Previously we have demonstrated an apoptosis inducing activity for a rat hepatocyte conditioned medium (CM) eventually mediated by acidic isoferritins (Bresgen et al., 2004). Since growth inhibitory effects have been reported for acidic isoferritins, in particular placental isoferritins (PLF) which reveal immunomodulatory activities, primary cultures of rat hepatocytes were used to elucidate the apoptosis inducing potential of isoferritins purified from rat hepatocyte culture supernatants. In addition, the role of p53 and FasL in isoferritin mediated apoptosis induction as well as the influence of proliferative stimulation (by EGF) and dexamethasone on this novel property of isoferritins in hepatocytes was also investigated. Results. Isoferritins purified from different rat hepatocyte CM as well as commercial ferritin, purified from rat liver tissue significantly (P≤0.05) stimulated apoptosis in primary rat hepatocytes. In addition, isoferritins released from hepatocytes in vitro contain a 43kDa ferritin subunit comparable to immunosuppressive PLF, and are homologous to placental immunomodulatory ferritin (PLIF). Remarkably, analysis of FasL expression and experiments with neutralizing anti-FasL antibodies, which significantly (P≤0.05) suppressed apoptosis induction by purified CM point at the involvement of the Fas pathway in isoferritin mediated apoptosis induction. In addition, isoferritin containing CM also shifted the level of p53 in primary rat hepatocytes. Furthermore, epidermal growth factor (EGF) and dexamethasone (DEX) almost completely inhibited apoptosis induced by isoferritins purified from the CM. Since EGF and DEX have been reported to inhibit the activation of caspase 8 and Bid and also upregulate antiapoptotic bcl-xL in hepatocytes, this finding suggests that proapoptotic mitochondrial pathways are also involved in isoferritin mediated apoptosis. Conclusion. Our findings provide strong evidence that primary rat hepatocytes release isoferritins with homology to PLIF, which stimulate apoptosis based on a mechanism which involves p53, activation of the Fas pathway and downstream targeting of proapoptotic mitochondrial pathways.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]