335 VEGF-A GENE SILENCING AND IMMUNOSTIMULATION COMBINED IN ONE SIRNA INCREASED ANTI TUMORAL THERAPY EFFICACY IN AN HCC ORTHOTOPIC FIBROTIC TUMOR MODEL

Abstract

Four HBV associated-HCC cell lines, SNU-739, SNU-761, SNU-878 and SNU-886, which had been established from HCC patients with chronic HBV infection, were used to study the expression of PPARr and they were cultured in the 10 mmol/L to 100 mmol/L concentration of PPARr synthetic agonists, troglitzone, pioglitazone, rosiglitazone, and its natural ligand, 15deoxy-D12, 14-prostaglandin J2 (15d-PGJ2). Cell growth was determined via MTT assay in 24, 48 and 72 hour intervals. The cell cycle was analyzed by flow cytometry. DNA fragments were measured for apoptosis assay. Caspase 3, 8, and 9 activities were also determined by colorimetric protease assay. The amount of bcl-2 and bax mRNA was quantified by RT-PCR. The expression of PPARr was all found in the above HBV-associated HCC cell lines. Cell growth was inhibited by all the PPARr agonists doseand time-dependently. Especially, the effect of pioglitazone and 15d-PGJ2 was prominent. Cell cycle analysis revealed an increased proportion of cells in sub-G1 phase only by pioglitazone and 15d-PGJ2. In apoptosis assay, DNA fragments were also increased in all the HCC cell lines when treated with pioglitazone and 15d-PGJ2, but the effect of troglitazone and rosiglitazone was minimal. Pioglitazone and 15d-PGJ2 were found to increase caspase-3 and 9 activities but not caspase-8 and they decreased the level of expression of antiapoptotic factor, bcl-2, but not the proapoptotic factor, bax. Apoptosis induced by pioglitazone and 15d-PGJ2 was revealed caspasedependent. The pancaspase inhibitor and capase-3 inhibitor inhibited the apoptosis induced by various PPARr agonists dose-dependently. In conclusion, these findings suggest that pioglitazone and 15d-PGJ2 have antineoplastic effects on HBV-associated HCC cells by induction of apoptosis through a caspase dependent intrinsic pathway.