The growth of solid tumours and their metastases is dependent on the development
of new blood vessels (angiogenesis). Therefore angiogenesis inhibitors are
potential antitumour drugs.
In our previous studies it was found that the angiogenesis inhibitor TNP-470
given to transplantable melanoma-bearing hamsters can decrease the rate of
the tumour growth, although the survival time of the animals treated was not
significantly affected. It was found finally that TNP-470 given in the vicinity of
the growing tumour can cause complete remission of the melanoma in hamsters
treated in this way. To check what side-effects could be evoked by such
treatment, an examination of the morphology of the blood vessels of the lungs,
kidneys and livers of the treated animals was carried out. It was found that the
angiogenesis inhibitor applied did not cause any changes which could be observed
by light and electron microscopes in the structure of the examined blood
vessels of the treated animals

Koszałka, Patrycja

Kubasik-Juraniec, Jolanta

Myśliwski, Andrzej

Szmit, Ewa

English

Via Medica Journals

Folia Morphol. Vol. 63, No. 1, pp. 5–9Copyright © 2004 Via MedicaISSN 0015–5659www.fm.viamedica.plO R I G I N A L  A R T I C L E5The effect of angiogenesis inhibitor TNP-470on the blood vessels of the lungs, kidneys andlivers of treated hamstersAndrzej Myśliwski1, Jolanta Kubasik-Juraniec2, Patrycja Koszałka3, Ewa Szmit11Department of Histology and Immunology, Medical University, Gdańsk, Poland2Laboratory of Electron Microscopy, Medical University, Gdańsk, Poland3Intercollegiate Faculty of Biotechnology, Medical University, Gdańsk, Poland[Received 26 August 2003; Accepted 29 November 2003]The growth of solid tumours and their metastases is dependent on the develop-ment of new blood vessels (angiogenesis). Therefore angiogenesis inhibitors arepotential antitumour drugs.In our previous studies it was found that the angiogenesis inhibitor TNP-470given to transplantable melanoma-bearing hamsters can decrease the rate ofthe tumour growth, although the survival time of the animals treated was notsignificantly affected. It was found finally that TNP-470 given in the vicinity ofthe growing tumour can cause complete remission of the melanoma in ham-sters treated in this way. To check what side-effects could be evoked by suchtreatment, an examination of the morphology of the blood vessels of the lungs,kidneys and livers of the treated animals was carried out. It was found that theangiogenesis inhibitor applied did not cause any changes which could be ob-served by light and electron microscopes in the structure of the examined bloodvessels of the treated animals.Key words: TNP-470, melanoma, hamsters, blood vesselsAddress for correspondence: Prof. Andrzej Myśliwski, Department of Histology and Immunology, Medical University, 80–210 Gdańsk,ul. Dębinki 1, Poland, tel: +48 58 349 14 30, fax: +48 58 349 14 36, e-mail: anmys@amg.gda.plINTRODUCTIONIt is well recognised that angiogenesis has es-sential significance for the growth of solid tumours[4, 5, 10]. Therefore many angiogenesis inhibitorshave been examined as potential antitumour drugs. Oneof the most promising as a future antitumour drug ap-pears to be the synthetic fumagilin analogue TNP-470[3, 6–8]. In our previous studies on that compoundwe achieved some effect in the suppression of thegrowth of the transplantable melanoma in hamsters.The growth rate of this tumour was significantlydecreased but the survival time of the tumour-bear-ing animals was prolonged only slightly [12, 13]. Inour last published studies we found that TNP-470given in the vicinity of a growing tumour can causecomplete remission of Bomirski Ab amelanotic mel-anoma and prevent the development of its metastas-es [14]. The success of such treatment for a fast-growing tumour that is able to kill a hamster in sev-eral weeks indicates that TNP-470 can be one of themost effective antitumour drugs. However, there