Human Agonistic Antibody to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 2 Induces Cytotoxicity and Apoptosis in Prostate Cancer and Bladder Cancer Cells

Basic Science Human Agonistic Antibody to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 2 Induces Cytotoxicity and Apoptosis in Prostate Cancer and Bladder Cancer Cells Osamu Shimada, Xiuxian Wu, Xinghua Jin, Mohammed Ahmed Abdel-Muneem Nouh, Michele Fiscella, Vivian Albert, Tadashi Matsuda, and Yoshiyuki Kakehi OBJECTIVES

METHODS

RESULTS

CONCLUSIONS

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. In this study, we investigated the susceptibility of human prostate cancer and bladder cancer cells to HGS-ETR2, a human monoclonal agonistic antibody specific for TRAIL-R2. The cell surface expression of TRAIL-R1 and TRAIL-R2 on prostate cancer and bladder cancer cells was determined using flow cytometry. Cytotoxicity was assessed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay, and caspase activities were measured by a quantitative colorimetric assay. HGS-ETR2 effectively induced apoptotic cell death in DU145, PC3, and LNCaP human prostate cancer cells and J82 and T24 human bladder cancer cells. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell surface expression of the two TRAIL receptors, in that TRAIL-R2, but not TRAIL-R1, was frequently expressed in the prostate cancer and bladder cancer cells. HGS-ETR2 significantly activated the caspase cascade, including caspase-3, -6, -8, and -9, which were the downstream molecules of the death receptors in prostate cancer cells. Caspase-3, -6, and -9 were also significantly activated with HGS-ETR2-induced apoptosis in the bladder cancer cells. These findings suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent against prostate cancer and bladder cancer. UROLOGY 69: 395– 401, 2007. © 2007 Elsevier Inc.

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umor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a proapoptotic member of the TNF superfamily,1 has a potential as an effective anticancer agent, because it selectively induces apoptosis in a variety of tumor cells, yet is relatively nontoxic to normal cells.2– 4 In a previous study, we showed that TRAIL combined with adriamycin induced significant apoptosis in human renal cell carcinoma (RCC) cells. However, TRAIL monotherapy was not as effective.5 TRAIL triggers apoptosis by binding to two receptors: TRAIL-R1 (DR4) and TRAIL-R2 (DR5).6,7 The activation of these receptors results in a signal transFrom the Departments of Urology and Biochemistry, Kagawa University Faculty of Medicine, Kagawa, Japan; Department of Urology, Kansai Medical University, Osaka, Japan; and Antibody Development Department, Human Genome Sciences, Rockville, Maryland Reprint requests: Yoshiyuki Kakehi, M.D., Ph.D., Department of Urology, Kagawa University Faculty of Medicine, 1750-1 Oaza Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: [email protected] Submitted: January 24, 2006; accepted (with revisions): December 11, 2006

© 2007 Elsevier Inc. All Rights Reserved

duction cascade that initiates both intrinsic and extrinsic apoptotic pathways.8 In addition, TRAIL binds to two other receptors, TRAIL-R3 (DcR1)6 and TRAIL-R4 (DcR2),9 which lack a functional cytoplasmic death domain, and to a secreted TNF receptor homologue, osteoprotegerin.10 These receptors have been proposed to inhibit TRAIL-induced apoptosis by acting as decoy receptors. Potentially, the degree of TRAIL-R1 and TRAIL-R2-mediated apoptosis induced by TRAIL might be lowered in the presence of DcR1 and DcR2 activation. Transfection of decoy receptors into TRAIL-sensitive cells renders them TRAIL resistant.6,11 Thus, using a specific activator of TRAIL-R1 or TRAIL-R2 is preferable to exclude interference from competition with decoy receptors. Most normal tissues and cells do not show a detectable cell surface expression of TRAIL-R2 and are resistant to TRAIL-R2-mediated apoptosis, although TRAIL-R2 mRNA is detectable in some normal tissues.12,13 In contrast, a large number of tumor tissues and 0090-4295/07/$32.00 doi:10.1016/j.urology.2006.12.007

