Abstract B226: Induction of growth arrest DNA damage-inducible gene 45 beta (GADD45b) expression contributes to sorafenib-induced apoptosis in hepatocellular carcinoma (HCC) cells

Abstract

Abstract GADD45b, which is associated with cellular stress response and apoptosis regulation, was frequently under-expressed in HCC. In a previous study we found GADD45b belongs to a group of sorafenib-regulated genes that are independent of mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK)/ extracellular signal-regulated kinase (ERK) signaling pathway. The present study explored the role of sorafenib-induced GADD45b expression in HCC cells. HCC cell lines tested included HepG2, Hep3B, Huh-7, and Huh-7R, a sorafenib-resistant cell line established by continuously exposure of Huh-7 cells to sorafenib. GADD45b expression was examined by quantitative real-time PCR and western blotting. Control of GADD45b transcription was examined by luciferase reporter assay using a series of reporter plasmids with deletions or site-directed mutants of the 5′-flanking region of GADD45b promoter. Apoptosis was analyzed by measuring the subG1 fraction and annexin V staining using flow cytometry. Both the mRNA and the protein levels of GADD45b was increased after sorafenib but not after the MEK inhibitor CI-1040 or U0126 treatment, suggesting that GADD45b induction was independent of cellular MEK/ERK activity. GADD45b induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6–7 µM) but less so prominent in sorafenib-resistant HCC cells (Hep3B and Huh-7R, IC50 12–15 µM). Knockdown of GADD45b expression by siRNA partially abrogated the pro-apoptotic effects of sorafenib in Huh-7 and HepG2 cells but not Hep3B cells. The region −339/−267 in the 5′-flanking region of GADD45b promoter, which contained AP-1 (−298/−292) and Sp1 (−285/−277) binding sites, was found crucial for GADD45b induction by sorafenib. Binding of c-Jun and Sp1 to GADD45b promoter was confirmed by chromatin immunoprecipitation. Sorafenib also increased the phosphorylation of JNK in sorafenib-sensitive but not in sorafenib-resistant HCC cells. Sorafenib-induced GADD45b expression can be alleviated by treatment with the specific JNK inhibitor SP600125. Our data indicated that induction of GADD45b expression, which is mediated by cellular JNK/c-Jun signaling pathway but not MEK/ERK signaling, contributes to sorafenib-induced apoptosis in HCC cells. Failure of GADD45b induction may confer sorafenib resistance. GADD45b may be used as a predictive biomarker for sorafenib sensitivity in HCC. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, NSC98-3112-B-002-037) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B226.