Differences in TRA-8 anti-DR5 antibody response with Adriamycin by modulation of Akt, cell cycle and apoptotic pathways.

LB-228

Introduction: TRA-8 anti-DR5 antibody induces apoptosis in a variety of cancer cell lines both in vitro and in vivo. Breast cancer cell lines have varying sensitivities to TRA-8-mediated apoptosis, but Adriamycin can enhance sensitivity and in some cell lines reverse resistance to TRA-8. We previously reported that TRA-8 induced caspase-3, 8 and 9 cleavage in sensitive 2LMP and LCC6 breast cancer cells, but not in resistant BT474 cells. TRA-8 combined with Adriamycin or Velcade generated caspase cleavage, PARP cleavage and reduced Bid in each cell line. We explored the mechanistic differences among breast cancer cell lines after TRA-8, Adriamycin or combination treatment via modulation of Akt, cell cycle and additional apoptotic pathways. Methods: Cytotoxicity against 2LMP, a TRA-8 sensitive subclone of the MDA-MB-231 cell line, and BT474 resistant breast cancer cells, was detected using an ATPLite assay. Protein expression was detected by Western blot. JC-1 assays measured changes in mitochondria membrane potential. FACS detection of PI staining determined cell cycle phase. Results: Comparisons of phospho-Akt levels between breast cancer cells revealed p-Akt in BT474 with no detection in 2LMP and LCC6 cells. In BT474 cells, p-Akt was reduced with combination TRA-8 and Adriamycin treatment. However, treatment with two Akt inhibitors, including LY294002, did not sensitize BT474 cells to TRA-8 cytotoxicity. Adriamycin induced an accumulation of cells in the G2/M phase of the cell cycle in 2LMP and LCC6 cells. TRA-8 resistant BT474 cells showed S and G2/M arrest in response to Adriamycin. A higher Sub-G1 or apoptotic fraction was detected in the combination Adriamycin and TRA-8 treated cells. Adriamycin increased expression of S and G2 phase cyclins, A and B1 in 2LMP and BT474 cells. However, a cdk/cyclin activating phosphatase, cdc25C was phosphorylated and decreased by Adriamycin in BT474 cells, as well as c-myc. BT474 cells treated with TRA-8 or Adriamycin alone failed to activate apoptotic pathways, but combination treatment did activated apoptosis. Deregulation of the cell cycle by Adriamycin may have allowed for intrinsic apoptotic activation by TRA-8 to proceed with combination treatment. Reduced mitochondrial membrane potential was detected in 2LMP and LCC6 cells treated with TRA-8 alone, with a greater reduction in combination with Adriamycin and only with combination treatment in BT474 cells. Adriamycin reduced expression of Bcl-XL and Mcl-1 in BT474 cells alone and in combination with TRA-8, which removes inhibition of intrinsic apoptosis. Conclusions: Adriamycin reduced constitutive p-Akt in BT474 TRA-8 resistant cells, but inhibition of Akt was not sufficient to sensitize cells. In BT474 breast cancer cells, Adriamycin caused cell cycle arrest, decreased anti-apoptotic proteins and mitochondrial membrane potential, and sensitized cells to TRA-8 induced apoptosis.