SYNERGISTIC ANTITUMOR ACTIVITY BY COMBINED TREATMENT WITH GEMCITABINE AND ANTISENSE OLIGODEOXYNUCLEOTIDE TARGETING CLUSTERIN GENE IN AN INTRAVESICAL ADMINISTRATION MODEL AGAINST HUMAN BLADDER CANCER KOTCC-1 CELLS

0022-5347/04/1716-2477/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 171, 2477–2481, June 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000118382.89456.f7

SYNERGISTIC ANTITUMOR ACTIVITY BY COMBINED TREATMENT WITH GEMCITABINE AND ANTISENSE OLIGODEOXYNUCLEOTIDE TARGETING CLUSTERIN GENE IN AN INTRAVESICAL ADMINISTRATION MODEL AGAINST HUMAN BLADDER CANCER KOTCC-1 CELLS HIDEAKI MIYAKE,* HIROSHI ETO, ISAO HARA, ALAN SO, DANBIN LI

AND

MARTIN E. GLEAVE

From the Departments of Urology, Hyogo Medical Center for Adults, Akashi and Kobe University School of Medicine, Kobe, Japan, and Division of Urology, University of British Columbia and Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, Canada

ABSTRACT

Purpose: We investigated whether antisense (AS) oligodeoxynucleotide (ODN) targeting the clusterin gene enhances the cytotoxic effect of gemcitabine in human bladder cancer KoTCC-1 cells in vitro and in vivo, and evaluated the usefulness of the combined administration of AS clusterin ODN and gemcitabine using an intraperitoneal tumor cell injection model. Materials and Methods: The cytotoxic effect of combined treatment with AS clusterin ODN and gemcitabine on in vitro KoTCC-1 growth was examined using the MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) assay. The in vivo growth inhibitory effects of combined AS clusterin ODN and gemcitabine therapy on subcutaneous KoTCC-1 tumor was also examined. The intraperitoneal tumor cell injection model, which mimics intravesical administration therapy against bladder cancer, was used to evaluate the efficacy of combined AS clusterin ODN and gemcitabine therapy. Results: AS clusterin ODN treatment of KoTCC-1 cells significantly enhanced gemcitabine chemosensitivity in a dose dependent manner, decreasing gemcitabine IC50 by approximately 90%. In vivo systemic administration of AS clusterin ODN and gemcitabine significantly decreased subcutaneous KoTCC-1 tumor volume compared with scramble control ODN plus gemcitabine. Furthermore, combined administration of AS clusterin ODN plus gemcitabine resulted in significantly delayed formation of hemorrhagic ascites compared with scramble control ODN plus gemcitabine in an intraperitoneal tumor cell injection model. Conclusions: These findings suggest that AS clusterin ODN may be useful for enhancing the cytotoxicity of gemcitabine in patients with bladder cancer, particularly as a novel therapeutic strategy for intravesical instillation therapy. KEY WORDS: bladder; bladder neoplasms; administration, intravesical; gemcitabine; clusterin

Bladder cancer is the second most common malignancy of the genitourinary tract and the fourth or fifth leading cause of cancer related deaths in men in Western industrialized countries. At presentation 70% of bladder cancers are superficial and able to be managed conservatively by an endoscopic procedure, although 60% to 70% of superficial tumors recur and 20% to 30% of recurrent disease progresses to higher stage or grade.1 The high progression rate and unpredictable pattern of disease progression have resulted in the widespread use of intravesical therapy using biological or chemotherapeutic agents. An advantage of this approach is that comparatively high doses of agents can be administered because of the lack of absorption.2 However, intravesical therapy using classic chemotherapeutic agents has not been shown to be effective for high grade superficial disease.3 Furthermore, bacillus Calmette-Guerin (BCG), the most effective agent used for intravesical therapy, induces a response in only two-thirds of patients and a third of responders have recurrent disease.4 Thus, it is important to develop a novel therapeutic strategy for patients with superficial bladder cancer resistant to standard intravesical therapy and consequently improve the survival of such patients. Clusterin, also known as testosterone-repressed prostate

