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SYNERGISTIC INHIBITION OF TUMOR GROWTH AND METASTASIS BY COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN A HUMAN BLADDER CANCER KOTCC-1 MODEL
0022-5347/04/1724-1485/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 172, 1485–1489, October 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000133653.74536.43
SYNERGISTIC INHIBITION OF TUMOR GROWTH AND METASTASIS BY COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN A HUMAN BLADDER CANCER KOTCC-1 MODEL MOTOTSUGU MURAMAKI,* HIDEAKI MIYAKE, ISAO HARA, GAKU KAWABATA AND SADAO KAMIDONO From the Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe and Department of Urology, Hyogo Medical Center for Adults, Akashi, Japan
ABSTRACT
Purpose: TNP-470 (O-(chloroacetylcarbamoyl)fumagillol) is a potent inhibitor of angiogenesis that was reported to enhance synergistically the antitumor effects of cytotoxic agents. We evaluated the effectiveness of combined treatment with TNP-470 and gemcitabine in vitro and in vivo using highly metastatic human bladder cancer KoTCC-1 cells. Materials and Methods: The in vitro growth inhibitory and apoptotic effects of gemcitabine and/or TNP-470 on KoTCC-1 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay and TUNEL staining, respectively. We then evaluated the combined effect of gemcitabine and TNP-470 therapy after subcutaneous and orthotopic injection of KoTCC-1 cells into athymic nude mice. Established tumors were analyzed by TUNEL staining and immunohistochemical staining of CD31 to quantify microvessel density. Results: In vitro TNP-470 enhanced the cytotoxic effect of gemcitabine on KoTCC-1 cells, decreasing its IC50 by more than 50%. This combined treatment significantly induced apoptosis compared with treatment with either agent alone. In vivo combined treatment with TNP-470 and gemcitabine significantly decreased tumor growth after subcutaneous and orthotopic injection into athymic nude mice and significantly decreased the incidence of lymph node metastasis after arthotopic injection compared with either agent alone. Immunohistochemical analysis of the subcutaneous tumor after each treatment demonstrated that gemcitabine administration significantly induced apoptotic cell death. In contrast, a significant decrease in microvessel density was observed after TNP-470 treatment. Conclusions: These findings demonstrate that TNP-470 and gemcitabine act synergistically to inhibit tumor growth and metastasis by enhancing apoptosis and suppressing angiogenesis. KEY WORDS: bladder, bladder neoplasms, O-(chloroacetylcarbamoyl)fumagillol, gemcitabine
Bladder cancer is the second most common malignancy of the genitourinary tract and the fourth or fifth leading cause of cancer related death in men in Western industrialized countries. The prognosis in patients with invasive and/or metastatic bladder cancer is still extremely poor despite recent therapeutic advances.1 The mainstay of treatment for advanced bladder cancer is cisplatin based combination chemotherapy. The most commonly used regimen is methotrexate, vinblastine, doxorubicin and cisplatin and the disease-free survival rate in patients undergoing methotrexate, vinblastine, doxorubicin and cisplatin therapy is only 3.7% at 6 years.2 Therefore, there is substantial need for the development of a novel therapeutic strategy in the care of these patients. Recently gemcitabine (2⬘, 2⬘-difluorodeoxycytidine), a deoxycytidine analogue, has been proved to have activity in several solid tumors in clinical trials.3 The therapeutic efficacy of gemcitabine has been studied in patients with advanced bladder cancer.4 In these studies gemcitabine showed significant activity in advanced bladder cancer with minimal toxicity but provided a modest survival advantage. Thus, to achieve a more cytotoxic effect combined treatment with gemcitabine and other active agents could be required. It is well established that tumor growth and metastasis depend on the induction of a new blood supply.5 As deter-
mined by microvessel density (MVD), angiogenic activity has been shown to correlate with a worse prognosis in a number of solid tumors, including bladder cancer.6 Angiogenesis is mediated by the competing actions of proteins that stimulate angiogenesis and those that inhibit angiogenesis. Of the more than 20 proteins known today the relevance of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as stimulating factors for tumor angiogenesis has been confirmed in bladder cancer.7⫺9 Interaction between these factors and vascular endothelium is important to neovascularization in solid tumors.10 Accordingly anti-angiogenetic agents targeting these factors or vascular endothelial cells could be promising therapeutic modalities for tumor dormancy therapies.11 TNP-470 is an analogue of fumagillin, derived from Aspergillus fumigatus, which strongly inhibits vascular endothelial cell proliferation and migration.12 It was previously reported that TNP-470 has an inhibitory effect on the growth and metastasis of a number of human cancers, including bladder cancer, through inhibition of the growth of vascular endothelial cells.13 Moreover, several combinations of TNP470 with other cytotoxic agents have reported to have synergistic effects in delaying tumor growth in animal studies14 and in a clinical trial.15 From these findings it is suggested that the combination of gemcitabine and TNP-470 may provide a promising inhibitory effect on the progression of bladder cancer. Therefore, we report the effect of combined treatment with gemcitabine and
Accepted for publication April 23, 2004. * Requests for reprints: Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, 7–5-1, Kusunoki-cho, Chuo-ku, 650-0017 Kobe, Japan. 1485
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TNP-470 in in vitro and in vivo experimental models using the human bladder cancer cell line KoTCC-1. MATERIALS AND METHODS
