Function and regulation of death-associated protein kinase1 (DAPK) in colon cancer cells and macrophage-like cell line : identifcation of p38 as a novel DAPK interacting partner during TNF»-mediated apoptosis in colon cancer
Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signalling upon cytokine exposure. Recently, it has been found that a high DAPK expression in colorectal tumor cells and tumor-associated macrophages (TAMs) was correlated with a high apoptotic rate in tumor cells but not in TAMs suggesting a new function of DAPK in apoptosis induction during the interaction between colorectal tumor cells and TAMs. However, the role of DAPK regulation in macrophage-associated tumor cell death is still elusive. We used a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and HCT116 colorectal tumor cells to recapitulate DAPK-associated tumor cell death that might reflect the in vivo tumor setting. We measured Cytokines in differentiated/activated macrophage supernatants ELISA. DAPK expression and DAPK activity were determined by real-time RT-PCR, Western Blotting, and DAPK in vitro kinase assay. Co-immunoprecipitation and triple immunofluorescence labelling were used to show protein interactions. For detection of protein-dependent apoptosis induction, we used the Annexin V-binding assay, caspase 3/7 activity ELISA, Western Blotting and siRNA-transfection. Here, we show that in vitro DAPK induction and apoptosis in tumor cells was due to TNF-á release from the activated macrophages. p53 was found to act down-stream of DAPK in this scenario. Simultaneously, an early phosphorylation of p38 MAPK was observed. We identified for the first time that p-p38 MAPK co-localizes and interacts with DAPK and triggers DAPK-mediated apoptosis in the HCT116 cells. We addressed the physiological relevance of our findings and showed that supernatants of freshly isolated human macrophages were also able to induce DAPK, p-p38, and caspase 3 cleavage in HCT116 cells. We further verified the co-localization of DAPK and p-p38 proteins by immunohistochemistry analysis of human colon cancer slices. Moreover, the observed apoptosis resistance in the macrophages was mediated by DAPK. Finally, we suggest that, the cytoprotective effect of DAPK impacts the mitochondrial signalling pathway by inhibiting the caspase 3 cleavage. Altogether, we are the first to show p-p38 triggering of DAPK-activation in TNFalpha-induced apoptosis. Our findings highlight the mechanisms underlying DAPK regulation in tumor cell death evoked by immune cells.