isthe problem of side-effects, which in the case of ef-fective antitumour drugs is usually very serious. Inour successful studies TNP-470 was given in the vi-cinity of a growing tumour, so the dose of this com-pound applied could be comparatively low. Even so,the systemic action of TNP-470 was inescapable. Wetherefore decided to test the effect of the strongantiangionesis inhibitor TNP-470 on the blood ves-sels of the lungs, kidneys and livers of the treated6Folia Morphol., 2004, Vol. 63, No. 1hamsters. Since the action of TNP-470 was found tobe directed at the endothelial cells [3, 9], our atten-tion was mainly concentrated on the endotheliumof the vessels examined.MATERIAL AND METHODSTNP-470 was the kind gift of Takeda Chemical In-dustries (Osaka, Japan). The chemical structure of thecompound has been presented by Ingbar et al. [8].Forty random-bred Syrian (golden) male hamsterswere used in the study. They were obtained fromthe animal colony of the Silesian Medical University(Katowice, Poland) and maintained in our colony ona standard diet and water ad libitum. The care andtreatment of the animals were in accordance withthe “European Convention for the Protection of Ver-tebrate Animals Used for Experimental and OtherScientific Purposes”. The study was approved by theLocal Committee for Animal Experiments, Gdańsk,Poland.Bomirski melanoma variant Ab was maintainedby serial passage in the hamsters using the suspen-sion method of tumour transplantation. The naturalhistory and characteristics of this melanoma are de-scribed by Bomirski et al. [2]. The tumour cells werealways implanted on the right flank of the animalsby subcutaneous (s.c.) injection.TNP-470 in a suspension vehicle composed of 1%ethanol and 5% Arabic gum in a saline was givens.c. at a dose of 30 mg/kg (about 3.9 mg/animal in500 mL of vehicle) in the vicinity of the palpable tu-mour every day for 21 days. The control tumour-bearing animals received injections of vehicle. Allanimals were sacrificed on the 28th day from thebeginning of the experiment.Sample preparation for histological examinationSmall blocks of the organs under examinationwere put into a 4% formaldehyde solution in PBS.After being washed in water and dehydrated in eth-anol, the tissue was embedded in paraffin. Sectionsof the examined tissues were stained with eosineand haematoxylin. Photographs were taken witha Nikon E800 microscope.Sample preparation for the electron microscopeThe hamsters were anaesthetised by an intraperi-toneal injection of pentobarbital sodium (50 mg/kg).The animals were perfused transcardially with 50 mlof 0.9% saline containing 500 heparin units, and thenperfused with a fixative composed of 4% paraform-aldehyde and 1% glutaraldehyde in a 0.05 M phos-phate buffer (pH 7.4) containing 0.03% CaCl2. Blocksof tissues were rapidly removed and post-fixed inthe same fixative overnight at 4oC. Next each spec-imen was briefly rinsed in a 0.1 M phosphate buff-er containing CaCl2 at room temperature, and thenpost-fixed for 1 hour in 1% osmium tetroxide. Afterbeing dehydrated in increasing concentrations of al-cohol and  propylene oxide, the specimens were em-bedded in Epon 812. The ultrathin sections werestained with uranyl acetate and lead citrate and ex-amined with a JEM 1200EX II electron microscope.RESULTSLungsThe structure of blood vessels of various sizesfrom hamsters, both healthy and tumour-bearingones, which had been treated with TNP-470 did notdiffer when examined by light microscope fromthose of untreated animals (Fig. 1). Endothelial cellsdid not show any symptoms of histological lesions.Also the ultrastructure of the endothelial cells ofthe lung vessels from the treated hamsters wasnormal (Fig. 2).KidneysThe capillaries of the renal glomerules from ani-mals treated with TNP-470 did not differ from thoseof the untreated animals, both under a light micro-scope (Fig. 3, 4) and under an electron microscope(Fig. 5, 6). The larger blood vessels