Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-alpha2b in rat preneoplastic liver. Quiroga A, Alvarez ML, Parody JP, Ronco MT, Francés D, , Pisani GB, Carnovale CE, Carrillo MC.

QUIROGA A; ALVAREZ ML; PARODY JP; RONCO MT,; FRANCéS D; PISANI GB; CARNOVALE CE; CARRILLO MC

Los Angeles, California, Estados Unidos

Congreso; American Association for Cancer Research. Annual Meeting.; 2007

American Association for Cancer Research.

Interferon-á2b (IFN-á2b) is used in the preventive treatment of patients with severe hepatic illness such as hepatitis B or C and hepatocarcinomas. In a previous work, using a rat liver preneoplastic model, we have demonstrated that IFN-á2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-â1. In this study, it was analyzed the involvement of reactive oxygen species (ROS) on the apoptotic mechanism induced by IFN-á2b in rat preneoplastic liver. For in vitro studies, male Wistar rats were subjected to a 2-phase model of hepatic preneoplasia (initiation-promotion). Hepatocytes were obtained and cultured at different times: a) without any treatment, b) with IFN-á2b, c) with IFN-á2b plus anti-TGF-â1, d) with IFN-á2b plus ascorbic acid (ASC). IFN-á2b induced increase of TGF-â1 mRNA levels at 4 h of culture and a two-fold increase of TGF-b1 secretion at 7 hours of culture. ASC totally abolished the increase of TGF-â1 mRNA levels, and TGF-â1 levels in the culture media remained unchanged. IFN-á2b-treatment increased hepatocytes apoptosis since 20 h of culture onwards. ASC or anti-TGF-b1 treatments completely abolished it. After IFN-á2b treatment there were 2 peaks of ROS at 1 h and 9 h of culture. Anti-TGF-â1 did not block the production of the first peak of ROS whereas totally blocked the appearance of the second one. ASC abolished the production of both peaks. IFN-á2b induced NADPH oxidase activity increase at 1 h of culture, reaching a maximum at 9 h. Anti-TGF-â1 did not block activation at 1 h, but did at 9 h. In all cases, ASC totally blocked NADPH oxidase activation. Since ASC abolished all the apoptotic effects induced in vitro by IFN-á2b, we determined the relevance of ROS on the onset of the apoptotic process in vivo in the whole preneoplastic liver. Treatment of preneoplastic rats with IFN-á2b + ASC abolished the IFN-á2b apoptotic effects observed in IFN-á2b-treated rats. Results indicate that IFN-á2b induces hepatocytic TGF-â1 production and secretion  by induction of ROS formation through the activation of a membrane bound NADPH oxidase complex. TGF-â1, in turn, induces programmed cell death. On the other hand, it was also demonstrated that treatment of rats with IFN-á2b plus a ROS scavenger such as ascorbic acid, abolishes the apoptotic effect of IFN-á2b in rat preneoplastic livers, leading to an increase of the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling molecules in the IFN-á2b-induced apoptosis in hepatic preneoplastic cells.