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

TRAIL and Hsp90 inhibition
17‐AAG enhancement of TRAIL‐induced apoptosis is caspases‐dependent
To study the role of caspases in the synergistic interaction of TRAIL and 17‐AAG, the
expression of caspases‐8, ‐9, and ‐3 were visualized by Western blotting. Time‐course
experiments showed a slight increase in caspase‐8 cleavage after combined treatment
compared to TRAIL alone in both A549 and H460 cells after 3 h incubation (Fig. 4A and B).
An increase in caspase‐9 cleavage after 17‐AAG and TRAIL treatment was found in H460
cells from 3 h post‐treatment on, as indicated by enhanced detection of cleaved caspase‐9
or reduction of the procaspase form, which was not observed in A549 cells. Enhancement
of caspase‐3 and PARP cleavage by 17‐AAG could not be clearly detected in both cell lines.
Figure 4. 17‐AAG sensitizes for TRAIL in a caspase‐dependent manner. (A) and (C) Representative of
two independent Western blots of caspase‐8,‐9,‐3 and PARP of A549 cells after treatment with 100
ng/ml TRAIL, 100 nM 17‐AAG or the combination for the indicated time‐points. In a similar way,
H460 cells were examined after exposure to 10 ng/ml TRAIL or/and 100 nM 17‐AAG. As control for
protein loading, membranes were probed with an antibody recognizing β‐actin. (B) and (D) Sub‐G1
fractions of A549 cells pre‐treated with the pancaspase inhibitor zVAD‐fmk (20 µM), zIETD‐fmk (20
µM), or zLEHD‐fmk (20 µM) for 2 h and subsequent treatment with either TRAIL (100 ng/ml), 17‐AAG
(100 nM) or the combination of both drugs for 24 h. Similarly, H460 cells were treated with TRAIL
(10 ng/ml), 17‐AAG (100 nM) or the combination for 24 h. Means of triplicate determinations ±SEM.
The involvement of caspases in the sensitizing effect of 17‐AAG on TRAIL‐induced
apoptosis was further examined by using synthetic caspases inhibitors. The broad‐caspase
inhibitor z‐VAD‐FMK (20 µM) completely inhibited TRAIL‐induced apoptosis determined
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