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

Chapter 5
targets and subsequent proteasomal degradation thus removing possible anti‐apoptotic
proteins resulting in sensitization for apoptosis [15]. The small molecule, 17‐allylamino‐17‐
demethoxy‐geldanamycin (17‐AAG) is a potent Hsp90 inhibitor that induces cell cycle
arrest and apoptosis in a variety of tumor cells, including NSCLC [16].
In this study we investigated whether 17‐AAG enhances the apoptosis‐inducing effect of
TRAIL in NSCLC cells. Furthermore, the mode of action of the synergy of these two
compounds was studied.
MATERIALS & METHODS
Cell lines and reagents
The NSCLC cell lines, A549 and H460, were cultured in RPMI‐1640 supplemented with 10%
Fetal Calf Serum (FCS). The cells were maintained in a humidified incubator at 37 °C and in
a 5% CO2‐atmosphere. TRAIL (PeproTech EC Ltd, London, UK) was aliquoted in PBS at a
final concentration of 500 ng/ml and stored at ‐20 °C. 17‐AAG was diluted in DMSO (stock
solution of 1mM) and stored at ‐20 °C. The caspase inhibitors Z‐VAD‐fmk (general caspase
inhibitor), Z‐IETD‐fmk (caspase‐8) and Z‐LEHD‐fmk (caspase‐9) were purchased from
Bachem AG, dissolved in DMSO (10 mM or 20 mM) and stored at ‐20 °C.
Drug cytotoxicity assays
Drug cytotoxicity was determined using the methyl thiazole tetrazolium (MTT) assay. Cells
were seeded in 96‐wells plates with a cell density of 9,000 cells/well. After 24 h, enabling
attachment, cells were exposed (in triplicate) to increasing concentrations of the two
drugs for 24 h. Next, the medium was removed and 50 µl MTT (10% diluted in HBSS
buffer) per well was added and incubated for 1‐3 h at 37 °C. After the incubation, 150 μl
DMSO per well was added and following 10‐15 min slowly shaking, the absorbance of the
plate was measured at 540 nm with a TECAN plate reader by using the XFLUOR4 program.
IC50 values were determined from the graphs. Synergy of TRAIL and 17‐AAG was
determined by calculation of the combination index (CI) using Calcusyn (Calcusyn Biosoft,
Cambridge, UK) as described earlier [17]. For calculation of the CI, only values above a
fraction affected (FA) of 0.5 were used, equivalent to 50 ‐ 100% growth inhibition. A CI <
0.9 indicates synergism and >1.1 antagonism.
Cell cycle and cell death measurements
Cells were plated at a density of 200,000 cells/well in 6‐wells plates. After 24 h enabling
attachment, the medium was replaced by medium containing the drug(s) as indicated.
When indicated, caspase inhibitors were added to a final concentration of 20 µM, 2 h
prior to drug treatment. After drug exposure cells were trypsinized, resuspended in
medium collected from the matching sample and centrifuged for 5 min at 1200 rpm. After
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