Enhancement of Death Receptor 4Mediated Apoptosis and Cytotoxicity in Renal CellCarcinoma Cells by Subtoxic Concentrations of DoxorubicinXinghua Jin, Xiu-Xian Wu,* Mohammed Ahmed Abdel-Muneem Nouh and Yoshiyuki KakehiFrom the Departments of Urology and Biochemistry (XJ), Faculty of Medicine, Kagawa University, Kagawa, Japan

Purpose: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells byengaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediatedapoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific fordeath receptor 4.Materials and Methods: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassay. Synergy was assessed by isobolographic analysis.Results: Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil,vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined withdoxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line,Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity withHGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p �0.05). Doxorubicin remarkablyincreased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin andHGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream moleculesof death receptors.Conclusions: These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediatedapoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy ofdoxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.

Key Words: carcinoma, renal cell; doxorubicin; apoptosis; receptors, death domain

Renal cell carcinoma remains one of the most drugresistant malignancies because of the lack of effectivetherapy.1 Although biological response modifiers such

as interferon-�, interferon-� and interleukin-2 are relativelyeffective against RCC, the overall response rate induced bythese drugs is only 10% to 20%.2 Therefore, the developmentof novel and effective therapies, including targeted thera-pies, is clearly needed.

TRAIL, a pro-apoptotic member of the TNF superfamily,has potential as an effective anticancer agent because itselectively induces apoptosis in various tumor cells and yetit is relatively nontoxic to normal cells.3,4 TRAIL triggersapoptosis by binding to the 2 receptors TRAIL-R1 (DR4) andTRAIL-R2 (DR5).5 Activation of these receptors results in asignal transduction cascade that initiates intrinsic and ex-trinsic apoptotic pathways.6 In addition, TRAIL binds toanother 2 receptors, TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2),which lack a functional cytoplasmic death domain, and to the

Submitted for publication June 23, 2006.* Correspondence: Department of Urology, Faculty of Medicine,

Kagawa University, 1750-1 Oaza Ikenobe, Miki-cho, Kita-gun,Kagawa 761-0793, Japan (telephone: 087-891-2202; FAX: 087-891-2203; e-mail: wuxian@med.kagawa-u.ac.jp).

See Editorial on page 1606.

0022-5347/07/1775-1894/0THE JOURNAL OF UROLOGY®

Copyright © 2007 by AMERICAN UROLOGICAL ASSOCIATION

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secreted TNF receptor homologue osteoprotegerin.5,7 Thesereceptors were proposed to inhibit TRAIL induced apopto-sis. Potentially the degree of TRAIL-R1 and TRAIL-R2mediated apoptosis induced by TRAIL might be lowered inthe presence of TRAIL-R3 and TRAIL-R4 activation.Therefore, using a specific activator of TRAIL-R1 orTRAIL-R2 is preferable to exclude interference from com-petition with DcRs.

It was reported that mouse or rabbit mAbs to humanTRAIL-R1 or TRAIL-R2 have antitumor activities in vitroand in vivo.8,9 These agonistic antibodies work by activatingTRAIL mediated apoptotic pathways in a manner similar toTRAIL as TRAIL-R1 antibody induced poly(adenosinediphosphate-ribose) polymerase cleavage and as TRAIL-R2antibody induced activation of caspases and c-Jun N-termi-nal kinase/p38 in tumor cells.9,10 Recently Pukac et al re-ported that HGS-ETR1, a fully human agonistic mAb spe-cific for TRAIL-R1, decreased the viability of multiple typesof tumor cells in vitro and inhibited tumor growth in vivo.11

Using fully human antibodies would be preferable for clini-cal applications to avoid the immunogenicity associated withmouse and rabbit antibodies.12

We recently reported that the human TRAIL-R1 mAbHGS-ETR1 induced apoptotic cell death in only 1 of 11

human RCC cell cultures, whereas HGS-ETR2 was effec-

Vol. 177, 1894-1899, May 2007Printed in U.S.A.

DOI:10.1016/j.juro.2007.01.018

DEATH RECEPTOR 4 MEDIATED APOPTOSIS AND CYTOTOXICITY IN RENAL CELL CANCER 1895

tive in 10 RCC cell cultures.13 Thus, it is essential todevelop an enhancer of HGS-ETR1 sensitivity. We inves-tigated whether the cytotoxic effect of HGS-ETR1 wouldbe enhanced in combination with chemotherapeutic drugsclinically used in RCC therapy, such as ADR, 5-FU, VBLor gemcitabine. Furthermore, we explored possible mech-anisms that may be involved in the reversal of drug re-sistance.

