Microvascular Research 54, 178 182 (1997) Article No. MR972036 BRIEF COMMUNICATION Inhibition of Angiogenesis by Oral Ingestion of Powdered Shark Cartilage in a Rat Model 1 Paul F. Davis,* Yi He,* Richard H. Furneaux, Peter S. Johnston, Beate M. Rüger,* and George C. Slim,2 *Department of Medicine and Department of Surgery, Wellington School of Medicine, P.O. Box 7343, Wellington, New Zealand; and Industrial Research Ltd. P.O. Box 31-310, Lower Hutt, New Zealand Received January 6, 1997 INTRODUCTION kawa et al., 1990). However, the links between oral ingestion of cartilage and its anti-angiogenic and anti-tumour properties have yet to be convincingly The oral consumption of dried powdered shark demonstrated. cartilage has been widely promoted as a natural Clinical trials of powdered shark cartilage as an health remedy for the treatment of cancer (Wilson, anti-cancer agent have been initiated in Mexico and 1994; Lane and Comac, 1992, 1996). It is proposed to the United States but the results have yet to be pubact by preventing the angiogenesis required by solid lished. In this study we have examined whether the tumours to grow larger than 2 3 mm (Folkman, oral ingestion of powdered shark cartilage by rats 1995). has any effect on the angiogenesis induced in mesen- There is some evidence for the presence of anti- teric windows by mast cell stimulation. angiogenic factors in shark cartilage. Implantation of polymer pellets containing a shark cartilage extract alongside tumours in rabbit corneas inhibited tumour neovascularization (Lee and Langer, 1983). Injection MATERIALS AND METHODS of a suspension of shark cartilage reduced angiogenesis in tumours implanted in mice (Cataldi and Osbourne, 1995) and anti-angiogenic factors Shark cartilage. Dried powdered samples of carti- could be partially purified from shark cartilage (Oilage from two commercial batches (designated A and B) manufactured principally from blue shark were supplied by McFarlane Laboratories Ltd. (Auckland, 1 Supported by the New Zealand Foundation of Science, Research New Zealand). and Technology, the New Zealand Lottery Grants Board and the Induced angiogenesis. A modification of the rat Wellington Medical Research Foundation. We thank Mr. John Croft mesenteric-window assay (Norrby et al., 1990) was of McFarlane Laboratories (NZ) Ltd. for providing samples of shark cartilage. used. Sprague Dawley rats (6 weeks old, equal num- 2 To whom correspondence should be addressed. bers of male and female) were assigned to one of 178 0026-2862/97 $25.00
Brief Communication 179 TABLE 1 Percentage of Rat Mesenteric Window Area Occupied by Blood Vessels 16 and 25 Days after Stimulation with Compound 48/80 Normal / Shark cartilage A / Shark cartilage B Days after Animal induction grouping n %Area n %Area P:2a n %Area P:2a 16 All 52 50.2 { 2.6 42 23.4 { 2.6 õ0.0001 45 32.3 { 2.1 õ0.0001 16 Male 26 49.7 { 4.2 21 25.5 { 2.4 õ0.0001 23 36.1 { 2.1 õ0.04 16 Female 26 50.5 { 3.3 21 21.2 { 4.7 õ0.0007 22 28.3 { 3.7 õ0.0003 25 All rats 80 37.7 { 2.2 79 17.5 { 1.9 õ0.001 nd 25 Male 37 32.9 { 3.0 38 13.0 { 2.3 õ0.0001 nd 25 Female 43 41.8 { 3.1 41 21.6 { 2.9 õ0.0001 nd Note. Each of the five groups comprised eight rats (four male and four female). Shark cartilage was administered at 6 g/kg of food. All data are means { SEM. Statistical significance was assessed by Student s t test. nd, not determined; n, number of windows. Diet three groups and fed ground rat food (Diet 86, cooled and the mesenteric windows were surgically Sharpes Grain and Seeds Ltd., Lower Hutt, New removed, mounted on glass slides, and viewed with Zealand) which was freely available. The groups a light microscope. The images were digitised and were fed an unsupplemented diet or a diet supplemented the vascularized area was outlined manually and cal- with either Shark Cartilage A or B (up to 15 culated using the programme Image 1.59 (National g/kg of food). Most frequently 6 g/kg of food was Institutes of Health, Bethesda, MD). employed. This equates to about 0.6 g of shark carti- Approval for these experiments was obtained from lage/kg body weight if the rats consumed all their the Animal Ethics Committee of the Wellington food, which is less than that generally recommended School of Medicine. for human use by the manufacturers of powdered shark cartilage (1 g/kg body weight) (Lane and Comac, 1992). There was õ2% difference in weight gain amongst the three groups, demonstrating that RESULTS the supplementations had no evident detrimental effect. Angiogenesis was induced in the virtually avas- Sixteen days after the commencement of Compound cular mesenteric windows of rats by intraperitoneal 48/80 administration, the percentage area injection of the mast cell secretagogue Compound that was vascularized in each mesenteric window 48/80 (Sigma Chem. Co., St. Louis, MO) twice daily from rats fed shark cartilage was significantly less for 4.5 days (Norrby et al., 1990). Feeding of shark than that in rats on an unsupplemented diet (Fig. 1, cartilage commenced 2 weeks prior to the induction Table 1). Although Cartilage A appeared to be more of angiogenesis and continued until sacrifice. Water effective than B, this difference was not statistically was available ad libitum. The animals were sacrificed significant. A similar effect was seen 25 days after after16or25daysbyexposuretoco 2 and injected the commencement of stimulation (Table 1). At this with India ink through the superior mesenteric artery time, Compound 48/80 was more effective at stimu- (Mattsby-Baltzer et al., 1994). The carcasses were lating angiogenesis in female rats than in male rats, FIG. 1. Vascularization of rat mesenteric windows; (a) without mast cell stimulation, (b, c, d) animals sacrificed 16 days after mast cell stimulation. (b) Control animal; (c) animal fed Shark Cartilage A (6 g/kg of food), commencing 2 weeks prior to stimulation; (d) animal fed Shark Cartilage B (6 g/kg of food), commencing 2 weeks prior to stimulation.
