Retinoic acid-induced apoptosis is prevented by serum albumin and enhanced by Lipiodol in human hepatoma Hep3B cells

The effects of retinoic acid (RA) on the cell growth and viability of human hepatoma Hep3B cells were examined. We showed that removal of serum in the presence of RA results in cell death in a dose-dependent manner in human hepatoma Hep3B cells. Time-course cell death analysis showed that RA at a dose of 10 μM induces a rapid (48–72 h) fall in cell viability (>95%). The drug-induced cell death was RA-specific, since three RA analogs (retinol, retinal and retinol acetate) did not show any cytocidal activity at an equimolar dose. Fluorescence microscopy and DNA fragmentation analysis showed that Hep3B cells treated with RA underwent a death process highly reminiscent of apoptosis, with chromatin condensation, nuclear fragmentation and the presence of a 180–200 bp DNA fragment ladder. Additionally, we found that RA-induced apoptosis was reduced by 70–80% when the medium was supplemented with serum albumin (human and bovine) at a concentration of 0.05%. However, a variety of known growth factors were ineffective in preventing RA-induced apoptosis. Preincubating serum and serum albumin with Lipiodol restored the apoptotic effects of RA demonstrated in serum-free systems. These data suggest that the binding of RA by serum albumin may have reduced the bioavailability of RA, restricting its apoptotic effects on Hep3B cells. Blocking RA–albumin interactions with a lipid lymphographic contrast medium (Lipiodol) may improve the bioavailability of RA and significantly enhance its apoptotic effect on human hepatoma Hep3B cells.