MATERIALS AND METHODS

ReagentsThe human TRAIL-R1 mAb HGS-ETR1 is a fully humanmonoclonal antibody (IgG1) that was isolated using phagedisplay technology.11 Enzyme-linked immunosorbent assayand/or Biacore® analysis was used to determine that HGS-ETR1 is highly specific for binding to TRAIL-R1. PE conju-gated anti-TRAIL-R1 mAb (Genzyme Techne, Minneapolis,Minnesota) was used.

RCC Cell Lines and Primary RCC CellsThe 2 human RCC cell lines ACHN and Caki-1 (AmericanType Culture Collection, Rockville, Maryland) were used.Primary RCC cells were separated from the surgical speci-mens of 5 patients with untreated RCC, as described previ-ously.14 All patients were diagnosed with RCC of the alveo-lar type and clear cell subtype by histological examination.Pathological stage and grade were consistent with 2000WHO criteria, including, T2N4M0 grade 2 in patient 1,T2N0M0 grade 2 in patient 2, T2N0M0 grade 1 in patient 3,T2N0M0 grade 1 in patient 4 and T3N1M0 grade 2 in pa-tient 5.

Cytotoxicity AssaysCytotoxicity was assessed by MTT assay, as described pre-viously.14,15 Briefly, a 100 �l suspension of 2 � 104 cells wasseeded into a 96-well flat bottom microtiter plate. Afterincubation for 24 hours 100 �l drug solution or medium(control) were added to the plates in triplicate and each platewas incubated for an additional 24 hours. Subsequently20 �l MTT working solution (Sigma) (5 mg/ml) were addedfor 4 hours and 100 �l isopropanol supplemented with 0.05N hydrochloric acid were added for 30 minutes. A was mea-sured using a microplate reader (Bio-Rad®) at 570 nm witha 630 nm reference. Percent cytotoxicity was calculated us-ing the equation, cytotoxicity � [1 � (A of experimentalwells/A of control wells) � 100].

In sequential treatment experiments cells were pre-incu-bated for 8 hours with medium only, HGS-ETR1 or ADR,washed 3 times with medium and exposed to HGS-ETR1and/or ADR for 16 hours. Cytotoxicity was measured by theMTT assay.

Cell viability was determined by the trypan blue dyeexclusion test. Cells were seeded in a 6-well plate at 3 � 105

cells per well and cultured for 24 hours. Cells were thentreated in duplicate with HGS-ETR1 and/or ADR for 24hours. After treatment the cells were harvested and viablecells were counted with 0.5% trypan blue dye (Sigma).

Flow Cytometric AnalysisCell surface expression of TRAIL-R1 in RCC cells was de-

termined by flow cytometry with an EPICS® XL.13 Briefly,

RCC cells were seeded in 60 mm dishes at 5 � 105 cells perdish and cultured for 24 hours. Cells then were treated withADR at 0.1 to 10 �g/ml for 3 to 24 hours. After treatment thecells were harvested from the substrate using 0.05% trypsinand 0.02% ethylenediaminetetraacetic acid, and washedtwice in phosphate buffered saline containing 0.2% fetalbovine serum and 0.01% NaN3. The number of cells wascounted and 2 � 105 cells were incubated with PE conju-gated anti-TRAIL-R1 mAb at 4C for 30 minutes, washed andanalyzed.

Caspase Activity and Caspase Inhibition AssaysCaspase-3, 6, 8 and 9 activities were measured by a quan-titative colorimetric assay with Caspase-3, 6, 8 and 9 color-imetric protease assay kits (MBL, Nagoya, Japan), as de-scribed previously.15 Briefly, cells were treated with HGS-ETR1 and/or ADR homogenized in 200 �l cell lysis buffer,incubated for 10 minutes on ice and centrifuged at 10,000 �gravity for 1 minute at 4C. The supernatant was recoveredand the protein concentration was determined by a Bio-RadDC protein assay. The 50 �l of cell lysate corresponding to200 �g total protein, 50 �l 2 � reaction buffer and 5 �l 4 mMAsp-Glu-Val-Asp-, Val-Glu-lle-Asp-, lle-Glu-Thr-Asp- orLeu-Glu-His-Asp-pNA substrates were added to each well of96-well plates and the plates were incubated at 37C for 24hours. The A value of each well was measured with a micro-plate reader at 405 nm.