also had a nor-mal structure, both for the treated and the untreat-ed animals.LiversThe structure of the blood vessels of various sizes,lobular and interlobular (Fig. 7), from the livers of thetreated animals was similar to those of the untreatedanimals and no symptoms of histological lesions werenoticeable. The ultrastructure of the endothelium ofthe examined vessels also seemed to be normal (Fig. 8).The presence of the tumour in animals had noeffect on the structure of the vessels of the exam-ined organs.DISCUSSIONThe results of our studies indicate that the an-giogenesis inhibitor TNP-470, which is able to causea complete remission of a fast-growing tumour, ledneither to changes in the structure, nor probably thefunction, of the blood vessels of such important or-gans as the lungs, kidneys and livers. The period oftreatment with TNP-470 was 21 days, which is short7Andrzej Myśliwski et al., Effect of TNP-470 on blood vesselsin comparison to the duration of the administrationof the antitumour drugs applied in oncology. Thiswas, in any case, sufficient time for recovery for ham-sters bearing transplantable melanoma. The goodstate of the structure of the endothelium and otherparts of the blood vessel walls examined by light andelectron microscopes did not preclude changes inthe function of these vessels. However, if such chang-es had occurred, they were reversible, since the ani-mals which survived treatment with TNP-470 wereexamined 5 months after the last injection of thatdrug and were found to be healthy [14]. The angio-genesis inhibitor TNP-470, therefore, appeared in ourstudies to be both effective as an antitumour drugand harmless to the animals treated. These resultswere achieved with an animal model and, at present,it is difficult to make any claims as to whether sucha good effect would be possible in humans. TNP-470is already undergoing pre-clinical investigations [1, 6]but reports about the side-effects of such treatmentFigure 1. Lung of a melanoma-bearing hamster treated withTNP-470. Cross-section of a branch of the pulmonary artery.Both the endothelium and the thin media are normal in structure;bv — blood vessel, p — lung parenchyma. Staining with eosineand haematoxylin. Bar: 50 mm.1Figure 3. Renal corpuscle of the kidney of a melanoma-bearinghamster treated with TNP-470. Glomerular vessels have normalstructure; g — renal glomerule, p — renal parenchyma. Stainingwith eosine and haematoxylin. Bar: 50 mm.3Figure 4. Blood vessel in the renal cortex of a melanoma-bear-ing hamster treated with TNP-470. Both endothelium and me-dia demonstrate normal structure; bv — blood vessel, p — re-nal parenchyma. Staining with eosine and haematoxylin. Bar:50 mm.4Figure 2. Lung of a melanoma-bearing hamster treated withTNP-470. Cross sections of blood capillaries (b cap) in the lungparenchyma. Blood capillaries demonstrate normal ultrastructureof the endothelium. Bar: 1 mm.2 1 mm8Folia Morphol., 2004, Vol. 63, No. 1in humans are rather scanty and limited to such ob-servations as the occurrence of nausea and fatigue,and the presence of some neurological symptomswhich disappeared after therapy was discontinued[6]. Examinations of the effect of TNP-470 on en-dothelial cells were mainly carried out on endothe-lial cells cultured in vitro [3]. In our studies TNP-470was given in the vicinity of a growing tumour, sothat the dose applied of that compound could bequite low. The systemic action of TNP-470 was un-Figure 6. Renal corpuscle of the kidney of a melanoma-bearinghamster treated with TNP-470. Endothelium of the glomerularcapillary (g cap) demonstrates normal ultrastructure;p — podocyte. Bar: 2 mm.6Figure 7. Liver of a melanoma-bearing hamster treated withTNP-470.  