一氧化氮供体经由caspase依赖途径诱导SMMC-7721细胞凋亡

Nitric oxide donor induced apoptosis in SMMC-7721 cells by caspase-dependent

目的研究NO供体——新型偶氮鎓二醇盐衍生物(JS-K)体外对人肝癌SMMC-7721细胞增殖抑制作用。方法用噻唑兰法检测JS-K对细胞增殖的影响;倒置显微镜观察24 h后细胞形态学变化;以Annexin V-FITC/PI双染法检测细胞凋亡率;以DAF-FM DA荧光探针检测细胞中NO水平;以蛋白免疫印迹法观察JS-K对B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(caspase-9)蛋白表达变化。结果 JS-K能显著抑制细胞增殖,呈一定的剂量、时间依赖性。倒置显微镜观察到JS-K组出现变圆、皱缩等形态学变化;3个浓度JS-K和对照组(5-FU 100μmol·L^(-1))作用细胞24 h,凋亡率分别为(19.12±0.07)%,(40.81±0.03)%,(75.53±0.12)%,(56.70±0.07)%,均高于空白组的(2.72±0.01)%,差异有统计学意义(P<0.05)。3个浓度JS-K作用细胞24 h后,细胞内NO水平分别是(151.2±0.1)%,(248.6±0.2%),(347.6±0.4)%,均高于空白组100%,差异有统计学意义(P<0.05);加入Carboxy-PTIO后,JS-K组NO水平由(318.6±0.2)%降至(307.6±0.3)%,差异有统计学意义(P<0.05)。Bax/Bcl-2比值也升高,3个浓度JS-K和对照组依次为(0.54±0.02)%,(1.19±0.03)%,(1.32±0.02)%,(1.20±0.01)%,均高于空白组的(0.20±0.02)%,差异有统计学意义(P<0.05)。加入caspase-9抑制剂(Z-LEHD-FMK)和caspase-3抑制剂(Z-VAD-FMK)后,可显著减少JS-K诱导的细胞凋亡。结论 JS-K可通过caspase依赖性途径诱导SMMC-7721细胞凋亡。

Objective To investigate the apoptosis induction effects of JS-K, {O2 （2, 4 - dinitrophenyl） 1 - [（4 - ethoxycarbonyl） piperazin - 1 - yl ] diazen - 1 - ium - 1,2 - diolate } , a nitric oxide do- nor, on human hepatoma SMMC -7721 cell line. Methods Cell proli- feration was measured by 3 - （4,5 - dimethyhhiazol - 2 - yl） - 2,5 - di- phenyhetrazolium bromide （MTF） assay. Morphological changes after in- cubation for 24 h were observed under inverted microscope. The apopto- tic rate of cells was determined with AnnexinV - FITC/PI double stai- ning. And the NO levels were tested with DAF- FM DA fluorescent probe. The expression of B - cell lymphoma - 2 （ Bcl - 2）, Bcl - 2 asso- ciated X protein （Bax）, cleaved cysteinyl aspartate specific proteinase - 3 （caspase -3 ）, and cleaved cysteinyl aspartate specific proteinase -9 （caspase- 9 ） were detected by Western blot. Results JS -Ksignificantly inhibited the growth of SMMC -7721 ceils, and showed a certain dose- and -time dependent manner. Cells morphology became mellow and smaller in experimental group under inverted microscope. With the increase of JS -K concentration, the apoptosis ratio increased gradually which were （19. 12 ± 0. 07 ）%, （40. 81± 0. 03）%, （75.53 ± 0. 12）%, and the apoptosis ratio in control group was （56.70 ± 0. 07 ）%, the four groups higher than the blank group （2. 72±0. 01 ）% （P 〈 0. 05 ）. The intracellular NO levels were rising in three concentrations test groups with （151.2±0. 1）%, （248.6±0. 2）%, （347.6±0. 4）%, higher than the blank group with 100% （P 〈0. 05 ）. The NO levels of JS- K group decreased from （318.6 ± 0. 2）% to （307.6 ± 0.3 ）% after adding Carboxy- PTIO （ P 〈 0. 05 ）. The ratio of Bax/Bcl - 2 in three concentrations test groups and control group were （0. 54± 0. 02 ） %, （1.19 ± 0.03）%, （1.32 ± 0.02）%, （1.20 ± 0.01）%, respectively, higher than the blank group with （0. 20 ± 0.02 ）% （all P 〈 0. 05; ）. Also demonstrated that in the presence of caspase -9 inhibitor （Z -LEHD -FMK 50 μmol · L^- 1 ）, and the easpase - 3 inhibitor （ Z - DEVD - FMK 50 μmol · L ^- 1 ） , JS - K induced apoptosis were signi- ficantly reduced. Conclusion JS -K as a NO donor can significantly induce apoptosis through a caspase -dependent manner in SMMC - 7721 cells.