Journal of Neuro-Oncology 58: 131136, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Antitumor effect of TNP-470 is not associated to decrease of angiogenesis in an experimental malignant neuroectodermic tumor
C. Morales, M. Zurita and J. Vaquero Laboratory of Experimental Neuro-Oncology, Neuroscience Research Unit of the Mapfre-Medicine Foundation, Puerta de Hierro Clinic, Autonomous University, Madrid, Spain

Key words: angiogenesis, experimental tumor, TNP-470 Summary The hypothesis that tumor growth depends on neovascularization has been broadly used in oncology research. TNP-470 is a fumagillin synthetic analog that is isolated from Aspergillus fumigatus, and experimental studies suggested that it shows antitumor effect mediated by its strong antiangiogenic effect. Because limited experience exists about the antitumoral effect of TNP-470 in cerebral tumors, we have carried out a study in order to evaluate the effect of TNP-470 on tumor growth and the vascular area in an experimental malignant neuroectodermic tumor growing in the subcutaneous space of immunocompetent Wistar rats. Our results showed a signicant tumor growth inhibition in animals treated with TNP-470 when compared to those in the control group (intratumoral injections were administered in 30 mg/kg dose, three times a week on alternate days during four consecutive weeks). Since the quantitative analysis of tumor vascular parameters number of microvessels and total intratumor vascular area in the experimental groups did not show signicant statistical differences, we conclude that TNP-470 has a signicant antitumor effect on our neuroectodermic tumor, but this effect is mediated by other antineoplastic mechanisms that are independent of its previously described angiostatic capacity. Introduction In recent years, one of the main focuses of attention in the treatment of neoplasms is based on the search of angiogenesis inhibitory substances. The hypothesis that tumor growth depends on neovascularization and that inhibition of angiogenesis could inhibit tumor growth was formulated by Folkman in the 1970s. The neovascularization in a malignant tumor is the result of a balance between several angiogenic and antiangiogenic factors that are secreted by tumor cells, endothelial cells and/or macrophages [14]. In humans, malignant brain tumors are neoplasms that exhibit a great angiogenic activity, which is necessary for their growth. Thereby antiangiogenic treatment could be a potential therapy for these tumors, and several experimental model systems in vivo and in vitro, and clinical trials, have shown promising results [59]. During the last decade, TNP-470 (AGM-1470) has been described as one of the most potent angiogenesis inhibitors. This agent is a synthetic analog to fumagillin, a naturally secreted antibiotic from the Aspergillus fumigatus fungus. The agent inhibits proliferation of endothelial cells and some tumor cells, and it was found to suppress tumor growth in vivo and in vitro [10,11]. The antineoplastic effect of TNP-470 has been studied on several experimental models of neural tumors in nude mice: schwanomas, neurobromas and neurobrosarcomas [12], gliosarcoma 9L [13], glioblastomas [1416], meningiomas [17], medulloblastomas [18] and neuroblastomas [1923]. In the present study, we have investigated the effects of TNP-470 on tumor growth in an experimental model of malignant neuroectodermic tumor in immunocompetent rats. The tumor was obtained in our laboratory from a neoplasm induced by etil-nitrosourea (ENU) after transplacentary administration in Wistar rats. Its biological characterization supplies an useful model of malignant neuroectodermic tumor [24].

