase 1(NQO1)mRNA levels in the Keap1/Nrf2/antioxidant response elements(ARE)signaling pathway;Western blot was used to detect the protein levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Keap1,Nrf2 and NQO1.Results In the concentration gradient of piperine from 0μmol·L^(-1) to 300μmol·L^(-1),the survival rates of A431 and SCL-1 cells decreased with the increase of piperine concentration and treatment time;compared with the control group,after treated with 50μmol·L^(-1) and 100μmol·L^(-1) piperine,cell viability and invasiveness decreased,apoptosis rate increased,caspase-3 activity increased,Bax/Bcl-2 ratio increased,the mRNA and protein expression of Keap1 were inhibited,mRNA levels of Nrf2 and NQO1 increased,and the protein expressions of N-Nrf2 and NQO1 increased.After transfection of si-Nrf2 or pcDNA-Keap1,the inhibitory effects of piperine on cell viability and invasion ability,the promotion of apoptosis and the activation of Keap1/Nrf2/ARE pathway were reversed.Conclusion Piperine activates the Keap1/Nrf2/ARE pathway to promote the apoptosis of human skin squamous cell carcinoma cells.