³[H] Thymidine was purchased from Bhaba Atomic Research Centre, Mumbai, India. Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin and Trypsin-EDTA were purchased from In-vitrogen, USA. Endothelial Growth Medium (EGM-2) was obtained from Cambrex Bioscience, Walkersville, USA. The antibodiy PECAM-1 and ABC reagent for CD 31 staining were procured from Santa Cruz Biotechnology, California USA. Matrigel was purchased from Chemicon International, CA, USA.

The seaweeds Amphiroa fragilissima (Delile) Trevisan, Asparagopsis taxiformis (Linnaeus) Lamouroux, Caulerpa peltata J.V. Lamouroux, Caulerpa racemosa (Forsskal) J. Agardh, Caulerpa taxifolia (Vahl) C. Agardh, Chlorodesmis fastigiata (C.Agardh) Ducker, Codium elongatum (Turner) C. Agardh, Dictyopteris australis Lamouroux, Dictyopteris deliculata Lamouroux, Sargassum marginatum (C. Agardh) J.Agardh, Stoechospermum marginatum (C.Agardh) Kutzing, Padina Gymnospora (Kützing) Sonder, Padina tetrastromatica Hauck, Spatoglossum asperum J. Agardh, Spatoglossum variabile Figari and De Notaris were collected from in and around the coasts of Goa and Maharashtra, India. The seaweeds were botanically identified by a taxonomist and the identification was confirmed by comparing with the already available voucher specimens at the Herbarium of the National Institute of Oceanography, India.

Shade dried seaweed powder (1kg) was extracted with 4 Ltr methanol successively three times after keeping at room temperature for 24 h. The organic extracts obtained were filtered and evaporated to dryness using a rotary evaporator (Roteva, India) in order to obtain a semisolid residue. The methanolic extracts obtained were further diluted in appropriate cell culture media in order to obtain the required concentrations needed for further analysis. The Stoechospermum marginatum extract was designated as SME for future reference.

HUVEC (Human Umbilical Vein Endothelial) cells were obtained from Cambrex Bioscience, Walkersville, USA and cultured in EGM-2 medium with 2% FBS, 0.04% hydrocortisone, 0.1% long R3-human Insulin like growth factor (IGF-1),0.1% ascorbic acid, 0.4% human fibroblast growth factor (bFGF), 0.1% VEGF, 0.05% gentamycin and 0.05% amphotericin-B according to the manufacturer's protocol. BeWo (Choriocarcinoma) cells were obtained from the National Center for Cell Science, Pune, India and cultured in DMEM Ham's F-12 medium (Supplemented with 10% FBS, 1% Penicillin-Streptomycin and Gentamycin). HEK 293 (Non-transformed Human embryonic kidney) cells were obtained from the National Center for Cell Science, Pune, India. EAT cells (Mouse mammary carcinoma) were maintained in the Laboratory of Molecular Oncology, Department of Studies in Biotechnology, University of Mysore, Mysore, India and used routinely for in-vivo transplantation. Both EAT and HEK 293 cells were maintained in DMEM with 10% FBS and 1% Penicillin- Streptomycin. All cells were grown in a humidified atmosphere, containing 5% CO₂ at 37℃. When the cells reached confluency they were passaged by trypsinization using 0.025% trypsin/0.01% EDTA. Cells from passages 2-5 were used for the experiments.

Female swiss albino mice (8-10 weeks old) and male Wistar rats weighing 300-350 g were obtained from the animal house, Department of Zoology, University of Mysore, Mysore, India. Fertilized eggs were acquired from a government poultry farm in Bangalore, India. All experiments were approved by the committee for the purpose of control and supervision of experiments on animals (CSPCA), Government of India, and performed strictly according to the NIH guidelines for the care and use of laboratory animals (IACUC Registration No: 122/99/IACUC).