The caspase inhibition assay was performed with thegeneral caspase inhibitor Z-VAD-FMK (ICN Pharmaceuti-cals, Aurora, Ohio). ACHN cells were pretreated withZ-VAD-FMK (50 or 100 �M) for 1 hour and then they wereexposed to 1 �g/ml ADR and 100 ng/ml HGS-ETR1 for 23hours. Cell viability was assessed by the MTT assay.

Apoptosis AssaysApoptosis was determined in 2 ways. 1) Following incuba-tion with HGS-ETR1 and/or ADR for 24 hours floating andadherent cells were harvested, stained with 1 �g/ml Hoechst33258 (Wako, Tokyo, Japan) at 37C for 30 minutes and ob-served under a BX51 fluorescence microscope (Olympus™).2) For the DNA fragmentation assay DNA was extracted fromprepared cells with an Apoptosis Ladder Detection Kit (MBL)according to manufacturer instructions. Extracted DNAsamples were resolved by electrophoresis on 2% agarose geland stained with ethidium bromide.

Statistical AnalysisAll determinations were done at least 3 times and resultsare presented as the mean � SD of 3 experiments. Signifi-cance was analyzed by Student’s t test with p �0.05 consid-ered significant. Synergistic cytotoxicity was determined byisobolographic analysis, as described previously.16

RESULTS

Synergistic Cytotoxicity ofHGS-ETR1 and ADR Against RCC CellsWe examined whether treatment of the ACHN human RCCcell line with HGS-ETR1 combined with ADR, 5-FU, VBL orgemcitabine would result in synergistic cytotoxic activity.When ACHN cells were treated with a combination of HGS-

ETR1 (1 to 100 ng/ml) and ADR (0.1 to 10 �g/ml) for 24

DEATH RECEPTOR 4 MEDIATED APOPTOSIS AND CYTOTOXICITY IN RENAL CELL CANCER1896

hours, significant potentiation of cytotoxicity and synergywas achieved (fig. 1, A and B). However, there was no syn-ergistic effect of HGS-ETR1 (1 to 100 ng/ml) in combinationwith 5-FU, VBL or gemcitabine (0.1 to 10 �g/ml) (data notshown). A similar synergistic effect was noted in anotherRCC cell line, Caki-1, by combination treatment with HGS-ETR1 and ADR (fig. 1, C and D). This synergy was confirmedby the trypan blue dye exclusion test (fig. 1, E).

To determine whether synergy was a reflection of theproperties of established cancer cell lines we tested for syn-ergy in 5 samples of freshly derived RCC cells. In all casessignificant synergy was achieved irrespective of the sensi-tivity of RCC cells to ADR or HGS-ETR1 when each wasused alone (fig. 2).

Together these findings clearly demonstrated that treat-ment of RCC cell lines or freshly derived RCC cells with acombination of ETR1 and ADR resulted in the potentiationof cytotoxicity. In all cases synergy was achieved with sub-toxic concentrations of ADR.

FIG. 1. Synergistic cytotoxicity of HGS-ETR1 and ADR in humanRCC cell lines ACHN (A, B and E) and Caki-1 (C and D) treated for24 hours with 1 to 100 ng/ml HGS-ETR1 alone, 0.1 to 10 �g/ml ADRalone or combination. Cytotoxicity was measured by MTT assay.Synergy was assessed by isobolographic analysis (B and D) and cellnumber was determined by trypan blue dye exclusion test aftertreatment with 100 ng/ml HGS-ETR1 and/or 1 �g/ml ADR for 24

hours (E). Asterisk indicates significantly lower vs each agent alone(p �0.01).

Effect of EPI, THP andAMR on Synergy With HGS-ETR1Three derivatives of ADR, that is EPI, THP and AMR, werealso tested for their cytotoxic effects on ACHN cells whenused in combination with HGS-ETR1. Synergy was clearlyachieved with EPI, THP and AMR in combination withHGS-ETR1 (fig. 3).

