姜黄素和乳香酸对人口腔表皮样癌KB细胞中花生四烯酸代谢通路COX-2和5-LOX的作用

The effects of curcumin and boswellic acid on arachidonic acid metabolism enzymes COX-2 and 5-LOX in oral epidermoid carcinoma cell line KB

目的:观察姜黄素和乳香酸对人口腔表皮样癌细胞系KB中花生四烯酸代谢通路环氧化物酶(cyclooxygenase,COX)-2和5-脂氧化物酶(lipoxygenase,LOX)的作用。方法:培养人口腔表皮样癌KB细胞,采用不同浓度的姜黄素和乳香酸及二者联合处理KB细胞24、48、72h,光镜下观察不同浓度药物作用时,细胞形态的变化及贴壁生长的情况,MTT方法检测其对细胞增殖的影响;AnnexinV-FITC/PI染色法检测对KB细胞凋亡的影响;RT-PCR及Western-blotting法检测花生四烯酸代谢通路中COX-2、5-LOX表达。结果:光镜下观察发现,姜黄素和乳香酸能一定程度上抑制KB细胞的贴壁生长。MTT结果显示,姜黄素和乳香酸均能抑制KB细胞增殖,呈现一定的浓度和时间依赖关系。流式细胞检测结果表明,姜黄素和乳香酸均能促进KB细胞凋亡,呈现一定的浓度依赖关系。两种药物联合应用抑制细胞增殖和促进凋亡的作用明显强于单独作用。RT-PCR及Western-blotting结果提示,姜黄素能抑制花生四烯酸代谢通路中限速酶COX-2和5-LOX的表达,乳香酸可以抑制5-LOX表达,且成一定的剂量相关性,两者联合应用时对COX-2和5-LOX的表达均有抑制作用,尤其是对5-LOX通路的抑制,联合应用较任何一种药物单独应用作用显著增强。结论:姜黄素能抑制人口腔表皮样癌细胞KB的增殖,促进凋亡,并能抑制炎症代谢通路中COX-2和5-LOX的表达,乳香酸能抑制人口腔表皮样癌细胞KB的增殖,促进凋亡,且能抑制5-LOX表达,提示天然植物药姜黄素和乳香酸可能通过抑制花生四烯酸代谢通路中COX-2、5-LOX的表达而发挥一定的口腔癌化学预防作用。

Objective：To explore the inhibitory effects of curcumin and acetyl-11-keto-beta-boswellic acid （AKBA） in arachidonic acid （AA） metabolism enzymes COX-2 and 5-LOX and investigate the proliferation and apoptosis effects of both agents in oral epidermoid carcinoma cell line （OECC） KB.Methods：OECC cell line KB were cultured and treated with low to high doses of curcumin and AKBA separatedly or combined for 24,48,72 hours.The cells morphology was observed under light microscope .The cells proliferation was measured by MTT assay.Cells apoptosis was examined by flow cytometry with Annexin-V FITC/PI staining.The expression of COX-2 and 5-LOX was detected by RT-PCR and Western-blotting.Results：Light microscope displayed that curcumin and AKBA could slow down the growth of KB cells.The MTT results showed that both curcumin and AKBA could inhibit the proliferation of KB cells in a dose-dependent and time-dependent manner.The apoptotic assay with Annexin V-FITC/PI showed that curcumin and AKBA could induce OECC line apoptosis in a dose-dependent manner.The cells growth inhibition and apoptosis inducing effect with combined treatment of the two compounds is much better than that of either single agent.The RT-PCR and Western-blotting showed that curcumin could inhibit the expression of COX-2 and 5-LOX,AKBA could inhibit the expression of 5-LOX,and the combined treatment of the two agents decreased the expression of 5-LOX much more than that of either one.Conclusions：Curcumin and AKBA could be served as chemopreventive agents in oral epidermoid cancer cells by inhibiting arachidonic acid （AA） metabolism pathway.