Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 200-205

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 200-205

Anti-Angiogenesis and Anti-Tumor Effect of Shark  Cartilage Extract*

WANG Feng , WANG Yitao, XIE Liping, ZHANG Rongqing **

Department of Biological Sciences and Biotechnology, Center for Ocean Science and Engineering,  Tsinghua University, Beijing 100084, China

* Supported by the National High Technology Research and Development Program of China (No. 819-05-01 and No. 819-Z-05)  
**To whom correspondence should be addressed

Received: 2000-01-17; revised: 2000-03-07

Code Number: ts01066

Abstract:   

The effect of shark cartilage extract (SCE), purified in this laboratory, on angiogenesis in chick chorioallantoic membrane (CAM), on the activity of collagenase IV and on human umbilical vein endothelial cell (ECV-304) proliferation and apoptosis was investigated in vitro. The results showed that SCE caused a decline in CAM blood vessels and significantly prevented collagenase-induced collagenolysis. Moreover, SCE produced a dose-dependent decline in ECV-304 proliferation and altered its normal cell cycle. These results suggest that the anti-angiogenesis and anti-tumor effects of shark cartilage may be due to inhibition of endothelial cells as well as collagenolysis. 

Key  words:  shark cartilage extract; angiogenesis; chorioallantoic membrane; collagenase; endothelial cell

Introduction   

The progression of a tumor cell from benign delimited proliferation to malignant and metastatic growth is the major cause of poor clinical outcomes of cancer therapy. Angiogenesis in the host is a characteristic phenomenon of most malignant tumors. With adequate blood supply, tumor cells will not only grow, but also acquire metastatic potential[1]. This process may involve the production of tumor-derived mediators of endothelial cell migration and proliferation through enzymes (such as collagenase) that facilitate angiogenesis by affecting the endothelial cell basement membrane[2]. Inhibition of angiogenesis is a potentially powerful cancer therapy that may cause regression of the primary tumor as well as arrest its metastasis[3].

Shark cartilage is a vascular tissue that is rarely invaded by neoplasm. Unlike mammals, sharks have an endoskeleton composed entirely of cartilage, which is about 6% of the shark's body weight[4], so it provides a plentiful source of cartilage. Many research projects and clinical studies have proved the anti-tumor effect of shark cartilage. One group isolated a fraction from shark cartilage with a relative molecular mass between  1x10³-10x10³that contained the majority of the anti-angiogenesis activity[5]. Another group characterized an angiogenic inhibitor, U-995, from shark (Prionace glauca)[2]. These findings suggested that shark cartilage might have anti-angiogenesis activity and might inhibit the proliferation of endothelial cells. The present study demonstrates the effect of shark cartilage extract (SCE) on chorioallantoic membrane (CAM) and ECV-304 cell lines. Further investigation showed that SCE also significantly decreased the activity of collagenase IV which degrades the predominant component collagen IV of basement membrane and enhances apoptosis of ECV-304. These studies may yield a new understanding of the anti-tumor effect of SCE.

1 Materials and Methods   

1.1 Materials and reagents

Fertilized chick eggs were purchased from Stud Fowl Center, China Agriculture University. Collagen IV and Collagenase IV were purchased from Sigma (St. Louis, MO). Medium 199 and fetal bovine serum (FBS) were obtained from Gibico (Grand Island, NY). Other chemicals were obtained from the Beijing YiLi Refined Chemical Co., Ltd, (China).

1.2 Cell culture

The human umbilical vein endothelial cell line (ECV-304) was kindly provided by Prof. J. F. Stoltz, Faculté de Médecine-54500 Vandoeuvre-lés-NANCY, France. The cells were grown in medium M199 containing  2.2 mg/mL  sodium bicarbonate supplemented with 10% FBS, penicillin (100 mg/mL) and streptomycin (100 mg/mL), at 37 °C with 5% CO₂ . Cells were passaged and harvested for experiments before reaching confluence.

1.3 Preparation of SCE from shark cartilage 

The shark cartilage powder was extracted in a solution containing 1 mol/L NaCl at 4 °C for 48 hours with slight stirring, followed by centrifugation at 18 000 r/min for 20 min. The supernatant was dialyzed against deionized water in a dialysis tube with a 10x10³ cutoff and desiccated with polyethylene (PEG) 12000. The extract was centrifuged at 18 000 r/min for 20 min again and the supernatant was lyophilized. The final product was dissolved in 0.02 mol/L Tris-HCl buffer (pH=7) for later experiments.

1.4 CAM assay

The CAM assay was conducted using the method previously described by Sheu[2]. Fertilized eggs were incubated at 37 °C with 50%-60% humidity. On day 2 and day 9 of incubation, the eggs were prepared for experiment. A small hole was made through the shell of the egg directly over the air sac using a small craft drill. A second hole was drilled on the broad side of the egg over the embryonic blood vessels. Negative pressure was applied to the original hole, which resulted in the CAM pulling away from the shell membrane and creating a false air sac. A  1 cm x 1 cm  square window was made to directly observe the underlying CAM. Twenty microlitres SCE (2 mg/mL) was applied locally to methylcellulose film prepared previously with 5% methylcellulose and sterilized by UV, with an equal volume of sterilized deionized water applied to the control eggs. The windows were temporarily sealed with laboratory films. At the end of another 3-day incubation, the vessels were observed. Six eggs were tested for each group and the assay was performed twice to ensure reproducibility.