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tumor cell lines express high levels of TRAIL-R2 and are susceptible to TRAIL-R2-mediated apoptosis.14 Therefore, selective targeting of TRAIL-R2 might provide a novel and promising therapeutic strategy for human cancers. We recently showed that HGS-ETR2, a human TRAIL-R2 monoclonal antibody, induced apoptotic cell death in human RCC cell cultures and effectively suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice.15 In the present study, we investigated the cell surface expression of TRAIL-R1 and R2 on the prostate cancer and bladder cancer cells by flow cytometric analysis. TRAIL-R2 was frequently expressed on the prostate cancer and bladder cancer cells, but not TRAIL-R1. We also demonstrated that HGS-ETR2 effectively induced cytotoxicity and apoptosis in both prostate cancer and bladder cancer cells.

ously.17 In brief, a 100-␮L suspension of 5000 cells was seeded into a 96-well flat-bottom microtiter plate. After incubation for 24 hours, 100 ␮L of HGS-ETR2 or medium (control) was added to the plates, and each plate was incubated for an additional 24 to 72 hours, followed by the addition of 20 ␮L of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide working solution (5 mg/mL, Sigma Chemical, St. Louis, Mo) for 4 hours, and then 150 ␮L of isopropanol supplemented with 0.05 N hydrochloric acid for 30 minutes. Absorbance (A) was measured using a microplate reader (Bio-Rad, Tokyo, Japan) at 570 nm with a 630 nm reference: percentage of cytotoxicity ⫽ [1 ⫺ (A of experimental wells/A of control wells)] ⫻ 100. Cell viability was evaluated by trypan blue dye staining. The cells were seeded in 60-mm dishes (5 ⫻ 105 cells/dish). After incubation for 24 hours, the cells were treated with or without HGS-ETR2 at a concentration of 1 ␮g/mL for 24 to 48 hours. The cells were then harvested, and the viable cells were counted with 0.5% trypan blue dye (Sigma) under a phasecontrast microscope.

MATERIAL AND METHODS

Caspase Activity Assay

Cell Lines and Cultures

Caspase-3, -6, -8, and -9 activities were measured using a quantitative colorimetric assay with the Caspase-3, -6, -8, and -9 Colorimetric Protease Assay Kits (MBL, Nagoya, Japan) as described previously.18 In brief, the cells treated with HGSETR2 were homogenized in 200 ␮L cell lysis buffer, incubated for 10 minutes on ice, and then centrifuged at 10,000g for 1 minutes at 4°C. The supernatant was recovered, and the protein concentration was determined using a Bio-Rad DC protein assay. The 50 ␮L of the cell lysate corresponding to 200 ␮g of total protein, 50 ␮L of 2 ⫻ reaction buffer and 5 ␮L of the 4 mM Asp-Glu-Val-Asp-pNA, Val-Glu-lle-Asp-pNA, lle-GluThr-Asp-pNA, or Leu-Glu-His-Asp-pNA substrates were added to each well of the 96-well plates, and then the plate was incubated at 37°C for 24 hours. The A value of each well was measured with a microplate reader at 405 nm.

LNCaP, DU145, and PC3 human prostate cancer cell lines and J82 and T24 human bladder cancer cell lines were purchased from the American Type Culture Collection (Rockville, Md). These cells were cultured in Roswell Park Memorial Institute1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 U/mL penicillin, and 100 ␮g/mL streptomycin at 37°C in a humidified 5% carbon dioxide atmosphere.

Reagents The human TRAIL-R2 mAb (HGS-ETR2) was kindly provided by Human Genome Sciences (Rockville, Md). HGSETR2 is a fully human monoclonal antibody (IgG1) isolated using phage-display technology in collaboration with Cambridge Antibody Technology.16 Enzyme-linked immunosorbent assay and/or BIAcore analyses determined that HGS-ETR2 is highly specific for binding to TRAIL-R2. Phycoerythrin (PE)conjugated anti-human TRAIL-R1 (mouse IgG1) and TRAIL-R2 (mouse IgG2b) mAbs for flow cytometry were purchased from Genzyme Techne (Minneapolis, Minn).