Cell culture. The human bladder cancer cell line KoTCC-1 was established at our laboratory16 and maintained in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, Maryland) supplemented with 10% fetal calf serum. Chemotherapeutic and antiangiogenic agents. Gemcitabine gemcitabine powder was dissolved in saline to a final stock concentration of 40 mg/ml. The structure of TNP-470 has been reported previously.12 TNP-470 was suspended in a vehicle composed of 1% ethanol plus 5% gum arabic in saline. MTT assay. The in vitro growth inhibitory effects of gemcitabine and/or TNP-470 on KoTCC-1 cells were assessed using the MTT assay, as described previously. Briefly, 1 ⫻ 104 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. Cells were then treated with various concentrations of gemcitabine or gemcitabine plus 0.1 g/ml TNP-470 for in vitro combined treatment. After 48 hours of incubation 100 l MTT (0.5 mg/ml) in serum-free conditioned medium (Dulbecco’s modified Eagle’s medium [MEM]/F12, Life Technologies, Inc.) was added to each well and incubation continued for 4 hours at 37C. Formazan crystals were then dissolved in 50 l dimethyl sulfoxide. Absorbance was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, New Jersey) at 540 nm. Absorbance values were normalized to values obtained for vehicle treated cells to determine the percent of survival. Each assay was performed in triplicate. Quantitative measurements of bFGF, IL-8 and VEGF. A total of 5,000 KoTCC-1 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. Medium was exchanged for 10% fetal bovine serum supplemented MEM containing various concentration of gemcitabine and/or TNP-470. The cells were washed twice with phosphate buffered saline and added to 200 l MEM supplemented with 5% bovine serum. After 48 hours the amounts of bFGF, IL-8 and VEGF in cell-free supernatants were determined using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, Minnesota). The percent of protein production per factor was then determined by comparing absorbance with that of cells treated with vehicle alone. Combined treatment was performed with various concentration of gemcitabine plus a 75% bFGF productive dose of TNP-470 (10 g/ml). In vitro TUNEL staining. The in vitro apoptotic effect of gemcitabine and/or TNP-470 was evaluated using the TUNEL staining technique. First 5 ⫻ 104 KoTCC-1 cells were seeded in each well of a 2-well glass slide (Nalge Nunc International Co., Naperville, Illinois) and allowed to attach overnight. Cells were then treated with gemcitabine (10 g/ml) and/or TNP-470 (10 g/ml) for 24 hours. Each concentration of gemcitabine and TNP-470 was the IC50 determined by MTT assay for KoTCC-1 cells treated with each single agent. TUNEL staining was performed using the ApopTag In Situ Apoptosis Detection System (Serologicals Co., Norcross, Georgia) according to manufacturer directions. The apoptotic index is expressed as the average percent of positively stained cells per total number of cells per high power field in 5 random fields. Animal studies. Athymic 6 to 8-week-old female 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. KoTCC-1 cells were trypsinized, washed twice with phosphate buffered saline and injected subcutaneously (5 ⫻ 106 cells) into the right flank or directly administered (1 ⫻ 105 cells) into the bladder wall, as described previously.16
Seven days after injection mice were randomly selected for treatment with vehicle, gemcitabine alone, TNP-470 alone or gemcitabine plus TNP-470. After randomization 60 mg/kg gemcitabine were injected intraperitoneally once weekly into each mouse for 21 days and 15 mg/kg TNP-470 were injected subcutaneously daily for 21 days. Control animals were treated with vehicle alone, which consisted of 1% ethanol and 5% gum arabic in normal saline. Tumor volume was measured twice weekly and calculated by the formula, length ⫻ width ⫻ depth ⫻ 0.5236. Five weeks after the injection of tumor cells into the bladder wall the mice were sacrificed and all abdominal organs were examined for metastases. TUNEL staining and microvessel quantification of the subcutaneous tumors. Subcutaneous tumors were collected 45 days after injection of the KoTCC-1 cells. To evaluate the apoptotic effect of gemcitabine and TNP-470 in vivo TUNEL staining of subcutaneous tumor specimens was performed as described. The percent of positively stained cells per total number of cells per high power field in 5 random fields, where no necrotic cells were observed, was counted and averaged. For microvessel quantification immunohistochemical staining was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, California). Sections were deparaffinized and incubated with a 1:200 dilution of antimouse CD31 rat monoclonal antibody (MEC 13.3, Santa Cruz Biotechnology, Inc., Santa Cruz, California) for 45 minutes, followed by biotinylated rabbit antirat IgG[H⫹L] (Vector Laboratories). Sections were then incubated with horseradish-peroxidase conjugated avidin for 30 minutes. Absorbed peroxidase was visualized by incubating with 0.05% 3,3⬘-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) and counterstained with methyl green. Intratumoral MVD was quantified in each harvested tumor by enumerating CD31 positive vessels under a microscope using a previously described method.17 For TUNEL staining and MVD quantification counting was performed using a 25-point Chalkley eyepiece by 2 independent observers. The counting of each section was performed in triplicate. There were no significant differences in intra-observer and interobserver variability. Statistical analysis. All data were evaluated with the chisquare test or Student t test using the Stat View software packages (SAS Institute, Inc., Cary, North Carolina). Probability values less than 5% were considered significant.
FIG. 1. Effect of combined treatment with TNP-470 and gemcitabine on KoTCC-1 cell growth. KoTCC-1 cells were treated with various concentrations of gemcitabine alone or various concentrations of gemcitabine plus 0.1 g/ml TNP-470. After 48 hours of incubation number of viable cells was determined by MTT assay. Data points represent mean ⫾ SD of triplicate analyses. Single asterisk indicates Student’s t test p ⬍0.05 vs treatment with gemcitabine alone. Double asterisks indicate Student’s t test p ⬍0.01 vs treatment with gemcitabine alone.
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TABLE 1. In vitro induction of apoptosis by treatment with gemcitabine and/or TNP-470 in KoTCC-1 cells Mean Apoptotic Index ⫾ SD (%)
Treatment
Control (1% ethanol ⫹ 5% gum arabic/saline) Gemcitabine (10 g/ml) TNP-470 (10 g/ml) Gemcitabine (10 g/ml) ⫹ TNP-470 (10 g/ml) Apoptotic index is expressed as average percent of positively stained staining.
