Background: Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. Bortezomib, a proteasome inhibitor, has been approved by FDA for refractory multiple myeloma. In this study, we showed that bortezomib sensitizes resistant HCC cells to TRAIL-induced apoptosis.

Methods: TRAIL resistant human HCC cells, including HuH7, Hep3B and Sk-Hep1, were studied. Cells were exposed to various concentrations of bortezomib or TRAIL or their combinations for 24 hours. Apoptosis was examined by both flow cytometry for sub-G1 fraction and cell death ELISA for fragmented DNA. The signaling change of PI3K/Akt pathway was assessed by western blot for phospho-Akt.

Results: HCC cells, including HuH7, Hep3B and Sk-Hep1, show significant resistance to TRAIL-induced apoptosis (up to 1000 ng/ml). The combination of bortezomib (as low as 50 nM) and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found PI3K/Akt pathway played a very important role in mediating TRAIL sensitization of bortezomib. Our first evidence showed that bortezomib downregulated phospho-Akt (serine 473) in a dose- and time-dependent manner in TRAIL-treated HCC cells. Secondly, we found that LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis, indicating the importance of PI3K/Akt pathway in mediating TRAIL resistance in HCC cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib.

Conclusions: Bortezomib sensitizes resistant HCC cells to TRAIL-induced apoptosis at clinical achievable concentrations, and this effect is at least partly mediated via the inhibition of PI3K/Akt pathway. Supported by NTUH95A11, NSC95-2314-B-002-072, NTU95R0066-BM-1-02, DOH94-TD-B-111-001 and DOH95-TD-B-111-001

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA