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

Chapter 3
inhibitors and TRAIL receptor targeting agents may be an opportunity for cancer therapy
[10‐16]. In the present study, we have examined TRAIL‐induced activation of the MAPKs
p38 and JNK in TRAIL sensitive and resistant NSCLC cells. The consequences and
mechanism of activation have been examined indicating opposing effects of these two
kinases on TRAIL‐induced apoptosis.
MATERIALS & METHODS
Cell Culture
NSCLC cells A549, H460, H1299, H1975, H322, and SW1573 were cultured as monolayers
in RPMI 1640 medium supplemented with (v/v) 10% FBS, 100 units/ml penicillin, and 100
µg/ml streptomycin. Cells were maintained in a humidified 5% CO2 atmosphere at 37°C.
Cell death measurement
Cell death measurements were performed by FACS analysis as described previously [9].
Cells were seeded in 6‐well plates at a density of 300,000 cells/well. After drug exposure,
the adherent NSCLC cells were trypsinized, resuspended in medium collected from the
matching sample and centrifuged for 5 min at 1200 rpm. Next, cells were stained with
propidium iodide (PI) buffer (50 µg/ml PI, 0.1 mg/ml RNAse A, 0.1% Triton‐X, 0.1% (Tri‐)
Sodium Citrate dissolved in PBS) in the dark on ice. DNA content of the cells was analyzed
by a FACSCalibur flowcytometer (Becton Dickinson, Immunocytometry Systems, San Jose,
CA, USA) with an acquisition of 10,000 events. The sub‐G1 peak was used to determine the
extent of cell death.
Western blotting
Western blotting was performed as described previously [9]. Briefly, cells were exposed to
TRAIL for different time points or pre‐incubated with p38 and JNK inhibitors for 30 min
prior to TRAIL treatment. The cells were washed twice with ice‐cold PBS and resuspended
in lysis buffer (Cell Signalling Technology Inc.) supplemented with 0.04% protease inhibitor
cocktail (Roche, Almere, the Netherlands) and Na2VO3. Cell lysates were scraped,
transferred into a vial and centrifuged at 11,000 g at 4 °C for 10 min. Protein
concentrations were determined by the Bio‐Rad assay, according to the manufacturer’s
instruction (Bio‐Rad Laboratories, Veenendaal, the Netherlands). The following primary
antibodies were used all from Cell Signaling Technology Inc. (Danvers, MA, USA) at a
1:1000 dilution: Anti‐caspase 3 (#9662), anti‐caspase 8 (#9746), anti‐caspase 9 (#9502),
anti‐cleaved caspase 3 (#9661), phospho‐SAPK/JNK (Thr183/Tyr185, #9251), SAPK/JNK
(#9252), phospho‐p38 (Thr180/Tyr182, #9211), p38 (#9212), Mcl‐1 (#4572), RIP1 (#3493),
PARP (#9542), Bid (#2002), Bcl‐2 (#2872), Bcl‐xL (#2762). As secondary antibodies
(1:10,000 goat‐α‐mouse‐IRDye (800CW;#926‐32210 and 680;#926‐32220) or goat‐α‐
‐ 42 ‐