We have tested for the effect of LPS alone, which had no effect on the viability of the Bosutinib molecular weight colon tumor cells (supplemental Figure S1 at http://ajp.amjpathol.org) using the MTT and the cytotoxicity assays. The apoptosis in tumor cells was assessed after 24 to 72 hours of incubation with the macrophage-conditioned media by Annexin-V-FITC staining and after 48 hours by caspase-3/7 activity assays. There was a significant apoptosis induction (2.5-fold) in tumor cells treated with actM supernatants, starting early at 24 hours (6.37% to 15.87%) and continuing until 72 hours compared with untreated HCT116 tumor cells (Figure 1A). In contrast, diffM supernatants induced only an early increase in the percentage of apoptotic HCT116 tumor cells at 24 hours (6.37% to 17.87%), but cell death diminished at later time points (Figure 1A).

Considering the 48 hours time point, these findings were in accordance with caspase activity data, showing threefold up-regulation in caspase 3/7 activity in HCT116 tumor cells treated with actM supernatants (Figure 1B). Thus, our data suggest that actM may release a soluble factor into the media, which might be responsible for apoptosis induction in HCT116 tumor cells. To investigate whether DAPK is involved in the observed apoptosis induction, we examined DAPK mRNA and protein expression. Real-time RT-PCR revealed that the steady state level of DAPK mRNA remained unchanged during the whole experimental setup (data not shown). However, there was an increase in the DAPK protein level even after 6 hours of incubation with supernatants from diffM and actM (Figure 1C).

While the DAPK protein level was found to be increased only slightly at later time points after incubation with diffM supernatants, the DAPK protein level continued to increase markedly after incubation with actM supernatants, similar to the time course of apoptosis induction (Figure 1C). These data suggest that macrophage-mediated secretion of cytotoxic factors by actM might induce DAPK protein accumulation without affecting its gene transcription. Figure 1 Macrophage-induced cell death in HCT116 cells is accompanied by DAPK up-regulation. A: Annexin-V measurements of HCT116 cells (ctrl) and HCT116 cells subjected to diffM supernatant (sup.) or actM supernatant. B: HCT116 subjected to diffM supernatant or … Similar results were obtained using freshly isolated PBMCs treated with 10 ng/ml LPS for 4 hours.

The supernatants were given to HCT116 tumor cells. Indeed, we observed an up-regulation of DAPK after 6 hours and a further reinforcement after 24 hours (Figure 1D). Simultaneously, after 6 hours and 24 hours cleaved fragments of caspase 3 could be detected as signs of apoptosis induction (Figure 1D). Increase in TNF�� and IFN�� Release on Macrophage Activation Earlier studies showed that TNF�� and IFN�� promote DAPK-dependent apoptosis.2,1 These cytokines might be the cytotoxic Cilengitide factors secreted by actM in our experimental setting.