ABSTRACT
OBJECTIVE To investigate the effects of nitric oxide (NO) donor, O2-{2,4-dinitro-5-[4-(N-methylamino) benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) on apop-tosis in human hepatocarcinoma cells. METHODS Proliferation of HepG2 cells treated with PABA/NO 7.5, 15.0 and 30.0μmol · L-1 was measured with Cell Counting Kit-8 (CCK-8) assay, the morphological features were observed under fluorescence microscopy, the level of NO was measured by DAF-FM DA staining, the apoptosis rate was determined by Annexin Ⅴ-FITC staining, mitochondrial membrane potential was determined by Rhodamine 123 staining, and protein expressions of Bcl-2, Bax, cleaved caspase 3, cytochrome c (Cyt c) and apoptosis inducing factor (AIF) were measured by Western blotting analysis. RESULTS Compared with cell control group, PABA/NO could obviously inhibit the proliferation of HepG2 cells (P<0.01). IC50 value of HepG2 cells treated with PABA/NO for 24 h was (10.8±0.6)μmol·L-1. The cells became round, deformed and appeared shrunken.The level of NO was increased and the fluores-cence intensity was 121 ± 9 (P<0.05), 174 ± 31 (P<0.05) and 230 ± 43 (P<0.01). The apoptosis rate was increased from (2.9 ± 0.5)％ to (17.0 ± 4.5)％ (P<0.01), (39.8 ± 5.4)％ (P<0.01) and (74.3 ± 45.2)％ (P<0.01). The fluorescence intensity of Rh123 was reduced from 668±69 to 605±73, 420±65 (P<0.05) and 242±47 (P<0.01). Compared with cell control group, PABA/NO down-regulated Bcl-2, up-regulated Bax and activated cleaved caspase 3. Meanwhile, the expression of Cyt c in the cytoplasm was increased from 0.15±0.04 to 0.27±0.06 (P<0.05), 0.38±0.07 (P<0.01) and 0.82±0.16 (P<0.01). The expression of AIF in the nucleus was increased from 0.183±0.032 to 0.231±0.011, 0.682±0.020 (P<0.01) and 0.966± 0.090 (P<0.01). Addition of carboxy-PTIO (NO scavenger) 50 μmol · L- 1 blocked PABA/NO-induced down-regulation of Bcl-2, up-regulation of Bax and cleaved caspase 3 activation. Additionally, up-regu-lation of Cyt c in the cytoplasm and up-regulation of AIF in the nucleus were also blocked by carboxy-PTIO in PABA/NO-treated HepG2 cells (P<0.01). CONCLUSION PABA/NO may induce HepG2 cell apoptosis through a mitochondrial pathway.