Flow Cytometric Analysis of TRAIL-R1 and TRAIL-R2 The cell surface expression of TRAIL-R1 and TRAIL-R2 on prostate cancer and bladder cancer cells was determined by flow cytometry15 with EPICS XL (Beckman Coulter, Miami, Fla). In brief, the cells were seeded in 60-mm dishes (5 ⫻ 105 cells/dish) and cultured for 24 hours. The cells were then harvested from the substrate using 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid, washed twice in phosphate-buffered saline containing 0.2% FBS and 0.01% NaN3, and the number of cells was counted. Then, 2 ⫻ 105 cells were incubated with PEconjugated anti-human TRAIL-R1 or TRAIL-R2 mAbs at 4°C for 30 minutes, washed, and analyzed. The control consisted of cells in a separate tube treated with PE-labeled mouse IgG1 or mouse IgG2b (Southern Biotech, Birmingham, Ala).

Cytotoxicity Assays Cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, as described previ396

Apoptosis Assay Apoptosis was determined by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (MBL).19 In brief, DU145 or J82 cells were treated with HGS-ETR2 at 1000 ng/mL for 8 to 48 hours, washed twice with cold phosphate-buffered saline, and resuspended in a binding buffer at a concentration of 5 ⫻ 105 cells/mL. Subsequently, 5 ␮L Annexin V-FITC and 5 ␮L propidium iodide were added, and cells were analyzed by flow cytometry with EPICS XL (Beckman Coulter).

Statistical Analysis All determinations were repeated three times, and the results are expressed as the mean ⫾ SD. Quantitative experiments were analyzed using the Student t test. P ⬍0.05 was considered statistically significant.

RESULTS Expression of TRAIL-R1 and TRAIL-R2 To identify the molecular mechanisms of HGS-ETR2-mediated apoptosis, the cell surface expression of TRAIL-R1 and TRAIL-R2 was examined by flow cytometry. The expression of TRAIL-R1 was detected in less than 10% of either prostate cancer or bladder cancer cells. In contrast, UROLOGY 69 (2), 2007

Figure 1. Cell surface expression of TRAIL-R1 and TRAIL-R2. (A) DU145, (B) PC3, and (C) LNCaP human prostate cancer cells and (D) J82 and (E) T24 human bladder cancer cells were incubated with mouse IgG or mouse anti-human TRAIL-R1 or TRAIL-R2 mAb and analyzed by flow cytometry. Shaded and unshaded peaks correspond to specific and control staining, respectively. Left and right panels were TRAIL-R1 and TRAIL-R2 expressions, respectively. These figures were representative of three different experiments.

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Conce ntration of ETR2 (ng/ml) 0.1

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Figure 2. Dose and time-dependent cytotoxic effects of HGS-ETR2. Circles indicate 24 hours; triangles, 48 hours; and squares, 72 hours. (A) DU145, (B) PC3, and (C) LNCaP human prostate cancer cells and (D) J82 and (E) T24 human bladder cancer cells treated with serially diluted concentrations of HGS-ETR2 for 24 to 72 hours. Cytoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Results derived from three different experiments.

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C TRAIL-R2 was strongly expressed in the prostate cancer and bladder cancer cells tested (Fig. 1). Cytotoxic Effects of HGS-ETR2 Because both prostate cancer and bladder cancer cells frequently expressed TRAIL-R2, we next examined their susceptibility to the effect of HGS-ETR2. Treatment with varying concentrations of HGS-ETR2 for 24 to 72 hours resulted in concentration-dependent and time-dependent cytotoxicities in DU145 human prostate cancer cells (Fig. 2A). Similar cytotoxic effects were obtained in the other human prostate cancer cells, PC3 (Fig. 2B) and LNCaP (Fig. 2C), and human bladder cancer cells, J82 (Fig. 2D) and T24 (Fig. 2E). The cytotoxicity was specific for the HGS-ETR2, because IgG (Sigma) had no cytotoxic effect (Table 1). The cytotoxic effects of HGSETR2 were confirmed by a trypan blue dye exclusion test 398

(data not shown). These results clearly demonstrated that treatment of prostate cancer and bladder cancer cells with HGS-ETR2 resulted in potentiation of cytotoxicity. To examine the effect of androgen on the cytotoxicity of HGS-ETR2 against LNCaP cells, the cytotoxicity was also determined in phenol red-free RPMI medium in 10% charcoal-stripped FBS. No differences were found in the cytotoxic effects of HGS-ETR2 on the LNCaP cells in the presence of FBS or charcoal-stripped FBS (data not shown). HGS-ETR2 Activates Caspases and Triggers Apoptosis Caspase activation is the final common molecular event required for executing apoptosis in most biologic systems.20 To investigate caspase activation, cell lysates obtained after treating DU145 and J82 cells with HGSUROLOGY 69 (2), 2007

