3 ��M) for different time periods; then protein levels of c-Abl, caspase 8, … Protection of HCT116 cells against TRAIL by selleck chemical Brefeldin A STI571 is associated with JNK and p38 signaling Since JNK and p38 MAPK are important in inducing apoptosis, we investigated their involvement in TRAIL-induced cell death, and their linkage to the action of STI571. As a result, TRAIL alone significantly induced JNK and p38 phosphorylation, but did not affect ERK activation. Pretreatment with STI571 resulted in reductions in JNK and p38 activation (Figure (Figure4A,4A, left panel). Moreover, we found that SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) could partially reverse TRAIL-induced cell death, but did not produce further increased protection in combination with STI571 (Figure (Figure4A,4A, right panel).

Nevertheless, in LNCaP and PC3 cells, neither SB203580 nor SP600125 treatment, either alone or in combination, altered TRAIL-induced cytotoxicity (Figure 4B, C, right panel). And unlike the effects in HCT116 cells, STI571 cannot alter TRAIL-induced p38 and JNK activation (Figure 4B, C, left panel). These results suggest the involvement of JNK and p38 in TRAIL-induced cell death in colon cancer cells, and the protective mechanism of STI571 might be related to both kinases. Figure 4 Protection of HCT116 cells against TRAIL by STI571 is associated with JNK and p38 signaling. HCT116 (A, D), LNCaP (B), and PC3 cells (C) were treated with TRAIL (50 ng/ml), STI571 (0.3 ��M) and/or anisomycin (10 ��M) for the time indicated. …

Following observing the ability of STI571 to inhibit TRAIL-activated stress kinases in HCT116 cells, we were wondering the stimuli-specific action of STI571. Thus we tested effects of STI571 on stress kinase activation caused by anisomycin, which is known to be a potent inducer of JNK and p38 [36]. Results revealed that anisomycin rapidly activated JNK and p38 phosphorylation in HCT116 cells, and the extents of activation were not affected by STI571 (Figure (Figure4D,4D, left panel). Moreover, anisomycin alone induced cell death, but this effect was not reversed by pretreatment with STI571, SB203580, or SP600125 (Figure (Figure4D,4D, right panel). These results suggest that STI571-elicited attenuation of stress kinase activation is not a general action, but is specific in colon cancer cells in response to the extrinsic death inducer, TRAIL.

Reduced cell susceptibility to TRAIL by STI571 is dependent on c-Abl and p73 To understand the role of c-Abl in STI571′s action, we used RNA-silencing technology. Results showed that TRAIL-induced cytotoxicity GSK-3 was reversed by c-Abl siRNA (Figure (Figure5A),5A), and under this condition, STI571-induced protection was no longer observed. Moreover, c-Abl siRNA reduced p38 and JNK activations after TRAIL treatment compared to cells transfected with scrambled control siRNA (Figure (Figure5B).5B).