TRAIL recombinant adenovirus triggers robust apoptosis in multidrug-resistant HL-60/Vinc cells preferentially through death receptor DR5

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic because of its highly selective apoptosis-inducing action on neoplastic versus normal cells. However, some cancer cells express resistance to recombinant soluble TRAIL. To overcome this problem, we used a TRAIL adenovirus (Ad5/35-TRAIL) to induce apoptosis in a drug-sensitive and multidrug-resistant variant of HL-60 leukemia cells and determined the molecular mechanisms of Ad5/35-TRAIL-induced apoptosis. Ad5/35-TRAIL did not induce apoptosis in normal human lymphocytes, but caused massive apoptosis in acute myelocytic leukemia cells. It triggered more efficient apoptosis in drug-resistant HL-60/Vinc cells than in HL-60 cells. Treating the cells with anti-DR4 and anti-DR5 neutralizing antibodies (particularly anti-DR5) reduced, whereas anti-DcR1 antibody enhanced, the apoptosis triggered by Ad5/35-TRAIL. Whereas Ad5/35-TRAIL induced apoptosis in both cell lines through activation of caspase-3 and caspase-10, known to link the cell death receptor pathway to the mitochondrial pathway, it triggered increased mitochondrial membrane potential change (m) only in HL-60/Vinc cells. Ad5/35-TRAIL also increased the production of reactive oxygen species, which play an important role in apoptosis. Therefore, using Ad5/35-TRAIL may be an effective therapeutic strategy for eliminating TRAIL-resistant malignant cells and these studies may provide clues to treat and eradicate acute myelocytic leukemias.

FIG. 1.

Dose-dependent TRAIL- and Ad5/35-TRAIL-induced apoptosis in the drug-sensitive HL-60 cell line and its P-gp-overexpressing MDR variant, HL-60/Vinc. (A) Western blot analysis of endogenous P-gp levels in HL-60 and HL-60/Vinc cells. Western blotting was performed as described in Materials and Methods. (B) Effect of TRAIL on apoptotic cell death in HL-60 and HL-60/Vinc cells. Cells (5 × 10⁵) were treated for 24 hr with or without TRAIL at 10, 20, 50, and 100 ng/ml, and apoptosis was measured by annexin V-binding assay. Significant differences between 10- and 100-ng/ml TRAIL treatments were obtained (^*p < 0.003, ^**p < 0.001). (C) Effect of Ad5/35-TRAIL on apoptotic cell death in HL-60 and HL-60/Vinc cells. Cells (5 × 10⁵) were treated for 24 hr with or without 5, 10, 20, and 50 MOI of Ad5/35-TRAIL, and apoptosis was measured by annexin V-binding assay. Significant differences between 5 and 50 MOI of Ad5/35-TRAIL treatments were obtained (^*p < 0.002, ^**p < 0.001).

FIG. 2.

TRAIL- and Ad5/35-TRAIL-induced apoptosis in the drug-sensitive HL-60 cell line and its P-gp-expressing MDR variant, HL-60/Vinc. (A) Effect of TRAIL and Ad5/35-TRAIL on apoptotic cell death in HL-60 and HL-60/Vinc cells. Cells (5 × 10⁵) were treated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, and 50 MOI of Ad5/35-TRAIL for 24 hr, and apoptosis was measured by annexin V-binding assay. Significant differences between various treatments were obtained (^*p < 0.004, ^**p < 0.0008). (B) Analysis of trimeric, dimeric, and monomeric TRAIL expression in cell lysate. HL-60 and HL-60/Vinc cells (5 × 10⁶) were incubated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr followed by protein extraction. Aliquots (50 μg of protein per lane) were separated by 5–15% SDS–PAGE and subjected to Western blot analysis as described in Materials and Methods. Equivalent loading was confirmed by reprobing the blot with anti-β-actin. Lanes 1–4 in each panel are HL-60/control, Ad5/35 vector, TRAIL, and Ad5/35-TRAIL, respectively. Lanes 5–8 in each panel are HL-60/Vinc control, Ad5/35 vector, TRAIL, and Ad5/35-TRAIL, respectively.

FIG. 3.

Effect of anti-TRAIL receptor antibodies on (A) TRAIL (^*p < 0.01, ^**p < 0.004, ^***p < 0.09) and (B) Ad5/35-TRAIL (^*p < 0.007, ^**p < 0.0001, ^***p < 0.06)-induced apoptosis. HL-60 and HL-60/Vinc cells were incubated with anti-DR4, -DR5, or -DcR1 neutralizing antibodies for 3 hr at 37°C, and then the cells were treated with or without 50 MOI of Ad5/35 or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr; apoptosis was measured by annexin V binding assay as described in Materials and Methods.