1.5 Collagenase IV inhibitor assay by capillary electrophoresis

Uncoated capillaries, 47 cm longx75 mm I.D., were mounted into a Beckman P/ACE 5510 capillary electrophoresis apparatus. The capillary was flushed with 0.1 mol/L NaOH and deionized water for 10 min in turn. Then the capillary was filled with 50 mmol/L phosphoric-boric acid buffer (pH=8.0), previously degassed by ultrasonic waves for 5 hours. Substrate collagen IV was dissolved in 5 mmol/L HAc to a concentration of 2 mg/mL, the collagenase IV was dissolved in Tris-HCl to a concentration of 30 mg/mL, and SCE was dissolved in Tris-HCl to a concentration of 2 mg/mL. Five reaction systems: (1) Collagenase IV 10 mL, HAc 10 mL and Tris-HCl 10 mL. (2) Collagen IV 10 mL and Tris-HCl 20 mL. (3) SCE 30 mL. (4) Collagen IV 10 mL, collagenase IV 10 mL and Tris-HCl 10 mL. (5) Collagen IV 10 mL, collagenase IV 10 mL and SCE 10 mL were added to the capillary by pressure. All Tris-HCl buffers used in the experiments had pH  7.4 . Eight kilovolts was applied to the capillary and the absorption at  253 nm  was recorded every 15 s for 5 min ( 25 °C , 120 mA).BT(2+1*4  1.6 Growth inhibition assay of ECV-304 cells by MTT ECV-304 cells were cultured at 10⁴ cells/mL in a flat-bottomed 96-well plate for 24 hours. Different volumes, 10, 20, 30, 40, and 50 mL of SCE (2 mg/mL) were applied to the separate wells with sterile deionized water as the control. After 72 hours of cultivation, 20 mL of 3-(4,5-dimethyl-2-thia-zolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT, 5 mg/mL) were added to each well, and the cells were recultured for a further 4 hours. The culture supernatants were then removed from each well. The formazan crystals were dissolved by adding 150 mL of dimethyl sulfoxide. The plate was read on a microplate reader at 490 nm.

1.7 Cell cycles and apoptosis assay of ECV-304 cells

ECV-304 cells were cultured in flasks for 24 hours. The cells were harvested by trypsinization at 24, 48, and 72 hours after addition of SCE (2 mg/mL). After they were washed with PBS two times and fixed with ice-cold 70% ethanol, the cells were incubated with RNAse-A (50 mg/mL) for 30 min, then dyed with propidium iodine. After filtration through nylon mesh, cells were analyzed by flow cytometry technology as described previously[6]. The percentage of cells in each phase and the apoptosis rate were calculated. Cells with DNA content less than that of cells in the G1 phase were deemed to have undergone apoptosis.

1.8 Statistical analysis

The results were expressed as the  mean±SEM  accompanied by the number of tests. The Student's t-test was performed to determine the level of significance between groups; P<0.05 was considered significant. 

2 Results   

2.1 Inhibition of angiogenesis in chick  chorioallantoic membrane (CAM)

The anti-angiogenesis activity of SCE was tested by measuring the ability of SCE to disrupt the angiogenesis in CAM. The effect of SCE on the growing blood vessels in vivo was investigated with CAM on day 5 and day 12. SCE inhibited the growth of blood vessels during both the early and the late period, Fig.1 (b) and Fig.2 (b). The density, distribution and tortuosity of the blood vessels were less than those of the control group, Fig.1 (a) and Fig.2 (a).

2.2 Effect on the degradation activity of  collagenase IV

Capillary electrophoresis was used to dynamically study the effect of the degradation activity of collagenase IV. The solvent for all samples was a Tris-HCl buffer, which formed a peak appearing around  7.5-8.3  minutes. Figure 3 shows the absorption profile of collagenase IV at an experimental concentration of 10 mg/mL. We could not see its peak even at 100 mg/mL. Thus, its effect in the later analysis of the profiles in the more complex reaction system can be ignored. Figure 4 shows the profile of collagen IV at an experimental concentration of 670 mg/mL. The main peak of the protein appeared during  8.6 -10.2  minutes with an area of about  5.52 . The profile for SCE, Fig. 5, had no comparable peak around  8.6  minute where the main collagen IV peak appeared. Figures 6 and 7 show the reserved collagen IV peaks of the two systems, collagen IV-collagenase IV and collagen IV-collagenase IV-SCE, after 8 min reaction. The areas were  2.75  and  3.93 , respectively. Comparison of the reserved collagen IV peak areas of these systems in Fig. 8 shows that the system with SCE preserved more substrate, collagen IV, than the substrate-enzyme system. This result indicates that SCE can cause anti-collagenolysis.

