For antigen retrieval, pretreatment was performed by microwave heating in 1 mmol/L sodium citrate puffer (30 minutes, 600 W, pH 6.0). ABT888 Incubation of each one series with anti-DAPK (mouse monoclonal antibody, dilution 1:500, BD Transduction Laboratories, Heidelberg, Germany), anti-p-p38 MAPK (rabbit monoclonal antibody, 1:100, Cell Signaling Technology, Boston, MA), and anti-M30 CytoDeath (mouse monoclonal antibody dilution 1:75, Roche Diagnostics, Basel, Switzerland) was conducted at room temperature for 12 hours and followed by PBS-washing. Positive immunohistochemical reactions were revealed using the iVIEW DAB Detection Kit (Ventana, Germany) as chromogen substrate. Specimens were counterstained with hematoxylin and mounted with DEPEX. Samples were examined by two different reviewers blinded to other data.
All aspects of this study were reviewed and approved by the Ethics Committee of the Medical Faculty of Magdeburg and all patients provided informed consent. Semiquantitative Assessment of Immunohistochemical Protein Expression and Apoptosis For DAPK and p-p38 protein expression, cytoplasmic staining intensity, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (1 = <10%; 2 = 10 to 50%; 3 = 51 to 80%; 4 = >80%) were scored semiquantitatively, resulting in an immunoreactive score (staining intensity �� positive cells) with a possible maximum of 12 points. For DAPK, an immunoreactive score of 0 to 3 points was considered as ��low expressing�� compared with samples with an immunoreactive score of 4 to 12 points, which where referred to ��high expressing.
�� Apoptosis was determined as a percentage of M30-labeled tumor cells and scored as 0 (<1%), 1 (1% to 5%), 2 (5% to 10%), 3 (10% to 15%), or 4 (>15%). Statistical Analysis Data are presented as the means of triplicates from three separate experiments �� SEM. Statistical comparisons of the amounts of cytokines and immunohistochemical protein expression between various subgroups were performed by Student��s t-test. The level of statistical significance was set at P < 0.05. Results Macrophage-Mediated Batimastat Killing of HCT116 Tumor Cells and Up-Regulation of DAPK Expression In our previous studies, DAPK protein expression in colorectal tumor cells and their tumor-associated macrophages was found to be highly correlated. Furthermore, the increase in the DAPK level was associated with higher apoptosis rate in colorectal tumor cells.19 To determine whether tumor-associated macrophages and the secretion of their cytotoxic mediators might induce apoptosis in these tumor cells, we used a cell culture assay to simulate the events that might occur in the in vivo tumor setting.