TABLE OF CONTENTS 6.1. INTRODUCTION 6.2. MATERIALS AND METHODS

Transcription

1 CHAPTER 6 EFFECT OF VERNOLIDE-A ON RADIATION INDUCED HYPOXIA MEDIATED TUMOUR ANGIOGENESIS

2 TABLE OF CONTENTS 6.1. INTRODUCTION 6.2. MATERIALS AND METHODS Animals Cell line Chemicals Irradiation Drug administration Determination of the effect of Vernolide-A on solid tumour reduction Determination of the effect of Vernolide-A on vascular endothelial growth factor (VEGF) level (in vivo) Determination of the effect of Vernolide-A on vascular density by Immunohistochemistry Determination of the effect of Vernolide-A on the gene expressions of VEGF and HIF-1α Determination of the effect of Vernolide-A on cell proliferation Determination of the effect of Vernolide-A on clonogenic survival by Soft agar assay Determination of the effect of Vernolide-A on vascular endothelial growth factor (VEGF) level (in vitro) Determination of the effect of Vernolide-A on collagen matrix invasion Determination of the effect of Vernolide-A on MMP activation by gelatin Zymography Determination of the effect of Vernolide-A on gene expressions of VEGF, HIF-1α and VEGF receptor (Flk-1) 6.3. RESULTS Effect of Vernolide-A on Solid tumour reduction Effect of Vernolide-A on vascular endothelial growth factor level (in vivo) Effect of Vernolide-A on vascular density Effect of Vernolide-A on gene expressions of VEGF and HIF-1α Effect of Vernolide-A on cell proliferation Effect of Vernolide-A on clonogenic survival Effect of Vernolide-A on vascular endothelial growth factor level (in vitro)

3 Effect of Vernolide-A on collagen matrix invasion Effect of Vernolide-A on MMP activation Effect of Vernolide-A on the expression of VEGF, HIF-1α and VEGF receptor (Flk-1) genes 6.4. DISCUSSION

4 6.1. INTRODUCTION Radiation therapy is a widely used conventional treatment modality for local control of solid tumours. Hypoxia is an important phenomenon in the tumour microenvironment which plays a critical role in malignant tumour progression and treatment resistance. This occurs in solid tumours as a result of an inadequate supply of oxygen, due to exponential cellular proliferation and an inefficient vascular supply (Shannon et al., 2003). Hypoxic cancer cells are highly resistant to radiotherapy which is one of the major reasons for the treatment failure. The cellular response to hypoxia is mediated through the hypoxia inducible transcription factor-1 (HIF-1). HIF-1 is critically important for tumour progression and angiogenesis. In fact, HIF-α is overexpressed in 70% of human cancers and their metastases. Thus, agents that inhibit angiogenesis and tumour growth via inhibition of HIF-1α represent an attractive target for cancer treatment (Escuin et al., 2004). Tumour cell invasion involves the proteolytic degradation of extracellular matrix (ECM) components by tumour cell-secreted matrix metalloproteinases (Basset et al., 1997; Rao, 2003). Type IV collagen is an important protein of the basement membrane. Elevated levels of Type IV collagenases, MMP-2 (gelatinase A), and MMP-9 (gelatinase B) have been found in many tumours and are believed to play an important role in cellular invasion and metastasis (Björklund and Koivunen, 2005). These enzymes are secreted as inactive pro-mmps (Stetler-Stevenson et al., 1989) and are activated by proteolytic cleavage (Mignatti et al., 1986). There is a direct correlation between ionizing radiation and MMP activation. Studies revealed that ionizing radiation can enhance tumour cell invasion and angiogenesis by modulating MMP, VEGF, and VEGF receptor expression in tumour bearing animals (Kaliski et al., 2005). One of the genes upregulated by HIF-1 is vascular endothelial growth factor (VEGF), a potent mediator of angiogenesis that enhances endothelial cell survival, invasion and also induces vasodilation. VEGF expression leads to hyperproliferation of blood vessels, which should improve oxygenation within tumours. Both HIF-1α and VEGF have been tested as potential targets for improving the radiation response (Karar and Maity, 2009). Studies show that ionizing radiation can induce vascular endothelial growth factor (VEGF) secretion by B16 melanoma 134

