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

Chapter 6
TRAIL‐dependent cell death. At 72 h, levels of dead cells were not higher than with TFT
alone. This seems to contrast with the synergistic effect measured in the MTT assay.
However, the sub‐G1 level is an indication of the ratio between apoptotic and non‐
apoptotic cells, while the MTT assay also takes growth inhibition into account.
The contribution of caspase‐dependent cell death was investigated by adding the broad‐
range caspase inhibitor zVAD to the cultures. This resulted in a partial inhibition of around
30 to 40% of TFT‐induced cell death, indicating that caspase‐independent cell death is a
major contributor to cell death (Fig. 3B). TRAIL‐induced apoptosis, as expected, was
completely prevented by zVAD. In the first 24 h of combined treatment, the induction of
cell death was completely inhibited by zVAD further indicating that at this time‐point TFT
enhanced or sensitized TRAIL‐dependent cell death in both H460 and A549 cells. After
another 48 h incubation with TFT alone, cell death was partially prevented by zVAD in line
with the observations in TFT alone treated cells, suggesting that TFT‐dependent
cytotoxicity contributed to cell death at longer exposure times.
A
B
Figure 3. Caspase‐dependent and ‐independent cell death activation. (A) TRAIL, TFT and the combination were
examined for time‐dependent cell death activation by determining the percentage of sub‐G1 cells in PI‐stained
cells. (B) Comparison of cell death activation in the presence or absence of zVAD‐fmk. Combined treatments of
24 and 48 h included a 24 h combination of TRAIL and TFT followed by 48 or 24 h period in drug free medium
(DFM), respectively. TFT at 5 µM, TRAIL (10 ng/ml in H460), TRAIL (150 ng/ml in A549) were used for single and
combined treatments. Values are means of three independent experiments ± SEM.
‐ 108 ‐