Article Figures & Data
Figures
- Figure 1.
Biochemical characterization of mouse anti-DR4 AY4 mAb. A, size exclusion elution profile of AY4 injected at 0.5 mg/mL (≈3.3 μmol/L) and monitored at 280 nm. The arrows indicate the elution positions of molecular weight standards (alcohol dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa), including blue dextran 2000 (∼2,000 kDa) as a void volume marker. B, binding specificity of AY4 and TRAIL analyzed by ELISA. ELISA plates were coated with 10 μg/mL of DR4-ECD, DR5-ECD, DcR1-ECD, DcR2-ECD, mTR-ECD, or CD95-ECD and then incubated with 1 μg/mL TRAIL (black columns) or 1 μg/mL AY4 (white columns). C, kinetic interactions of AY4 with DR4-ECD determined by SPR analyses. SPR sensograms were obtained from injections of serially diluted DR4-ECD at 100, 50, 25, 12.5, 6.25, and 3.125 nmol/L (solid lines from top to bottom) over an AY4 immobilized surface at 2,400 response units. The injection of DR5-ECD (long dashed line), DcR1-ECD (dotted line), and DcR2-ECD (dash-dotted line) at 2 μmol/L exhibited negligible binding to AY4. D, competitive ELISA. Binding activity of AY4 (1 μg/mL) to DR4 in the presence of serially diluted TRAIL (from 800 to 1.56 μg/mL) was assessed in plates coated with DR4-ECD. B and D, bars, SD.
- Figure 2.
AY4 alone induces cell death of various human cancer cells through cell surface–expressed DR4 without significant cytotoxicity on normal human hepatocytes. A and B, cell killing activities of TRAIL (left) and anti-DR4 AY4 mAb (right) in various TRAIL-sensitive and TRAIL-resistant tumor cells (A) and in normal human hepatocytes (B). Cells were incubated with the indicated concentrations of TRAIL or AY4 for 24 h. C, inhibition of cell death induced by TRAIL (left) or AY4 (right) by the soluble competitor DR4-ECD (black columns) or DR5-ECD (white columns). Cells were treated with 0.5 μg/mL TRAIL (≈30 nmol/L) or 10 μg/mL AY4 (≈67 nmol/L) for 24 h in the presence of the indicated concentrations (0, 0.04, and 1 μg/mL) of DR4-ECD or DR5-ECD. Bars, SD.
- Figure 3.
Dual staining of Annexin V-FITC and PI for Jurkat, Ramos, and HL60 cells treated with medium only for 12 h, 0.5 μg/mL TRAIL for 3.5 h, or 10 μg/mL AY4 for 6 and 12 h (as indicated in each panel). Samples were then analyzed by flow cytometry. In the dot plots, Annexin V-FITC+/PI− cells (bottom right quadrant, % shown) and Annexin V-FITC+/PI+ cells (top right quadrant, % shown) are considered as “early apoptotic” and “dead” cells, respectively.
- Figure 4.
AY4 induces caspase-dependent apoptotic cell death of AY4-sensitive cells. A, detection of proteolytic processing of apoptotic molecules by Western blotting analyses in HCT116 (left), Jurkat (middle), and HL60 cells (right), which were untreated (control) or treated with either 0.5 μg/mL TRAIL for 3.5 h or the indicated concentrations of AY4 for 24 h. Proforms (black arrowhead) and cleaved forms (white arrowhead) of caspase-8, Bid, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) are indicated. The β-actin protein levels are included as a control for protein loading. B and C, effects of a pan-caspase inhibitor, Z-VAD-fmk, on the cell viability of HCT116, H460, Ramos, and Jurkat cells treated with either TRAIL (B) or AY4 (C). Cells untreated (black columns) or pretreated (white columns) with Z-VAD-fmk (10 μmol/L) for 0.5 h were further incubated with either 0.5 μg/mL TRAIL or 10 μg/mL AY4 for 24 h. Bars, SD.
- Figure 5.
AY4-stimulated DR4 signaling requires FADD and caspase-8 and can transduce the extrinsic and/or intrinsic apoptotic pathways depending on the cell types. A, cell viability of Jurkat wild-type and its FADD-deficient (FADD-def.), caspase-8–deficient (Caspase-8-def.), or RIP1-deficient (RIP1-def.) cells, which were incubated with the indicated concentrations of TRAIL (left) or AY4 (right) for 24 h. B, HCT116 and its Bax-deficient (Bax−/−) or p53-deficient (p53−/−) cells were incubated with the indicated concentrations of TRAIL (left) or AY4 (right) for 24 h. C, effects of a caspase-9 inhibitor, Z-LEHD-fmk, on the cell viability of HCT116, H460, Ramos, Jurkat, and HL60 cells treated with either TRAIL (left) or AY4 (right). Cells untreated (black columns) or pretreated (white columns) for 0.5 h with Z-LEHD-fmk (20 μmol/L) were further incubated with either 0.5 μg/mL TRAIL or 10 μg/mL AY4 for 24 h. Bars, SD.
- Figure 6.
Antitumor effect of anti-DR4 AY4 on growth of preestablished H460 tumors in athymic nude mice. A to C, comparisons of tumor growth during the treatment (A) and isolated tumor weight (B) and tumor size (C) at the end of treatment (after 22 d of tumor inoculation) between mice treated with AY4 and IC antibody. After preestablishing of H460 cells with volume of ∼100 mm3 (day 7), mice (n = 6 per group) were administered i.v. with five doses of AY4 or IC antibody at a single dosage of 5 mg/kg/injection (100 μg/injection) every 3 d from day 7 (as indicated by white arrowheads). Bars, SD. Significance was determined using the Student's t test versus control IC–treated group. *, P < 0.0001; **, P < 0.01.
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Volume 8, Issue 8
