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Keywords:

  • AGM-1470;
  • bladder carcinoma;
  • N-butyl-N-(4-hydroxybutyl) nitrosamine;
  • angiogenesis;
  • endothelial cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

To examine the antitumour effects of the angiogenesis inhibitor AGM-1470 (TNP-470) on rat urinary bladder tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN).

Materials and methods

Fischer-344 rats were allocated to one of four groups of 15 rats each; in group 1, rats were administered AGM-1470 intraperitoneally, with group 2 acting as the control and given only saline; in group 3, AGM-1470 was instillation intravesically and group 4 acting as control (intravesical saline). All rats were given 0.05% BBN in their drinking water for 8 weeks and then given water with no BBN. AGM-1470 was administered at a dose of 30 mg/kg every other day for 6 weeks in group 1 and at 15 mg/kg once a week for 6 weeks in group 3. This treatment was commenced at 21 weeks after the start of BBN treatment, when tumorigenesis was apparent in all rat bladders; ≈70–80% of the tumours were carcinomas. All rats were killed in the 27th week. The antitumour effects of AGM-1470 on the BBN-induced bladder tumours were evaluated macroscopically and histologically. The inhibitory effect of AGM-1470 on endothelial cell proliferation was assessed in groups 1 and 2 by immunohistochemical staining for Factor VIII-related antigen and by counting the microvessels.

Results

The number and volume of bladder tumours were significantly less in group 1 than group 2. In the latter, at least one bladder tumour developed in each of the 15 rats. Histologically, transitional cell carcinoma (TCC) was found in 13 rats and papilloma in two, with invasive cancer in three of the 13 TCCs. Bladder tumours developed in only four of the 15 rats in group 1. Carcinomas were found in three of these four rats and no invasive cancer was detected. The rats in group 1 had significantly fewer microvessels than the controls. The rats in group 4 also showed slightly but insignificantly less tumour growth and fewer carcinomas. In neither experiment were any major side-effects seen except for mild weight loss after AGM-1470 treatment.

Conclusion

AGM-1470 inhibited the growth and malignant progression of BBN-induced bladder tumours in rats, apparently mainly by the inhibition of tumour vessel development. The intraperitoneal administration of AGM-1470 produced better results than did intravesical instillation. These results suggest that the angiogenesis inhibitor AGM-1470 is a promising agent for the treatment of human bladder cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Angiogenesis, the formation of new blood vessels, is essential for the development and growth of solid tumours [1]. Fumajillin is a compound isolated from Aspergillus fumigatus and is known to be an antibiotic against amoebiasis. Fumajillin and its synthetic analogue, AGM-1470, have been found to be potent angiogenesis inhibitors, although AGM-1470 is 50 times more effective and is much less toxic than the parent compound [2]. AGM-1470 has been shown to inhibit the growth of solid tumours in vivo, but not that of the same tumour cells in vitro, indicating that the inhibitory effect of AGM-1470 on tumour growth is induced by the inhibition of angiogenesis [2]. Recent reports have shown that AGM-1470 inhibits tumour growth and metastasis in a nude-mouse model with subcutaneously implanted human cancer cell lines [3], and in a variety of rodent tumours [4]. Thus, AGM-1470 could inhibit not only tumour growth but also metastasis.

Bladder carcinoma is the second most common malignant tumour of the genitourinary area. Most bladder cancers are confined within the mucosal layer; however, some show deeper penetration into the muscle layer from the mucosa and such patients have a poor outcome even after radical treatment [5]. Frequent recurrence is another clinical feature of this tumour [6]. Recent clinicopathological studies have shown that the higher vascular density that exists in bladder cancer tissues increases the risk of lymph node metastasis [7] and mortality [8]. From this perspective, anti-angiogenic therapy against bladder cancer could be a new strategy for improving the prognosis of patients with bladder cancer.

