TRA-8, an agonistic mouse monoclonal antibody to human DR5, generates apoptosis in a variety of human tumor cell lines and inhibits tumor growth in vivo (Nature Med 7:954-60, 2001 and Clin Cancer Res 9:3731-41, 2003). In this study, we investigated the response of breast cancer cells and xenografts to combination treatment with TRA-8 and chemotherapy and the mechanisms associated with the induction of apoptosis. Cytotoxicity was detected using an ATPLite assay. Treatment responses were compared in 2LMP, a TRA-8 sensitive subclone of the MDA-MB-231 cell line, LCC6, a subclone of the MDA-MB-435 cell line with intermediate sensitivity, and BT474 resistant cells, via protein expression changes detected by Western blot analysis. Adriamycin or Velcade, a proteosome inhibitor, in combination with TRA-8 produced additive cytotoxicity against TRA-8 sensitive 2LMP breast cancer cells and synergism against LCC6 cells and the BT474 resistant line. In vivo the same combinations inhibited the growth of LCC6 xenografts. TRA-8 induced caspase-3, 8 and 9 cleavage in 2LMP and LCC6 cells at 2-3 h, but no activation of caspases was seen in BT474 cells at 3 h. Caspase activation correlated with TRA-8 cytotoxicity in each line. When cells were pretreated with Adriamycin or Velcade for 24 h, activation of these caspases was seen in all three cell lines with a greater induction in combination with TRA-8 treatment. Cleavage of poly (ADP-ribose) polymerase (PARP), a product of caspase-3 activity, was seen in 2LMP and LCC6 cells with TRA-8 alone and when combined with either drug. BT474 cells showed PARP cleavage only with the combination treatment. X-linked inhibitor of apoptosis (XIAP) remained unmodulated in 2LMP and LCC6 cells, but was decreased by combination treatment with TRA-8 and Adriamycin or Velcade in BT474 cells. Another IAP, survivin, showed no change with TRA-8 treatment alone, but increased with combined Adriamycin or Velcade treatment in BT474 cells. Velcade alone and combined with TRA-8 increased the p27 level in BT474 cells. No change in expression was seen for Bax, Bcl-2 or Bcl-XL with the individual or combination treatments. In summary, TRA-8 in combination with Adriamycin or Velcade produced additive or synergistic cytotoxicity and activation of caspases 3, 8 and 9 in breast cancer cells. TRA-8 resistant BT474 cells showed synergistic sensitization by Adriamycin and Velcade. This sensitization was detected by caspase activation and XIAP modulation. Future studies will further elucidate the mechanisms by which TRA-8 kills cells in combination with Adriamycin, Velcade and other chemotherapeutic agents. Supported in part by DOD DAMD-17-02-1-0264, NCI P50 CA8019 and Sankyo Co. Ltd.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]