Dual induction of caspase 3- and transglutaminase-dependent apoptosis by acyclic retinoid in hepatocellular carcinoma cells

BACKGROUND: Hepatocellular carcinoma has a high mortality rate due to its rate of recurrence. Acyclic retinoid prevents recurrence of hepatocellular carcinoma in patients after surgical removal of their primary tumors by inducing apoptosis in hepatocellular carcinoma cells, although the molecular mechanisms of action are not understood. METHODS: Human hepatocellular carcinoma cells in culture, as well as nude mice transplanted with hepatocellular carcinoma cells and rats given with N-diethylnitrosamine were treated with acyclic retinoid. Changes in activated caspase 3 and transglutaminase 2 (TG2) levels, Sp1 cross-linking and its activities, expression of epidermal growth factor receptor, and apoptotic levels were measured. RESULTS: Acyclic retinoid simultaneously stimulated the activation of caspase 3, and the expression, nuclear localization and crosslinking activity of TG2, resulting in crosslinking and inactivation of the transcription factor, Sp1, thereby reducing expression of epidermal growth factor receptor and cell death in three hepatocellular carcinoma cell lines. These effects were partially restored by a caspase inhibitor, transfection of antisense TG2, restoration of functional Sp1, or an excess of epidermal growth factor. Nuclear expression of TG2 and crosslinked Sp1, as also activated caspase 3 were found in both hepatocellular carcinoma cells transplanted into nude mice and cancerous regions within the liver in N-diethylnitrosamine-induced hepatocarcinogenesis model in rats, following treatment of animals with acyclic retinoid. CONCLUSIONS: Treatment with acyclic retinoid produces a dual activation of caspase 3 and TG2 induced apoptosis of hepatocellular carcinoma cells via modification and inactivation of Sp1, resulting in reduced expression of epidermal growth factor receptor.