Sensitization of RCC Cells toDR4 Mediated Cytotoxicity by ADRTo explore the underlying mechanisms in this synergisticcytotoxicity with HGS-ETR1 and ADR the effect of sequen-tial treatment with these 2 agents was examined. Pretreat-ment of ACHN cells with ADR (1 �g/ml) for 8 hours, followedby treatment with HGS-ETR1 (100 ng/ml) for 16 hours,induced significantly more cytotoxicity than reverse treat-ment or simultaneous treatment using these 2 agents(fig. 4). This sequential effect was also observed in 1 sampleof freshly derived RCC cells (patient 3) and with differentconcentrations of ADR (data not shown). These findingsindicated that ADR sensitized RCC cells to DR4 mediated

FIG. 2. Synergistic cytotoxicity of HGS-ETR1 and ADR in freshlyisolated RCC cells derived from patients 1 to 5 (A to E, respectively).RCC cells were separated from surgical specimens and treated for24 hours with 1 to 100 ng/ml HGS-ETR1 alone, 0.1 to 10 �g/ml ADRalone or combination. Cytotoxicity was measured by MTT assay andsynergy was assessed by isobolographic analysis (F).

cytotoxicity.

DEATH RECEPTOR 4 MEDIATED APOPTOSIS AND CYTOTOXICITY IN RENAL CELL CANCER 1897

Induction of TRAIL-R1 Expression by ADRTo further assess the molecular mechanisms underlying thesynergy of ADR and HGS-ETR1 in RCC cells the effect ofADR on TRAIL-R1 expression was examined by flowcytometric analysis. TRAIL-R1 expression was detected in5.6% of untreated ACHN cells and it increased remarkablyafter treatment with ADR (fig. 5, A). Figure 5, B shows thetime course of ACHN cells in response to ADR. TRAIL-R1levels increased to 34.5% after 3 hours of treatment withADR and to 93.5% after 24 hours of treatment. The doseresponse to ADR was done at the 24-hour time point(fig. 5, C). The induction of TRAIL-R1 by ADR was alsoobserved in Caki-1 and in 5 samples of freshly derived RCCcells (fig. 1, D). The up-regulation of TRAIL-R1 was alsoachieved after ACHN cells were treated with EPI or THP(data not shown).

FIG. 3. Synergistic cytotoxicity in ACHN cells treated for 24 hourswith 1 to 100 ng/ml HGS-ETR1 alone, 0.1 to 10 �g/ml ADR deriva-tives alone or combination. Cytotoxicity was measured by MTTassay and synergy was assessed by isobolographic analysis.

FIG. 4. Effect of the sequence of treatment with HGS-ETR1 andADR on cytotoxicity in ACHN cells. Cells were pre-incubated for8 hours (8h) with medium only, 100 ng/ml HGS-ETR1 or 1 �g/mlADR, washed 3 times with medium and exposed to 100 ng/mlHGS-ETR1 and/or 1 �g/ml ADR for 16 hours (16h). Cytotoxicity wasmeasured by MTT assay. F. T., treatment 1. S. T., treatment 2.Asterisk indicates significantly higher vs HGS-ETR1 or ADR alone(p �0.01). Pound sign indicates significantly higher vs first HGS-

ETR1 treatment, second ADR treatment or simultaneous treatmentusing these 2 agents (p �0.05).

Activation of CaspaseCascades and Induction of ApoptosisActivation of TNF receptor families, including TRAIL-R1,initially leads to the activation of caspase-8, which initiatesa cascade in which other caspases, including caspase-6 and3, are activated, ultimately resulting in the irreversible com-mitment of cells to undergo apoptosis.8,10 Treatment ofACHN cells with the combination of HGS-ETR1 and ADRresulted in significant activation of caspase-8, 9, 6 and 3,although ADR alone activated caspase-8 and 3 (fig. 6, A). Incontrast, HGS-ETR1 alone did not activate caspase-8, 9, 6 or3. Caspase activation was also observed when the treatmenttime of HGS-ETR1 and ADR was shortened from 24 to 12hours, although it was not observed when treatment wasshortened to 6 hours (data not shown).

We further examined the effect of the general caspase in-hibitor Z-VAD-FMK on the cell death caused by HGS-ETR1and ADR. The synergistic cytotoxicity of HGS-ETR1 and ADRwas significantly inhibited by Z-VAD-FMK (fig. 6, B).