Table 1. Cytotoxic effect of IgG for prostate and bladder cancer cells Cancer Cells

0

1

DU145 PC3 LNCaP J82 T24

0 0 0 0 0

4.77 ⫾ 4.5 1.87 ⫾ 8.3 5.10 ⫾ 4.0 5.2 ⫾ 6.0 1.67 ⫾ 8.8

Percent Cytotoxicity of IgG (ng/mL) 10 100 4.64 ⫾ 4.8 0.92 ⫾ 3.2 4.53 ⫾ 5.2 2.40 ⫾ 6.8 3.11 ⫾ 5.7

2.82 ⫾ 4.9 12.6 ⫾ 4.0 6.72 ⫾ 4.1 5.10 ⫾ 5.9 8.59 ⫾ 5.0

1000 7.36 ⫾ 5.2 8.24 ⫾ 5.5 8.10 ⫾ 4.4 7.14 ⫾ 6.6 5.47 ⫾ 5.5

Data presented as mean ⫾ SD.

ETR2 were incubated with each of the chromogenic substrates for caspase-3, -6, -8, and -9. An increase in activity with time was found for all of these caspases in DU145 cells in a synchronous manner (P ⬍0.025; Fig. 3A). Among these caspases, caspase-3 activity was the most increased after treatment with HGS-ETR2. Caspase-3, -6, and -9, but not caspase-8, were also activated by HGS-ETR2 in J82 cells (P ⬍0.025; Fig. 3B). The ability of HGS-ETR2 to induce cell death in the prostate cancer and bladder cancer cells was evaluated by flow cytometry using DU145 and J82 cells. Flow cytometric analysis after Annexin V-FITC and propidium iodide staining showed the HGS-ETR2 induced apoptosis. Early and late apoptotic cells were found in DU145 (Fig. 3C) and J82 (Fig. 3D) cell cultures after treatment with 1000 ng/mL HGS-ETR2. These data show that the cytotoxicity of HGS-ETR2 is mediated by apoptosis.

COMMENT TRAIL-R mAb-mediated treatment has been shown to be effective in some cancer cell lines,21,22 and it has been highlighted by the recent introduction of TRAIL-R agonistic antibodies into human Phase I trials.23 In this study, we examined the effects of a new agonistic human mAb against TRAIL-R2, HGS-ETR2, on established human prostate cancer and bladder cancer cell lines. The results of our study revealed that HGS-ETR2 induced apoptosis in both cell types. Also, the efficacy of HGSETR2 for both prostate cancer and bladder cancer cell cultures is likely to depend on the cell surface expression of TRAIL-R2. Finally, the activation of caspases was involved in HGS-ETR2-induced apoptosis. It remains unclear whether the efficacy of HGS-ETR2-induced apoptosis results from a specific cell surface expression of TRAIL-R2, because TRAIL-R1-positive plus TRAILR2-negative cells have not been available. These findings, however, suggest that the HGS-ETR2-based treatment might be an effective strategy for both prostate and bladder cancer. The cell surface expression of TRAIL-R1 or -R2 is essential for TRAIL-induced apoptosis, although tumor cells expressing these death receptors are not always sensitive to TRAIL because of intracellular mechanisms.24 –26 In a previous study, we showed that the surface levels of TRAIL-R1 and -R2 mainly qualify the susceptibility of human RCC cells to the TRAIL-R1 and UROLOGY 69 (2), 2007