³[H] Thymidine incorporation assay was performed as described previously [26]. In brief, the tumor cells were plated onto 12-well culture plates at 25 × 10³ cells/well and incubated at 37℃ in 5% CO₂ for 48 hrs in DMEM medium. Following incubation, seaweed extract at 100 µg/ml concentration was added to the wells in triplicate, leaving three wells as control prior to addition of ³[H] thymidine (1 µci/ mL) to all wells. After incubation for 48 hrs, the cells were washed with PBS and then fixed with 10% ice-cold trichloroacetic acid in order to precipitate the DNA. The Radioactivity was measured in scintillation solution using liquid scintillation spectrometry. The concentration of the sample was then plotted against the percentage cell survival.

The matrigel tube formation assay has been widely been used as an in-vitro measurement of endothelial cell differentiation and as such it was performed according to the manufactures instruction (In vitro angiogenesis assay kit, Millipore). In brief, matrigel (50 µl) was added to each well of a 96 well plate, which was then incubated at 37℃ for 1 hr allowing the gel to polymerize. Human Umbilical Vein Endothelial Cells (HUVECS) were suspended in endothelial growth medium and cells (1 × 10⁴) were seeded in to each well containing 150 µl of EGM. The cells were incubated with or without the test sample (SME) at 37℃ and 5% CO₂. All of the conditions were performed in triplicates. After incubation for 16 - 20 hrs, the tube formation was photographed at 40 × magnification using an inverted microscope (Olympus, Germany).

A wound-healing assay was performed for measurement of the in vitro effect of S. marginatum extract on the unidirectional migration of HUVECs according to Liang et al. [27]. In brief, HUVECs were seeded at 2 × 10⁵ cells per well in a 6-well plate, and incubated overnight with complete medium (EGM-2 with 1% FBS) at 37℃ in a humidified atmosphere of 5% CO₂. The cells were then serum starved overnight and a wound was scratched on the monolayer using a sterile pipette tip. The plates were washed with PBS twice for removal of any detached cells. For stimulation, VEGF (10 ng ml^-1) was added, with or without S. marginatum extract at 0.1mg ml^-1, and incubation continued for 3, 6, 18, 24, 36 and 42 hrs. The images were taken at the time of the wounding and at different time intervals thereafter using a phase-contrast microscope (Olympus, Germany).

CAM assay was performed according to the method of Gururaj et al. [28]. In brief, the fertilized eggs were incubated at 37℃ in a humidified and sterile atmosphere for 10 days. Under aseptic conditions, a window was made on the eggshell to check for proper development of the embryo. The window was resealed and further development was allowed. On the 12^lh day, saline, recombinant VEGF (50 ng per egg) or the extract (100 µl/ egg) was air dried on sterile glass cover slips. The window was reopened and the cover slip was inverted over the CAM. The window was closed again, and the eggs were returned to the incubator for two more days. The windows were opened on the 14^th day and inspected for changes in microvessel density in the area under the cover slip and photographed.

The rat corneal micro-pocket assay was performed as described previously [29]. In brief, hydron polymer (poly hydroxyl ethylmethacrylate was dissolved in ethanol to a final concentration of 12%. A 5 µl aliquot of this mixture was then pipetted onto Teflon pegs. Aliquots of 10 µl of 12% Hydron/Ethanol alone (group 1), with 1 µg of cytokine VEGF (group 2) and VEGF + 5 µg seaweed extract (group 3) was added to each pellet and allowed to dry under a laminar flow hood at room temperature for 2 hrs. The pellets were incubated at 4℃ overnight. All procedures were performed under sterile condition. Male Wistar rats weighing 300-350 g were anaesthetized with i.p with a combination of ketamine (87 mg kg^-1) and xylazine (13 mg kg^-1). A drop of 0.5% proparacaine was instilled into the eye and the globe was proptosed using a pair of 0.3 mm tissue forceps. Using a surgical microscope, a paracentral linear incision 1 mm from the center of the cornea, 1.5 nm in length and 50% of the corneal depth was made using a No. 11 surgical blade to create a corneal micropocket. The incision was bluntly dissected through the stroma to the limbal area using a curved iris spatula. A single pellet was advanced into the lamellar pocket to the limbus using corneal forceps. Postoperatively, gentamycin ointment was applied to the anterior surface of the operated eye. The rats were observed for 24-72 hrs for occurrence of non-specific inflammation and for localization of the pellets. On day 7, the rats were anaesthetized with ketamine and the corneas were observed under a stereo-binocular microscope with a CCD camera and photographed.