132 Material and methods Tumor Our experimental tumor is a primitive neuroepithelial neoplasm able to grow subcutaneously and without modifying its histologic characteristics in singenic Wistar rats. It is maintained by successive passes and cryopreservation at 80 C in essential minimum medium (EMM) for cell culture containing 10% dymethylsulfoxide. Animals and allotransplantation The study has been carried out on 20 Wistar rats. When rats were aged two days, a small fragment of cryopreserved tumor (1 mm in diameter) was implanted in epicranial subcutaneous area. Thirty days after implantation, all animals developed an average tumor volume of 0.5 cc. Animals were put at random into two experimental groups, TNP-470 and control group. Each group had 10 animals, and the study was started when tumors were approximately 0.30.5 cm in diameter. Preparation and administration of the TNP-470 The TNP-470 was kindly provided by Chemical Industries Takeda (Osaka, Japan). The compound had been stored at 20 C and later on was diluted in pure ethanol and stored in a solution stock of 100 mg/ml at 4 C. Immediately before its administration this solution stock was diluted in sterile saline serum in the appropiate concentrations to obtain a volume for injection of 0.1 cc. The animals treated with TNP-470 received a 30 mg/kg TNP-470 subcutaneous injection three times a week on alternate days during four consecutive weeks. The control animals received, on the same administration schedule, similar volumes of 2% ethanol diluted in sterile saline serum. Evaluation of animals and elaboration of tumor growth curves During treatment, animals health, body weight and tumor volume were daily evaluated in both experimental groups. Every day tumor perpendicular diameters were measured using a Vernier calliper. Each tumor volume was calculated according to the equation V (cm3 ) = d 2 (cm2 ) D (cm)/2, where d and D are the smallest and the largest tumor diameters, Immunohistochemical studies were carried out in order to know cell proliferation index of tumor. Primary monoclonal antibody Ki-67-MM1 (Novocastra Laboratories Ltd., Newcastle, UK) and kit Vectastain RTU (Vector Laboratories Inc., Burlingame, CA) were used. Histological sections were deparafnized and rehydrated, trypsinized for 15 min, and rinsed in phosphate-buffered saline (PBS), pH 7.5. They were incubated for 30 min in methanol containing 3% of H2 O2 , hydrated and treated with PBS three times for a total duration of 15 min, then the sections were placed in 10 mM citrate buffer (pH 6.0) and heated in the microwave oven for 12 min. The sections were incubated with a primary monoclonal antibody Ki-67-MM1 at 4 C overnight. The slides were again rinsed three times in PBS for 15 min. A 30-min incubation with biotinylated secondary antibody was followed by a standard PBS rinse. Another 30-min incubation with avidinperoxidase complex was succeded by another PBS rinse, followed by treatment with diaminobenzidine and contrasted with hematoxiline. The labeling index of Ki-67 for each experimental tumor was calculated with morphometric system (Optimas, 6.2 software package, Optimas Corporation, Bothell, WA, USA). respectively. The data were used to elaborate tumor growth curves in each experimental group. The antitumor activity was assessed by tumor growth curves, which were elaborated for each animal in both groups. Statistical analysis was performed using t -Student test to evaluate the differences between averaged values from each experimental group. Values of p < 0.05 were considered to be statistically signicant. The present study was performed in accordance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals produced by the American National Society for Medical Research and the National Academy of Sciences respectively. Histological studies At the end of the treatment, the animals were killed and histological studies of tumors were carried out. They were xed with 4% paraformaldehide and histological study with hematoxylineosin staining was performed. Antiproliferative effect of TNP-470

133 Study of tumor angiogenesis

300

Body weight curves

To evaluate possible changes in intratumor vessels in animals treated with TNP-470 in comparison to those in the control group, the number of vessels in each specimen of tumor tissue was counted and the vascular area was measured by morphometry. For it, histological slices belonging to each animal in TNP-470 and control group were studied by morphometry under microscope. The number of microvessels and the vascular area, in m2 , were counted in ve microscopical elds, at 250, and averaged. Then, for each microscopical eld, at 250, the mean number of microvessels and the vascular area from each experimental group was recorded. Statistical analysis The statistical analysis was carried out by means of the InStat statistical system (v 1.01, GraphPad Software Inc., San Diego, CA). In both experimental groups, the mean of tumor growth for different time points, number of microvessels, vascular area, and Ki-67 labeling index, were calculated. A t -Student test was used for comparisions. Differences were considered to be signicant for p < 0.05. Results All tumors allografts grew in the recipient immunocompetent rats and tumors had consistent growth rates in untreated rats. None of the animals receiving subcutaneous TNP-470 injections showed toxicity signs as a consequence of the drug during the treatment. There was no weight loss for the duration of therapy in any rats treated with TNP-470 (Figure 1). The administration of TNP-470 in our model of neuroectodermic tumor, at doses used, was able to inhibit tumor growth producing a signicant reduction of tumor volume in all treated animals. There was even a case of complete tumoral regression at the end of treatment. The mean volume of the tumors during the treatment in both TNP-470 and control groups is showed at the tumor growth curves (Figure 2). There are signicant differences between both groups (p < 0.05). This statistical difference could be veried within the rst week of treatment. Histologically, tumors treated with TNP-470 showed signs of tumoral involution such as large necrotic areas, but there was almost always areas with the malignancy

250 200 150 100 50 0 1 3 5 8 10 12 15 17 19 22 24 26 Days of treatment

Body weight (grs.)