Druckrey et al. [9] first described the carcinogenic effect of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) on the rat urinary bladder. Since then, this model system has been used by many investigators to study the histogenesis and the effects of various agents on the tumorigenesis and growth of bladder tumours [10[11]–12]. In a preliminary experiment, we found that when 0.05% BBN was administered to rats daily for 8 weeks, bladder tumours developed in all rats and by the 20th week, 70–80% of these tumours were carcinomas. We used this model system in the present study to investigate the effects of the angiogenesis inhibitor AGM-1470 on the growth and malignant progression of bladder tumours, and on the status of the angiogenesis in the mucosal tissue of the bladder.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Female Fischer-344 rats were obtained from Charles River Japan (Tokyo, Japan) and used at 7 weeks of age, when they weighed 120–140 g. They were housed five rats per cage at a temperature of 23±2°C in a controlled 12 h light:12 h dark environment with free access to water and standard rat chow. AGM-1470 (TNP-470; molecular weight 401.89) was kindly provided by Takeda Chemical Industries, Ltd (Osaka, Japan). Stock solutions of AGM-1470 were prepared in absolute ethanol and suspended in 5% gum arabic and normal saline.

Sixty rats were given 0.05% BBN in their drinking water for 8 weeks and then given water with no BBN for 12 weeks. In a preliminary study, tumour development and progression to TCC in the urinary bladder occurred in all and 70–80% of the rats, respectively, by the 20th week after starting the administration of the BBN. The 60 rats used in the present study were divided into four groups of 15 rats each. Starting at week 21, AGM-1470 was administered intraperitoneally to group 1, at a dose of 30 mg/kg in saline every other day for 6 weeks; group 2 rats received an equivalent volume of saline, also intraperitoneally. In group 3, AGM-1470 solution (15 mg/kg) was instilled into the rats once a week for 6 weeks through a catheter into the bladder while the rats were under Nembutal™ anaesthesia. The second group of control rats (group 4) received an equivalent volume of saline also instilled through a catheter. Before and 1 h after the instillation, the bladder contents were emptied by light abdominal massage to ensure that the solutions instilled were retained for only 1 h. All rats were killed at the end of week 27. The bladder was then immediately fixed by injection of ≈ 1.0 mL of 10% buffered formalin (pH 7.4) into the lumen. The body weight and major organ (heart, kidney, liver and spleen) weights were measured in each rat to detect any side-effects.

The bladder tumours that developed were counted macroscopically and the tumour diameters measured. The tumour volume was calculated as (width)2 ×(length)/2. The bladder was then cut into four serial sections which were paraffin-embedded and stained with haematoxylin and eosin, as described previously [13,14]. The histological changes that had occurred in the bladder epithelium were classed as simple hyperplasia (SH), papillary or nodular hyperplasia (PNH), papilloma or carcinoma. The pathological grade and stage of the carcinomas were evaluated according to the ‘General Rules for Clinical and Pathological Studies on Bladder Cancer in Japan’ [15].

Formalin-fixed paraffin-embedded sections (3 μm) of bladder tissue were used for immunohistochemistry. Capillary endothelial cells were detected using the immunohistochemical staining of Factor VIII-related antigen (von Willebrand’s factor) using a Universal DAKO LSAB 2 Rat Kit (K609, DAKO Co., Carpinteria, CA, USA). Briefly, the sections were dewaxed in xylene and then rehydrated in a graded alcohol series. The endogenous peroxidase activity in the tissue was blocked by treating the sections with 3% hydrogen peroxide for 5 min. The sections were then incubated with rabbit anti-rat Factor VIII-related antigen (diluted 1:200, L1809, DAKO) for 10 min at room temperature. After being washed three times in Tris-buffered saline (TBS, pH 7.6), the sections were incubated with the secondary antibody (biotinylated rabbit antimouse) for 10 min. The sections were then incubated with streptavidin conjugated with horseradish peroxidase for 10 min. After incubation with substrate chromogen (3,3’-diaminobenzidine tetrahydrochloride) solution, the sections were washed in double-distilled water, counterstained in Mayer’s haematoxylin, dried in ethanol and xylene, and then mounted.