ACHN cells were analyzed to determine if the synergisticcytotoxicity of HGS-ETR1 and ADR was mediated by apo-ptosis. Hoechst 33258 staining showed that ADR inducedapoptosis in a few cells. However, when ADR and HGS-ETR1 were used in combination, most cells showed apoptoticand typical morphological features of apoptosis, such asnuclear condensation and segmentation (fig. 6, C). Whenused alone, neither HGS-ETR1 nor ADR caused DNA frag-mentation. However, obvious DNA fragmentation was ob-served when cells were incubated with the 2 agents simul-taneously (fig. 6, D). These results indicate that thesynergistic cytotoxicity of HGS-ETR1 and ADR is realized byinducing apoptosis.

DISCUSSION

The current study shows that HGS-ETR1 and ADR had a

FIG. 5. Effect of ADR on TRAIL-R1 expression in RCC cells. ACHNcells were treated with medium only or 1 �g/ml ADR for 24 hours(A), for variable times (B) or at different concentrations (C). Caki-1and fresh RCC cells were treated with medium only or 0.1 �g/mlADR for 24 hours (D). Cells were harvested, incubated with PEconjugated anti-TRAIL-R1 mAb for 30 minutes at 4C and analyzedby flow cytometry. Gray areas represent IgG1 isotype staining. Thinhistogram indicates TRAIL-R1 staining. Thick histogram indicatesTRAIL-R1 staining after ADR treatment. Results are presented asmean � SD number of positive cells in 3 experiments. Asteriskindicates significantly different vs control (p �0.05).

synergistic effect on RCC cells, which were resistant to each

DEATH RECEPTOR 4 MEDIATED APOPTOSIS AND CYTOTOXICITY IN RENAL CELL CANCER1898

agent used alone. Synergy was achieved in the ACHN andCaki-1 human RCC cell lines and in 5 freshly derived RCCcell cultures. It required relatively low concentrations ofeach agent, thus, minimizing drug toxicity and maximizingpotential therapeutic applications in vivo. In addition, asynergistic effect was observed with HGS-ETR1 in combina-tion with the 3 ADR derivatives EPI, THP or AMR. Thesedata strongly suggest that combination treatment usingHGS-ETR1 and anthracyclines is promising from a clinicalperspective.

A synergistic effect is achieved by the reciprocal interac-tion of 2 agents. Our previous studies using anti-Fas mAbdemonstrated that combined treatment with ADR and anti-Fas mAb resulted in synergistic cytotoxicity against RCCcells.14,17 In that case anti-Fas mAb and ADR had reciprocalinteraction with each other. For example, ADR enhanced theexpression of Fas and anti-Fas mAb increased the cellularconcentration of ADR. We also reported that pretreatmentwith ADR followed by TRAIL could induce cytotoxicity sim-ilar to that of simultaneous treatment but the reverse se-quence of treatment induced significantly less cytotoxicity.15

Interestingly the current study shows that pretreatmentwith ADR followed by HGS-ETR1 could induce more signif-icant cytotoxicity than reverse treatment or simultaneoustreatment using these 2 agents. These findings suggest thatHGS-ETR1, TRAIL and anti-Fas mAb have a synergisticcytotoxic effect on RCC cells in a different manner. Addi-tionally, these different sequential effects might provide afoundation to optimize administration of these drugs forapplication in the clinical setting.

Cell surface expression of TRAIL-R1 or R2 is essential forTRAIL induced apoptosis, although tumor cells expressing

FIG. 6. Caspase activation and apoptosis induction by HGS-ETR1and ADR in ACHN cells treated for 24 hours with 100 ng/ml HGS-ETR1 alone, 1 �g/ml ADR alone or combination. A, Caspase-3, 6, 8and 9 activities were measured by a quantitative colorimetric assay(A). Synergistic cytotoxicity of HGS-ETR1 and ADR in cells treatedfor 24 hours with 100 ng/ml HGS-ETR1 plus 1 �g/ml ADR inabsence or presence of 50 or 100 �M Z-VAD-FMK (VAD) (B). Cyto-toxicity was measured by MTT assay. Asterisk indicates signifi-cantly different vs control (p �0.05). Fluorescence microscopy showsapoptotic cells stained with Hoechst 33258 (C). For DNA fragmen-tation assay DNA was extracted, separated by electrophoresis on2% agarose gel and stained with ethidium bromide (D). Lane M,DNA marker. Lane 1, medium only. Lane 2, HGS-ETR1. Lane 3,ADR. Lane 4, HGS-ETR1 plus ADR.