message-2 or sulfated glycoprotein-2, has been shown to have important roles in various pathophysiological processes, such as reproduction, lipid transport, complement regulation and apoptosis.5 Since clusterin expression is increased in various benign and malignant tissues undergoing apoptosis, it has been regarded as a marker of cell death.6 However, recent studies provide conflicting evidence concerning the relationship between clusterin up-regulation and increased apoptotic activity.7 We also reported the powerful anti-apoptotic activity of clusterin using several kinds of malignant tumor models.8 –10 Accordingly clusterin has been regarded as an optimal therapeutic target for inhibiting tumor progression using several strategies, including antisense (AS) oligodeoxynucleotide (ODN).11 In bladder cancer our previous study has shown that clusterin over expression is closely associated with disease recurrence and progression12 and AS ODN targeting the clusterin gene synergistically enhances the cytotoxic effects of cisplatin, resulting in the inhibition of tumor growth and metastasis in a human bladder cancer model system.13 Furthermore, Dalbagni et al recently reported substantial activity of gemcitabine as an intravesical agent in BCG refractory superficial bladder cancer.14 Considering these findings, we currently tested whether combined treatment with gemcitabine and AS clusterin ODN would cooperatively inhibit human bladder KoTCC-1 tumor growth using an intraperi-

Accepted for publication December 19, 2003. * Requests for reprints: Department of Urology, Hyogo Medical Center for Adults, 13–70, Kitaohji-cho, Akashi 673-8558, Japan. 2477

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toneal tumor cell injection model that has been shown to mimic intravesical administration therapy.15 MATERIALS AND METHODS

Tumor cell line. The human bladder cancer cell line KoTCC-1 was established at our laboratory16 and maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, Maryland), supplemented with 10% fetal calf serum. Chemotherapeutic agent. Methotrexate, etoposide, doxorubicin, cisplatin and gemcitabine (British Columbia Cancer Agency, Vancouver, British Columbia, Canada) were dissolved in phosphate buffered saline (PBS) at 1 mg/ml and diluted in medium before each experiment. AS clusterin ODN. The 2⬘-O-(2-methoxy)ethyl (2⬘-MOE) modified ODNs used in this study were synthesized as described previously.17 The sequence of AS clusterin ODN corresponding to the human clusterin translation initiation site was 5⬘-CAGCAGCAGAGTCTTCATCAT-3⬘. A scrambled ODN (5⬘-CAGCGCTGACAACAGTTTCAT-3⬘) for AS clusterin ODN served as a control in this study. Treatment of cells with ODN. Lipofectin, a cationic lipid (Life Technologies, Inc.) was used to increase cell ODN uptake. KoTCC-1 cells were treated with various concentrations of ODN after pre-incubation for 20 minutes with 3 ␮g/ml lipofectin in serum-free OPTI-MEM (Life Technologies, Inc.). Four hours after the beginning of incubation medium containing ODN and lipofectin was replaced with standard culture medium, as described. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Chemical Co., St. Louis, Missouri) assay. The in vitro growth inhibitory effects of AS clusterin ODN plus a chemotherapeutic agent on KoTCC-1 cells were assessed using the MTT assay, as described previously.15 Briefly, 1 ⫻ 104 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. Cells were then treated once daily with 500 nM ODN for 2 days. Following ODN treatment cells were treated with various concentrations of the chemotherapeutic agent (methotrexate, etoposide, doxorubicin, cisplatin or gemcitabine). After 48 hours of incubation 20 ␮l 5 mg/ml MTT in PBS was added to each well, followed by incubation for 4 hours at 37C. Formazan crystals were then dissolved in dimethyl sulfoxide. Optical density was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, New Jersey) at 540 nm. Absorbance values were normalized by the values obtained for vehicle treated cells to determine the percent of survival. Each assay was performed in triplicate. Animal studies. Six to 8-week-old female athymic nude mice (Balb/c nu/nu) (Clea Japan, Inc., Tokyo, Japan) were housed in a controlled environment at 22C on a 12-hour light/12-hour dark cycle. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Each experimental group consisted of 10 mice. Tumor cells were trypsinized, washed twice with PBS and injected subcutaneously with 1 ⫻ 106 cells in the flank or 5 ⫻ 104 cells were administered intraperitoneally into the intraperitoneal cavity, as previously described.15 Seven days after subcutaneous tumor cell injection mice were randomly selected for treatment with AS clusterin ODN alone, scramble control ODN alone, AS clusterin ODN plus gemcitabine or scramble control ODN plus gemcitabine. After randomization 10 mg/kg. AS clusterin or scramble control ODN was injected intraperitoneally once daily into each mouse on days 8 to 12 and 15 to 19, and 1 mg gemcitabine was injected intravenously on days 13, 14, 20 and 21. Tumor volume was measured once weekly and calculated by the formula, length ⫻ width ⫻ depth ⫻ 0.5236.8 Data points are reported as average tumor volume ⫾ SD. For the intraperitoneal tumor cell injection model 2 sets of

