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

Chapter 3
previously established H460‐derived cell line, H460‐CrmA [28]. In this cell line, the cowpox
virus‐encoded anti‐apoptotic protein cytokine response modifier A (CrmA), an inhibitor of
caspase‐8, is stably overexpressed. Apoptosis by TRAIL was efficiently prevented in CrmA
expressing cells and inhibition of p38 or JNK under these conditions had no effect, as
depicted in Fig. 4a. As expected, TRAIL‐induced caspase‐8 and RIP1 cleavage was not
observed in the H460‐CrmA cells as shown in Fig. 4b. Furthermore, inhibition of caspase‐8
activation did not affect p38 phosphorylation after TRAIL exposure (Fig. 4c). However,
TRAIL‐induced JNK phosphorylation was not detected in H460‐CrmA cells, indicating that
caspase‐8 activity is important for JNK activation by TRAIL.
Figure
4. TRAIL‐induced caspase‐8 activation is required for JNK phosphorylation. (a) H460 cells
overexpressing the caspase‐8 inhibiter CrmA (H460‐CrmA) were treated with and without TRAIL
alone, or in combination with SP600125 (10 µM) or SB203580 (10 µM) for 24 h. Cell death was
determined by FACS analysis of the sub‐G1 fraction. Means of triplicate experiments (± s.d.) are
shown. (b) Caspase‐8 and RIP1 cleavage by TRAIL in H460 and H460‐CrmA cells. (c) Western blots
displaying p38 and JNK and phosphorylated variants. The cells were treated with 50 ng/ml TRAIL for
3 h.
Mcl‐1
is a mediator of pro‐apoptotic and anti‐apoptotic effects of p38 and JNK,
respectively.
Anti‐apoptotic Mcl‐1 and Bcl‐2 are known transcriptional targets of both p38 and JNK
signaling [23;29;30]. Therefore, we studied the effect of p38 and JNK inhibition on Mcl‐1
and Bcl‐2 mRNA levels using qPCR in H460 cells. As shown in Fig. 5a, SP600125 treatment
decreased Mcl‐1 expression, whereas p38 inhibition by SB203580 enhanced transcription.
Bcl‐2 transcription was not affected by these treatments. Examination of protein levels by
Western blotting revealed a reduction in Mcl‐1 expression upon JNK inhibition, while p38
inhibition clearly enhanced Mcl‐1 expression (Fig. 5b). Similar results were obtained when
silencing JNK and p38 expressions with specific siRNAs (not shown). It thus appears that
normally JNK activity enhances Mcl‐1 expression, while in contrast p38 activity reduces
Mcl‐1 levels. To further explore the role of Mcl‐1 in this context its expression was silenced
in H460 cells using a specific siRNA (Fig. 5c). In these Mcl‐1 knockdown cells TRAIL‐induced
cell death was enhanced by approximately 100% when compared to control H460 cells.
‐ 50 ‐