p Value (Student’s t test)
2⫾1 31 ⫾ 12 Vs control ⫹ TNP-470 ⬍0.01 10 ⫾ 5 Vs control ⬍0.05 43 ⫾ 14 Vs control ⫹ TNP-470 ⬍0.01, vs gemcitabine ⬍0.05 cells per total number of cells per high power field in 5 random fields using TUNEL
RESULTS
In vitro growth inhibition by gemcitabine and/or TNP-470 in KoTCC-1 cells. To evaluate the in vitro growth inhibition of combined treatment with gemcitabine and TNP-470 the MTT assay was initially performed. Although TNP-470 alone at this concentration (0.1 g/ml) did not affect cell growth, it significantly enhanced the cytotoxic effect of gemcitabine, decreasing its IC50 by more than 50% (fig. 1). The apoptotic effect induced by gemcitabine and/or TNP-470 was then determined by TUNEL staining. Results are expressed as an average percent of positively stained cells per total number of cells per high power field in 5 random fields. Treatment with gemcitabine alone significantly induced KoTCC-1 cell apoptosis compared with the control and treatment with TNP-470 (p ⬍0.01). TNP470 alone slightly but significantly induced KoTCC-1 cell apoptosis compared with the control (p ⬍0.05). Moreover, the incidence of apoptosis was significantly higher with gemcitabine plus TNP-470 than with gemcitabine alone or TNP-470 alone (p ⬍0.05 and p ⬍0.01, respectively, table 1). In vitro inhibitory effect on production of angiogenic factors by gemcitabine and/or TNP-470 in KoTCC-1 cells. The effect of treatment with gemcitabine and TNP-470 on the production of angiogenic factors in KoTCC-1 cells was evaluated by ELISA. After incubation for 48 hours in conditioned medium the amount of bFGF, IL-8 and VEGF in cell-free supernatants was determined using commercial ELISA kits. Neither gemcitabine nor TNP-470 affected the production of IL-8 and VEGF in KoTCC-1 cells (data not shown). Gemcitabine also did not affect the production of bFGF, although TNP-470 inhibited the production of bFGF in KoTCC-1 cells in a dose dependent manner (fig. 2, A and B). Gemcitabine did not influence the inhibitory effect of TNP-470 on bFGF production when the 2 agents were administrated together (fig. 2, C). Synergistic inhibition of the growth and metastasis of
FIG. 2. In vitro inhibitory effect on bFGF production by treatment with TNP-470 and/or gemcitabine in KoTCC-1 cells. KoTCC-1 cells were treated with various concentrations of TNP-470 and/or gemcitabine. After 48 hours of incubation amount of bFGF in cell-free supernatants was determined by ELISA. A, TNP-470 inhibited bFGF production in dose dependent manner. B, gemcitabine did not affect bFGF production. C, gemcitabine did not influence inhibitory effect on bFGF production on combined treatment with TNP-470.
KoTCC-1 cells in vivo by combined treatment of gemcitabine and TNP-470. The efficacy of combined treatment with gemcitabine and TNP-470 for inhibiting the growth of subcutaneous KoTCC-1 tumors was evaluated. Seven days after the injection of tumor cells athymic nude mice bearing KoTCC-1 tumors were randomly selected for treatment with vehicle, gemcitabine alone, TNP-470 alone and gemcitabine plus TNP-470. Mean tumor volume was similar at the beginning of treatment in each group. Whereas there were slight and insignificant decreases in tumor volume in mice treated with gemcitabine alone and TNP-470 alone compared with those treated with vehicle, combined gemcitabine and TNP-470 treatment had synergistic growth inhibitory effects. At 45 days after tumor injection the tumor volume in mice treated with gemcitabine plus TNP-470 was 70.0%, 64.2% and 61.9% smaller than that in mice treated with vehicle, gemcitabine alone and TNP-470 alone, respectively (fig. 3). However, differences in the mean body weights of mice in each group were not significant (data not shown). We performed TUNEL staining of KoTCC-1 tumors to confirm the apoptotic effects induced by treatment with gemcitabine and/or TNP-470 in vivo. Treatment with TNP-470 alone induced a slight but significant increase in apoptotic cells in KoTCC-1 tumors compared with the control. Moreover, treatment with gemcitabine alone, and combined gemcitabine and TNP-470 treatment had a more significant apoptotic effect than treatment with vehicle or TNP-470 alone (fig. 4, A and B). We then performed immunohistochemical staining of CD31 to confirm the anti-angiogenic effects of these 2 agents. Intratumoral MVD was quantitated by enumerating CD31 positive vessels under a microscope. MVD was significantly lower in mice treated with TNP-470 alone or gemcitabine plus TNP-470 than in those treated with vehicle or gemcitabine alone (fig. 4, A and C). We finally examined the effects of combined gemcitabine
FIG. 3. Subcutaneous tumor growth of KoTCC-1 cells in athymic nude mice treated with vehicle, gemcitabine alone, TNP-470 alone and gemcitabine plus TNP-470. Tumor volume was measured twice weekly and calculated using formula, length ⫻ width ⫻ depth ⫻ 0.5236. Data points represent mean tumor volume ⫾ SD of 1 preparations. Asterisk indicates Student’s t test p ⬍0.01 vs other treatment group.
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COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN BLADDER CANCER
FIG. 4. Immunohistochemical analysis of subcutaneous tumors. Apoptosis induction and microvessels in subcutaneous tumors were evaluated by TUNEL staining and immunohistochemical staining of CD31, respectively. A, representative specimens of each treatment group on TUNEL and immunohistochemical staining of CD31. Scale bar represents 0.1 mm. B, percent of positively stained apoptotic cells was counted and averaged. Bars represent SD. Single asterisk indicates Student’s t test p ⬍0.01 vs control. Double asterisks indicate Student’s t test p ⬍0.05 vs control. C, intratumoral MVD was quantified by counting CD31 positive vessels under microscope. Single asterisk indicates Student’s t test p ⬍0.01 vs control or gemcitabine.
and TNP-470 treatment on metastatic features using a recently reported orthotopic injection model16 with the same treatment schedule described. Combined gemcitabine and TNP-470 treatment substantially suppressed primary tumor growth as well as the incidence of lymph node metastasis after orthotopic injection of KoTCC-1 cells compared with the other 3 treatment regimens (table 2). The weight of the primary KoTCC-1 tumor, and the incidence of retroperitoneal lymph node metastasis, mesenteric lymph node metastasis and hemorrhagic ascites were significantly lower in mice treated with gemcitabine plus TNP-470 than in those treated with vehicle, gemcitabine alone or TNP-470 alone.
