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

Chapter 5
by sub‐G1 levels in the absence and presence of 17‐AAG in A549 and H460 cells (Fig. 4C
and D). Inhibition of caspase‐8 by the synthetic inhibitor z‐IETD‐FMK (20 µM) also
prevented synergistic apoptosis activation by TRAIL and 17‐AAG in both cell lines.
However, the caspase‐9 inhibitor, z‐LEHD‐FMK (20 µM) decreased sub‐G1 levels after
TRAIL/ 17‐AAG exposure only in H460 cells and not in the A549 cell line. Overall, these
findings are in line with the caspase cleavage patterns observed in the Western blots.
Effects of 17‐AAG and TRAIL on client proteins of Hsp90
Hsp90 stabilizes multiple oncoproteins such as EGFR, Her‐2, NF‐κB, and Akt. In addition,
RIP1 has also been found to be a target of Hsp90 inhibitors [18;19]. We evaluated the
expression and phosphorylation status of a number of client proteins that could be
involved in mediating the synergistic effect of 17‐AAG and TRAIL. RIP1 is part of the
secondary signaling complex known to be involved in pro‐survival signaling by TRAIL [20].
The TRAIL/17‐AAG combination decreased the expression of RIP1 in A549 cells in
particular after 6 h incubation, which was accompanied with an increase in RIP1 cleavage.
After 24 h incubation this effect on RIP1 was reduced. H460 cells treated with TRAIL
resulted in RIP1 cleavage that was not clearly enhanced by addition of 17‐AAG. However,
as shown previously silencing of RIP1 expression with a short hairpin RNA did not sensitize
the resistant A549 cells for TRAIL‐induced apoptosis, making a role of RIP1 in 17‐AAG‐
dependent sensitization unlikely [21]. Activation of ERK, Akt and NF‐κB can negatively
regulate TRAIL‐induced apoptosis and activation of these pathways has been correlated
with TRAIL resistance in NSCLC cells as we and others have previously shown [10;11;21].
Therefore, we extended our study to examine the effect of 17‐AAG on Akt, ERK and
IĸB/NF‐κB. We observed TRAIL‐induced Akt and ERK phosphorylation in the resistant A549
cells and levels were attenuated by 17‐AAG to different extents (Fig. 5A). In H460 cells, p‐
ERK levels were decreased after 17‐AAG exposure for 6 and 24 h. TRAIL treatment had no
effect on p‐ERK levels in these sensitive cells. TRAIL treatment for 1 h induced IκBα
phosphorylation only in A549 cells, but co‐incubation with 17‐AAG did not abrogate the
phosphorylation of this kinase.
Next, we examined the expression of survivin in NSCLC cells. Survivin is a member of the
Inhibitor of Apoptosis (IAP) protein family and regulates apoptosis by inhibiting effector‐
caspases. Survivin, which interacts with Hsp90 [22], has been identified to play an
important role in the sensitizing effect of 17‐AAG in TRAIL‐induced apoptosis in malignant
gliomas, as 17‐AAG downregulated the expression of this protein [23].
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