ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND MEDIATED APOPTOSIS BY CISPLATIN AND CARBOPLATIN

0022-5347/01/1651-0263/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 165, 263–270, January 2001 Printed in U.S.A.

ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND MEDIATED APOPTOSIS BY CISPLATIN AND CARBOPLATIN YOICHI MIZUTANI, MASAHIRO NAKAO, OSAMU OGAWA, OSAMU YOSHIDA, BENJAMIN BONAVIDA AND TSUNEHARU MIKI From the Department of Urology, Kyoto Prefectural University of Medicine and Department of Urology, Faculty of Medicine, Kyoto University, Kyoto, Japan, and Department of Microbiology and Immunology, University of California-Los Angeles School of Medicine, University of California at Los Angeles, Los Angeles, California

ABSTRACT

Purpose: The development and acquisition of multiple drug resistance in cancer cells are a consequence of cancer chemotherapy and remain a major obstacle in treatment. Therefore, there is an obvious need for alternative approaches, such as immunotherapy and gene therapy. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of the tumor necrosis factor ligand families and it selectively induces apoptosis against cancer cells. Several cytotoxic anticancer drugs also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. We reasoned that combination treatment of cancer cells with TRAIL and drugs may overcome this resistance. We evaluated whether bladder cancer cells are sensitive to TRAIL mediated cytotoxicity and whether TRAIL may synergize with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells. Materials and Methods: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Results: Human T24 bladder cancer line was relatively resistant to TRAIL and TRAIL was not cytotoxic against normal bladder cells. Treatment of T24 cells with TRAIL in combination with 5-fluorouracil or mitomycin C did not overcome resistance to these agents. However, treatment of T24 cells with a combination of TRAIL and cisplatin resulted in a synergistic cytotoxic effect. Synergy was also achieved in the cisplatin resistant T24 line (T24/CDDP), 2 other bladder cancer lines and 3 freshly derived bladder cancer cells. The combination of TRAIL and carboplatin resulted in a synergistic cytotoxic effect on T24 cells. However, the combination of TRAIL and trans-diamminedichloroplatinum (II) resulted in an antagonistic cytotoxic effect. The synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Treating T24 cells with cisplatin enhanced the expression of bax but not bcl-2. Incubation of T24 cells with TRAIL increased the intracellular accumulation of cisplatin. Conclusions: This study demonstrates that combination treatment of bladder cancer cells with TRAIL and cisplatin overcomes their resistance. The sensitization obtained with established cisplatin resistant and freshly isolated bladder cancer cells required low subtoxic concentrations of cisplatin, supporting the in vivo potential application of a combination of TRAIL and cisplatin for treating TRAIL resistant and cisplatin resistant bladder cancer. KEY WORDS: bladder, bladder neoplasms, drug resistance, cisplatin, carboplatin

There have been many advances in cancer therapy since the introduction of anticancer chemotherapeutic agents. However, one of the consequences of chemotherapy is the development of multiple drug resistant phenotypes. Drug resistance remains a major obstacle in cancer chemotherapy. Hence, alternative approaches are necessary, such as immunotherapy and gene therapy. The cloning of biologically active cytotoxic molecules has been considered as a new potential therapy for drug resistant cancer cells. For example, some members of the tumor necrosis factor superfamily are characterized by the ability to induce apoptosis in cancer cells. Tumor necrosis factor-␣ is the first molecule to be tested for its anticancer activity, followed by Fas ligand. These 2 molecules are efficient in

killing various cancer cells, although they cause significant damage to normal tissue that results in life threatening toxicity.1, 2 The search for a cytotoxic molecule that is selective for cancer cells has continued until the recently discovered new member of the tumor necrosis factor superfamily, namely tumor necrosis factor related apoptosis inducing ligand (TRAIL/Apo-2L).3, 4 TRAIL has been shown to induce selectively apoptosis in cancer cells and it has minimal or no toxicity against normal tissues, as examined in vitro and in vivo in mice.5, 6 Therefore, TRAIL is a new agent with great potential for an in vivo anticancer effect. However, most cancer cells are not sensitive to TRAIL mediated apoptosis. Several anticancer chemotherapeutic drugs as well as TRAIL mediate apoptosis and may share common intracellular signaling pathways leading to apoptosis. We reasoned that cancer cells that are resistant to TRAIL and drugs may be sensitized by combination treatment with TRAIL and anticancer drugs. We investigated whether the resistance of bladder cancer cells to TRAIL and drugs may be overcome by