However, differences in the mean body weights of mice in each group were not significant (data not shown). DISCUSSION
Acquired drug resistance is a major problem in the treatment of advanced bladder cancer. Most cases of bladder cancer initially respond to cisplatin based combination chemotherapy, although the development of an acquired resistant phenotype is frequently observed with disease progression.1 The emergence of resistance depends on the genetic instability, heterogeneity and high mutational rate of cancer cells.18
TABLE 2. Changes in metastasis of KoTCC-1 cells injected into nude mice bladder wall after gemcitabine and/or TNP-470 treatment Treatment
No. Lymph Node Metastases (%) Retroperitoneal
Intra-Abdominal
No. Hemorrhagic Ascites (%)
Mean Primary Tumor Wt (mg)
Control 10 (100) 10 (100) 8 (80) 170.4 ⫾ 72.6 Gemcitabine 7 (70) 5 (50) 5 (50) 148.6 ⫾ 45.5 TNP-470 6 (60) 6 (60) 4 (40) 135.8 ⫾ 49.2 Gemcitabine ⫹ TNP-470 2 (20)* 1 (10)* 0* 43.8 ⫾ 12.3† Two weeks after tumor cell implantation 1% ethanol plus 5% gum arabic/saline or 15 mg/kg TNP-470 were injected subcutaneously daily for 2 weeks and 60 mg/kg gemcitabine were injected intraperitoneally once weekly for 2 weeks. * Significantly different vs vehicle, gemcitabine or TNP-470 alone (chi-square test p ⬍0.05). † Significantly different vs vehicle, gemcitabine or TNP-470 alone (Student’s t test p ⬍0.05).
COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN BLADDER CANCER
In contrast, endothelial cells are genetically stable and homogenous, and have a low mutational rate. Therefore, antiangiogenic therapy directed against endothelial cells of a tumor may not result in the acquisition of drug resistance.19 The efficacy of several combined treatments with other cytotoxic agents was investigated to enhance the therapeutic effect of anti-angiogenic agents such as TNP-470.14 In this study we evaluated the effectiveness of combined treatment with TNP-470 and gemcitabine, a promising chemotherapeutic agent for bladder cancer, using human bladder cancer KoTCC-1 cells in vitro and in vivo. In in vitro experiments treatment of KoTCC-1 cells with gemcitabine and TNP-470 decreased the IC50 of gemcitabine by greater than 50% and markedly enhanced gemcitabine induced apoptosis. We also evaluated in vitro the inhibitory effect of gemcitabine and/or TNP-470 on the production of several angiogenic factors. Although TNP-470 did not affect the production of VEGF and IL-8 in KoTCC-1 cells, it inhibited that of bFGF in a dose dependent manner. Previous studies have shown that TNP-470 decreases bFGF production, resulting in the inhibition of bFGF induced angiogenesis through the suppression of vascular endothelial cell growth and migration.20 These findings suggest that TNP-470 has an anti-angiogenic effect, at least in part, through the inhibition of bFGF induced angiogenesis. In in vivo experiments gemcitabine and TNP-470 acted synergistically to inhibit the growth of subcutaneous KoTCC-1 tumors. Immunohistochemical staining revealed that this synergistic effect was associated with an increase in apoptotic cells in subcutaneous tumors, which was mainly induced by gemcitabine, as well as a decrease in angiogenic activity in subcutaneous tumors, which was induced by TNP470. Combined treatment also suppressed the incidence of lymph node metastasis after orthotopic injection of KoTCC-1 cells, resulting in a significant delay in tumor progression. Moreover, the dose of gemcitabine used in the in vivo experiments was 60 mg/kg weekly, which was the half the maximum tolerated dose of gemcitabine determined in our preliminary animal experiments using KoTCC-1 cells (data not shown). Although it remains unclear whether this regimen directly affects the metastatic process or suppresses metastasis through the inhibition of orthotopic tumor growth, it is suggested that combined treatment with TNP-470 and a comparatively low dose of gemcitabine could provide an effective and feasible therapeutic strategy against advanced bladder cancer.
3. 4.
5. 6.
7. 8.
9.
10.
11. 12. 13.
14.
15. CONCLUSIONS
Findings in the current study suggest that combined treatment with TNP-470 and gemcitabine has synergistic inhibitory effects on tumor growth and metastasis in vivo. The apoptotic effect was induced mainly by gemcitabine. In contrast, the anti-angiogenic effect was achieved only with TNP470. These 2 effects would appear to work synergistically, enhancing therapeutic efficacy. The preclinical data presented provide a rational strategy for treating patients with advanced bladder cancer. Gemcitabine was provided by Lilly Research Center, Ltd., Indianapolis, Indiana. MTT was obtained from Sigma Chemical Co., St. Louis, Missouri. TNP-470 was obtained from Takeda Chemical Industries, Ltd., Osaka, Japan.
16.
17.
18. 19.
REFERENCES
1. Thurman, S. A. and DeWeese, T. L.: Multimodality therapy for the treatment of muscle-invasive bladder cancer. Semin Urol Oncol, 18: 313, 2000 2. Saxman, S. B., Propert, K. J., Einhorn, L. H., Crawford, E. D.,
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Tannock, I., Raghavan, D. et al: Long-term follow-up of a phase III intergroup study of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study. J Clin Oncol, 15: 2564, 1997 Heinemann, V.: Gemcitabine: progress in the treatment of pancreatic cancer. Oncology, 60: 8, 2001 von der Maase, H., Hansen, S. W., Roberts, J. T., Dogliotti, L., Oliver, T., Moore, M. J. et al: Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol, 18: 3068, 2000 Hanahan, D. and Folkman, J.: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86: 353, 1996 Bochner, B. H., Cote, R. J., Weidner, N., Groshen, S., Chen, S. C., Skinner, D. G. et al: Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis. J Natl Cancer Inst, 87: 1603, 1995 Allen, L. E. and Maher, P. A.: Expression of basic fibroblast growth factor and its receptor in an invasive bladder carcinoma cell line. J Cell Physiol, 155: 368, 1993 Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau, E. J., Dvorak, H. F. et al: Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol, 143: 1255, 1993 Dickinson, A. J., Fox, S. B., Persad, R. A., Hollyer, J., Sibley, G. N. and Harris, A. L.: Quantification of angiogenesis as an independent predictor of prognosis in invasive bladder carcinomas. Br J Urol, 74: 762, 1994 Sumi, Y., Muramatsu, H., Takei, Y., Hata, K., Ueda, M. and Muramatsu, T.: Midkine, a heparin-binding growth factor, promotes growth and glycosaminoglycan synthesis of endothelial cells through its action on smooth muscle cells in an artificial blood vessel model. J Cell Sci, 115: 2659, 2002 Streeter, E. H. and Harris, A. L.: Angiogenesis in bladder cancer–prognostic marker and target for future therapy. Surg Oncol, 11: 85, 2002 Ingber, D., Fujita, T., Kishimoto, S., Sudo, K., Kanamaru, T., Brem, H. et al: Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature, 348: 555, 1990 Beecken, W. D., Fernandez, A., Panigrahy, D., Achilles, E. G., Kisker, O., Flynn, E. et al: Efficacy of antiangiogenic therapy with TNP-470 in superficial and invasive bladder cancer models in mice. Urology, 56: 521, 2000 Inoue, K., Chikazawa, M., Fukata, S., Yoshikawa, C. and Shuin, T.: Docetaxel enhances the therapeutic effect of the angiogenesis inhibitor TNP-470 (AGM-1470) in metastatic human transitional cell carcinoma. Clin Cancer Res, 9: 886, 2003 Herbst, R. S., Madden, T. L., Tran, H. T., Blumenschein, G. R., Jr., Meyers, C. A., Seabrooke, L. F. et al: Safety and pharmacokinetic effects of TNP-470, an angiogenesis inhibitor, combined with paclitaxel in patients with solid tumors: evidence for activity in non-small-cell lung cancer. J Clin Oncol, 20: 4440, 2002 Miyake, H., Hara, I., Yamanaka, K., Gohji, K., Arakawa, S. and Kamidono, S.: Overexpression of Bcl-2 enhances metastatic potential of human bladder cancer cells. Br J Cancer, 79: 1651, 1999 Fox, S. B., Leek, R. D., Weekes, M. P., Whitehouse, R. M., Gatter, K. C. and Harris, A. L.: Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol, 177: 275, 1995 Moscow, J. A., Morrow, C. S. and Cowan, K. H.: Multidrug resistance. Cancer Chemother Biol Response Modif, 13: 91, 1992 Boehm, T., Folkman, J., Browder, T. and O’Reilly, M. S.: Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature, 390: 404, 1997 Minischetti, M., Vacca, A., Ribatti, D., Iurlaro, M., Ria, R., Pellegrino, A. et al: TNP-470 and recombinant human interferon-alpha2a inhibit angiogenesis synergistically. Br J Haematol, 109: 829, 2000
Vol. 172, 1485–1489, October 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000133653.74536.43
SYNERGISTIC INHIBITION OF TUMOR GROWTH AND METASTASIS BY COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN A HUMAN BLADDER CANCER KOTCC-1 MODEL MOTOTSUGU MURAMAKI,* HIDEAKI MIYAKE, ISAO HARA, GAKU KAWABATA AND SADAO KAMIDONO From the Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe and Department of Urology, Hyogo Medical Center for Adults, Akashi, Japan
ABSTRACT
Purpose: TNP-470 (O-(chloroacetylcarbamoyl)fumagillol) is a potent inhibitor of angiogenesis that was reported to enhance synergistically the antitumor effects of cytotoxic agents. We evaluated the effectiveness of combined treatment with TNP-470 and gemcitabine in vitro and in vivo using highly metastatic human bladder cancer KoTCC-1 cells. Materials and Methods: The in vitro growth inhibitory and apoptotic effects of gemcitabine and/or TNP-470 on KoTCC-1 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay and TUNEL staining, respectively. We then evaluated the combined effect of gemcitabine and TNP-470 therapy after subcutaneous and orthotopic injection of KoTCC-1 cells into athymic nude mice. Established tumors were analyzed by TUNEL staining and immunohistochemical staining of CD31 to quantify microvessel density. Results: In vitro TNP-470 enhanced the cytotoxic effect of gemcitabine on KoTCC-1 cells, decreasing its IC50 by more than 50%. This combined treatment significantly induced apoptosis compared with treatment with either agent alone. In vivo combined treatment with TNP-470 and gemcitabine significantly decreased tumor growth after subcutaneous and orthotopic injection into athymic nude mice and significantly decreased the incidence of lymph node metastasis after arthotopic injection compared with either agent alone. Immunohistochemical analysis of the subcutaneous tumor after each treatment demonstrated that gemcitabine administration significantly induced apoptotic cell death. In contrast, a significant decrease in microvessel density was observed after TNP-470 treatment. Conclusions: These findings demonstrate that TNP-470 and gemcitabine act synergistically to inhibit tumor growth and metastasis by enhancing apoptosis and suppressing angiogenesis. KEY WORDS: bladder, bladder neoplasms, O-(chloroacetylcarbamoyl)fumagillol, gemcitabine
Bladder cancer is the second most common malignancy of the genitourinary tract and the fourth or fifth leading cause of cancer related death in men in Western industrialized countries. The prognosis in patients with invasive and/or metastatic bladder cancer is still extremely poor despite recent therapeutic advances.1 The mainstay of treatment for advanced bladder cancer is cisplatin based combination chemotherapy. The most commonly used regimen is methotrexate, vinblastine, doxorubicin and cisplatin and the disease-free survival rate in patients undergoing methotrexate, vinblastine, doxorubicin and cisplatin therapy is only 3.7% at 6 years.2 Therefore, there is substantial need for the development of a novel therapeutic strategy in the care of these patients. Recently gemcitabine (2⬘, 2⬘-difluorodeoxycytidine), a deoxycytidine analogue, has been proved to have activity in several solid tumors in clinical trials.3 The therapeutic efficacy of gemcitabine has been studied in patients with advanced bladder cancer.4 In these studies gemcitabine showed significant activity in advanced bladder cancer with minimal toxicity but provided a modest survival advantage. Thus, to achieve a more cytotoxic effect combined treatment with gemcitabine and other active agents could be required. It is well established that tumor growth and metastasis depend on the induction of a new blood supply.5 As deter-
mined by microvessel density (MVD), angiogenic activity has been shown to correlate with a worse prognosis in a number of solid tumors, including bladder cancer.6 Angiogenesis is mediated by the competing actions of proteins that stimulate angiogenesis and those that inhibit angiogenesis. Of the more than 20 proteins known today the relevance of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as stimulating factors for tumor angiogenesis has been confirmed in bladder cancer.7⫺9 Interaction between these factors and vascular endothelium is important to neovascularization in solid tumors.10 Accordingly anti-angiogenetic agents targeting these factors or vascular endothelial cells could be promising therapeutic modalities for tumor dormancy therapies.11 TNP-470 is an analogue of fumagillin, derived from Aspergillus fumigatus, which strongly inhibits vascular endothelial cell proliferation and migration.12 It was previously reported that TNP-470 has an inhibitory effect on the growth and metastasis of a number of human cancers, including bladder cancer, through inhibition of the growth of vascular endothelial cells.13 Moreover, several combinations of TNP470 with other cytotoxic agents have reported to have synergistic effects in delaying tumor growth in animal studies14 and in a clinical trial.15 From these findings it is suggested that the combination of gemcitabine and TNP-470 may provide a promising inhibitory effect on the progression of bladder cancer. Therefore, we report the effect of combined treatment with gemcitabine and
