Evidence for Two Modes of Development of Acquired Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance: INVOLVEMENT OF Bcl-xL

Jae J Song,Jee Young An,Yong Tae Kwon,Yong J Lee

doi:10.1074/jbc.m608065200

Abstract

Previous studies have shown that repeated application of TRAIL induces acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using human prostate adenocarcinoma DU-145 and human pancreatic carcinoma MiaPaCa-2 cells as a model, we now demonstrate for the first time that two states of acquired TRAIL resistance can be developed after TRAIL treatment. Data from survival assay and Western blot analysis show that acquired TRAIL resistance was developed within 1 day and gradually decayed within 6 days after TRAIL treatment in both cell lines. After TRAIL treatment, the level of Bcl-xL increased and reached a maximum within 2 days and gradually decreased in both cell lines. Bcl-xL-mediated development of acquired TRAIL resistance was suppressed by knockdown of Bcl-xL expression. Protein interaction assay revealed that during the development of TRAIL resistance, Bcl-xL dissociated from Bad and then associated with Bax. Overexpression of mutant-type Bad (S136A), which prevents this dissociation, partially suppressed the development of acquired TRAIL resistance. Thus, our results suggest that (a) dissociation of Bad from Bcl-xL and (b) an increase in the intracellular level of Bcl-xL are responsible for development of acquired TRAIL resistance.

Highlights

Previous studies have shown that repeated application of TRAIL induces acquired resistance to tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)
Using human prostate adenocarcinoma DU-145 and human pancreatic carcinoma MiaPaCa-2 cells as a model, we demonstrate for the first time that two states of acquired TRAIL resistance can be developed after TRAIL treatment
Development and Decay of Acquired TRAIL Resistance—We investigated whether TRAIL resistance develops after TRAIL treatment

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Survival Assay—Human prostate adenocarcinoma DU-145 cells and human pancreatic cancer MiaPaCa-2 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (HyClone, Logan, UT) and 26 mM sodium bicarbonate for monolayer cell culture. The cells were maintained in a humidified atmosphere containing 5% CO2 and air at 37 °C. For trypan blue exclusion assay [34], trypsinized cells were pelleted and resuspended in 0.2 ml of medium, 0.5 ml of 0.4% trypan blue solution, and 0.3 ml of phosphate-buffered saline solution (PBS). The samples were mixed thoroughly, incubated at room temperature for 15 min, and examined

RNA Interference by siRNA of

RESULTS

Data from survival assay and Western blot analysis show that

DISCUSSION

Previous studies have shown that