Carbon monoxide protects primary mouse hepatocytes from induced apoptosis by activating the nitric oxide pathway

Background and Aims: The anti-inflammatory, anti-viral and anti-apoptotic enzyme heme oxygenase-1 (HO-1) cleaves free heme by producing iron, biliverdin and carbon monoxide (CO). The anti-apoptotic effect of HO-1 has been attributed to its product CO, but the mechanism is not yet clear. Since HO-1 is over-expressed in many types of cancer, including hepatocellular carcinoma (HCC), we hypothesize that the HO-1 product CO provides survival advantages in cancer cells by preventing apoptosis. Therefore, the aim of our investigations is to identify and specifically inhibit CO-dependent anti-apoptotic signalling pathways. Methods: Freshly isolated primary mouse hepatocytes were preincubated with the CO-donor methylene chloride (MC) for six hours and with specific pathway inhibitors. Apoptosis was induced by subsequent coincubation with the transcriptional inhibitor Actinomycin D (ActD) and TNF. Cytotoxicity was measured by lactate dehydrogenase (LDH) release 20 hours after apoptosis induction. Results: CO significantly reduced cytotoxicity of ActD and TNF in mouse hepatocytes. Inhibition of inducible nitric oxide synthase (iNOS) or cGMP-dependent protein kinase (PKG) partly abolished the protective effects of CO. Likewise, NO was able to protect from ActD/TNF induced cytotoxicity. Conclusions: CO and NO protect mouse hepatocytes from apoptosis in vitro. We postulate a mechanism by which CO induces iNOS, producing NO, which activates soluble guanylate cyclase (sGC), resulting in an elevation of the second messenger cGMP. The anti-apoptotic effect might therefore be provided by the NO/cGMP/PKG cascade.

apoptosis - carbon monoxide - signal transduction