17-DMAG体外对结肠癌HT-29细胞增生和凋亡的影响

Effects of heat shock protein 90 inhibitor 17-DMAG on proliferation and apoptosis of colon cancer cell line HT-29

目的观察17-二甲胺乙胺基-17-去甲氧基格尔德霉素（ 17- dimethylmninoeihylanino- 17- de-methoxygehtanamyein, 17- DMAG ）对人结肠癌HT-29细胞增生抑制及诱导其凋亡的作用。方法用CCK-8检测17-DMAG时结肠癌HT-29细胞增生的影响；DAPI染色在倒置荧光显做镜下观察17-DMAG作用于HT-29细胞24h后细胞核的形态；AnnexinV—FITC／PI蚁染法流式细胞检测细胞捌亡率；免疫印迹实验分析Caspase-3蛋白表达水平变化。结果17-DMAG呈时间-剂量-依赖性抑制HT-29细胞增牛。0．1μmol／L、0．25μmoL／L、0．5μmol／L、1．0μmol／L、2．5μmol／L和0．5μmol／L作用于HT-29细胞24h后，细胞增生抑制率分别为（14．36±0．95）％、（22．17±1．15）％、（28．45±1．16）％、（35．04±1．58）％、（46．85±2．44）％和（57．19±2．06）％；作用48h后，细胞增生抑制率分别为（20．80±1．17）％、（27．55±0．65）％、（33．33±1．23）％、（46．20±4．76）％、（55．45±4．47）％和（61．75±2．72）％；作用72h，细胞增生抑制率分别为（29．62±2．27）％、（39．19±1．74）％、（44．29±2．00）％、（50．66±2．17）％、（58．84±3．18）％和（70．74±2．65）％。DAPI染色倒置荧光显微镜观察0．25μmol／L、0．5μmol／L、1．0μmol／L与2．5μmol／L的17-DMAG作用HT-29细胞24h，均可见到细胞凋亡的改变。Annexin V-FITC双染法检测结果显示，正常对照组HT-29细胞24h的自然总凋亡率（早期±晚期）为（2．72±0．57）％，0．25μmol／L、0．5μmol／L、1．0μmol／L和2．5μmol／L17-DMAG干预24h后细胞总凋亡率分别为（5．38±0．46）％、（6．88±0．52）％、（10．44±0．32）％与（17．87±4．66）％，不同浓度组的细胞凋亡率比较差异有统计学意义（P〈0．05）。17-DMAG药物作用HT-29细胞24h，随着药物剂量的增加，cleavedCaspase-3的表达有增加趋势。结论17-DMAG体外呈时间-剂量依赖性抑制HT-29细胞增生，诱导其通过Caspase-3途径凋亡。

Objective To investigate the effects of Hsp90 inhibitor 17-DMAG on proliferation and apoptosis of human colon cancer cell line HT-29. Methods HT-29 cells were treated with 17-DMAG. The cell proliferation inhibition rate was evaluated by CCK-8 assay. Apoptosis of HT-29 cells by 17-DMAG was delineated by DAPI stai- ning assay and Annexin V PI double labeling FCM was used to determine cell apoptotic rate. Fmthermore, Westen blotting analysis was used to determine easpase-3 and cleaved caspase-3 protein expression. Results 17-DMAG time-dose-dependently inhibited the proliferation of HT-29 cells. After 0.1μmol/L, 0.25 μmol/L, 0.5μmol/L, 1.0 μmol/L, 2.5 μmol/L and 5 μmol/L 17-DMAG exposured for 24 hours, the cell proliferation inhibition rate was （14.36±0.95）%, （22. 17 ±1. 15）%, （28.45±1. 16）%, （35.04±1.58）%, （46.85 ±2.44）%, （57.19 ± 2.06）% respectively, after exposured for 48 hour, the cell proliferation inhibition rate was increased to （20.80 ± 1.17）%, （27.55 ±0.65）%, （33.33 ± 1.23）%, （46.20 ±4.76）%, （55.45 ±4.47）%, （61.75 ± 2.72 ） % respectively, after exposure for 72 hours, the cell proliferation inhibition rate was to （29.62 ± 2.27 ） % , （39.19 ± 1.74）%, （44.29 ±2.00）%, （50.66 ±2.17）%, （58.84 ±3.18）%, （70.74 ±2.65）%. DAPI stai- ning showed that HT-29 ceils treated with 17-DMAG displayed chromatin condensation and nuclear fragmentation which are typical changes of apoptosis. Annexin V PI double labeling FCM showed that when HT-29 cells were ex- posed to 0, 0.25, 0.5, 1.0 and 2.5（μmol/L） 17-DMAG for 24 hours, the total apoptotic rate for 24 hours was （2.72 ±0.57）%, （5.38 ±0.46）%, （6.88 ±0.52）%, （10.44 ±0.32）% and （17.87 ±4.66）% respective- ly. （P 〈0.05）. In addition, the expression of procaspase-3 decreased, while cleaved caspase-3 increased in the presence of 17-DMAG at different concentrations for 24 hours. Conclusion 17-DMAG can time-dose-dependently inhibit proliferation and promote apoptosis of HT-29 cells in vitro.