The crude extract of the seaweed S. marginatum selected for the study was tested for its effect on EAT cell growth in-vivo [30]. EAT cells (5 × 10⁶) were injected intraperitoneally (i.p) into mice (two groups of mice, six in each group) and growth was recorded each day from the day of transplantation. To determine whether the seaweed extract supressed tumor growth and angiogenesis mediated by EAT cells in-vivo, the extract (l00 µg of SME) was injected into the peritoneum of EAT bearing mice each day from the sixth day of transplantation. The body weight of the mice was monitored from the first day until the twelfth day. On the twelfth day, the animals were sacrificed and the volume of the ascites formed in both untreated and treated mice was recorded. The pelleted cells were counted using the trypan blue dye exclusion method using a hemocytometer. The animals were then dissected in order to observe the effect of the extract on peritoneal angiogenesis.

To determine whether the SME inhibited the microvessel density, the effect of the extract was verified on the angiogenic response induced by cytokine VEGF in EAT bearing mice. EAT bearing mice were treated regularly with the extract from the sixth day of transplantation. On the twelfth day, the animals were sacrificed and the peritoneum from treated or untreated mice was fixed in 10% formalin. Sections (5 µm) were made from paraffin embedded peritoneum using an automatic microtome (SLEE Cryostat) and stained with hematoxylin and eosin. Microvessel counts and image photography were performed using a Leitz-DIAPLAN microscope with an attached CCD camera.

The effect of SME on proliferating endothelial cells was determined by staining the paraffin sections of the peritoneum of treated or untreated mice with anti-CD 31 anti-bodies [31]. Peritoneum sections were processed according to the protocol supplied by the manufacturer (Santa Cruz Biotechnology, CA, USA). In brief, sections were dewaxed in xylene three times for 5 minutes each. The sections were rehydrated in descending concentrations of ethanol (100% ethanol for 5 minutes, 95% for 2 minutes and 80% for 2 minutes) and washed in distilled water. For antigen retrieval the sections were heated at 95 ℃ for 15 minutes in a humidified atmosphere. The sections were treated with 3% H₂O₂ in PBS to block endogenous peroxidase activity. They were blocked in blocking serum for 30 minutes in order to reduce non specific binding and were then incubated with anti-CD 31 (PECAM-1) anti-bodies for 2 hrs. Following washing with PBS, slides were incubated with secondary anti-body (biotinylated rabbit anti- mouse IgG) for 1 hr at room temperature. The slides were washed in PBS for 5 minutes and incubated with the substrate (100 µl/ section) followed by ABC reagent for 45 minutes (2 ml histo buffer + 20 µl Avidin solution + 20 µl Biotin solution). After incubation, the slides were washed in PBS for 5 minutes. Antigen and antibody complexes were detected using substrate (DAB, 100 µl/ section) for 5 minutes. The sections were washed thee times for 2 minutes in tap water and twice for 2 minutes in distilled water. Subsequently, the slides were counter stained with 2% hematoxylin for 5-7 minutes and washed again in tap water three times for 5 minutes each. The slides were washed effectively for two minutes each in 50% ethanol, 80% ethanol and absolute alcohol. After xylene wash, the slides were mounted using entellan mountant solution and the sections were scored using DIAPLAN light microscope and photographed.

All experiments were performed in triplicate (n = 3) at least and values are expressed as mean ± SD. STATISTICA software (Statsoft, 1999) was utilized for comparison of the mean values of each treatment). Data were analyzed by one way analysis of variance (ANOVA) for determination of significant differences. P-values < 0.05 were considered significant and P-values < 0.001 highly significant.