Accepted for publication July 21, 2000. Supported in part by Grant-in-Aid 12470336 from the Japanese Ministry of Education, Science and Culture, and grants-in-aid from Setsuro Fujii Memorial Osaka Foundation for Promotion of Fundamental Medical Research, Ichiro Kinbara Foundation for Medical Research and Suzuki Foundation for Urological Research. 263

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a combination of TRAIL and anticancer drugs used clinically against bladder cancer. MATERIALS AND METHODS

Tumor cells. The T24, J82 and HT1197 human bladder cancer cell lines were maintained in monolayers on plastic dishes in RPMI-1640 medium supplemented with 25 mM. HEPES, 2 mM. L-glutamine, 1% nonessential amino acid, 100 units per ml. penicillin, 100 ␮g./ml. streptomycin and 10% heat inactivated fetal bovine serum, hereafter referred to as complete medium.7, 8 The T24/CDDP line is a cisplatin resistant subline of the T24 cell line.9 Fresh bladder cancer cells from 3 patients were separated from surgical specimens as previously described.10, 11 The histological diagnosis revealed that all patients had transitional cell carcinoma of the bladder. Histological classification and staging according to the TNM classification were stage TaN0M0 grade 1, stage T1N0M0 grade 2 and stage TaN0M0 grade 2 in patients 1 to 3, respectively. Briefly, cell suspensions were prepared by treating finely minced tumor tissues with 3 mg./ml. collagenase. After washing 3 times in RPMI-1640 medium the cell suspensions were layered on discontinuous gradients consisting of 2 ml. of 100%, 2 ml. of 80% and 2 ml. of 50% Ficoll-Hypaque in 15 ml. plastic tubes, and centrifuged at 400 ⫻ gravity for 30 minutes. Lymphocyte rich mononuclear cells were collected from the 100% interface, and tumor and mesothelial cells were obtained from the 80% interface. Cell suspensions enriched with tumor cells were sometimes contaminated by monocyte macrophages, mesothelial cells or lymphocytes. To eliminate further contamination of host cells we layered the cell suspensions on a discontinuous gradient containing 2 ml. each of 25%, 15% and 10% Percoll in complete medium in 15 ml. plastic tubes and centrifuged them for 7 minutes at 25 ⫻ gravity at room temperature. Tumor cells depleted of lymphoid cells were collected from the bottom, washed and suspended in complete medium. To remove further contamination from mesothelial cells and monocyte macrophages we incubated the cell suspension in plastic dishes for 30 to 60 minutes at 37C in a humidified 5% carbon dioxide atmosphere. After incubation nonadherent cells were recovered, washed and suspended in complete medium. Usually the nonadherent cells contained mainly tumor cells with less than 5% contaminating nonmalignant cells, as judged by morphological examination of Wright-Giemsa stained smears, and they were more than 93% viable according to the trypan blue dye exclusion test. Cells with less than 5% contamination with nonmalignant cells were accepted for use as cancer cells. Urine sediment cells from healthy donors were grown in vitro and these cultured cells were of urothelial origin.12, 13 We used the short-term cultured cells of exfoliated cells from the urine of healthy volunteers as normal bladder cells. The reagents used were recombinant human TRAIL, cisplatin, trans-diamminedichloroplatinum (II) (TDDP), carboplatin, mitomycin C and 5-fluorouracil. Cytotoxicity assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to determine tumor cell lysis as previously described.14, 15 Briefly, 100 ␮L. of target cell suspension (2 ⫻ 104 cells) were added to each well of 96-well flat bottom microtiter plates and each plate was incubated for 24 hours at 37C in a humidified 5% carbon dioxide atmosphere. After incubation 100 ␮l. of drug solution or complete medium for control were distributed in the 96-well plates and each plate was incubated for 24 hours at 37C. Following incubation 20 ␮l. of MTT working solution (5 mg./ml.) were added to each culture well and the cultures were incubated for 4 hours at 37C in a humidified 5% carbon dioxide atmosphere. The culture medium was removed from the wells and replaced with 100 ␮l. of isopropanol supplemented with 0.05 N. hydrochloride. The absorbance of each

