The purpose of this study was to evaluate the effect of mouse TRA-8 anti-DR5 monoclonal antibody or TRA-8 combined with gemcitabine on pancreatic carcinoma cell lines and xenografts and to explore their antitumor mechanisms. MIA PaCa-2 and S2-013 cells were treated with gemcitabine (0-300 nM) for 24 h followed by TRA-8 (0-1,000 ng/ml) alone or in combination with gemcitabine for 24 h. Cell survival was calculated from ATP levels measured using the ATPLite assay. Cells were also treated with TRA-8 (300 ng/ml), gemcitabine (300 nM) or the combination for 24 h then probed for caspase 3, phospho IκB-α, transcription factor NF-κB (c-Rel, p65), Bcl-XL, Bax and XIAP by Western blot analysis. Cytotoxicity results showed that MIA PaCa-2 cells were sensitive to TRA-8 but resistant to gemcitabine. In contrast, S2-013 cells were sensitive to TRA-8 and gemcitabine. Combined treatment showed additive cytotoxicity with TRA-8 plus gemcitabine against MIA PaCa-2 cells but synergistic cytotoxicity against S2-013 cells. Activation of caspase 3 in MIA PaCa-2 cells was induced by TRA-8, and this effect was enhanced by combination therapy. Activation of caspase 3 in S2-013 cells was only seen with the combined treatment. TRA-8 or TRA-8 plus gemcitabine decreased the expression of Bcl-XL and increased Bax in MIA PaCa-2 cells, thus the ratio of Bcl-XL/Bax was decreased. No change in Bcl-XL was found in S2-013 cells, while gemcitabine or the combination treatment decreased Bax levels. In S2-013 cells, gemcitabine or the combination treatment inhibited phosphorylation of IκB-α and decreased the levels of both c-Rel and p65. Anti-apoptotic protein XIAP was also down-regulated in these samples. The inhibition of XIAP, c-Rel and p65 was greatest in S2-013 cells treated with TRA-8 plus gemcitabine. There was increased phosphorylation of IκB-α in MIA PaCa-2 cells treated with TRA-8 and gemcitabine but no change in NF-κB levels. In MIA PaCa-2 cells, anti-apoptotic protein XIAP was down-regulated only by combination therapy. In conclusion, 1) TRA-8 and TRA-8 plus gemcitabine treatment were effective in inducing apoptosis of pancreatic carcinoma cell lines. 2) In MIA PaCa-2 cells, apoptosis induced by TRA-8 or combination treatment was correlated with downregulation of anti-apoptotic proteins Bcl-XL or XIAP and upregulation of Bax. 3) Synergistic cytotoxicity of TRA-8 and gemcitabine in S2-013 cells was correlated with inhibition of NF-κB. The mechanism by which TRA-8 enhanced the gemcitabine-induced inhibition of NF-κB in S2-013 cells is unknown and needs further investigation. Supported by SPORE in Pancreatic Cancer Grant # P50 CA10195 and Sankyo Co., Ltd.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]