Control Group TNP-470 Group

Figure 1. Body weight curves of animals with tumor allografts in TNP-470 and control groups. Mean values do not show statistically signicative differences between both experimental groups (p > 0.05).

characteristic of tumor, such as mitosis and cell pleomorphism. In treated tumors, a signicant decrease in the number of intratumor microvessels was not found (Figure 3). Morphometric studies showed that the mean of tumor microvessels per microscopical eld, at 250, was 7.4 2.6 for treated tumors and 8.6 3 for nontreated tumors. The mean of vascular area in m2 , per microscopical eld, at 250, was 23.4 12.1 for TNP-treated tumors, and 24.3 14.1 for control tumors. The statistical analysis by means of a t -Student test did not reect signicant differences neither in the number of microvessels, nor in the total vascular area between the two groups (p > 0.05). Mean values of Ki-67 index were 32.5 5.76 for TNP-treated tumors and 32.28 4.24 for control tumors. A statistical analysis by a t -Student test does not reect signicant differences between TNP-470 treated animals and those in control group (p > 0.05). Discussion The angiogenesis inhibitors are considered to be new antitumoral agents in the treatment of solid tumors.

134
Tumor Growth Curves of TNP-470 and Control Groups
6000 Control Group 5000
Tumor Volume (cc)

TNP-470 Group

4000 3000 2000 1000 0 1 5 10 15 19 24 29 33 Days of treatment

Figure 2. Tumor growth curves of tumor allografts in control group and TNP-470 group. TNP-470 was administered in doses of 30 mg/kg three times a week on alternate days, during four consecutive weeks. On the same administration schedule, animals in control group received similar volumes of 2% ethanol in saline. Since the eighth day of treatment, mean values showed signicant statistically differences between both experimental groups (p = 0.008).

Figure 3. Histological aspects of tumors in TNP-treated tumors and controls (H&E., 40). A shows the histological aspect of our experimental tumor. High degree of cellularity and microvessels can be seen. In B, the histological aspect of TNP-470 treated tumor can be seen. In treated tumors, necrotic areas are present, but the number of microvessels is similar to non-treated tumors.

TNP-470 is a fumagillin synthetic derivative, but more active and less toxic than the antibiotic naturally secreted by Aspergillus fumigatus [11]. Besides it has shown a potent inhibitory action on human endothelial cell proliferation and on neoangiogenesis induced in tumors [10,11]. The inhibition of angiogenesis constitutes a promising therapy for cerebral tumors. Previous studies have shown TNP-470 to have an in vivo inhibitory effect on tumor growth. Several of the tumor types inhibited were human cerebral tumor lines growing in nude mice, included xenotransplant of glioblastomas [1416], meningiomas [17], medulloblastomas [18] and neuroblastomas [1922]. The TNP-470 antitumor effect has also been described in experimental tumor models of gliosarcoma [13] and neuroblastoma allotransplant [23] in immunocompetent animals. The present study showed that TNP-470 has a signicant antitumor effect on an experimental neuroectodermic tumor after subcutaneously allotransplanted in immunocompetent Wistar rats, because tumor growth rate in TNP-470 group was signicantly lesser than in control group. On the other hand, treated animals

did not show systemic adverse effects. Besides there were no differences neither in body weight nor in general health between TNP-470 and control group, our present resuts suggest that TNP-470, being used in doses described, has antitumor activity and low toxicity. The histological study allowed to nd some differences between TNP-470 group and control group. In the former, as it is the case when other chemotherapeutic agents are used, signs of cytotoxic effect on tumor cells were found. These effects were mainly the presence of large intratumoral necrotic areas and lesser cellularity than in control group. Nevertheless, no signicant decrease in angiogenesis was noticed when TNP-470 was administered in this experimental model. In our study, we were not able to nd signicant histological changes in angiogenesis between treated animals and those in control group, and quantitative analysis of vascular parameters (number of microvessels and total intratumoral vascular area) did not show signicant statistical differences. Therefore, we could conclude that TNP-470 has antitumoral effect on our experimental tumor, but it is not associated to antiangiogenic activity. It is probable that