FIG. 4.

RT-PCR, real-time PCR, and Western blot analysis of c-FLIP expression in HL-60 and HL-60/Vinc cells. (A) Expression of c-FLIP_L and c-FLIP_S before and after treatment. Lanes 1–4 show c-FLIP_L mRNA levels in HL-60/control, HL-60/Ad5/35-TRAIL, HL-60/Vinc/control, and HL-60/Vinc/Ad5/35-TRAIL, respectively. Lanes 5–8 show c-FLIP_S mRNA levels in HL-60/control, HL-60/Ad5/35-TRAIL, HL-60/Vinc/control, and HL-60/Vinc/Ad5/35-TRAIL, respectively. (B) Expression levels of c-FLIP_L and c-FLIP_S mRNA with or without treatments were quantified by real-time PCR. Values represent means ± SD of three independent experiments. (C) Western blot shows little c-FLIP_L and c-FLIP_S before and after treatment in HL-60 and HL-60/Vinc cells. Lanes 1–4 are HL-60/control, (HL-60)/Ad5/35 vector, (HL-60)/TRAIL, and (HL-60)/Ad5/35-TRAIL, respectively. Lanes 5–8 are HL-60/Vinc/control, (HL-60/Vinc)/Ad5/35 vector, (HL-60/Vinc)/TRAIL, and (HL-60/Vinc)/Ad5/35-TRAIL, respectively.

FIG. 5.

TRAIL and Ad5/35-TRAIL trigger caspase activation in HL-60 and HL-60/Vinc cells: (A) caspase-8, (B) caspase-10, (C) caspase-9, and (D) caspase-3. Aliquots (50 μg of protein per lane) were separated by 10% SDS–PAGE and subjected to Western blot analysis as described in Materials and Methods. Lanes 1–4 in each panel are HL-60/control, HL-60/Ad5/35 vector, HL-60/TRAIL, and HL-60/Ad5/35-TRAIL, respectively. Lanes 5–8 in each panel are HL-60/Vinc/control, HL-60/Vinc/Ad5/35 vector, HL-60/Vinc/TRAIL, and HL-60/Vinc/Ad5/35-TRAIL, respectively.

FIG. 6.

Effect of (A) caspase-3 and (B) caspase-10 inhibitors on TRAIL- and Ad5/35-TRAIL-induced apoptosis. HL-60 and HL-60/Vinc cells were incubated in growth medium in the presence or absence of 50 μM caspase-3 inhibitor Ac-DEVD-CHO for 3 hr at 37°C, and then the cells were treated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr, and apoptosis was measured by annexin V binding assay as described in Materials and Methods. (A) ^*p < 0.005, ^**p < 0.02, ^***p < 0.03; (B) ^*p < 0.008, ^**p < 0.001.

FIG. 7.

Effect of Ad5/35-TRAIL on mitochondrial potential change (Δψ_m). Cells (5 × 10⁵) were treated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine (JC-1) was used as a probe as described in Materials and Methods. JC-1 membrane potential-related fluorescence was recorded with an FL1 photomultiplier tube (PMT) and a FACSCalibur flow cytometer. This experiment was performed in triplicate and results are shown as means ± SD.

FIG. 8.

Analysis of reactive oxygen species (ROS) in TRAIL- and Ad5/35-TRAIL-triggered apoptosis. Cells (5 × 10⁵) were treated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr. After treatment, the cells were incubated for 30 min at 37°C with 5 μM dichlorodihydrofluorescein diacetate (DCFH-DA) and analyzed with a FACSCalibur flow cytometer as described by the manufacturer (*p < 0.005, **p < 0.0008).

FIG. 9.

NAc effect on TRAIL- and Ad5/35-TRAIL-induced apoptosis: (A) HL-60 cells; (B) HL-60/Vinc cells. Cells (5 × 10⁵) were incubated with 10 mM NAc for 3 hr before being treated with or without 50 MOI of Ad5/35, TRAIL at 20 ng/ml, or 50 MOI of Ad5/35-TRAIL at 37°C for 24 hr; apoptosis was determined by annexin V flow cytometry as described in text. NAc decreases Ad5/35-TRAIL-induced apoptosis: (A) *p < 0.004, **p < 0.005; (B) *p < 0.005, **p < 0.001.