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

TRAIL and Hsp90 inhibition
17‐AAG induces cell cycle arrests and enhances TRAIL‐induced cell death
In order to examine the mechanism underlying TRAIL and 17‐AAG cytotoxicity cell cycle
progression was studied of treated cells by flowcytometric analysis of PI stained cells.
TRAIL exposure alone in H460 and A549 cells did not alter cell cycle progression (Fig. 2).
After 17‐AAG treatment however, an increase of around 15% in the portion of A549 cells
in the G2/M phase was observed compared to untreated cells; this coincided with a
decrease in the proportion of S phase cells (Fig. 2A). In H460 cells no such G2/M arrest by
17‐AAG was seen, but rather an approximately 10% increase of cells in the G1 phase was
detected together with a similar decrease in cells in S phase (Fig. 2B).
Figure 2. Effect of 17‐AAG and TRAIL on cell cycle progression. (A) A549 cells were incubated with
100 ng/ml TRAIL, 100 nM 17‐AAG or the combination for 24 h. (B) The H460 cell line was treated for
24 h with 10 ng/ml TRAIL, 100 nM 17‐AAG or the combination of these two drugs. Values are means
of at least 2 independent experiments ±s.d.
Cell death was also measured by determining the sub‐G1 fraction. A549 cells were treated
with TRAIL (50 ng/ml and 100 ng/ml) for 24 h showing only at around 5% of cell death.
Simultaneously treatment with 100 nM 17‐AAG resulted in an approximately 3‐fold
increase in sub‐G1 cells, whereas 17‐AAG alone hardly affected cell death (Fig. 3A).
Synergistic cell killing by TRAIL and 17‐AAG was also observed in H460 cells, where the
percentage of cells in the sub‐G1 increased around 3‐fold at a low concentration of TRAIL
(10 ng/ml) and showed an approximately 1.7‐fold increase upon combination with 50
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