莪术油对乳腺癌MCF-7细胞端粒酶活性及DNA损伤反应的调控作用

Regulation of Telomerase Activity and DNA Damage Response by Zedoary Turmeric Oil in Breast Cancer Cell Line MCF-7

目的研究莪术油通过hTert作为下游靶基因对乳腺癌MCF-7细胞端粒酶活性及DNA损伤反应的调控作用。方法以300μg/mL莪术油处理MCF-7细胞,采用MTT法测定细胞增殖活性,绘制生长曲线,筛选最佳药物处理时间。于加药后48 h,实时定量RT-PCR检测端粒酶催化亚单位hTert表达水平、TRAP Assay检测端粒酶活性变化;收集加药前以及加药后24,48,72 h细胞,采用免疫荧光及Western Blot检测DNA损伤反应相关53BP1蛋白水平。结果莪术油处理的MCF-7细胞24 h后增殖活力开始降低,至48 h生长明显减缓,实时定量RT-PCR结果表明,加药后48 h,hTert表达水平降低为对照细胞的2.43倍(2-△△Ct),TRAP Assay结果显示端粒酶活性降低为对照细胞的2.27倍,免疫荧光及Western Blot结果显示莪术油处理的细胞53BP1蛋白磷酸化水平自转染24 h后开始升高,至48 h左右达到高峰,至72 h时仍保持在较高水平。结论莪术油可以hTert作为下游靶分子,通过调控端粒酶活性及DNA损伤反应,抑制乳腺癌MCF-7细胞的增殖。

Objective To study the regulation of telomerase activity and DNA damage response by Zedoary Turmeric oil （ZTO） with hTert as the downstream target gene in breast cancer cell line MCF-7. Methods MCF-7 cell was treated by 300μg/mL of ZTO, the effect on cell proliferation was observed by methyl thiazolyl tetrazolium（MTY）assay, and cell growth curve was made for screening the optimal treatment time. The hTert expression level of MCF-7 cell was detected by real-time quantitative RT-PCR 48 hours after treatment. Meanwhile telomerase activity was checked by TRAP Assay. Before treatment and 24, 48 and 72 hours after treatment, cells were collected for checking the DNA damage response related 53BP1 protein level by immunofluorescence and western blot methods. Results MCF-7 cell showed a decreased proliferation activity 24 hours after ZTO treatment, and showed an obvious growth repression in 48 hours. The result of real-time quantitative RT-PCR showed that the hTert expression level was re- pressed by 2.43 folds （2-△△Ct）. TRAP Assay results showed that telomerase activity was also decreased by 2.27 folds as compared with the control group. Immunofluorescence and western Blot results showed that the protein phosphorylation level of 53BP1 started to increase in 24 hours, reached the peak around the 48th hour and still retained at high level 72 hours after treatment. Conclusion ZTO can regulate telomerase activity and DNA damage response by targeting hTert as the downstream molecule, and can repress the proliferation activity in breast cancer cells MCF-7.