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

Chapter 5
DISCUSSION
In the present study, we found synergistic activity between 17‐AAG and TRAIL in TRAIL‐
sensitive H460 and TRAIL‐resistant A549 cells. Hsp90 is amongst others a chaperone of
proteins regulating cell cycle progression [25]. Cell cycle arrest in the G2‐M phase by
Hsp90 inhibitors has been related to Plk1 [26]. In Hodgkin’s lymphoma cells, 17‐AAG
decreased the expressions of Plk1 and cyclin B1, which regulates the transition of G2‐M
phase to mitosis and is activated by Plk1 [27]. Although 17‐AAG affected cell cycle
progression differently in A549 and H460 cells, inducing a G2/M‐arrest versus a G1 arrest,
respectively, synergistic activity was the result of enhanced apoptosis activation.
17‐AAG enhanced TRAIL‐induced cell death in H460 cells, and sensitized resistant A549
cells for TRAIL. The synergistic cytotoxicity was caspase‐dependent; 17‐AAG stimulated
caspase‐8 activation in both cell lines. In H460 cells, enhanced caspase‐9 activation was
also involved in increased apoptosis activation. In order to obtain insight in the underlying
mechanism of apoptosis sensitization by 17‐AAG we have examined several Hsp90
chaperones that are known to regulate apoptosis.
RIP1 and IκB are two essential components of the NF‐κB pathway and are client proteins
of Hsp90 [28]. RIP1 expression was reported to decrease upon Hsp90 inhibition leading to
sensitization for TRAIL‐induced apoptosis in breast and lung cancer cells [18;19]. Inhibition
of NF‐κB as a molecular mechanism of 17‐AAG‐dependent TRAIL sensitization has been
described in colon and lung cancer cells [18;24]. In the present study, 17‐AAG resulted in a
decrease in RIP expression in A549 cells that was accompanied by an increase in RIP
cleavage. This was not observed in H460 cells that already demonstrate RIP1 cleavage
following TRAIL treatment. Since RIP1 silencing in A549 cells does not lead to sensitization
for TRAIL [21], RIP1 is unlikely to be the primary mediator of the effect of 17‐AAG on
TRAIL sensitivity in our study. TRAIL‐dependent phosphorylation of IκBα, leading to
degradation of this inhibitory protein and activation of the NF‐κB pathway was observed
only in TRAIL resistant A549 cell line. Several studies have shown that the activation of NF‐
κB confers resistance to TRAIL‐induced apoptosis, including NSCLC, and inhibition of NF‐κB
can lead to TRAIL‐sensitization [11;29;30]. However, 17‐AAG did not attenuate the
phosphorylation of IκBα induced by TRAIL, making this pathway an unlikely effector of 17‐
AAG activity in our study.
IAP family members, in particularly XIAP and survivin have also been linked to resistance
to TRAIL‐induced apoptosis [31]. In resistant glioma cells, 17‐AAG sensitized for TRAIL by
reducing survivin levels through enhanced proteasomal degradation [23]. Furthermore,
the 17‐AAG/TRAIL combination has been reported to cause down‐regulation of XIAP,
leading to enhanced caspase‐3 activation in colon cancer cells [24]. In the present study,
we found no differences in survivin and XIAP expression in NSCLC cell lines between single
and combined treatment with 17‐AAG and TRAIL.
Examination of Akt revealed an inhibitory effect of 17‐AAG on Akt phosphorylation only in
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