NOSH‐aspirin a dual nitric oxide‐and hydrogen sulfide‐releasing hybrid reciprocally regulates NF‐κB: S‐nitrosylation vs S‐sulfhydration

Abstract

Recently we reported the design of a hybrid aspirin with the ability to release nitric oxide (NO) and hydrogen sulfide (H2S), two important signalling molecules of physiological relevance, we have dubbed this new agent NOSH‐aspirin. It has been shown that NO can directly modify sulfhydryl residues of proteins through Snitrosylation and induce apoptosis. S‐sulfhydration of proteins by H2S may be analogous to that of protein S‐nitrosylation by NO leading to hydropersulfide (‐SSH) formation. In vitro: aspirin and NOSH‐ASA inhibited HT‐29 human colon cancer cell growth, the IC50s being >; 5000 μM and 0.05 ± 0.003 μM at 24h, respectively. NOSH‐aspirin reduced NF‐κB protein levels, and activated caspase‐3 enzyme in a dose‐and time‐dependent manner. Based on the biotin switch assay, which detects S‐NO and S‐SH proteins depending on presence or absence of ascorbate, we observed that a number of proteins from NOSH‐aspirin treated HT‐29 cells showed increased S‐nitrosylation and decreased S‐sulhydration. Pretreatment of the cells with carboxy‐PTIO and NaF, abrogated these effects. NOSH‐aspirin also S‐nitrosylated and S‐sulfhydrated NF‐κB p65 and denitrosylated and desulfhydrated caspase‐3 in a time‐dependent manner in these cells. In vivo: Liver lysates from rats treated with NOSH‐ASA showed dose‐dependent increases in S‐nitrosylation and decreases in S‐sulfhydration of NF‐κB p65. Thus, sulfhydration and nitrosylation both appear to regulate NF‐κB p65 in a reciprocal manner. These data provide a posttranslational mechanistic role for NO and H2S, and a rational for the chemopreventive effects of NOSH‐aspirin. Supported in part by NIH grant R24 DA018055