well was measured with a microculture plate reader at 540 nm. The percent cytotoxicity was calculated by the formula, percent cytotoxicity ⫽ (1 ⫺ [absorbance of experimental wells/absorbance of control wells]) ⫻ 100. Chromatin staining with Hoechst 33258. Apoptosis was determined by chromatin staining with Hoechst 33258 as previously described.16 T24 cells in a chamber slide were incubated with TRAIL at 100 ng./ml. in the absence or presence of cisplatin at 10 ␮g./ml. for 12 hours at 37C in a humidified 5% carbon dioxide atmosphere. After incubation the supernatant was discarded and T24 cells were fixed with 1% glutaraldehyde in phosphate buffered saline (PBS) for 30 minutes at room temperature, washed 4 times with PBS and exposed to Hoechst 33258 at 10 ␮M. for 30 minutes at room temperature. Cell preparations were examined under ultraviolet illumination with a fluorescence microscope. Apoptosis was defined as the observation of apoptotic bodies, chromatin condensation and/or fragmented nuclei. DNA ladder assay. Apoptosis was also determined by DNA ladder assay as previously described.9 T24 cells were treated with 100 ng./ml. TRAIL and/or 10 ␮g./ml. cisplatin for 12 hours at 37C in a humidified 5% carbon dioxide atmosphere. The cells were lysed in 200 ␮l. of DNA lysis buffer (100 mM. sodium chloride, 10 mM. tris, 10 mM. ethylenediaminetetraacetic acid [EDTA], 0.5% sodium dodecyl sulfate and 0.5 ␮g./ml. proteinase K) and incubated for 3 hours at 37C. Lysates were extracted with an equal volume of 1:1 phenol: chloroform and precipitated with 2 volumes of isopropanol. Cell pellets were resuspended in 100 ␮l. of tris-EDTA with 50 ␮g./ml. ribonuclease A and incubated for 15 minutes at 37C. Sample buffer (25 ␮l. of 5 ⫻ buffer, 50% glycerol, 0.1 M. tris, 0.1% sodium dodecyl sulfate, 0.1 M. EDTA, 0.01% xylene cyanole and 0.01% bromphenol blue) was added and samples were incubated for 10 minutes at 68C. Samples were loaded onto a 1% agarose gel, electrophoresed overnight at 20 to 30 mA. and stained with ethidium bromide to visualize DNA. Immunocytochemical detection of bcl-2 and bax expression. The expression of bcl-2 and bax in T24 cells was examined by immunocytochemical staining using a DAKO LSAB* kit, lots 117 to 4. T24 cells incubated with 10 ␮g./ml. cisplatin for 12 hours were treated with trypsin EDTA, followed by attachment to glass slides with centrifugation in a cytospin. The cells were fixed with 10% formaldehyde for 1 hour and rehydrated in 70% ethanol. The streptavidin biotinylated immunoperoxidase method was used for immunocytochemical staining. The content of cisplatin in T24 cells was determined by flameless atomic absorption spectrophotometry using a spectrophotometer as previously described in detail.17, 18 Statistical analysis. All determinations were made in triplicate and results are expressed as the mean plus or minus standard deviation. Statistical significance was determined by Student’s t test with p ⱕ0.05 considered significant. Calculations of synergistic cytotoxicity were determined by isobolographic analysis as described by Berenbaum.19, 20 Whether any particular dose combination was additive, synergistic or antagonistic is shown by whether the point representing that combination lies on, below or above the straight line joining the doses of the 2 drugs that, when given alone, produced the same effect as that combination on isobolographic analysis. RESULTS

Synergistic cytotoxicity against bladder cancer cells after combination treatment with TRAIL and cisplatin. We examined the cytotoxic effect of TRAIL in combination with commonly used anticancer agents, such as cisplatin, mitomycin C and 5-fluorouracil, against the T24 bladder cancer cell line. The T24 line was resistant to TRAIL mediated cytotoxicity. There was an additive cytotoxic effect of TRAIL in combina* DAKO Corp., Carpinteria, California.