TRAIL-R2 mAbs, as well as TRAIL.15 Supporting our data, it has been shown that the efficacy of TRAIL correlates with the cell surface expression of TRAIL-R1 and /or -R2 in leukemia cells.27 Furthermore, upregulation of TRAIL-R1 and -R2 by adriamycin,5 2-methoxyestradiol,28 or interferon-alpha29 enhanced the responsiveness of certain cancer cells to TRAIL. Consequently, these observations, including ours, suggest that TRAIL-R1 or TRAIL-R2 expression might be a reliable indicator of the degree of cytotoxicity that could be achieved by TRAIL or TRAIL-R mAbs with certain tumors. It is possible that HGS-ETR2 has an anticancer effect for both prostate cancer and bladder cancer because TRAIL-R2 is frequently expressed in both prostate cancer and bladder cancer cells. To our knowledge, this is the first report showing that a TRAIL-R2 mAb induces apoptosis and cytotoxicity on prostate cancer and bladder cancer cells. The present study clearly demonstrated that HGS-ETR2 was effective in the androgen-insensitive prostate cancer cells, DU145 and PC3, as well as the androgen-sensitive prostate cancer cells, LNCaP. Furthermore, bladder cancer cells were also sensitive to HGS-ETR2-induced cytotoxicity. In accordance with our data, it has been reported that human TRAIL-R2 mAb was cytotoxic to human melanoma cells30 and myeloma cells.31 A recent study has demonstrated that in addition to eliminating most TRAIL-sensitive tumor cells, the agonistic TRAIL-R2 can induce tumor-specific effector and memory T cells that can eradicate even TRAIL-resistant tumor variants and provide long-term protection against tumor recurrence in vivo.22 Furthermore, it has recently been reported that TRAIL-R2 mAb induced cell death in a variety of lymphoma cells and enhanced the killing effect of doxorubicin and bortezomib.32 These results suggest that TRAIL-R2 mAbbased therapy is promising for treating cancer. It is difficult to examine an isolated TRAIL-mediated signal transduction because various receptors complicate signal transduction. Using the specific mAb, HGS-ETR2, we were able to evaluate caspase involvement specifically in TRAIL-R2-mediated apoptosis. We found that HGSETR2 significantly activated initiative caspases such as caspase-9 and -8 and effective caspases, including caspase-6 and -3 in prostate cancer cells. Furthermore, the caspase-9, -6, and -3 were also significantly activated 399

Figure 3. Activation of caspases and apoptosis induced by HGS-ETR2. White bars indicate control (medium only); stippled bars, HGS-ETR2 for 1 hour; striped bars, HGS-ETR2 for 3 hours; and black bars, HGS-ETR2 for 6 hours. (A) DU145 human prostate cancer cells and (B) J82 human bladder cancer cells treated with HGS-ETR2 (1000 ng/mL) for indicated times, and caspase-9, -8, -6, and -3 activity measured by quantitative colorimetric assay. Results derived from three different experiments (*P ⬍0.025, Student’s t test). (C) DU145 and (D) J82 cells were treated with medium or HGS-ETR2 (1000 ng/mL) for 24 hours and stained with Annexin V-FITC and propidium iodide. Annexin V-FITC-positive and propidium iodide-positive cells were measured by flow cytometry. Left and right panels were medium and HGS-ETR2 treatment, respectively.

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in HGS-ETR2-induced apoptosis in bladder cancer cells. These results suggest that the caspase cascade plays an important role in TRAIL-R2 mAb-mediated apoptosis in both prostate cancer and bladder cancer cells.

CONCLUSIONS The present results indicate that the TRAIL-R2 mAb, HGS-ETR2, effectively induces apoptosis and cytotoxicity in both prostate cancer and bladder cancer cells, in which TRAIL-R2 is frequently expressed. These findings provide a foundation for the development of TRAIL-R2 mAb treatment regimens in both prostate cancer and bladder cancer. Acknowledgment. To Kouichi Yube of the Research Equipment Center, Kagawa University Faculty of Medicine, for his technical assistance. References 1. Griffith TS, and Lynch DH: TRAIL: a molecule with multiple receptors and control mechanisms. Curr Opin Immunol 10: 559 – 563, 1998. 2. Walczak H, Miller RE, Ariail K, et al: Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5: 157–163, 1999. 3. Van Valen F, Fulda S, Truckenbrod B, et al: Apoptotic responsiveness of the Ewing’s sarcoma family of tumours to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Int J Cancer 88: 252–259, 2000. 4. Hao C, Song JH, Hsi B, et al: TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice. Cancer Res 64: 8502– 8506, 2004. 5. Wu XX, Kakehi Y, Mizutani Y, et al: Enhancement of TRAIL/Apo2Lmediated apoptosis by adriamycin through inducing DR4 and DR5 in renal cell carcinoma cells. Int J Cancer 104: 409 – 417, 2003. 6. Pan G, Ni J, Wei YF, et al: An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277: 815– 818, 1997. 7. Pan G, O’Rourke K, Chinnaiyan AM, et al: The receptor for the cytotoxic ligand TRAIL. Science 276: 111–113, 1997. 8. Ashkenazi A, and Dixit VM: Death receptors: signaling and modulation. Science 281: 1305–1308, 1998. 9. Ashkenazi A, and Dixit VM: Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11: 255–260, 1999. 10. Emery JG, McDonnell P, Burke MB, et al: Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 273: 14363– 14367, 1998. 11. Degli-Esposti MA, Dougall WC, Smolak PJ, et al: The novel receptor TRAIL-R4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7: 813– 820, 1997. 12. Ichikawa K, Liu W, Fleck M, et al: TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis. J Immunol 171: 1061–1069, 2003. 13. Miranda-Carus ME, Balsa A, Benito-Miguel M, et al: Rheumatoid arthritis synovial fluid fibroblasts express TRAIL-R2 (DR5) that is functionally active. Arthritis Rheum 50: 2786 –2793, 2004. 14. Ichikawa K, Liu W, Zhao L, et al: Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nat Med 7: 954 –960, 2001.

