towards improved death receptor targeted therapy for ... - TI Pharma

Chapter 3
P38 and JNK have opposing effects on TRAIL‐induced apoptosis
P38 and JNK have been described to mediate both pro‐ and anti‐apoptotic activities
depending on the stimulus applied and the cell type studied [23]. In order to explore their
role in TRAIL‐induced apoptosis in NSCLC cells we employed selective chemical JNK and
p38 inhibitors. As shown in Fig. 2a, JNK inhibition with SP600125 resulted in enhanced
TRAIL‐induced cell death in H460 cells, indicating anti‐apoptotic activity of JNK. P38
inhibition with SB203580, on the other hand, decreased the level of cell death, indicating
that p38 has pro‐apoptotic activity in TRAIL signaling (Fig. 2b). The chemical inhibitors
SP600125 and SB203580 have been generated against the ATP binding pockets of JNK and
p38, respectively, and do not affect the phosphorylation status of the kinases themselves
or only partially in case of autophosphorylation [24;25]. As a control and in line with this
we did not observe a decrease in TRAIL‐dependent phosphorylation of JNK and p38 in the
presence of these inhibitors (Fig. 2f). Furthermore, at concentrations lower than 25 µM
hardly any significant cross‐inhibition of other MAPKs have been reported, which has been
linked to the unique binding properties of these chemicals in the ATP‐binding sites of the
enzymes [26]. Nonetheless, to further corroborate our findings we also employed specific
siRNAs against JNK and p38 that upon transfection strongly reduced the expression of
these kinases in H460 cells when compared to control siRNA transfected cells (Fig 2c, d). In
resistant A549 cells, JNK and p38 inhibition did not significantly affect TRAIL‐dependent
apoptosis (Fig. 2a, b). Next, the effect of kinases inhibition on TRAIL‐induced caspases
activation was studied by Western blotting following 3 or 24 h treatment. JNK inhibition by
SP600125 appeared to stimulate caspase‐8, Bid, RIP1, caspase‐9, caspase‐3 and PARP
cleavage, as judged by more intense cleaved products and/ or a decrease in the full length
proteins (Fig. 2e). Inhibition of p38 with SB203580 in combination with TRAIL hardly
affected cleavage of caspase‐8, Bid and RIP1, whereas cleavage of caspase‐9, caspase‐3
and PARP was somewhat decreased compared to TRAIL alone.
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