ABT-737 induces expression of the death receptor 5 and sensitizes human cancer cells to TRAIL-induced apoptosis

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.

FIGURE 1.

ABT-737 synergizes with TRAIL to induce apoptosis. A, PV10 cells and KRC/Y cells were treated with ABT-737 at the indicated doses in the absence or presence of TRAIL (100 ng/ml) for 24 h. Cell viability was determined by an acid phosphatase assay (see “Experimental Procedures”) (mean ± S.D., n = 4). B, phase-contrast microscopy of PV10 cells and KRC/Y cells after combined treatment with ABT-737 (10 μm) and TRAIL (100 ng/ml) for 3 h. C, Western blot of extracts of PV10 cells treated with ABT-737 at the indicated doses in the absence or presence of TRAIL (100 ng/ml) for 3 h. Arrows indicate caspase (Casp) or PARP cleavage products. D, Western blot analysis of KRC/Y cells treated with DMSO, 100 ng/ml TRAIL, 10 μm ABT-737, or the combination. Arrows indicate caspase and PARP cleavage products.

FIGURE 2.

ABT-737 enhances TRAIL-induced apoptosis in renal, prostate, and lung cancer cells but not in normal kidney cells. A, renal cancer (A498, ACHN, and 786-0), prostate cancer (PC3R and LNCaP), and human embryonic kidney cells 293 (HEK293) were treated with DMSO, 100 ng/ml TRAIL, 10 μm ABT-737, or a combination of the two agents for 24 h. Cell viability was determined by an acid phosphatase assay (mean ± S.D., n = 4). B, cleavage of caspase (Casp)-8, -9, and -3 and Bid was examined on the Western blots. Arrows denote procaspase-8 (p55 and p53), first cleavage fragments (p47 and p43), and the active p18 form of caspase-8; procaspase-9 (p47) processed to produce the active p37 and p35 forms; procaspase-3 (p32) processed to produce active p21 and p17 products; and full-length Bid (p22) and p15 truncated Bid. C, percentages of cell death in response to ABT-737, TRAIL, and ABT-737 plus TRAILs were assessed by the trypan blue exclusion assay. Each cell line was treated with either DMSO, 100 ng/ml TRAIL, 10 μm ABT-737, or the combination for 24 h (mean ± S.D., n = 4).

FIGURE 3.

BH3 proteins play a role in controlling resistance to ABT-737 and TRAIL. A, expression patterns determined by Western blot analysis of Bcl-2 family and apoptosis pathway proteins in 14 cell lines. B, PV10 cells were treated individually with siRNAs to either Bcl-2, Bcl-xl, or Mcl-1, and extracts of these cells were examined by Western blotting for the levels of these proteins. C, PV10 cells transfected with either control, Bcl-2, Bcl-xl, or Mcl-1, and siRNA duplexes were then treated with DMSO, 100 ng/ml TRAIL, ABT-737 10 μm, or the combination for 24 h. Cell viability was measured by the acid phosphatase assay (mean ± S.D., n = 4).

FIGURE 4.