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15. Zeng Y, Wu XX, Fiscella M, et al: Monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo. Int J Oncol 28: 421– 430, 2006. 16. Pukac L, Kanakaraj P, Humphreys R, et al: HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumour types in vitro and in vivo. Br J Cancer 92: 1430 –1441, 2005. 17. Wu XX, Mizutani Y, Kakehi Y, et al: Enhancement of Fas-mediated apoptosis in renal cell carcinoma cells by adriamycin. Cancer Res 60: 2912–2918, 2000. 18. Wu XX, Kakehi Y, Mizutani Y, et al: Activation of caspase-3 in renal cell carcinoma cells by anthracyclines or 5-fluorouracil. Int J Oncol 19: 19 –24, 2001. 19. Wu XX, Ogawa O, and Kakehi Y: Enhancement of arsenic trioxideinduced apoptosis in renal cell carcinoma cells by [scap]l[r]-buthionine sulfoximine. Int J Oncol 24: 1485–1497, 2004. 20. Green DR, and Evan GI: A matter of life and death. Cancer Cell 1: 19 –30, 2002. 21. Chuntharapai A, Dodge K, Grimmer K, et al: Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. J Immunol 166: 4891– 4898, 2001. 22. Takeda K, Yamaguchi N, Akiba H, et al: Induction of tumorspecific T cell immunity by anti-DR5 antibody therapy. J Exp Med 199: 437– 448, 2004. 23. Reed JC: Apoptosis-targeted therapies for cancer. Cancer Cell 3: 17–22, 2003. 24. Amantana A, London CA, Iversen PL, et al: X-linked inhibitor of apoptosis protein inhibition induces apoptosis and enhances chemotherapy sensitivity in human prostate cancer cells. Mol Cancer Ther 3: 699 –707, 2004. 25. Johnson TR, Stone K, Nikrad M, et al: The proteasome inhibitor PS-341 overcomes TRAIL resistance in Bax and caspase 9-negative or Bcl-xL overexpressing cells. Oncogene 22: 4953– 4963, 2003. 26. Wang CY, Mayo MW, Korneluk RG, et al: NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281: 1680 –1683, 1998. 27. Liu Q, Hilsenbeck S, and Gazitt Y: Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9, and synergy with APO2/TRAIL. Blood 101: 4078 – 4087, 2003. 28. LaVallee TM, Zhan XH, Johnson MS, et al: 2-Methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway. Cancer Res 63: 468 – 475, 2003. 29. Shigeno M, Nakao K, Ichikawa T, et al: Interferon-alpha sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-kappa B inactivation. Oncogene 22: 1653– 1662, 2003. 30. Griffith TS, Rauch CT, Smolak PJ, et al: Functional analysis of TRAIL receptors using monoclonal antibodies. J Immunol 162: 2597–2605, 1999. 31. Menoret E, Gomez-Bougie P, Geffroy-Luseau A, et al: Mcl-1L cleavage is involved in TRAIL-R1 and TRAIL-R2 mediated apoptosis induced by HGS-ETR1 and HGS-ETR2 human mAb in myeloma cells. Blood 108: 1346 –1352, 2006. 32. Georgakis GV, Li Y, Humphreys R, et al: Activity of selective fully human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells: induction of apoptosis and enhancement of doxorubicin- and bortezomib-induced cell death. Br J Haematol 130: 501–510, 2005.

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