these death receptors are not always sensitive to TRAIL due

to intracellular mechanisms.18 In a previous study we ob-served that the surface levels of TRAIL-R1 and R2 mainlyqualify the susceptibility of human RCC cells to TRAIL-R1and TRAIL-R2 mAbs as well as TRAIL.13 It was reportedthat the efficacy of TRAIL correlates with cell surface ex-pression of TRAIL-R1 and/or R2 in leukemia cells.19 Thecurrent study shows that ADR up-regulated TRAIL-R1 ex-pression in RCC cells in a dose and time dependent manner.These results indicate that TRAIL-R1 has a critical role inthe enhancement of DR4 mediated apoptosis by ADR.

Caspases are critical protease mediators of apoptosistrigged by TRAIL.6 However, it is difficult to examine iso-lated TRAIL mediated signal transduction because variousreceptors complicate signal transduction. Using the specificTRAIL-R1 mAb HGS-ETR1 we evaluated caspase involve-ment specifically in DR4 mediated apoptosis. We found thatthe HGS-ETR1 and ADR combination significantly activatedinitiative caspases, such as caspase-8 and 9, and effectivecaspases, including caspase-6 and 3, in RCC cells. Further-more, the synergistic cytotoxicity of HGS-ETR1 and ADRwas significantly inhibited by the general caspase inhibitorZ-VAD-FMK. These findings suggest that the caspase cas-cade has an important role in the synergistic cytotoxicity ofHGS-ETR1 and ADR in RCC cells.

TRAIL may have an important role in T-cell mediatedand natural killer cell mediated cytotoxicity and apoptosisagainst cancer cells.20 This study demonstrates that treat-ment with HGS-ETR1 and ADR resulted in significant po-tentiation of cytotoxicity and apoptosis against RCC cells. Inaddition, a previous study demonstrated that treatment offreshly derived RCC cells with ADR enhanced their suscep-tibility to lysis by peripheral blood lymphocytes and tumorinfiltrating lymphocytes.14 These results suggest that theenhancement of DR4 mediated apoptosis following ADRtreatment might be a mechanism responsible for the en-hancement of susceptibility of ADR treated RCC cells tocytotoxic lymphocytes and a combination of ADR and immu-notherapy might be an alternative approach to the treat-ment of ADR/immunotherapy resistant RCC.

CONCLUSIONS

The chemotherapeutic drug resistance of RCC cells remainsa major obstacle to successful treatment and more effectivetherapy is needed. The current study clearly demonstratesthat HGS-ETR1 and ADR had a synergistic cytotoxicity inhuman RCC lines and freshly derived RCC cells at lowconcentrations of ADR (less than 10 �g/ml) that were lowerthan the range used clinically. The synergistic cytotoxicity ofHGS-ETR1 and ADR was realized by inducing apoptosisupon up-regulating TRAIL-R1 expression and activating thecaspase cascade. These findings suggest that treatment forRCC using HGS-ETR1 combined with ADR is promising fora potential clinical application.

ACKNOWLEDGMENTS

Drs. Michele Fiscella and Vivian Albert, Human GenomeSciences provided HGS-ETR1 and Kouichi Yube, ResearchEquipment Center, Faculty of Medicine, Kagawa Universityprovided technical assistance. HGS-ETR1 was provided byHuman Genome Sciences, Rockville, Maryland. HGS-ETR1

was isolated in collaboration with Cambridge Antibody

DEATH RECEPTOR 4 MEDIATED APOPTOSIS AND CYTOTOXICITY IN RENAL CELL CANCER 1899

Technology. ADR was obtained from Sigma®. EPI was ob-tained from Kyowa Hakkou, Tokyo, Japan. THP was obtainedfrom Meiji Pharmaceutical, Tokyo, Japan. AMR was obtainedfrom Sumitomo Pharmaceutical, Osaka, Japan.

Abbreviations and Acronyms

5-FU � 5-fluorouracilA � absorbance

ADR � doxorubicinAMR � amrubicinDcR � decoy receptorDR � death receptor

EPI � epirubicinMTT � 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromidePE � phycoerythrin

RCC � renal cell carcinomaTHP � pirarubicin

TRAIL � tumor necrosis factor-related apoptosis-inducing ligand

VBL � vinblastine

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