Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1

Abstract

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells; however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti-TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab-induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab-induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or γ-irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.

Keywords: DR4; Fab; TRAIL; TRAIL receptor 1; TRAIL-resistance; agonistic human antibody; cancer cells; mitochondria; p38.

Figure 1

Amino acid sequences of heavy (V_H) and light (V_L) chains and visualization of the purified DR4-4 Fab. The amino acid sequences of the V_H (A) and V_L (B) regions of DR4-4 Fab are available from European Molecular Biology Laboratory/GenBank under accession numbers JN030159 (V_H) and JN030158 (V_L). The purified DR4-4 Fab (1 µg/mL for immunoblotting and 10 µg/mL for Coomassie blue staining) was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with anti–human IgG (Fab specific) monoclonal antibody (Ca) and Coomassie blue staining (Cb). DR4-4 Fab is presented by an arrow at approximately 45 kDa.

Figure 2

Specific binding of DR4-4 Fab to DR4 antigen. Direct-binding (A) and competitive (B) enzyme-linked immunosorbent assay (ELISA) for specific binding of DR4-4 Fab to DR4. Recombinant DR4 and DR5 were coated onto the wells of ELISA plates at 5 µg/mL, followed by incubation with DR4-4 Fab (A) or DR4-4 Fab preincubated with competitor, DR4 or DR5 (B) (data presented as mean ± standard deviation). (C) Binding affinity of recombinant human DR4 antigen for DR4-4 (1 µM) immobilized on a nitrile triacetic acid chip as measured by Biacore surface plasmon resonance. (D) Fluorescence-activated cell sorting analysis of the cellular binding of DR4-4 Fab. Cells (5 × 10⁵) were incubated with fluorescein isothiocyanate (FITC)-labeled DR4-4 Fab for 30 min at 4 °C without (a) or with (b) pretreatment with unlabeled DR4-4 Fab. Dot plot presents the profile of forward scatter (FSC)/side scatter (SSC) of control cells. P is a gate of cells which were used for the analysis. (C,D) are representative results among triplicate experiments.

Figure 3

Specific cytotoxic effects of DR4-4 Fab on cancer cells. Cells (2.5 × 10⁴) were treated with the indicated concentrations of TRAIL (A) or DR4-4 Fab (B) for 48 h. (C) Viability (as assessed by MTT assay) of normal human fibroblasts (1 × 10⁵) treated with TRAIL or DR4-4 Fab for 48 h. (D) Cell morphology of normal human fibroblasts treated with TRAIL or DR4-4 Fab for 48 h was evaluated by phase contrast microscopy at 100×. Data presented as mean ± standard deviation.

Figure 4

Caspase-independent and mitochondrial-induced cytotoxicity of DR4-4 Fab. (A) Jurkat cells (2.5 × 10⁴) were treated with TRAIL (125 ng/mL) or DR4-4 Fab (100 µg/mL) for the indicated time and cell viability was measured by MTT assay. (B) Cells (1 × 10⁶) were treated with TRAIL (125 ng/mL) or DR4-4 Fab (100 µg/mL) for the indicated time with or without a 3 h pretreatment with z-VAD (20 µM). Caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP), and cleaved PARP were detected by immunoblotting with specific antibodies. β-actin was used as a control to quantify the relative amount of sample loaded in each well. (C) MTT assay after treatment of cells with DR4-4 Fab (30 min pretreatment with or without z-VAD (20 µM), Nec-1 (50 µM), and N-acetyl-l-cysteine (NAC; 5 mM)) or TRAIL (30 min pretreatment with or without z-VAD (20 µM)). (D) Cells were treated with DR4-4 Fab (100 µg/mL) for 6 h in the presence or absence of NAC (5 mM), stained with annexin V-FITC/PI, and analyzed by flow cytometry (1 × 10⁶ cells were counted). (E) Mitochondrial reactive oxygen species generation was measured by flow cytometry after treatment with DR4-4 Fab (100 µg/mL) for the indicated time (0–48 h) and 5 µM MitoSOX staining. (F) Cells were treated with 100 µg/mL DR4-4 Fab (0, 16, 24, 32, and 48 h) and stained with JC-1 to analyze mitochondrial membrane potential (MMP). The ratio of red to green forms of JC-1 was measured by flow cytometry. (G) Cells were treated with 100 µg/mL DR4-4 Fab for 48 h with or without wortmannin (WM) and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA; 10 µM). After staining with JC-1, cells were analyzed by flow cytometry for MMP. Data presented as mean ± standard deviation (* p < 0.05, ** p < 0.005, versus control).

Figure 5

Morphological observation of cells treated with DR4-4 Fab. (A) Representative transmission electron microscopy images taken after THP-1 cell treatment with 50 µg/mL DR4-4 Fab for 0 h (a), 24 h (b), or 48 h (c,d). Scale bar, 2.5 µm. (B) Representative images showing morphological changes in the mitochondrial network of HeLa cells treated with 50 µg/mL DR4-4 Fab for 24 h observed by confocal microscopy (100×) after MitoTracker Red FM staining of mitochondria. Scale bar, 5 µm. The parts of the images were enlarged and presented in yellow squares.

Figure 6

Immunoblot analysis of signaling molecules induced by DR4-4 Fab. Immunoblotting performed after the indicated length of treatment of Jurkat cells (1 × 10⁶) with TRAIL (125 ng/mL) or DR4-4 Fab (100 µg/mL). (A) P-p38 and p38 were detected with specific antibodies. (B) P-JNK and IκBα were detected after the treatment of TRAIL or DR4-4 Fab. P-ERK and ERK were analyzed only after the treatment with DR4-4 Fab. (C) C-FLIP, tBID, Bcl-xL, Bcl-2, Mcl-1, and Bax were analyzed with specific antibodies after the treatment of TRAIL or DR4-4 Fab. α-tubulin and β-actin were used to quantify the relative amount of sample loaded into each well. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma extra-large; c-FLIP, cellular FADD-like IL-1β-converting enzyme–inhibitory protein; ERK, extracellular signal-regulated kinase; IκBα, nuclear factor κB inhibitor α; JNK, c-Jun N-terminal kinase; Mcl-1, myeloid cell leukemia 1; p-, phosphorylated protein; t-Bid, truncated cytosolic BH3 interacting-domain death agonist.

Figure 7

Enhanced rates of cell death following cotreatment with DR4-4 Fab and TRAIL or γ-irradiation. (A) Cytotoxicity of cotreatment with DR4-4 Fab and TRAIL as measured by MTT assay. Cell viability for single treatment with TRAIL is also presented with concentrations indicated (upmost line). (B) Cells pre-irradiated with γ-radiation (absorbed dose = 2 Gy) were treated with DR4-4 Fab (100 µg/mL) for 24 h. Cell death was measured by MTT assay. Data presented as mean ± standard deviation (* p < 0.05, ** p < 0.005, versus control).