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FIG. 1. Synergistic cytotoxic effect of TRAIL and cisplatin used in combination against T24 cells. Results are derived from 3 experiments. A, assessed by 1-day MTT assay. Asterisks indicate that values for combined treatment were significantly higher than those for treatment with cisplatin only (p ⬍0.05). B, estimated by isobolographic analysis. TABLE 1. Cytotoxic effect of TRAIL and cisplatin used in combination on triplicate samples of normal bladder cells Cisplatin (mg./ml.)

Mean % Cytotoxicity ⫾ SD 0 Ng./Ml. TRAIL

1 Ng./Ml. TRAIL

10 Ng./Ml. TRAIL

100 Ng./Ml. TRAIL

0 0 ⫺1.7 ⫾ 0.5 3.5 ⫾ 2.2 ⫺1.7 ⫾ 3.1 0.1 0.6 ⫾ 1.1 6.8 ⫾ 1.5 2.5 ⫾ 2.7 3.7 ⫾ 3.7 1 8.6 ⫾ 0.5 8.8 ⫾ 4.6 14.9 ⫾ 3.2 11.6 ⫾ 2.5 10 18.5 ⫾ 2.3 21.3 ⫾ 2.9 16.5 ⫾ 2.3 16.8 ⫾ 3.1 The cytotoxic effect of TRAIL and cisplatin on normal bladder cells was assessed by a 1-day MTT assay. Similar results were obtained with normal bladder cells from another normal donor.

tion with mitomycin C or 5-fluorouracil on T24 cells (data not shown). When T24 cells were treated with a combination of TRAIL and cisplatin, significant potentiation of cytotoxicity and synergy was noted (fig. 1). However, TRAIL was not cytotoxic against normal bladder cells and synergy was not observed when normal bladder cells were used as target cells (table 1). To confirm the synergy we then examined the sensitivity of the T24 cisplatin resistant variant cell line T24/CDDP to

combination treatment with TRAIL and cisplatin. Significant synergy was achieved by combination treatment with TRAIL and cisplatin (fig. 2). Furthermore, synergy was also achieved in 2 other bladder cancer lines, J82 and HT1197 (data not shown). Based on these findings with established bladder cancer cell lines we assessed synergy on 3 freshly derived bladder cancer cells. In all 3 cases significant synergy was achieved irrespective of the baseline sensitivity of the cancer cells to cisplatin or TRAIL used alone (fig. 3). These findings demonstrate that combination treatment with TRAIL and cisplatin resulted in synergistic cytotoxic activity against bladder cancer cells. Synergy was achieved at low concentrations of cisplatin and TRAIL was not toxic to normal bladder cells, thus, minimizing toxicity and maximizing the therapeutic application in vivo. Effect of the sequence of treatment with TRAIL and cisplatin on synergy. These findings indicate that simultaneous treatment of T24 cells with TRAIL and cisplatin resulted in synergy. The effect of sequential treatment with TRAIL and cisplatin was examined and compared with treatment with each agent added together. T24 and freshly isolated bladder cancer cells from patient 1 were treated for 6 hours with 1

FIG. 2. Synergistic cytotoxic effect of TRAIL and cisplatin used in combination against T24/CDDP cells. Results were derived from 3 experiments. A, assessed by 1-day MTT assay. Asterisks indicate that values for combined treatment were significantly higher than for treatment with cisplatin only (p ⬍0.05). B, estimated by isobolographic analysis.

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FIG. 3. Synergistic cytotoxic effect of TRAIL and cisplatin used in combination against fresh bladder cancer cells derived from patients 1 to 3 was assessed by 1-day MTT assay and synergy was evaluated by isobolographic analysis. Results were derived from triplicate samples. Values for combined treatment were significantly higher than for treatment with cisplatin only (p ⬍0.05).