Preliminary screening of SME demonstrated potent antiproliferative ability against EAT cells (Fig. 1); therefore, it was selected for further anti-angiogenic studies. BeWo cells and HEK-293 cells were used to determine whether SME inhibits proliferation of tumor/normal cells in-vitro. SME extract inhibited proliferation of BeWo cells in a dose-dependent manner. However, no potent effect was seen in untransformed normal HEK-293 cells. As shown in Fig. 2A, inhibition of proliferation was 80.55, 83.54, 90.79, 95.43, and 96.36% on the proliferation in BeWo cells and 4.62, 5.76, 5.85, 6.71, and 7.40% was recorded, while in HEK 293 cells at 0.005, 0.025, 0.050, 0.075, and 0.1 mg ml^-1 concentrations of SME with P < 0.001 (Fig. 2B).

Tube formation assay was performed in-vitro in order to substantiate the effect of SME on formation of blood vessels by HUVEC's. In the positive control group stimulated with VEGF (10 ng), the HUVEC's adhered to the matrigel surface within 20 to 24 hrs and formed a branching anastomising network of capillary like tubes with multi-centric junctions over 24 hrs. However, treatment with SME prevented VEGF stimulated tube formation of HUVEC's, in a dose dependent manner (Fig. 3). Complete inhibition was achieved at 5 mg ml^-1of SME. At the latter concentration, most of the cells appeared as unorganized cell aggregates.

To determine whether cell migration was inhibited by SME, in-vitro wound healing assays was performed using HUVEC cells, as described in the Materials and Methods section. Cell migration of HUVECs treated with VEGF increased in control wells while, migration in cells treated with SME at 100 µg ml^-1 was effectively inhibited in the wound (A) healing assay (Fig. 4).

The CAM and rat cornea assays form a part of the conventional angiogenesis assays commonly used for in-vivo validation of the angio-suppressive efficacy of anti-angiogenic molecules. Results shown in Fig. 5A and Fig. 5B indicated that SME had a direct effect on inhibition of angiogenesis in an in-vivo model system. When compared to the extensive angiogenesis seen in VEGF treated CAM and rat cornea, angiogenesis at the site of application of SME was significantly reduced.

The treated as well as control animals were observed for the degree of vascularization occurring in the peritoneal cavity. The results shown in Fig. 6A demonstrate that control EAT bearing mice showed a gradual increase in body weight of 8 to 10 g when 5 × 10⁶ EAT cells were injected on day zero. In mice treated with/ without SME, a significant decrease was observed in groups treated with SME when compared to the control, indicating the effect of SME in preventing the growth of tumor cells (P < 0.05). In a fully grown ascites tumor, a volume of 6.25 ± 0.22 ml of ascites was generated during the tumor growth period of 12 days. In SME treated mice, the volume of ascites was approximately 2.6 ± 0.32 ml with P < 0.001 (Fig. 6B). The numbers of viable cells in fully grown EAT bearing mice was 3.25 ± 0.24 × 10⁸/ mouse, while this number was reduced to 0.64 ± 0.06 × 10⁸/ mouse with P < 0.001 in SME treated mice (Fig. 6C), indicating a considerable reduction when compared with the control. These results indicate the potential anti-tumor activity of SME. In a fully grown ascites tumor in-vivo, there is extensive peritoneal angiogenesis. However, in SME treated mice, a significant decrease in peritoneal angiogenesis was also observed in-vivo (Fig. 6D).

In addition, the effect of SME on survival of EAT bearing animals was tested. Upon intraperitoneal transplantation of 5 × 10⁶ cells/ mice, the EAT-bearing mice succumbed to the tumor burden 13 days after tumor transplantation, due to increase in the number of tumor cells with passage of time, whereas the animals treated with SME (4 mg kg^-1 body weight/dose, every day) extended the survival time of EAT-bearing mice from 13 days up to one month (data not shown).

Results of H & E staining indicated that there was a reduction in the number of newly formed microvessels in the peritoneum of SME treated peritoneum of EAT bearing mice, compared to the control (Fig. 7A). The proliferation of endothelial cells can be confirmed using CD31 marker. Our results of CD31 staining, showed a marked reduction in the number of proliferating endothelial cells in the peritoneum of SME treated EAT-bearing mice (Fig. 7B), corroborating the results shown in the inhibition of peritoneal angiogenesis in-vivo.