180 Brief Communication FIG. 1 Continued
Brief Communication 181 FIG. 1 Continued
182 Brief Communication cross the intestinal wall (Warshaw et al., 1974), but the majority will be digested to smaller peptides in the gut. However, GAGs, including chondroitin sulphate (Conte et al., 1995; Ronca and Conte, 1993), the major GAG present in cartilage, have been shown to be absorbed through the intestine largely intact when taken orally (Volpi, 1996). Work is continuing to determine which of the components of shark cartilage is responsible for the orally available antiangiogenic activity observed here. FIG. 2. Inhibition of mesenteric window angiogenesis induced by mast cell stimulation showing effectiveness of different doses of Shark Cartilage A. Dietary supplementation commenced 2 weeks prior stimulation and rats were sacrificed 16 days after the initiation of mast cell stimulation. The percentages are calculated from the means of the vascularised areas. For the control (no cartilage), 118 windows (5 rats); for 1.5 g/kg of food, 107 windows (5 rats); for 3 g/kg of food, 106 windows (5 rats); for 6 g/kg of food, 105 windows (5 rats); for 9 g/kg of food, 83 windows (4 rats); for 12 g/kg of food, 40 windows (2 rats); for 15 g/kg of food, 39 windows (2 rats). as previously reported (Norrby et al., 1990), but shark cartilage was as effective at reducing angiogenesis in malesasinfemales. There was a direct relationship between the inhibition of angiogenesis and the dose of Shark Cartilage A included in the rats diet up to an optimal level of 6 g/kg of food (Fig. 2). DISCUSSION These data demonstrate for the first time that the oral ingestion of powdered shark cartilage has a po- tent inhibitory effect on angiogenesis. The inhibitory factor must be stable in the gut and absorbed by the gastrointestinal tract in order for it to affect angiogenesis in the mesentery. This raises the question as to what is the likely identity of the inhibitory factor. The major components of powdered shark cartilage are protein (Ç40%) and glycosaminoglycans (GAGs, Ç5 20%), with the remainder being principally calcium salts. Small portions of ingested proteins can REFERENCES Cataldi, J. M., and Osbourne, D. L. (1995). Effects of shark cartilage on mammary neovascularisation in-vivo and cell proliferation invitro. FASEB J. 9, A135. Conte, A., Volpi, N., Palmieri, L., Bahous, I., and Ronca, G. (1995). Biochemical and pharmacokinetic aspects of oral treatment with chondroitin sulfate. Arzneimittelforschung 45, 918 925. Folkman, J. (1995). Seminars in medicine of the Beth Israel Hospital, Boston Clinical applications of research on angiogenesis. N. Engl. J. Med. 333, 1757 1763. Lane, I. W., and Comac, L. (1992). Sharks Don t Get Cancer: How Shark Cartilage Could Save Your Life. Avery, New York. Lane, I. W., and Comac, L. (1996). Sharks Still Don t Get Cancer. Avery, New York. Lee, A., and Langer, R. (1983). Shark cartilage contains inhibitors of tumour angiogenesis. Science 221, 1185 1187. Mattsby-Baltzer, I., Jakobsson, A., Sorbo, J., and Norrby, K. (1994). Endotoxin is angiogenic. Int. J. Exp. Pathol. 75, 191 196. Norrby, K., Jakobsson, A., and Sorbo, J. (1990). Quantitative angiogenesis in spreads of intact rat mesenteric windows. Microvasc. Res. 39, 341 348. Oikawa, T., Ashino-Fuse, H., Shimamura, M., Koide, U., and Iwaguchi, T. (1990). A novel angiogenic inhibitor derived from Japanese shark cartilage (I). Extraction and estimation of inhibitory activities toward tumour and embryonic angiogenesis. Cancer Lett. 51, 181 186. Ronca, G., and Conte, A. (1993). Metabolic fate of partially depolymerized shark chondroitin sulphate in man. Int. J. Clin. Pharm. Res. 13, 27 34. Volpi, N. (1996). Physico-chemical properties and the structure of dermatan sulfate fractions purified from plasma after oral administration in healthy human volunteers. Thromb. Haemostasis 75, 491 496. Warshaw, A. L., Walker, W. A., and Isselbacher, K. J. (1974). Protein uptake by the intestine: Evidence for absorption of intact macro- molecules. Gastroenterology 66, 987 992. Wilson, J. L. (1994). Shark cartilage: A review of background literature and research. Townsend Lett. Doctors 133, 864 872.