Longitudinal section of interlobular blood vessel (bv)and interlobular bile duct (bd) surrounded by liver parenchyma(p). The blood vessel demonstrates normal structure. Stainingwith eosine and haematoxylin. Bar: 50 mm.7Figure 5. Renal corpuscle of the kidney of a healthy hamster;g cap — glomerular capillary, p — podocyte. Bar: 2 mm.5Figure 8. Liver of a melanoma-bearing hamster treated withTNP-470. Cross-section of the liver sinusoid (ls) surrounded byliver parenchyma (p). The endothelium of the sinusoid demon-strates normal ultrastructure. Bar: 2 mm.82 mm2 mm2 mm9Andrzej Myśliwski et al., Effect of TNP-470 on blood vesselsavoidable. We decided, therefore, to test the influ-ence that a strong antiangiogenesis inhibitor couldexert on the blood vessels of the lungs, kidneys andliver of the hamsters treated with TNP-470. The prob-lem of the possible side-effects of the treatment withTNP-470 is very important since the only partly ef-fective action of that drug in pre-clinical trails in-duced some research groups to apply a combinedtreatment with other antitumour drugs, which them-selves sometimes cause severe side-effects [6, 11].The results of our studies and those of other re-search groups [3, 6] supply evidence that the side-effects evoked by TNP-470 are fairly limited and re-versible after treatment, which indicates that thisanalogue of fumagilin — TNP-470 is a potentiallyvaluable antitumour drug.REFERENCES1. Bhargava P, Marshall JL, Rizivi N, Dahut W, Yoe,Figuera M, Phipps K, Ong VS, Kato A, Hawkins (1999)A phase I and pharmacokinetic study of TNP-470 ad-ministrated weekly to patients with advanced cancer.Clin Cancer Res, 5: 1989–1995.2. Bomirski A, Slominski A, Bigda J (1988) The naturalhistory of a family of transplantable melanomas inhamsters. Cancer Metastasis Rev, 7: 95–118.3. Castronovo V, Belotti D (1996) TNP-470 (AGM-1470):Mechanism of action and early clinical development.Eur J Cancer, 32A: 2520–2527.4. Folkman J (1990) What is the evidence that tumors areangiogenesis dependent? J Natl Cancer Inst, 82: 4–6.5. Folkman J (1992) Tumor angiogenesis and tissue fac-tor. Nature Med, 2: 167–168.6. Gervaz P, Fontolliet M (1998) Therapeutic potential ofthe anti-angiogenesis drug TNP-470. Int J Exp Pathol,79: 359–362.7. Hotz HG, Reber HA, Hotz B, Sanghavi PC, Yu T, Foitzik T,Buhr HJ, Hines OJ (2001) Angiogenesis inhibitor TNP-470reduces human pancreatic cancer growth. J GastrointestSurg, 5: 131–138.8. Ingbar D, Fujita T, Kishimoto S, Sudo K, Kanamaru T,Brem H, Folkman J (1990) Synthetic analogues of fum-agilin that inhibit angiogenesis and suppress tumorgrowth. Nature, 348: 555–557.9. Kusaka M, Sudo K, Matsutani E, Kozai Y, Marui S,Fujita T, Ingbar D, Folkman J (1994) Cytostatic inhibi-tion of endothelial cell growth by angiogenesis inhib-itor TNP-470 (AGM-1470). Br J Cancer, 69: 212–216.10. Mahadevan V, Hart IR (1990) Matastasis and angio-genesis. Acta Oncol, 29: 97–103.11. Masunaga S, Ono K, Nishimura Y, Kanamori S, Saga T,Suzuki M, Kinashi Y, Takagaki M, Kasai S, Nagasawa H,Uto Y, Hori H (2000) Combined effects of tirapazamineand mild hyperthermia on anti-angiogenic agent(TNP-470) treated tumors-reference to the effect onintratumor quiescent cells. Int J Radiation OncologyBiol Phys, 47: 799–807.12. Myśliwski A, Szmit E, Szatkowski D, Sosonowska D(1998) Suppression of growth of Bomirski Ab melano-ma and its metastasis in hamsters by angiogenesis in-hibitor TNP-470. Anticancer Res, 18: 441–444.13. Myśliwski A, Bigda J, Koszałka P, Szmit E (2000)Synergistic effect of the agniogenesis inhibitor TNP--470 and Tumor Necrosis Factor (TNF) on BomirskiAb melanoma in hamsters. Anticancer Res, 20:4643–4648.14. Myśliwski P, Koszałka P, Bigda J, Szmit E (2002) Com-plete remission of Bomirski Ab amelanotic melanomain hamsters treated with the angiogenesis inhibitorTNP-470. Neoplasma, 49: 319–322.

The effect of angiogenesis inhibitor TNP-470 on the blood vessels of the lungs, kidneys and livers of treated hamsters

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The effect of angiogenesis inhibitor TNP-470 on the blood vessels of the lungs, kidneys and livers of treated hamsters

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