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

关键词:  青天葵甲醇提取物  鼻咽癌  CNE-2  ERK  凋亡

Key words:  Nervilia Fordii methanol extracts  nasopharyngeal carcinoma  CNE-2  ERK  apoptosis

摘要:

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。

Abstract:

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.