experiment were performed. Seven days after tumor cell injection mice were randomized into 4 groups as described. AS clusterin (10 mg/kg) or scramble control ODN was injected intraperitoneally once daily into each mouse on days 8 to 12 and 15 to 19, and 0.5 mg or 1 mg gemcitabine was injected intraperitoneally on days 13, 14, 20 and 21 in the first and second sets of the experiment, respectively. The formation of hemorrhagic ascites, which was confirmed by puncture with an 18 gauge needle, was regarded as the end point of these experiments. RESULTS

It has already been demonstrated that AS clusterin ODN used in the current study can inhibit the expression of clusterin mRNA in KoTCC-1 cells in a dose dependent manner.13 We initially examined the growth inhibitory effects of combined treatment of KoTCC-1 cells with AS clusterin ODN and several kinds of chemotherapeutic agents, including methotrexate, etoposide, doxorubicin, cisplatin and gemcitabine. The IC50 of each agent was significantly decreased by combined treatment with AS clusterin ODN. Of these 5 agents the most powerful synergistic effect promoting the growth inhibition of KoTCC-1 cells was observed when cells were treated with AS clusterin ODN plus gemcitabine (see table). To analyze further the cytotoxic effects of combined AS clusterin ODN plus gemcitabine KoTCC-1 cells were treated with 500 nM AS clusterin ODN or scramble control ODN once daily for 2 days and incubated with medium containing various concentrations of gemcitabine for 2 days. The MTT assay was then performed to measure the number of viable cells. AS clusterin ODN treatment significantly enhanced chemosensitivity to gemcitabine in a dose dependent manner, decreasing the IC50 of cisplatin by approximately 90% (fig. 1, A). Dose dependent synergy between AS clusterin ODN and gemcitabine was also observed by increasing the AS clusterin ODN concentration when the gemcitabine concentration was fixed at 5 ␮g/ml (fig. 1, B). The efficacy of a regimen combining AS clusterin ODN and gemcitabine for inhibiting the growth of subcutaneous KoTCC-1 tumors was then evaluated. Seven days after tumor cell implantation athymic nude mice were randomly selected for treatment with AS clusterin ODN alone, scramble control ODN alone, AS clusterin ODN plus gemcitabine or scramble control ODN plus gemcitabine. Mean tumor volume was similar at the beginning of treatment in each group. While changes in tumor volume in mice treated with AS clusterin ODN alone were similar to changes in those with scramble control ODN, combined AS clusterin ODN and gemcitabine therapy showed synergistic growth inhibitory effects compared with the effects of scramble control ODN plus gemcitabine therapy. At 50 days after tumor cell injection tumor volume in mice treated with AS clusterin ODN plus gemcitabine was 68.7%, 64.5% and 38.8% smaller than in mice treated with AS clusterin ODN alone, scramble control ODN alone and scramble control ODN plus gemcitabine, respectively (fig. 2). The efficacy of combined AS clusterin ODN and gemcitabine therapy was then evaluated using an intraperitoneal

Chemotherapeutic agent IC50 changes by additional treatment with AS clusterin ODN in KoTCC-1 cells Concentration Agent