The individual microvessels were counted in ×200 fields; any brown-stained endothelial cell or endothelial cluster was considered to be a single, countable microvessel [16]. The mean total number of microvessels in the mucosa throughout the bladder wall in the rats in group 1 was compared with that in group 2. Vessel lumina in the elements of the urothelium were not counted.

The mean number and volume of the tumours, and the results of microvessel counts were assessed using the Mann—Whitney U-test. A chi-square test was used to analyse the incidence of carcinoma and invasion. Differences were considered significant when P<0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The macroscopical findings and histological changes occurring in the rat urinary bladders are summarized in Table 1. The mean tumour number and volume in group 1 was significantly lower than that in group 2 (P<0.001). SH and PNH were observed in most of the rats in all groups. Carcinomas developed in three of the rats in group 1 and in 13 of the control rats (P=0.02). All of the carcinomas observed were TCCs and all of those in group 1 were superficial bladder tumours (pTa or pT1); no invasive cancer was observed. Muscle-invasive carcinoma (pT2 or pT3) was found in three of the 13 carcinomas in group 2 (Fig. 1) but the difference was not significant; two of these three carcinomas were grade 3. Although there was mild weight loss in the rats in group 1, no major side-effects were seen and no rats died during the experimental period.

Table 1.  The effect of intraperitoneal injection or intravesical instillation of AGM-1470 in rats Thumbnail image of
image

Figure 1. Grade 2 transitional cell carcinoma of the bladder epithelium in a control rat. Haematoxylin and eosin, original ×100.

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Neither the mean tumour number nor tumour volume in the rats in group 3 was significantly different from that in group 4 (Table 1). Eight of the 15 rats in group 3 had TCC, but none were invasive. In the control group, carcinoma was found in 13 of the 15 rats (not significant), and invasive carcinoma in two (not significant). During this experiment, the anaesthesia given for the intravesical instillation caused death in one rat; however, except for mild weight loss in the rats in group 3, no other major side-effects were seen.

The mean (sd) microvessel number in the mucosa throughout the bladder wall in group 1, at 189.0 (68.0), was significantly lower than that in the control group, at 362.8 (109.4) (Mann–Whitney U-test, P=0.001; Fig. 2).

image

Figure 2. Immunohistochemical staining for Factor VIII-related antigen in the mucosa throughout the bladder wall. (a) Many microvessels are markedly stained in the control rat (original ×100). (b) Less marked angiogenesis is seen in a rat treated with AGM-1470. Original ×100.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

AGM-1470 has been shown to be a potent inhibitor of endothelial cell proliferation in vitro[2] and of capillary-like tube formation of endothelial cells, with a minimal effect on non-endothelial cell growth, in the rat blood-vessel organ-culture assay [17]. Its has also been show to be anti-angiogenic in vivo; AGM-1470 inhibited capillary growth and induced the development an avascular zone in a chorioallantoic membrane assay, and suppressed the number and length of new blood vessels in a rat corneal assay [17]. Angiogenesis is a key factor in the development and growth of solid tumours, and the inhibition of angiogenesis could be a new strategy for cancer treatment. Indeed, the inhibition of angiogenesis has been reported to correlate strongly with the inhibition of tumour growth and metastasis [3,4,18[19]–20].

Previous studies of urological cancer have shown the antitumour effects of AGM-1470 on a human prostate cancer cell line, PC-3 [18] and on the murine RCC Renca tumour [19,20]. In these studies, the effects of AGM-1470 were examined in tumours generated by injections given either subcutaneously or under the renal capsule. In the present study, the BBN-induced rat urinary bladder tumour model was used, as we consider it to be similar to clinical bladder tumours. Intraperitoneal AGM-1470 strongly inhibited tumour growth, reducing the incidence of carcinoma and of muscle invasion compared with that found in the control group. We suggest that this effect was a result mainly of the inhibition of angiogenesis, as the bladder mucosa treated with AGM-1470 contained fewer microvessels than in the controls. In this animal model, it has been shown that BBN-induced tumorigenesis progresses from PNH or papilloma to carcinoma and that the formation of new blood vessels develops simultaneously with tumour growth [21]. The present results indicate that AGM-1470 inhibited not only tumour growth but also malignant progression, by suppressing angiogenesis. Furthermore, there were no toxic deaths during the period of the study. A previous study using mice, in which AGM-1470 was administered subcutaneously at a dose of 30 mg/kg every other day for >100 days, revealed no severe side-effects, e.g. hair loss, intestinal disturbance or drug-associated death [2].

