紫苏醇对肺癌A549细胞增殖和侵袭的抑制作用机制的研究

Anti-lung cancer effect and anti-angiogenesis therapy study of perillyl alcohol

目的:探讨紫苏醇(Perillyl alcohol,PA)对肺癌A549细胞增殖和侵袭的抑制作用机制,并阐明其对肿瘤血管生成信号的影响。方法:不同浓度PA和厄洛替尼加入到A549细胞中,利用溴化3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑(MTT)法测定药物组对A549细胞的抑制作用,Transwell法检测PA对A549细胞侵袭的抑制作用,采用间接荧光标记法测定细胞内活性氧(ROS)水平的变化;分光光度法检测PA对细胞凋亡蛋白Caspase-3活性的影响,Western blot法检测A549中VEGF、HIF-1α及COX-2的表达,凝胶迁移或电泳迁移率实验测定A549细胞中NF-κB活性。结果:与空白对照组比较,随着浓度的增加(10、50、100μg/ml),PA和厄洛替尼对A549细胞生长的抑制率在不断地增加,差异均有统计学意义(P<0.05),A549细胞侵袭能力呈现不断下降的趋势,差异均有统计学意义(P<0.05),A549细胞内活性氧水平随厄洛替尼浓度的增加变化不大,而ROS水平随着PA的浓度的增加而增加,在100μg/ml浓度的PA下引起的ROS百分率达到了(80.43±6.92)%,差异均有统计学意义(P<0.05)。细胞活力检测结果显示,随着PA和厄洛替尼浓度的增加和作用时间的延长,A549细胞中凋亡蛋白Caspase-3活性明显增加(P<0.05),随着紫苏醇浓度的增加,COX-2、VEGF和HIF-1α的表达呈不断降低的趋势,EMSA检测结果显示,随着PA浓度的增加,NF-κB的条带面积不断减少。结论:PA可能参与并促进了ROS的生成和Caspase-3活性增加,最终诱导A549细胞的凋亡,PA可能通过降低NF-κB的表达,进而诱导COX-2、VEGF等的表达减少,使血管生成滞后,能够有效地使细胞的穿透能力下降和凋亡发生。

Objective： To investigate the inhibitory effect of perillyl alcohol （PA） on the proliferation and invasion of lung cancer cell A549, and the influence of PA on tumor angiogenesis was studied. Methods： Different concentrations of PA and erlotinib were added into lung cancer cell A549, the inhibiting effect of drug group on lung cancer cell A549 was found by MTr assay. The inhibiting effect of PA on lung cancer cell A549 invasion was measured by Transwell assay. ROS changes of PA on lung cancer cell A549 was detected by fluorescent. Influence of PA on Caspase-3 activity of lung cancer cell A549 was measured by spectrophotometry, VEGF, HIF-1 ct, COX-2 expression in lung cancer cell A549 was measured by Western blot, and the NF-KB activity of lung cancer cell A549 was measured by EMSA. Results： Compared with blank control group, cell growth inhibition rate of PA and erlotinib on lung cancer cell A549 was increasing with the increased concentrations （10,50,100 μg/ml）, the difference was statistically significant （P〈 0. 05 ）, the invasion ability of lung cancer cell A549 was decreased continuously, the difference was statistically significant （ P〈0. 05 ）. The ROS level of lung cancer cell A549 had no obvious change with the increasing density of erlotinib, but obviously increased with the increasing concentrations of PA （10,50,100 μg/ml）. With the increasing concentrations of PA, the expression of COX-2, VEGF and HIF-lot were continuously decreased. EMSA assay showed that NF-κB was continuously decreased with the increasing concentrations of PA. Conclusion： The antitumor mechanism of PA on lung cancer cell A549 might be related to increase the expression level of ROS and reduce the expression of activity of NF-κB, COX-2, VEGF and HIF-1α with angiogenesis signaling pathway.