Accepted for publication April 23, 2004. * Requests for reprints: Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, 7–5-1, Kusunoki-cho, Chuo-ku, 650-0017 Kobe, Japan. 1485
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TNP-470 in in vitro and in vivo experimental models using the human bladder cancer cell line KoTCC-1. MATERIALS AND METHODS
Cell culture. The human bladder cancer cell line KoTCC-1 was established at our laboratory16 and maintained in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, Maryland) supplemented with 10% fetal calf serum. Chemotherapeutic and antiangiogenic agents. Gemcitabine gemcitabine powder was dissolved in saline to a final stock concentration of 40 mg/ml. The structure of TNP-470 has been reported previously.12 TNP-470 was suspended in a vehicle composed of 1% ethanol plus 5% gum arabic in saline. MTT assay. The in vitro growth inhibitory effects of gemcitabine and/or TNP-470 on KoTCC-1 cells were assessed using the MTT assay, as described previously. Briefly, 1 ⫻ 104 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. Cells were then treated with various concentrations of gemcitabine or gemcitabine plus 0.1 g/ml TNP-470 for in vitro combined treatment. After 48 hours of incubation 100 l MTT (0.5 mg/ml) in serum-free conditioned medium (Dulbecco’s modified Eagle’s medium [MEM]/F12, Life Technologies, Inc.) was added to each well and incubation continued for 4 hours at 37C. Formazan crystals were then dissolved in 50 l dimethyl sulfoxide. Absorbance was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, New Jersey) at 540 nm. Absorbance values were normalized to values obtained for vehicle treated cells to determine the percent of survival. Each assay was performed in triplicate. Quantitative measurements of bFGF, IL-8 and VEGF. A total of 5,000 KoTCC-1 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. Medium was exchanged for 10% fetal bovine serum supplemented MEM containing various concentration of gemcitabine and/or TNP-470. The cells were washed twice with phosphate buffered saline and added to 200 l MEM supplemented with 5% bovine serum. After 48 hours the amounts of bFGF, IL-8 and VEGF in cell-free supernatants were determined using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, Minnesota). The percent of protein production per factor was then determined by comparing absorbance with that of cells treated with vehicle alone. Combined treatment was performed with various concentration of gemcitabine plus a 75% bFGF productive dose of TNP-470 (10 g/ml). In vitro TUNEL staining. The in vitro apoptotic effect of gemcitabine and/or TNP-470 was evaluated using the TUNEL staining technique. First 5 ⫻ 104 KoTCC-1 cells were seeded in each well of a 2-well glass slide (Nalge Nunc International Co., Naperville, Illinois) and allowed to attach overnight. Cells were then treated with gemcitabine (10 g/ml) and/or TNP-470 (10 g/ml) for 24 hours. Each concentration of gemcitabine and TNP-470 was the IC50 determined by MTT assay for KoTCC-1 cells treated with each single agent. TUNEL staining was performed using the ApopTag In Situ Apoptosis Detection System (Serologicals Co., Norcross, Georgia) according to manufacturer directions. The apoptotic index is expressed as the average percent of positively stained cells per total number of cells per high power field in 5 random fields. Animal studies. Athymic 6 to 8-week-old female 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. KoTCC-1 cells were trypsinized, washed twice with phosphate buffered saline and injected subcutaneously (5 ⫻ 106 cells) into the right flank or directly administered (1 ⫻ 105 cells) into the bladder wall, as described previously.16
Seven days after injection mice were randomly selected for treatment with vehicle, gemcitabine alone, TNP-470 alone or gemcitabine plus TNP-470. After randomization 60 mg/kg gemcitabine were injected intraperitoneally once weekly into each mouse for 21 days and 15 mg/kg TNP-470 were injected subcutaneously daily for 21 days. Control animals were treated with vehicle alone, which consisted of 1% ethanol and 5% gum arabic in normal saline. Tumor volume was measured twice weekly and calculated by the formula, length ⫻ width ⫻ depth ⫻ 0.5236. Five weeks after the injection of tumor cells into the bladder wall the mice were sacrificed and all abdominal organs were examined for metastases. TUNEL staining and microvessel quantification of the subcutaneous tumors. Subcutaneous tumors were collected 45 days after injection of the KoTCC-1 cells. To evaluate the apoptotic effect of gemcitabine and TNP-470 in vivo TUNEL staining of subcutaneous tumor specimens was performed as described. The percent of positively stained cells per total number of cells per high power field in 5 random fields, where no necrotic cells were observed, was counted and averaged. For microvessel quantification immunohistochemical staining was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, California). Sections were deparaffinized and incubated with a 1:200 dilution of antimouse CD31 rat monoclonal antibody (MEC 13.3, Santa Cruz Biotechnology, Inc., Santa Cruz, California) for 45 minutes, followed by biotinylated rabbit antirat IgG[H⫹L] (Vector Laboratories). Sections were then incubated with horseradish-peroxidase conjugated avidin for 30 minutes. Absorbed peroxidase was visualized by incubating with 0.05% 3,3⬘-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) and counterstained with methyl green. Intratumoral MVD was quantified in each harvested tumor by enumerating CD31 positive vessels under a microscope using a previously described method.17 For TUNEL staining and MVD quantification counting was performed using a 25-point Chalkley eyepiece by 2 independent observers. The counting of each section was performed in triplicate. There were no significant differences in intra-observer and interobserver variability. Statistical analysis. All data were evaluated with the chisquare test or Student t test using the Stat View software packages (SAS Institute, Inc., Cary, North Carolina). Probability values less than 5% were considered significant.
