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

p38 and JNK induction by TRAIL
Notably, Mcl‐1 knockdown cells were not responding to either the pro‐apoptotic effect of
JNK inhibition or anti‐apoptotic effect of p38 inhibition, respectively (Fig. 5d). This
indicates that Mcl‐1 is a mediator of the apoptosis modulatory effects of p38 and JNK.
Figure 5. Mcl‐1 is a mediator of the anti‐apoptotic effect of JNK and pro‐apoptotic effect of p38 in
TRAIL signaling. (a) Quantitative RT‐PCR was performed to determine mRNA expression of Mcl‐1 and
Bcl‐2 after exposure to 10 µM SP600125 or 10 µM SB203580 for 24 h in H460 cells. (b) Mcl‐1 and Bcl‐
2 expression in H460 cells treated with TRAIL (50 ng/ml) ± SP600125 (10 µM) or ± SB203580 (10 µM)
for 24 h. (c) Mcl‐1 expression was silenced using siRNA in H460 cells and the expression levels were
determined by Western blotting and compared with control shRNA transfected cells. (d) H460‐
shCTRL and H460‐shMcl‐1 cells were treated with TRAIL (50 ng/ml) with and without SP600125 (10
µM) or SB203580 (10 µM) and effect on apoptosis was determined.
P38
and JNK activation in a panel of NSCLC cells
Finally, the effect of p38 and JNK inhibition on TRAIL sensitivity was examined in an
extended panel of NSCLC cells. TRAIL sensitivity was variable in these cells (Fig. 6a). H1299
and SW1573 cells showed less than 5% apoptosis when corrected for background
apoptosis levels. H322 cells displayed intermediate sensitivity with approximately 10%
apoptosis, whereas H1975 cells with around 25% apoptosis were most sensitive. Co‐
incubation with the JNK inhibitor SP600125 led to an increase in the amount of cell death,
mainly in the H1975 and H322 cell lines, in which the increase in sub‐G1 levels was around
8% and 6%, respectively. The increase in TRAIL‐induced cell death by SP600125 was only
2% in H322 cells and 3% in SW1573 cells. The p38 inhibitor SB203850 did have little or no
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