agent and the medium was removed. The second agent was subsequently added for 18 hours and cells were tested for viability. Results show that the highest percent cytotoxicity was obtained when cisplatin was given initially or with cisplatin but higher cytotoxicity was achieved when TRAIL was added first (table 2). Similar results were observed when TRAIL and cisplatin were used at different concentrations (data not shown). These findings demonstrate that the sequence of treatment with TRAIL and cisplatin may be critical for obtaining synergy. Effect of the cisplatin related cytotoxic drugs, carboplatin and TDDP, on synergy with TRAIL. The 2 closely related compounds of cisplatin, carboplatin and TDDP, were tested for the cytotoxic effect against T24 cells when used in combination with TRAIL. Synergy was achieved with carboplatin in combination with TRAIL (fig. 4, A and B). In contrast, the combination of TRAIL and TDDP had an antagonistic cytotoxic effect (fig. 4, C and D). Induction of apoptosis. Since TRAIL and cisplatin each mediate apoptosis, we examined by Hoechst 33258 staining whether the synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Apoptosis was deTABLE 2. Effect of sequence of treatment with TRAIL and cisplatin in 3 experiments on cytotoxic activity against T24 and fresh bladder cancer cells 6-Hr. Pretreatment

18-Hr. Treatment

Mean % Cytotoxicity ⫾ SD T24

Pt. 1

Medium TRAIL 12.3 ⫾ 2.7 20.3 ⫾ 2.0 Medium Cisplatin 31.7 ⫾ 2.1 38.2 ⫾ 5.0 Medium TRAIL ⫹ cisplatin*,† 61.2 ⫾ 3.2 84.8 ⫾ 5.9 TRAIL Cisplatin* 43.7 ⫾ 4.5 67.0 ⫾ 4.1 Cisplatin TRAIL*,† 59.1 ⫾ 3.7 81.6 ⫾ 4.3 T24 cells and fresh bladder cancer cells derived from patient 1 were pretreated with medium only, 100 ng./ml. TRAIL or 10 ␮g./ml. cisplatin for 6 hours. The medium was aspirated and bladder cancer cells were washed twice with RPMI medium. Cells were then incubated with 100 ng./ml. TRAIL and/or 10 ␮g./ml. cisplatin for 18-hour treatment. Cytotoxicity was assessed by 1-day MTT assay. * Values were significantly higher than for treatment with cisplatin only (p ⬍0.05). † Values for the combined treatment were significantly higher than for TRAIL given initially (p ⬍0.05).

fined as the observation of apoptotic bodies, chromatin condensation and/or fragmented nuclei. No apoptosis was noted in T24 cells cultured in medium. Cisplatin at a concentration of 10 ␮g./ml. mediated modest apoptosis (fig. 5, A). Likewise TRAIL at a concentration of 100 ng./ml. also mediated apoptosis slightly (fig. 5, B). When TRAIL and cisplatin were used in combination, we observed fragmented nuclei of T24 cells. Almost all T24 cells were apoptotic and there was synergy in apoptosis (fig. 5, C). These results indicated good correlation of cytotoxicity with apoptosis after treating T24 cells with a combination of TRAIL and cisplatin. Apoptosis was also evaluated by DNA ladder assay. Cisplatin mediated minimal apoptosis. When T24 cells were treated with TRAIL at a concentration of 100 ng./ml., DNA ladder was observed. Treating T24 cells with a combination of TRAIL and cisplatin resulted in stronger DNA ladder (fig. 6). Enhancement of bax expression in T24 cells by cisplatin. We examined whether treating T24 cells with cisplatin regulates the expression of bcl-2 and bax, which are associated with apoptosis. Treating T24 cells with cisplatin did not change the expression of bcl-2. However, bax expression in T24 cells was up-regulated after treatment with cisplatin (fig. 7). Intracellular accumulation of cisplatin. A possible mechanism of augmentation of cytotoxicity may be the result of increased accumulation of cisplatin in cells treated with TRAIL. When T24 cells were treated with a combination of cisplatin and TRAIL, cisplatin accumulation inside the cells increased modestly (table 3). DISCUSSION

