青天葵甲醇提取物通过ERK信号通路诱导鼻咽癌CNE-2细胞凋亡

Methanol Extract of Nervilia Fordii Induces Apoptosis of Nasopharyngeal Carcinoma CNE-2 Cells Through ERK Signaling Pathway

目的研究青天葵甲醇提取物(Nervilia fordii methanol extracts,NFME)体外对鼻咽癌CNE-2细胞的凋亡作用及其作用机制。方法MTT法检测不同浓度(0,0.25,0.5,1,2,3 mg·mL^(−1))的NFME处理CNE-2细胞24,48 h对CNE-2细胞生长抑制率的影响;克隆原形成试验观察NFME对CNE-2细胞克隆形成率的影响;Hoechst凋亡染色观察NFME对CNE-2细胞凋亡的影响;Western blotting测定NFME作用下caspase-3、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果MTT试验结果显示,与对照组相比,在给药24 h时0.5 mg·mL^(−1)的NFME就能抑制CNE-2细胞的增殖(P<0.05),抑制率为12.64%。而在给药48 h时0.25 mg·mL^(−1)的NFME就能抑制CNE-2细胞增殖(P<0.05),抑制率为22.43%;克隆原形成能力试验表明,0.25 mg·mL^(−1)的NFME能够抑制CNE-2细胞集落的形成(P<0.05);Hoechst33258凋亡染色观察到0.25 mg·mL^(−1)的NFME作用24 h能够观察到CNE-2细胞发生凋亡(P<0.05),凋亡率达到17.91%;Western blotting结果表明0.5 mg·mL^(−1)的NFME能使CNE-2细胞中caspase-3发生剪切,随着给药浓度增加其剪切作用越明显(P<0.01),同时0.5 mg·mL^(−1)的NFME能够降低CNE-2细胞中ERK1/2和c-Raf蛋白的磷酸化水平(P<0.05)。结论青天葵甲醇提取物能抑制鼻咽癌CNE-2细胞的增殖并诱导其发生凋亡,其作用机制可能与抑制ERK信号通路有关。

OBJECTIVE To investigate the apoptosis effect and mechanism of Nervilia fordii methanol extracts(NFME)on nasopharyngeal carcinoma CNE-2 cells in vitro.METHODS The growth inhibition rate of CNE-2 cells treated with different concentrations of NFME(0,0.25,0.5,1,2,3 mg·mL^(−1))for 24,48 h were detected by MTT.The effect of NFME on CNE-2 cell clone formation rate was observed by clonal test.The effect of NFME on CNE-2 cell apoptosis was observed by Hoechst apoptosis staining.The changes of phosphorylation level of caspase-3,ERK1/2 and c-Raf were detected by Western blotting.RESULTS The results of the MTT experiment showed that,compared with the control group,0.5 mg·mL^(−1) of NFME could inhibit the proliferation of CNE-2 cells at 24 h(P<0.05),and the inhibition rate reached 12.64%.At 48 h,0.25 mg·mL^(−1) of NFME could inhibit the proliferation of CNE-2 cells(P<0.05),and the inhibition rate reached 22.43%.The experiment of clonal ability showed that 0.25 mg·mL^(−1) of NFME could inhibit CNE-2 formation of cell colonies(P<0.05).Hoechst33258 apoptosis staining observed that 0.25 mg·mL^(−1) of NFME induced apoptosis in CNE-2 cells for 24 h(P<0.05),and the apoptosis rate reached 17.91%.The results of Western blotting showed that 0.5 mg·mL^(−1) of NFME caused caspase-3 to shear in CNE-2 cells,and the shear effect became more pronounced as the concentration of drug was increased(P<0.01).Meanwhile,NFME could cut down the phosphorylation level of ERK1/2 and c-Raf in CNE-2 cells at 0.5 mg·mL^(−1)(P<0.05).CONCLUSION NFME can inhibit nasopharyngeal carcinoma CNE-2 cells and induce apoptosis,its mechanism may be related to the inhibition of ERK signaling pathway.