Methotrexate Etoposide Doxorubicin Cisplatin Gemcitabine

Agent Alone

Agent ⫹ 500 nM AS Clusterin ODN

7 ␮g/ml 50 ␮g/ml 0.6 ␮g/ml 40 Mg/ml 50 ␮g/ml

4 ␮g/ml 15 ␮g/ml 0.2 ␮g/ml 10 Mg/ml 5 ␮g/ml

% IC50 Decrease 43 70 67 75 90

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FIG. 2. Effects of combined treatment with AS clusterin ODN plus gemcitabine on subcutaneous KoTCC-1 tumor growth. Seven days after subcutaneous tumor cell injection mice bearing KoTCC-1 tumor were randomly selected for treatment with AS clusterin ODN, scramble control (Co) ODN, AS clusterin ODN plus gemcitabine or scramble control ODN plus gemcitabine. After randomization 10 mg/kg. AS clusterin or scramble control ODN was injected intraperitoneally once daily into each mouse on days 8 to 12 and 15 to 19, and 1 mg gemcitabine was injected intravenously on days 13, 14, 20 and 21. Tumor volume was measured once weekly and calculated by formula, length ⫻ width ⫻ depth ⫻ 0.5236. Points represent mean tumor volume ⫾ SD per experimental group of 8 mice. Asterisk indicates significantly different from controls (Student’s t test p ⬍0.01).

FIG. 1. Effect of combined treatment with AS clusterin ODN and gemcitabine on KoTCC-1 cell growth. KoTCC-1 cells were treated daily with 500 nM AS clusterin ODN or scramble control (Co) ODN for 2 days (A). Following ODN treatment medium was replaced with medium containing various concentrations of gemcitabine. After 48 hours of incubation number of viable cells was determined by MTT assay. Data points represent mean ⫾ SD of triplicate analyses. Asterisk indicates significantly different from control (Student’s t test p ⬍0.01). Cells were treated daily with various concentrations of AS clusterin ODN or scramble control ODN for 2 days and then incubated for 48 hours with medium alone or medium containing 5 ␮g/ml gemcitabine (B). Number of viable cells was determined by MTT assay. Data points represent mean ⫾ SD of 3 independent experiments. Single asterisk indicates significantly different from control (Student’s t test p ⬍0.05). Double asterisks indicate significantly different from controls (Student’s t test p ⬍0.01).

tumor cell implantation model,15 which mimics intravesical administration against bladder cancer. In the first set of experiment with the lower gemcitabine dose the formation of hemorrhagic ascites in mice receiving AS clusterin ODN plus gemcitabine was significantly delayed compared with that in mice receiving AS clusterin ODN alone, scramble control ODN alone or scramble control ODN plus gemcitabine (fig. 3, A). In the second set of experiments using a higher gemcitabine dose hemorrhagic ascites were observed in only 1 mouse treated with AS clusterin ODN and gemcitabine. Combined administration of AS clusterin ODN and gemcitabine synergistically delayed the formation of ascites compared with that in the remaining 3 treatment groups (fig. 3, B). DISCUSSION

Gemcitabine is a novel deoxycytidine analogue with a broad spectrum of antitumor activity. After intracellular activation the active metabolite is incorporated into DNA, resulting in the inhibition of further DNA synthesis. Gemcitabine has been shown to be effective and well tolerated for