Bax is required for apoptosis induced by ABT-737 and TRAIL. A, activation of Bax or Bak was determined by FACS analysis using monoclonal N-Bax or N-Bak antibodies. PV10 or DU145 cells were treated with DMSO, 100 ng/ml TRAIL, 10 μm ABT-737, or the combination of TRAIL and ABT-737 for 24 h followed by FACS analysis. Isotype mouse IgG1 antibody was used as a negative control. PV10 cells were treated with 20 μm Z-VAD-fmk prior to the addition of ABT-737, TRAIL, or the combination as described in the first part of this panel. B, S100 cytosol fractions from PV10 and DU145 cells were isolated and examined by Western blot analysis for the release of cytochrome c (Cyt c) and Smac, levels of cleaved caspase-8 (Casp-8) and PARP, and β-actin. C, Bax-deficient (Bax^-/-) HCT116 cells and DU145 cells were treated with TRAIL in combination of various doses of ABT-737 for 24 h. Compared with nontreated control, cell viability was assessed by an acid phosphatase assay (mean ± S.D., n = 4). D, left panel, PV10 cells were transfected with either siRNA control (siCtrl) or siRNA Bax (siBax) for 36 h and then treated with DMSO, ABT-737 (10 μm), TRAIL (100 ng/ml), or the combination of ABT-737 and TRAIL for 24 h (mean ± S.D., n = 4). Changes in cell viability were determined by acid phosphatase assay. The level of Bax was examined in extracts of these cells by Western blot. Right panel, DU145 cells were transiently transfected with pcDNA3-Bax for 36 h and then treated with DMSO, ABT-737 (10 μm), TRAIL (100 ng/ml), or the combination of ABT-737 and TRAIL for 24 h (mean ± S.D., n = 4). Changes in cell viability were determined by the acid phosphatase assay and the level of Bax examined by Western blot.

FIGURE 5.

DR5 protein levels are up-regulated by ABT-737 treatment. A, effects of ABT-737 treatment of PV10 cells on the protein levels of key DISC proteins. Cells were treated with ABT-737 at the indicated dose for 24 h followed by Western blotting of cell extracts with specific antibodies. B, PV10, DU145, A549, HOP-62, and LNCaP cells were treated with DMSO or ABT-737 (5 or 10 μm) for 24 h, and the expression of cell surface DR4 and DR5 was determined by FACS analysis. Isotype-matched IgG1 monoclonal antibody was used as a negative control. C, PV10 cells were transfected with siRNA duplexes targeting DR5 for 36 h and then were treated with DMSO, 100 ng/ml TRAIL, 10 μm ABT-737, or the combination, and extracts were subjected to Western blotting with multiple antibodies. D, cells treated as in C and then were assessed for cell viability by an acid phosphatase assay (mean ± S.D., n = 4). E, RNA was extracted from PV10 cells treated with DMSO (0) or ABT-737 (5 or 10 μm) for 24 h. The level of DR5 mRNA was then determined by quantitative real time-PCR. F, PV10 cells were transfected with the vector PGVB2-DR5 (-1188)-luc encoding the upstream region of the DR5 promoter cloned in front of a luciferase reporter and then treated with DMSO or ABT-737 for 24 h (mean ± S.D., n = 3). DR5 luciferase activity was determined by dual luciferase assay (see “Experimental Procedures”).

FIGURE 6.

NF-κB activation by ABT-737 is required for DR5 up-regulation. A, PV10 cells were transfected with wild-type pGL3-DR5(-552)-luc (WT), pGL3-DR5(-552)-CHOPm, pGL3-DR5(-552)-NF-κBm, or pGL3-DR5(-552)-Elk-1m-luc. Luciferase activity was normalized to Renilla-luciferase activity (mean ± S.D., n = 3). B, PV10 cells were transfected with luciferase plasmids containing no insert empty vector (E.V.) (control), 1 (1×), or 5 (5×) copies of an NF-κB reporter plasmid pEF-Renilla-luc (RLuc), and firefly luciferase was normalized to RLuc. After 36 h, these cells were treated with DMSO or ABT-737 (10 μm) for 24 h prior to assay. C, PV10 cells were cotransfected with I-κBm (S/A) and PGVB2-DR5(-1188)-luc, and after 36 h were treated with DMSO or ABT-737 (10 μm) for an additional 24 h. Luciferase assays were carried out as described under “Experimental Procedures.” D, PV10 cells were transfected with empty vector (E.V.) control or pcDNA3-I-κBm, and after 36 h were treated with DMSO or ABT-737 (10 μm) for an additional 24 h. The expression of DR5, I-κBα, and GAPDH was determined by Western blotting. These Western blots were scanned, and the ratio of DR5 to GAPDH was determined by densitometry. E, again, cell surface DR5 expression was determined by FACS analysis. Isotype-matched IgG1 monoclonal antibody was used as a negative control.