北美香柏叶的乙醇提取物阻断A549细胞增殖并引起细胞凋亡的体外研究(英文)

Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro

目的:研究北美香柏叶的乙醇提取物对非小细胞肺癌A549细胞的抗肿瘤及抗增殖作用。方法:噻唑蓝法检验不同剂量北美香柏叶的乙醇提取物对细胞活性的影响。确定半数最大抑制浓度为282μg/mL,另外选择两个浓度188μg/mL和376μg/mL进行剂量依赖性检测。进行溴脱氧尿苷结合实验和细胞迁移实验检测药物的抗肿瘤细胞增殖活性。膜联蛋白V-异硫氰酸荧光黄-碘化丙啶双染色后采用荧光激活细胞分类分析仪对细胞早期凋亡进行检测。Hoechst 33258及吖啶橙-溴化乙啶荧光染色法进行DNA片段分析。间接酶联免疫吸附法分析Bax-Bcl2的调节和表达情况。逆转录聚合酶链反应检测caspase3基因表达情况,其活性和蛋白水平的表达则使用间接酶联免疫吸附法和蛋白印迹法进行检测。结果:A549的细胞活性在暴露于北美香柏叶的乙醇提取物24h后呈剂量依赖性下降。脱氧尿苷结合实验和细胞迁移实验表明细胞的增殖活性与暴露于药物的时间有时间依赖性关系。11.72%的细胞在双染色后呈阳性反应,表明药物引起了细胞的早期凋亡。药物作用24h后DNA片段彗星尾的出现以及Hoechst 33258荧光染色的增加提示显著的DNA缺口出现以及染色质凝聚。Bax的上调及Bcl2的下调表明了细胞凋亡的出现。逆转录聚合酶链反应、间接酶联免疫吸附法以及蛋白印迹法的检测结果表明caspase3的活性随着抗聚(腺苷二磷酸-核糖)聚合酶的表达的增加而增加。结论:北美香柏叶的乙醇提取物能够促进A549细胞凋亡并抑制其增殖活性。

OBJECTIVE： To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis （TO） on A549 non-small lung carcinoma cells in vitro. METHODS ： Cell viability was ascertained through 3- （4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay after deployment of TO in different doses. The half maximal inhibitory concentration （IC50） dose （282 μg/mL） was determined, and two other doses for dose-dependence study, one below the IC50 dose （IC35=188 μg/mL） and one above the IC50 dose （IC65=376 μg/mL） were selected. Bromodeoxyuridine （BrdU） incorporation assay and migration studies were performed to elucidate antiproliferative activity of the drug, if any. Fluorescence-activated cell sorting analysis after annexin V-fluorescein isothiocyanate and propidium iodide （annexin V-FITC-PI） dual staining method was done to ascertain early stage of apoptosis, if any. DNA fragmentation assay was done through Hoechst 33258 and acridine orange-ethidium bromide staining. DNA damage was quantified through comet assay. Bax-Bcl2 regulation and expression studies were performed through indirect enzyme-linked immunosorbent assay （ELISA）. Caspase 3 activity was measured at gene level through reverse transcription-polymerase chain reaction （RT-PCR） analysis. Its activation at protein level was analyzed through indirect ELISA and Western blot analysis. RESULTS. TO demonstrated a dose-dependent decrease in viability of A549 cells after 24 h of exposure. Cell proliferation was reduced in a time-dependent manner of drug exposure as revealed from BrdU incorporation and migration studies. Annexin-V-FITC positivity of cells up to 11.72% as compared to the untreated control revealed early state of TO-induced apoptosis. Occurrence of comet tail and increased fluorescence of Hoechst after 24 h of drug exposure revealed significantDNA nick generation and chromatin condensation. Bax up-regulation and Bcl-2 down-regulation suitably altered ratio of Bax/Bcl-2 in favor of apoptosis. From RT-PCR, indirect ELISA and Western blot studies, caspase 3 activity was also found to be significantly increased along with cleaved poly ADP-ribose polymerase expression. CONCLUSION： Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.