135 there is some relation between tumor reduction and cytotoxic effect on tumor cells, as it is the case for other chemotherapeutic agents. Recently, it has been described that TNP-470 has not only a selective cytotoxic effect on endothelial cells, but also a direct antitumoral effect on tumoral cells when high concentrations are used. It has been taken into account that in vitro studies with several cell lines such as of leukemia, myeloma, breast carcinoma and brosarcoma, TNP-470 has been shown to induce accumulation of cells in phase G0/G1 of the cell cycle [25]. In the present study, we have found that TNP-470 produced an inhibition of tumor growth, and from a histological viewpoint, there was an increment of necrotic areas, especially in the core of tumors in treated rats, but in outlying areas the histopatological characteristics of the experimental tumor remained. Furthermore, the studies of cell proliferation showed that Ki-67 labeling index of tumors treated with TNP-470 was not different from those in untreated group. It was similar as well to data obtained by other authors with PCNA antibody in a meduloblastoma model [13] and with BrdU and Ki-67 labeling index of malignant gliomas [15]. On the other hand, our present results differ from previous publications describing an inhibitory effect of TNP-470 on neovascularization in experimental models. In our present study, we started TNP-470 treatment four weeks after the implantation. Tumors were established and angiogenesis in progress, this is a situation that is often observed in clinical practice. In a previous study, treatment started two weeks after the implantation and the effect of TNP-470 on two human glioma lines xenografts in nude mice was reported, obtaining a signicant tumor growth suppression. In this report, no differences in vascular parameters between TNP-treated tumors and controls were described [16]. These observations suggest that the potential antiangiogenic effect of TNP-470 depends on tumor models, initial tumor volume and on whether or not the angiogenic process has started. An explanation why TNP-470 has not antiangiogenic effect in our present study may be that treatment stars once a critical number of tumor cells already exist. Another explanation could be that our experimental tumor might produce angiogenic factors or use angiogenic pathways that TNP-470 does not block. Although many researches have studied the potential mechanisms of TNP-470 inhibitory effect on endothelial cells proliferation [26,27], the specic mechanism has not been well described. Angiogenesis is the result of a balance between several inductory and inhibitory factors, direct as well as indirect. Therefore, different mechanisms to stimulate angiogenesis exist and some of them may not be sensitive to TNP-470 inhibition. Furthermore, it has been broadly demonstrated that angiogenesis can be activated in different ways and using different angiogenic factors that depend on the type of tumor. In cerebral neoplasms, vascular endothelial growth factor (VEGF) and basic broblastic growth factor (FGFb) have been shown to be important angiogenesis inductors, and in experimental glioma models angiogenesis signicantly decreased when these factors are blocked [6]. Previously, it had been described that TNP-470 inhibits the migration and cell proliferation induced by VEGF and bFGF [28] but TNP-470 effect on VEGF action can be contradictory. It has been recently described that TNP-470 causes in vitro a signicant decrease in VEGF expression [29], while an increase of VEGF expression has been described after TNP-470 administration in vivo, especially in cells that are adjacent to necrotic areas. Some report suggests that this could be a side effect of hypoxia secondary to the vascular damage following TNP-470 administration [30]. Another reason is that TNP-470 may lose its antiangiogenic effect. The causes could be related to FGFb. It has been recently published that TNP-470 interferes with FGFb action, for TNP-470 recognizes its endogenous receptors and competes with them [31]. However, in our experience, the administration of pentosan polysulfate a drug that inhibits FGFb-receptors neither altered angiogenesis nor inhibited tumor growth [32]. In conclusion, our study suggests that in an experimental model of malignant neuroectodermic tumor, the administration of TNP-470 did not cause signicant changes in tumor angiogenesis while inhibiting tumor growth. Therefore, we conclude that TNP-470 antitumor effect could be mediated by other antineoplastic mechanisms that are independent of its angiostatic capacity.

Acknowledgements The authors are grateful to Takeda Chemical Industries Ltd., Osaka, Japan, for generously providing the TNP-470. This work was partially supported by grants from Mapfre-Medicine Foundation, FIS 98/1018, and CAM 8.1/11/97.

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Address for offprints: J. Vaquero, Servicio de Neurocirug a, Cl nica Puerta de Hierro, San Mart n de Porrres, 4. 28035 Madrid, Spain; Fax: 34-913730535; E-mail: jvaqueroc@meditex.es