FIG. 1. Effect of combined treatment with TNP-470 and gemcitabine on KoTCC-1 cell growth. KoTCC-1 cells were treated with various concentrations of gemcitabine alone or various concentrations of gemcitabine plus 0.1 g/ml TNP-470. After 48 hours of incubation number of viable cells was determined by MTT assay. Data points represent mean ⫾ SD of triplicate analyses. Single asterisk indicates Student’s t test p ⬍0.05 vs treatment with gemcitabine alone. Double asterisks indicate Student’s t test p ⬍0.01 vs treatment with gemcitabine alone.
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TABLE 1. In vitro induction of apoptosis by treatment with gemcitabine and/or TNP-470 in KoTCC-1 cells Mean Apoptotic Index ⫾ SD (%)
Treatment
Control (1% ethanol ⫹ 5% gum arabic/saline) Gemcitabine (10 g/ml) TNP-470 (10 g/ml) Gemcitabine (10 g/ml) ⫹ TNP-470 (10 g/ml) Apoptotic index is expressed as average percent of positively stained staining.
p Value (Student’s t test)
2⫾1 31 ⫾ 12 Vs control ⫹ TNP-470 ⬍0.01 10 ⫾ 5 Vs control ⬍0.05 43 ⫾ 14 Vs control ⫹ TNP-470 ⬍0.01, vs gemcitabine ⬍0.05 cells per total number of cells per high power field in 5 random fields using TUNEL
RESULTS
In vitro growth inhibition by gemcitabine and/or TNP-470 in KoTCC-1 cells. To evaluate the in vitro growth inhibition of combined treatment with gemcitabine and TNP-470 the MTT assay was initially performed. Although TNP-470 alone at this concentration (0.1 g/ml) did not affect cell growth, it significantly enhanced the cytotoxic effect of gemcitabine, decreasing its IC50 by more than 50% (fig. 1). The apoptotic effect induced by gemcitabine and/or TNP-470 was then determined by TUNEL staining. Results are expressed as an average percent of positively stained cells per total number of cells per high power field in 5 random fields. Treatment with gemcitabine alone significantly induced KoTCC-1 cell apoptosis compared with the control and treatment with TNP-470 (p ⬍0.01). TNP470 alone slightly but significantly induced KoTCC-1 cell apoptosis compared with the control (p ⬍0.05). Moreover, the incidence of apoptosis was significantly higher with gemcitabine plus TNP-470 than with gemcitabine alone or TNP-470 alone (p ⬍0.05 and p ⬍0.01, respectively, table 1). In vitro inhibitory effect on production of angiogenic factors by gemcitabine and/or TNP-470 in KoTCC-1 cells. The effect of treatment with gemcitabine and TNP-470 on the production of angiogenic factors in KoTCC-1 cells was evaluated by ELISA. After incubation for 48 hours in conditioned medium the amount of bFGF, IL-8 and VEGF in cell-free supernatants was determined using commercial ELISA kits. Neither gemcitabine nor TNP-470 affected the production of IL-8 and VEGF in KoTCC-1 cells (data not shown). Gemcitabine also did not affect the production of bFGF, although TNP-470 inhibited the production of bFGF in KoTCC-1 cells in a dose dependent manner (fig. 2, A and B). Gemcitabine did not influence the inhibitory effect of TNP-470 on bFGF production when the 2 agents were administrated together (fig. 2, C). Synergistic inhibition of the growth and metastasis of
FIG. 2. In vitro inhibitory effect on bFGF production by treatment with TNP-470 and/or gemcitabine in KoTCC-1 cells. KoTCC-1 cells were treated with various concentrations of TNP-470 and/or gemcitabine. After 48 hours of incubation amount of bFGF in cell-free supernatants was determined by ELISA. A, TNP-470 inhibited bFGF production in dose dependent manner. B, gemcitabine did not affect bFGF production. C, gemcitabine did not influence inhibitory effect on bFGF production on combined treatment with TNP-470.
KoTCC-1 cells in vivo by combined treatment of gemcitabine and TNP-470. The efficacy of combined treatment with gemcitabine and TNP-470 for inhibiting the growth of subcutaneous KoTCC-1 tumors was evaluated. Seven days after the injection of tumor cells athymic nude mice bearing KoTCC-1 tumors were randomly selected for treatment with vehicle, gemcitabine alone, TNP-470 alone and gemcitabine plus TNP-470. Mean tumor volume was similar at the beginning of treatment in each group. Whereas there were slight and insignificant decreases in tumor volume in mice treated with gemcitabine alone and TNP-470 alone compared with those treated with vehicle, combined gemcitabine and TNP-470 treatment had synergistic growth inhibitory effects. At 45 days after tumor injection the tumor volume in mice treated with gemcitabine plus TNP-470 was 70.0%, 64.2% and 61.9% smaller than that in mice treated with vehicle, gemcitabine alone and TNP-470 alone, respectively (fig. 3). However, differences in the mean body weights of mice in each group were not significant (data not shown). We performed TUNEL staining of KoTCC-1 tumors to confirm the apoptotic effects induced by treatment with gemcitabine and/or TNP-470 in vivo. Treatment with TNP-470 alone induced a slight but significant increase in apoptotic cells in KoTCC-1 tumors compared with the control. Moreover, treatment with gemcitabine alone, and combined gemcitabine and TNP-470 treatment had a more significant apoptotic effect than treatment with vehicle or TNP-470 alone (fig. 4, A and B). We then performed immunohistochemical staining of CD31 to confirm the anti-angiogenic effects of these 2 agents. Intratumoral MVD was quantitated by enumerating CD31 positive vessels under a microscope. MVD was significantly lower in mice treated with TNP-470 alone or gemcitabine plus TNP-470 than in those treated with vehicle or gemcitabine alone (fig. 4, A and C). We finally examined the effects of combined gemcitabine
FIG. 3. Subcutaneous tumor growth of KoTCC-1 cells in athymic nude mice treated with vehicle, gemcitabine alone, TNP-470 alone and gemcitabine plus TNP-470. Tumor volume was measured twice weekly and calculated using formula, length ⫻ width ⫻ depth ⫻ 0.5236. Data points represent mean tumor volume ⫾ SD of 1 preparations. Asterisk indicates Student’s t test p ⬍0.01 vs other treatment group.