Our study demonstrates that treating established and freshly isolated bladder cancer cells with a combination of TRAIL and cisplatin resulted in the potentiation of cytotoxicity and apoptosis, and reversed resistance. Furthermore, synergy was observed in sensitive and resistant cancer cells. Comparable results have been achieved by others.21 Synergy was attained with subtoxic concentrations of cisplatin. Since high concentrations of cisplatin are toxic in vivo, the synergy obtained with low concentrations of the agent has clinical relevance. Furthermore, concentrations of cisplatin that sensitized cancer cells to TRAIL mediated apoptosis are within a range clinically achieved in patients with bladder cancer. Fas ligand and tumor necrosis factor-␣ as well as TRAIL are members of the tumor necrosis factor ligand family and induce apoptosis in cancer cells.22, 23 We have reported that treatment with cisplatin in combination with antiFas monoclonal antibody or tumor necrosis factor-␣ also resulted in synergistic cytotoxicity and apoptosis against bladder cancer cells.9, 24 These findings imply that cisplatin and tumor necrosis factor ligand family members with death domain may share common intracellular signaling pathways leading to apoptosis, such as the activation of caspases. The mechanisms responsible for cisplatin resistance in cancer cells are multifactorial. Alterations in the transmembrane transport of cisplatin in cancer cells result in decreased intracellular accumulation of cisplatin and resistance to cisplatin since the intracellular accumulation of cisplatin correlates with cisplatin resistance.25, 26 Cisplatin resistant cancer cells show increased levels of intracellular glutathione and/or sulfhydryl containing proteins, such as metallothionein.27, 28 Since cisplatin inhibits DNA synthesis by binding to DNA and forming intrastrand and interstrand cross links in DNA, augmenting DNA repair capability has a major role in cisplatin resistance.29 It has been reported that the increased ability to tolerate cisplatin damage is also responsible for cisplatin resistance.30 Several possible mechanisms of resistance to TRAIL mediated apoptosis have been reported, such as low expression of

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FIG. 4. Cytotoxic effect of TRAIL in combination with carboplatin or TDDP against T24 cells was assessed by 1-day MTT assay and estimated by isobolographic analysis. Results were derived from 3 experiments. A and B, in combination with carboplatin. Asterisks indicate that values for combined treatment were significantly higher than for treatment with carboplatin only (p ⬍0.05). C and D, in combination with TDDP.

FIG. 5. Fluorescence microscopic view of apoptosis in T24 cells treated with TRAIL and cisplatin. A, after treatment with 10 ␮g./ml. cisplatin. B, after treatment with 100 ng./ml. TRAIL. C, after treatment with 10 ␮g./ml. cisplatin and 100 ng./ml. TRAIL. Chromatin staining with Hoechst 33258, reduced from ⫻200.

TRAIL receptors, DR4 and DR5, and the enhanced expression of the antagonistic TRAIL receptors, DcR1 and DcR2.31, 32 The existence of multiple receptors for TRAIL suggests an unexpected complexity in the regulation of signaling by this cytokine. Anti-apoptotic molecules, such as bcl-2 and bcl-xL, may be potential resistant factors for TRAIL mediated apoptosis.33, 34 Since TRAIL induces apoptosis in cancer cells in a caspase dependent fashion, resistance to TRAIL mediated apoptosis may depend on the level of the

expression of caspases.35, 36 FLICE inhibitory protein was shown to bind to caspase 8 and prevent activation of the downstream events leading to apoptosis, including TRAIL mediated apoptosis.37, 38 Cisplatin mediates a significant inhibitory effect on DNA synthesis. Carboplatin, a closely related platinum analogue, has antitumor activity similar to that of cisplatin.39 In contrast, TDDP has much lower activity since the chloride and ammonium groups are in the transposition.40, 41 Treating