2.3 Inhibition of ECV-304 proliferation

Figure 9 shows the dose-dependent curve obtained 72 hours after cells were treated with SCE, at which time SCE reached its highest inhibition rate. The results show that SCE greatly inhibited cell proliferation compared with the control cells, which grew rapidly. The maximum inhibition rate of 58.7% was observed at a volume of 30 mL. The anti-proliferation activity was also observed with other cell lines (for example, the Hela cell line, data not shown).

2.4 Effect on ECV-304 cell cycle and apoptosis

The cell cycle patterns were examined by flow cytometric analysis to determine whether SCE treatment of ECV-304 altered cell cycle progression. Figures 10 and 12 showed that SCE could greatly reduce the volume of cells in the G2M phase while increasing the volume of cells in the S phase. However, Fig.11 shows that SCE had no regular effect on the cells in the G1 phase. More importantly, Fig. 13 shows that SCE can significantly increase the apoptosis rate of ECV-304, with the maximum effect on day 2 with a maximum apoptosis rate 5 times more than the control. The mechanisms by which SCE increases ECV-304 cell apoptosis needs further study.

3 Discussion   

The anti-tumor efficacy of shark cartilage has been investigated for a long time. More than seven anti-tumor factors have been isolated from shark cartilage[7]. Experimental results have suggested that its anti-tumor mechanisms include inhibiting the proliferation of tumor cells, restricting the growth of blood vessels[5], reducing mutation and damage induced by hydrogen peroxide[8] , inhibiting the production of a PG-like substance and stimulating the immune system. However, the exact structures of the active components of shark cartilage have not been obtained and its anti-tumor mechanism still remains unclear. The present paper studies its anti-angiogenesis and anti-tumor effects. The angiogenic process includes three main parts: (1) enzymatic degradation of the basement membrane[9], (2) endothelial cell locomotion[10, 11] , and (3) endothelial cell proliferation[12-14] , which is the emphasis of the present work.

Tumor growth and metastasis are angiogenesis-dependent[15]. A tumor relies on the growth of new capillary blood vessels to develop. Capillary blood vessels consist of endothelial cells and pericytes. These two types of cells carry all of the genetic information required for angiogenesis. Therefore, specific angiogenesis molecules can initiate this process and specific inhibitor molecules can stop it[3]. The angiogenesis of an embryo is similar to that of neoplasm. Our study showed that SCE could restrict blood vessel proliferation of CAM. This in vivo model provided direct evidence of the anti-angiogenesis activity of SCE. Collagens represent a family of closely related proteins that compose the framework of an extracellular matrix. A number of reports have shown that cartilage contains a collagenase inhibitor[16]. It has been suggested that collagenase inhibitors can play a critical role in the inhibition of angiogenesis or tumor cell invasion[17]. The assay of collagenolysis in this paper focused on collagen IV, which is the main component and skeleton of basement membrane and frequently combines with endothelial cells. When an endothelial cell contacts the collagen IV, it stops dividing and forms a capillary-like structure. Collagens are firstly degraded by the related collagenase, then by other proteases, such as trypsin. In the present paper, capillary electrophoresis was used to analysis the complex reaction systems and to dynamically observe their interaction. This method can provide more information than slab electrophoresis and some immune methods. Our study used collagen IV-collagenase IV-SCE as a substrate-enzyme-inhibitor system. The reserved substrate area represents the SCE inhibition effect. Figure 8 shows the relationship between the inhibition effects of SCE and that the inhibition rate can reach at least 40%. In addition, the SCE profile in Fig. 5 suggests that this extract is a kind of glucoprotein since slight variations of the glycosyl structure make the protein absorption vary as it passes the inspection window, which cause many small peaks.

The migration and proliferation of endothelial cells are important steps of angiogenesis[2]. It has been reported that SCE can inhibit the migration of endothelial cells as well as its DNA synthesis. In our experiment, the inhibition of endothelial cell growth by SCE indicated a dose-dependent effect with the highest inhibition rate of 58.7% obtained 48 h after cells were treated with SCE. We also tested the effect of SCE on cytocircle and cell apoptosis. The results show that SCE particularly acts on the cell DNA synthesis phase, so it can reduce cell mitosis by delaying or checking the cell division process from the S phase to the M phase. The results also showed that SCE could significantly increase cell apoptosis. Therefore, there are two ways through which SCE can inhibit endothelial cell growth as indicated by MTT assay.

In conclusion, SCE, a crude extract of shark cartilage, has shown significant anti-angiogenesis and anti-tumor activity in many aspects. Our laboratory has conducted vast research on mice, cultured tumor cell lines, immune organs and cells. We recently isolated two small anti-tumor proteins from shark cartilage and obtained their partial N-terminal amino acid sequences. Further molecular biology studies are being conducted.

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