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FIG. 4. Immunohistochemical analysis of subcutaneous tumors. Apoptosis induction and microvessels in subcutaneous tumors were evaluated by TUNEL staining and immunohistochemical staining of CD31, respectively. A, representative specimens of each treatment group on TUNEL and immunohistochemical staining of CD31. Scale bar represents 0.1 mm. B, percent of positively stained apoptotic cells was counted and averaged. Bars represent SD. Single asterisk indicates Student’s t test p ⬍0.01 vs control. Double asterisks indicate Student’s t test p ⬍0.05 vs control. C, intratumoral MVD was quantified by counting CD31 positive vessels under microscope. Single asterisk indicates Student’s t test p ⬍0.01 vs control or gemcitabine.
and TNP-470 treatment on metastatic features using a recently reported orthotopic injection model16 with the same treatment schedule described. Combined gemcitabine and TNP-470 treatment substantially suppressed primary tumor growth as well as the incidence of lymph node metastasis after orthotopic injection of KoTCC-1 cells compared with the other 3 treatment regimens (table 2). The weight of the primary KoTCC-1 tumor, and the incidence of retroperitoneal lymph node metastasis, mesenteric lymph node metastasis and hemorrhagic ascites were significantly lower in mice treated with gemcitabine plus TNP-470 than in those treated with vehicle, gemcitabine alone or TNP-470 alone.
However, differences in the mean body weights of mice in each group were not significant (data not shown). DISCUSSION
Acquired drug resistance is a major problem in the treatment of advanced bladder cancer. Most cases of bladder cancer initially respond to cisplatin based combination chemotherapy, although the development of an acquired resistant phenotype is frequently observed with disease progression.1 The emergence of resistance depends on the genetic instability, heterogeneity and high mutational rate of cancer cells.18
TABLE 2. Changes in metastasis of KoTCC-1 cells injected into nude mice bladder wall after gemcitabine and/or TNP-470 treatment Treatment
No. Lymph Node Metastases (%) Retroperitoneal
Intra-Abdominal
No. Hemorrhagic Ascites (%)
Mean Primary Tumor Wt (mg)
Control 10 (100) 10 (100) 8 (80) 170.4 ⫾ 72.6 Gemcitabine 7 (70) 5 (50) 5 (50) 148.6 ⫾ 45.5 TNP-470 6 (60) 6 (60) 4 (40) 135.8 ⫾ 49.2 Gemcitabine ⫹ TNP-470 2 (20)* 1 (10)* 0* 43.8 ⫾ 12.3† Two weeks after tumor cell implantation 1% ethanol plus 5% gum arabic/saline or 15 mg/kg TNP-470 were injected subcutaneously daily for 2 weeks and 60 mg/kg gemcitabine were injected intraperitoneally once weekly for 2 weeks. * Significantly different vs vehicle, gemcitabine or TNP-470 alone (chi-square test p ⬍0.05). † Significantly different vs vehicle, gemcitabine or TNP-470 alone (Student’s t test p ⬍0.05).
COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN BLADDER CANCER
In contrast, endothelial cells are genetically stable and homogenous, and have a low mutational rate. Therefore, antiangiogenic therapy directed against endothelial cells of a tumor may not result in the acquisition of drug resistance.19 The efficacy of several combined treatments with other cytotoxic agents was investigated to enhance the therapeutic effect of anti-angiogenic agents such as TNP-470.14 In this study we evaluated the effectiveness of combined treatment with TNP-470 and gemcitabine, a promising chemotherapeutic agent for bladder cancer, using human bladder cancer KoTCC-1 cells in vitro and in vivo. In in vitro experiments treatment of KoTCC-1 cells with gemcitabine and TNP-470 decreased the IC50 of gemcitabine by greater than 50% and markedly enhanced gemcitabine induced apoptosis. We also evaluated in vitro the inhibitory effect of gemcitabine and/or TNP-470 on the production of several angiogenic factors. Although TNP-470 did not affect the production of VEGF and IL-8 in KoTCC-1 cells, it inhibited that of bFGF in a dose dependent manner. Previous studies have shown that TNP-470 decreases bFGF production, resulting in the inhibition of bFGF induced angiogenesis through the suppression of vascular endothelial cell growth and migration.20 These findings suggest that TNP-470 has an anti-angiogenic effect, at least in part, through the inhibition of bFGF induced angiogenesis. In in vivo experiments gemcitabine and TNP-470 acted synergistically to inhibit the growth of subcutaneous KoTCC-1 tumors. Immunohistochemical staining revealed that this synergistic effect was associated with an increase in apoptotic cells in subcutaneous tumors, which was mainly induced by gemcitabine, as well as a decrease in angiogenic activity in subcutaneous tumors, which was induced by TNP470. Combined treatment also suppressed the incidence of lymph node metastasis after orthotopic injection of KoTCC-1 cells, resulting in a significant delay in tumor progression. Moreover, the dose of gemcitabine used in the in vivo experiments was 60 mg/kg weekly, which was the half the maximum tolerated dose of gemcitabine determined in our preliminary animal experiments using KoTCC-1 cells (data not shown). Although it remains unclear whether this regimen directly affects the metastatic process or suppresses metastasis through the inhibition of orthotopic tumor growth, it is suggested that combined treatment with TNP-470 and a comparatively low dose of gemcitabine could provide an effective and feasible therapeutic strategy against advanced bladder cancer.
3. 4.
5. 6.
7. 8.
9.
10.
11. 12. 13.
14.
15. CONCLUSIONS
Findings in the current study suggest that combined treatment with TNP-470 and gemcitabine has synergistic inhibitory effects on tumor growth and metastasis in vivo. The apoptotic effect was induced mainly by gemcitabine. In contrast, the anti-angiogenic effect was achieved only with TNP470. These 2 effects would appear to work synergistically, enhancing therapeutic efficacy. The preclinical data presented provide a rational strategy for treating patients with advanced bladder cancer. Gemcitabine was provided by Lilly Research Center, Ltd., Indianapolis, Indiana. MTT was obtained from Sigma Chemical Co., St. Louis, Missouri. TNP-470 was obtained from Takeda Chemical Industries, Ltd., Osaka, Japan.
16.
17.
18. 19.
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