the treatment of metastatic transitional cell carcinoma (TCC).18, 19 For example, a randomized phase III study showed that patients with unresectable TCC treated with gemcitabine plus cisplatin showed survival similar to that of those treated with methotrexate, vinblastine, doxorubicin and cisplatin but with a better safety profile and tolerability.19 Based on its excellent clinical efficacy and patient tolerability gemcitabine represents a optimal candidate for intravesical therapy. In fact, recent phase I trial of intravesical gemcitabine therapy for BCG refractory TCC of the bladder demonstrated that gemcitabine has substantial activity as an intravesical agent, that is 11 of 18 patients had a complete or mixed response without significant side effects,14 suggesting that gemcitabine could be an alternative to BCG for intravesical therapy for bladder cancer. Most cases of bladder cancer initially respond to combination chemotherapy, although the development of an acquired resistant phenotype is frequently observed with disease progression.1 Recently several studies indicated that over expression of antiapoptotic genes in bladder cancer cells, such as mutant-type p53 or Bcl-2, helps mediate drug resistance through the inhibition of apoptosis induced by chemotherapeutic agents.15, 20 This suggests that the approach of enhancing chemosensitivity by decreasing expression of the antiapoptotic gene appears to be a more rational strategy in patients with bladder cancer, including candidates for intravesical administration therapy, than the conventional approach of increasing the dose and combining several kinds of drugs. Furthermore, recent preclinical studies provide proof of principle evidence that targeting anti-apoptotic genes using antisense ODN enhances the cytotoxicity of conventional chemotherapy.9, 10, 13 Therefore, we examined whether AS clusterin ODN, which has been demonstrated to decrease clusterin expression,13 could enhance the cytotoxicity of gemcitabine in an intravesical tumor cell implantation model using human bladder cancer KoTCC-1 cells. We initially noted that 2⬘-MOE modified AS clusterin ODN, corresponding to the human clusterin translation ini-

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doses of gemcitabine this combined regimen synergistically delayed the formation of hemorrhagic ascites in mice compared with the administration of either agent alone. Particularly when injected with AS clusterin ODN and a higher dose of gemcitabine, hemorrhagic ascites were observed in only 1 mouse during the observation period. Accordingly if the relatively higher dose of gemcitabine were administered with AS clusterin ODN, this combined regimen may be markedly efficacious as an intravesical administration therapy. CONCLUSIONS

These findings suggest that decreasing clusterin mediated chemoresistance by AS clusterin ODN may provide a feasible strategy to enhance chemosensitivity in bladder cancer. The preclinical data presented provide proof of principle support for designing clinical studies investigating the efficacy of combined administration of AS clusterin ODN plus gemcitabine as an intravesical therapy for bladder cancer. REFERENCES

FIG. 3. Efficacy of combined intraperitoneal administration of AS clusterin ODN and gemcitabine in intraperitoneal tumor cell implantation model. Two sets of experiments were performed with different gemcitabine doses. Seven days after tumor cell injection mice bearing KoTCC-1 tumor were randomly selected for treatment with AS clusterin ODN, scramble control (Co) ODN, AS clusterin ODN plus gemcitabine or scramble control ODN plus gemcitabine. After randomization 10 mg/kg AS clusterin or scramble control ODN was injected intraperitoneally once daily into each mouse on days 8 to 12 and 15 to 19. Gemcitabine at 0.5 (A) or 1 (B) mg was injected intraperitoneally on days 13, 14, 20 and 21 in the first and second experiment sets, respectively. Hemorrhagic ascites formation, which was confirmed by puncture with 18 gauge needle, was considered experiment end point. Time to hemorrhagic ascites formation in mice receiving AS clusterin ODN plus gemcitabine was significantly delayed compared with that in mice treated with other 3 regimens (each experiment set Mantel-Cox log rank test p ⬍0.05).

tiation site, enhanced gemcitabine cytotoxicity in a dose dependent manner, decreasing its IC50 by 90%, although there was no growth inhibitory effect in KoTCC-1 cells treated with AS clusterin ODN alone. Furthermore, consistent with in vitro studies, a regimen combining AS clusterin ODN and gemcitabine synergistically inhibited the growth of subcutaneous KoTCC-1 tumors. These findings suggest that clusterin expression in bladder cancer cells may confer a phenotype resistant to cytotoxicity induced by chemotherapeutic agents. Therefore, despite the lack of a significant effect on cell proliferation in the absence of other apoptotic stimuli or cell death signals, the decrease in clusterin expression caused by AS clusterin ODN may enhance the sensitivity of cytotoxic chemotherapy for bladder cancer. We then examined the efficacy of combined treatment with AS clusterin ODN plus gemcitabine using an intraperitoneal tumor cell implantation model. In each experiment using 2

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