盐屋霉素A下调叉头框蛋白M1表达可抑制喉癌细胞恶性生物学行为

Down-regulation of forkhead box protein M1 by siomycin A can inhibit the malignant behaviors of laryngeal carcinoma cells

目的探讨盐屋霉素A下调叉头框蛋白M1(forkhead box protein M1,FoxM1)对喉癌细胞增殖、凋亡、侵袭能力等恶性生物学行为的影响。方法取处于对数生长期的Hep-2细胞,使用不同浓度盐屋霉素A进行处理,对照组不加盐屋霉素A。分别在培养24、48、72h后通过CCK-8法检测细胞活力;24、48h后通过CFSE染色法检测细胞增殖能力;48h后AnnexinⅤ-FITC/PI双染法检测细胞凋亡情况,Transwell小室法检测细胞侵袭能力。蛋白质印迹法检测FoxM1、Ki-67、cleaved caspase-3蛋白的表达变化,ELISA法检测细胞上清液中基质金属蛋白酶(MMP)-2、MMP-9的表达变化。结果 (1)盐屋霉素A作用后FoxM1蛋白的表达量降低(P<0.05)。CCK-8检测结果显示不同浓度盐屋霉素A对Hep-2细胞活力均有抑制作用,并随着盐屋霉素A浓度的增加和作用时间的延长,Hep-2的细胞活力逐渐下降,呈现浓度和时间依赖性。(2)1.3、1.5μmol/L盐屋霉素A处理后Hep-2细胞的增殖能力均降低(P<0.05),并呈现浓度依赖性;同时Ki-67蛋白的表达下调。(3)1.3、1.5μmol/L盐屋霉素A作用后,Hep-2细胞的凋亡率(10.60%,27.90%)均高于对照组(4.91%,P<0.05),并呈药物浓度依赖性;同时cleaved caspase-3蛋白的表达上调。(4)1.3、1.5μmol/L盐屋霉素A作用后Hep-2细胞的侵袭能力较对照组均下降(P<0.05),并呈药物浓度依赖性;同时MMP-2、MMP-9蛋白的表达下调。结论盐屋霉素A下调FoxM1可抑制喉癌细胞的增殖、侵袭等生物学行为。

Objective To investigate the effect of siomycin A-induced forkhead box protein M1（FoxM1） down-regulation on the malignant behaviors （proliferation, apoptosis, and invasive ability） of laryngeal carcinoma cells. Methods The experiment was performed with Hep-2 cells of the logarithmic growth phase. The experimental groups were treated with different doses of siomycin A, and the control group was treated without siomycin A. CCK-8 test was used to detect the viability of Hep-2 cells after treatment for 24, 48, and 72 h. CFSE staining was used to examine the proliferation ability of Hep-2 cells after treatment for 24 and 48 h. Annexin Ⅴ-FITC/PI dual staining was used to observe the siomycin A-induced apoptosis and Transwell test was used to examine the invasion ability of Hep-2 cells after treatment for 48 h. The expressions of FoxM1, Ki-67 and cleaved caspase-3 protein in Hep-2 cells were detected by Western blotting analysis. The supernatant levels of matrix metalloproteinase （MMP）-2 and MMP-9 were examined by ELISA assay. Results （1） Treatment with siomycin A significantly decreased the expression of FoxM1 protein compared with the control group （P〈0.05）. CCK-8 test found that siomycin A of different concentrations suppressed the viability of Hep-2 cells in a time- and dose-dependent manner. （2） Siomycin A at 1.3 and 1.5 μmol/L significantly suppressed the proliferation of Hep-2 cells in a dose-dependent manner （P〈0.05）, accompanied by down-regulated expression of Ki-67.（3）Siomycin A at 1.3 and 1.5 μmol/L also induced greater apoptosis of Hep-2 cells （10.6%, 27.9 %） in a dose-dependent manner compared with control group （4.91%, P〈0.05）, accompanied by up-regulated cleaved caspase-3 expression. （4） Siomycin A at 1.3 and 1.5 μmol/L suppressed the invasive ability of Hep-2 cells in a dose-dependent manner compared with control group （P〈0.05）, accompanied by down-regulated expression of MMP-2 and MMP-9. Conclusion Down-regulation of FoxM1 by siomycin A can inhibit the proliferation and invasive ability of laryngeal carcinoma cells.