Induction of apoptosis by S-nitrosoglutathione and Cu²⁺ or Ni²⁺ ion through modulation of bax, bad, and bcl-2 proteins in human colon adenocarcinoma cells

In this study, the amount of S-nitrosoglutathione (GSNO) was measured spectrophotometrically at 334 nm. A spontaneous decrease in absorbency at 334 nm was detected when GSNO was exposed to 37 °C and a high pH (pH 8.0). We investigated the catalytic roles of various metal ions on the decomposition of GSNO. The degradation of GSNO (0.5 mM) was enhanced by the presence of Cu²⁺ and Ni²⁺ ions. The amount of nitric oxide (NO) released from GSNO degradation was estimated by the Griess reaction based on nitrite accumulation. The results indicated that nitrite production was elevated at least twofold in the presence of Cu²⁺. Our study further indicated that Cu²⁺ enhanced GSNO-induced apoptosis in human colon adenocarcinoma HT 29 cells. We also found that copper ions modulated the expression of bad, bax, and bcl-2 in GSNO-treated HT 29 cells. The levels of bax and bad proteins in treated cells were significantly elevated about fourfold to sixfold when compared with mock-treated cells 24 h after combined treatment with GSNO plus Cu²⁺ or Ni²⁺. On the other hand, significant inhibition of bcl-2 occurred in HT 29 cells with simultaneous treatment of GSNO with Cu²⁺ (or Ni²⁺). It seemed that Cu²⁺ and Ni²⁺ can enhance the decomposition of GSNO, which liberates NO to activate the pathways. Our results demonstrated that the apoptotic effects induced by GSNO were promoted by Ni²⁺ and Cu²⁺ through two different mechanisms: depletion of intracellular glutathione and triggering of NO release from GSNO, which then promotes NO-induced apoptosis in human cells.