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2 major pathways to enhance TRAIL mediated apoptosis, including the suppression of anti-apoptotic molecules and the up-regulation of pro-apoptotic molecules. Bcl-2 and bcl-xL, which are major inhibitors of the mitochondrial apoptotic pathway, are regulated by anticancer agents.42, 43 Taxol decreases the activity of bcl-2 by inducing the phosphorylation of bcl-2.34, 44 Preliminary experiments showed that treating T24 cells with cisplatin enhanced the expression of p53, which induces apoptosis. Genotoxic drugs enhance the expression of the pro-apoptotic TRAIL receptor, DR5.45 Further studies are required to elucidate the effect of cisplatin on the various signaling molecules involved in the TRAIL apoptotic pathways. TRAIL and Fas ligand are each coexpressed on the cell surface of immune cells.46, 47 TRAIL as well as Fas ligand may have an important role in T-cell and natural killer cell mediated cytotoxicity and apoptosis against cancer cells.48, 49 Cells resistant to Fas mediated apoptosis are sensitive to TRAIL mediated apoptosis.50, 51 In this study and our previous report treatment with cisplatin in combination with TRAIL or antiFas monoclonal antibody resulted in significant potentiation of cytotoxicity and apoptosis against bladder cancer cells.9 In addition, treating freshly isolated bladder cancer cells with cisplatin enhanced susceptibility to lysis by autologous lymphocytes.9, 51 These findings suggest that the TRAIL mediated and Fas mediated apoptosis that is enhanced by cisplatin may be mechanisms responsible for the augmented susceptibility of cisplatin treated bladder cancer cells to cytotoxic lymphocytes. In addition, a combination of cisplatin and immunotherapy may be an alternative approach to the treatment of cisplatin and immunotherapy resistant bladder cancer. FIG. 6. Apoptosis in T24 cells treated with TRAIL and/or cisplatin revealed by DNA ladder assay. Each lane represents DNA of T24 cells stained with ethidium bromide. Marker, ␭-HindIII. 1, after treatment with medium only. 2, after treatment with 10 ␮g./ml. cisplatin. 3, after treatment with 10 ␮g./ml. cisplatin and 100 ng./ml. TRAIL. 4, after treatment with 100 ng./ml. TRAIL.

T24 cells with TRAIL in combination with cisplatin or carboplatin resulted in synergistic cytotoxicity, while the combination of TRAIL and TDDP did not. Treating T24 cells with TRAIL in combination with mitomycin C or 5-fluorouracil, which are also inhibitors of DNA synthesis, did not overcome resistance. These findings imply that the type of DNA crosslinking damage caused by cisplatin but not by mitomycin C or 5-fluorouracil may closely regulate or participate directly in the processes of TRAIL mediated cytotoxicity and apoptosis specifically. Although the up-regulation of bax expression by cisplatin and the increased intracellular accumulation of cisplatin by TRAIL are indicative of the synergistic cytotoxicity and apoptosis of TRAIL and cisplatin, to our knowledge the precise mechanism of synergy is not fully understood. There are

CONCLUSIONS

The overall response rate of patients with bladder cancer to current anticancer chemotherapeutic agents, including cisplatin, has improved. However, drug resistance and recurrent bladder cancer remain major problems and more effective therapies are necessary in these patients. The current study shows that combination treatment with TRAIL and cisplatin resulted in synergistic cytotoxicity and apoptosis against acquired and natural cisplatin resistant bladder cancer cells. Although in vivo studies are needed, these findings imply that treatment with a combination of cisplatin and TRAIL may be useful in patients with cisplatin and immuTABLE 3. Effect of TRAIL on the intracellular accumulation of cisplatin in 4 experiments in T24 cells Treatment

Mean Cisplatin ⫾ SD (ng./105 cells)

Medium 42.9 ⫾ 4.3 TRAIL 63.6 ⫾ 10.7, p ⬍0.05 T24 cells cells were treated with 10 ␮g./ml. cisplatin combined with medium or 100 ng./ml. TRAIL for 12 hours. The medium was aspirated and T24 cells were washed 3 times with RPMI medium. The intracellular concentration of cisplatin was measured by flameless atomic absorption spectrophotometry.

FIG. 7. Immunocytochemical staining shows that expression of bax in T24 cells was enhanced after treatment with cisplatin. A, medium only. B, 10 ␮g./ml. cisplatin. Reduced from ⫻200.

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notherapy resistant bladder cancer as a new form of therapy with more selective cytotoxicity and less collateral toxicity. Dr. Manabu Fukumoto provided advice on this investigation, recombinant human TRAIL was provided by Pepro Tech, Rocky Hill, New Jersey, cisplatin (lot Y22620) and TDDP (lot KI-1717) were provided by Nippon Kayaku Co., Ltd., Tokyo, Japan, carboplatin (lot PPV055) was provided by Bristol Myers Squib Co., Ltd., Tokyo, Japan, and mitomycin C (lot 624A11) and 5-fluorouracil (lot 469ABG) were supplied by Kyowa Hakkou Co., Ltd., Tokyo, Japan.

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