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

Chapter 5
ng/ml TRAIL (Fig. 3B). Apoptotic cell death was more precisely analyzed by determining
the level of phosphatidyl serine (PS) exposed on the cell surface following incubation with
annexin V‐FITC and together with determining PI uptake. 17‐AAG combined with TRAIL (2
concentrations) was able to enhance the level of annexin V‐FITC‐only positive cells by
around 3‐fold, indicative of early apoptosis, when compared to TRAIL alone treatment
(Fig. 3C). In H460 cells 17‐AAG addition resulted in an approximately more than 2‐fold or
1.5 fold increase in early apoptotic cells treated with a low and high concentration TRAIL,
respectively, and when compared to TRAIL alone (Fig. 3D). These apoptotic levels were
comparable to the fraction of cells detected in the sub‐G1 fraction (Fig. 3A and B).
Furthermore, under these conditions no significant differences between TRAIL and the
TRAIL/17‐AAG combination were detected in late apoptosis (white bars). Cells stained
with only PI that define necrotic cell death (grey bars) were not detected. Thus, 17‐AAG
can enhance apoptosis induced by TRAIL in these NSCLC cells.
Figure 3. 17‐AAG enhances TRAIL‐induced apoptosis. (A) A549 cells were treated with 50 ng/ml or
100 ng/ml TRAIL with or without 100 nM 17‐AAG and the percentage of sub‐G1 cells was measured
in PI‐stained cells following flow‐cytometry. (B) Sub‐G1 levels in H460 cells after 24 h incubation
with 10 ng/ml TRAIL, 50 ng/ml TRAIL, 17‐AAG (100 nM) or the combination. (C) A549 cells treated
with TRAIL (50 ng/ml and 100 ng/ml), 17‐AAG (100 nM) or the combination for 24 h were double‐
stained with annexin‐V/PI and analyzed by flow cytometry. Distinction can be made between early
apoptosis, late apoptosis and cell death. (D) Similar experiment with H460 cells exposed to TRAIL (10
ng/ml and 50 ng/ml), 17‐AAG (100 nM), or the combination. Values are means of at least two
independent experiments ±s.d.
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