Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-a2b in rat preneoplastic liver

QUIROGA, ARIEL DARIO; ALVAREZ, MARIA DE LUJAN; PARODY, JUAN PABLO; RONCO, MARIA TERESA; FRANCES, DANIEL ELEAZAR ANTONIO; PISANI, GERARDO BRUNO; CARNOVALE, CRISTINA ESTER; CARRILLO, MARÍA CRISTINA

Los Angeles,CA

Congreso; 98th American Asociation for Cancer Research Annual Meeting; 2007

Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-a2b in rat preneoplastic liver. Quiroga AD, Alvarez ML , Parody JP, Ronco MT, Francés DE, Pisani GB, Carnovale CE, Carrillo MC. Proceedings of the American Association for Cancer Research Vol. 48: 1336-1337; April 2007. Interferon-a2b (IFN-a2b) 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-a2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-b1. In this study, it was analyzed the involvement of reactive oxygen species (ROS) on the apoptotic mechanism induced by IFN-b2b 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-a2b, c) with IFN-a2b plus anti-TGF-¦b, d) with IFN-a2b plus ascorbic acid (ASC). IFN-a2b induced increase of TGF-b1 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-b1 levels in the culture media remained unchanged. IFN-a2b-treatment increased hepatocytes apoptosis since 20 h of culture onwards. ASC or anti-TGF-b1 treatments completely abolished it. After IFN-¦a2b treatment there were 2 peaks of ROS at 1 h and 9 h of culture. Anti-TGF-b1 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-a2b induced NADPH oxidase activity increase at 1 h of culture, reaching a maximum at 9 h. Anti-TGF-b1 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-a2b, 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-a2b apoptotic effects observed in IFN-a2b-treated rats. Results indicate that IFN-a2b induces hepatocytic TGF-b1 production and secretion  by induction of ROS formation through the activation of a membrane bound NADPH oxidase complex. TGF-b1, in turn, induces programmed cell death. On the other hand, it was also demonstrated that treatment of rats with IFN-a2b plus a ROS scavenger such as ascorbic acid, abolishes the apoptotic effect of IFN-a2b 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-a2b-induced apoptosis in hepatic preneoplastic cells.