EFFECTS OF SILIBININ ON ETHANOL-MEDIATED HEPATOCELLULAR CARCINOMA PROGRESSION IN MALE AND FEMALE MICE
Ethanol is a significant risk for the development of hepatic cirrhosis and progression to hepatocellular carcinoma (HCC). While females are more susceptible to the deleterious effects of ethanol, the incidence of cirrhosis and HCC is significantly higher in males than females. Previous reports identify a role for oxidative stress and inflammation in mediating the effects of ethanol on hepatocarcinogenesis, suggesting antioxidants may protect the liver from the deleterious effects of ethanol. Plant-derived antioxidants have been used for millennia to treat liver disease, yet rigorous scientific validation of efficacy is rare. Silibinin, a flavinoligand derived from Milk thistle (Silybum marianum), is one such compound indicated to possess potent antioxidant properties and historically cited as a hepatoprotectant. In these studies we sought to determine the effects of ethanol on HCC promotion and progression using in vitro and in vivo rodent models. Using dedifferentiated rat hepatoma cells (H4IIE) that constitutively express alcohol-metabolizing enzymes, we demonstrated ethanol enhanced cellular oxidative stress, and proliferation. Pre-treatment of cells with silibinin significantly inhibited ethanol metabolism (via cytochrome P4502E1), oxidative stress, and proliferation. These data indicate silibinin may be an effective antioxidant with which to inhibit ethanol-dependent HCC progression. We next employed an in vivo diethylnitrosamine (DEN) model of hepatocarcinogenesis in male and female mice (21-25 day old). Using this model foci develop within ~20 weeks and HCC within ~40 weeks. Mice were fed dietary silibinin (0.5%, (w/w), 9 weeks) with or without chronic alcohol feeding (10/20% (v/v) in drinking water, alternate days, 8 weeks) to coincide with foci or HCC development. Using this approach, ethanol preferentially promoted foci formation in males versus females, effects exacerbated during HCC progression. Ethanol-dependent increases in tumor incidence and volume in males occurred concomitant with increased CYP2E1 expression and activity, depleted glutathione/increased oxidative stress, and hepatic injury. Conversely, ethanol did not alter tumor incidence or volume in females, and changes in oxidative stress were not evidenced. Analysis of cellular apoptosis demonstrated increased cell death in female mice maintained on ethanol that developed DEN-induced hepatic foci. Inclusion of silibinin in the diet did not significantly reduce DEN-initiated foci formation or HCC progression in either males or females, with or without ethanol. Of note, measures of liver injury and oxidative stress were improved in animals fed silibinin during foci development, yet silibinin exacerbated the effects of ethanol during later-stage HCC progression. Finally, analysis of immune system signaling cascades demonstrated a net increase in cytokine expression and Th1/Th2 effectors in female, ethanol fed mice, effects inhibited by silibinin. In conclusion these data demonstrate silibinin effectively inhibits ethanol-dependent HCC cell growth in vitro. However, while chronic ethanol feeding promoted foci formation and HCC progression in male mice in vivo, dietary silibinin did not reverse these effects. Conversely, ethanol failed to significantly affect tumor incidence or burden in females. This lack of ethanol-dependent hepatocarcinogenesis enhancement in females is due, at least in part, to altered immune and apoptotic responses.