ABT-737 Induces Expression of the Death Receptor 5 and Sensitizes Human Cancer Cells to TRAIL-induced Apoptosis

Andrew S. Kraft,Karthikeyan Kandasamy,Jin H. Song

doi:10.1074/jbc.m802511200

Abstract

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

Highlights

Ptotic death of human cancer cells while sparing normal human cells (1– 4)
This loss of cell viability occurs with an increase in apoptosis in 12 different renal, prostate, and lung cancer cell lines (Fig. 2C)
To examine whether Mcl-1 functions in the renal can- whether the changes in Bax and Bak induced by ABT-737 cer cell line PV10, specific Small Interfering RNA (siRNA) duplexes targeting Bcl-2, Bcl- and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were associated with activation of the mitochonxL, and Mcl-1 were transfected into ABT-737-resistant cells drial apoptotic pathway, we measured the release of the mito

Summary

EXPERIMENTAL PROCEDURES

Cell Lines, Antibodies, and Reagents—Human cancer cell lines were grown in either Dulbecco’s modified Eagle’s medium or RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Pellets were washed with phosphate-buffered saline and incubated for 40 min at 4 °C with either mouse IgG1 antibody as a negative control, a mouse monoclonal antibody against amino acids 1–52 of Bak (AM03, clone TC100; Oncogene Research Products), or a mouse monoclonal antibody against amino acids 12–24 of Bax (clone 6A7; BD Biosciences). After washing with phosphate-buffered saline, the binding of antibody was visualized with fluorescein isothiocyanate-conjugated anti-mouse IgG (1:200) (Sigma). Silencing of Gene Expression with Small Interfering RNA (siRNA) and Gene Transfection—Gene silencing was achieved by transfecting cells with siRNA duplexes using the Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s instructions and siRNAs duplexes targeting human DR5 (5Ј-AACTACCAGAAAGGTATACCT-3Ј), Mcl-1 (5Ј-AAAAGTATCACAGACGTTCTC-3Ј), Bcl-2 (5Ј-AACCGGGAGATAGTGATGAAG-3Ј), Bcl-xl (5Ј-TAGGGTGGCCCTTGCAGTTCA-3Ј), Bax (5Ј-AACATGGAGCTGCAGAGGATGA-3Ј) or siRNA duplexes targeting human Bak 5Ј-AAGCGAAGTCTTTGCCTTCTC-3Ј. PCR detection system and iQ5 optical system software analysis were used for quantifying gene expression (Bio-Rad, version 2.0).

RESULTS

Regulation of Tumor Cell Sensitivity to Combined or Single Agent

DISCUSSION