Tanaka et al. [22] recently reported that AGM-1470 reduced N-methyl-N-nitrosourea-induced tumorigenesis in the heterotopically transplanted rat urinary bladder system. Together with the present results, this finding indicates that AGM-1470 could be a useful therapeutic agent for bladder tumours, inhibiting tumorigenesis, tumour growth and malignant progression.

The intravesical instillation of BCG or anticancer drugs has been used as prophylaxis against recurrences and for the clinical treatment of superficial bladder tumours [5]. This technique allows the agents to contact the tumours or preneoplastic lesions directly and lessens the systemic toxicity that may occur. For these reasons, we administered AGM-1470 intravesically to group 3; the tumour volume in this group was smaller than that of the control group, and the incidence of carcinoma and muscle invasion was also lower. Thus, intravesical AGM-1470 also appears to inhibit, to some extent, the growth and malignant progression of rat bladder tumours induced by BBN, although there was no significant difference between the results of group 3 and 4. The magnitude of the inhibitory effects was also weaker than that in group 1. The reasons underlying the difference in inhibition of tumour growth by AGM-1470 between groups 1 and 3 are unclear. The concentration of AGM-1470 in the capillaries and/or the contact time of AGM-1470 with the capillary endothelial cells in the bladder cancer tissues might be less when administered intravesically than intraperitoneally.

The mechanism underlying the anti-angiogenic effects of AGM-1470 was recently clarified. Kusaka et al. [23] showed that the specific anti-angiogenic action of AGM-1470 is a cytostatic rather than cytotoxic inhibition of the growth of endothelial cells. This cytostatic inhibition was confirmed, by flow cytometric analysis, to involve a selective suppression of DNA synthesis [23]. The specific effect of AGM-1470 on vascular endothelial cell growth arises from the suppression of cyclin D1 mRNA expression in the mid G1 phase [24]. Thus, AGM-1470 inhibits the growth of endothelial cells, which are major components of new blood vessels [25]. The immunohistochemical results of the present study show that AGM-1470 suppressed the growth of rat bladder tumours by inhibiting endothelial cell growth.

In a recent study using ribonuclease protection analysis, O’Brien et al. [26] reported that superficial and invasive bladder cancer are characterized by different angiogenic pathways. The expression of vascular endothelial growth factor (VEGF) RNA in superficial tumours was stronger than that in normal bladders, and the expression of platelet-derived endothelial cell growth factor (PDECGF) was higher in invasive cancers than in superficial tumours [26]. VEGF is a potent angiogenic factor, having a specific mitogen for endothelial cells [27]. In the animal model used in the present study the expression of VEGF or PDECGF RNA may also have been suppressed in the rats in group 3 and 4. Further studies using molecular analysis are needed to clarify whether this antitumour effect of AGM-1470 is related to angiogenic factors.

More recently, a Phase-I clinical trial involving patients with inoperable recurrent or metastatic squamous cell cancer of the uterine cervix was carried out using AGM-1470 (TNP-470) as a treatment [28]. Of 18 evaluable patients, one had a complete response and in three patients the disease stabilized. Neurotoxicity was dose-limiting, but appeared to be reversible and well tolerated. The initial dose tested was 9.3 mg/m2 and this was subsequently increased; an AGM-1470 dose of 60 mg/m2 as a 60-min intravenous infusion three times a week was recommended for further study [28]. The present results confirm that AGM-1470 could be a useful therapeutic agent for bladder cancer and a clinical study of AGM-1470 in the treatment of patients with advanced bladder cancer is expected to be performed in the future.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Takeda Chemical Industries Ltd for the generous gift of the AGM-1470 used in this study.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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