5 cells (Kaliski et al., 2005). In the present study, we evaluated the effect of Vernolide- A on ionizing radiation induced invasion and angiogenesis MATERIALS AND METHODS Animals C57BL/6 mice (4 6 weeks old, g body wt.) Cell line Human umbilical vein endothelial cells (HUVECs), B16F-10 melanoma cells. To reach and control hypoxic conditions, cells were placed in a modular incubator chamber (Billups Rothenberg, Inc., Del Mar, CA) and flushed for 10 minutes with a gas mixture of 5% CO 2-95% N 2. The chamber was then sealed and placed at 37 C in conventional cell incubator Chemicals DMEM with 10% FCS, Oligonucleotide primer sequences of VEGF, HIF-1α and Flk-1, Anti-mouse CD 31 antibody, Radioactive thymidine [ 3 H], Highly specific quantitative sandwich ELISA kit for mouse VEGF were used Irradiation Animals were exposed to a single acute dose of gamma-radiation of 6 Gy, 4Gy and 2Gy using the 60 Co-Theratron-Phoneix teletherapy unit (Atomic Energy Ltd., Mississauga, Ontario, Canada). The animals were kept immobilized in a specially designed, well-ventilated cage without any anesthesia and exposed to whole-body radiation at a rate of 1.41 Gy/min. PTW unidos dosimeter, 0.6 cc Thimble ionization chamber was used for measuring the exposure rate. The radiation field size was cm 2, and the cage housed 10 animals at a time. It was at a distance of 80 cm from the source Drug administration Vernolide-A was dissolved in minimum volume of ethanol and resuspended in 1% gum acacia and was given to animals intraperitoneally (i.p) at a concentration of 0.5mg/kg body weight. For in vitro studies 0.01, 0.05, 0.1 μg/ml of Vernolide-A were used. 135

6 Determination of the effect of Vernolide-A on solid tumour reduction C57BL/6 mice (6-8 weeks old) were divided into eight groups (8 animals /group). All the animals were injected with B16F-10 cells (10 6 cells per animal) subcutaneously on the right hind limb. Radiation or drug treatment was started only after the 8 th day of tumour induction. Group I was kept as untreated tumour control. Group II animals were treated with Vernolide-A at a concentration of 500 µg/kg body weight (i.p) for 10 days at 24 h intervals. Group III, IV and V animals were exposed to a single dose of 6Gy, 4Gy and 2Gy radiation respectively and kept as radiation control. Group VI, VII and VIII animals were exposed to 6Gy, 4Gy and 2Gy radiation respectively and all the animals were treated with Vernolide-A (500 µg/kg body weight/dose/animal, i.p) for 10 days, one hour after the radiation exposure. The tumour volume was measured at 24 th hour, 7 th day and 15 th day after radiation exposure. The solid tumour development was measured with vernier calipers and calculated using the formula V=4/3 Π r 2 1 r 2, where r 1 and r 2 are radii along two directions Determination of the effect of Vernolide-A on vascular endothelial growth factor (VEGF) level (in vivo) Blood was collected from the above group of animals by tail vein bleeding at 24 th hour, 7 th day and 15 th day after radiation exposure, serum was separated and used for the estimation of vascular endothelial growth factor (VEGF) level using ELISA kits purchased from R & D system, USA Determination of the effect of Vernolide-A on vascular density by Immunohistochemistry Tumour tissues were fixed in 10% neutral buffered formalin for 24 h, processed, and embedded in paraffin blocks. After deparaffinization and rehydration, 5 μm cross sections were treated overnight at room temperature with a monoclonal rat antimouse CD31 IgG antibody (1:100) (Abcam, Cambridge, MA), which specifically recognizes an epitope on the surface of endothelial cells. Secondary antibody incubation was made using HRP conjugated rabbit anti-mouse IgG antibody (1:200) (Banglore Genei) for one hour at room temperature. Diaminobenzidine (DAB; 136

7 Banglore Genei) was used as the chromagen and methyl green (Sigma) as the counterstain Determination of the effect of Vernolide-A on the gene expressions of VEGF and HIF-1α Total RNA was isolated from the tumour, and cdna was synthesized using Moloney murine leukemia virus reverse transcriptase. Amplification was performed using specific primers of vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF)-1α. The amplified products were electrophoresed on a 1.8% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light Determination of the effect of Vernolide-A on cell proliferation B16F-10 melanoma cells (5000 cells/well) were plated in a 96-well culture plate and incubated at 37 C in 5% CO 2 atmosphere. After 24 h, the cells were exposed to different doses of radiation (0.5, 1, 2, 3, 4, and 5 Gy) in the presence and absence of Vernolide-A (0.1μg/ml) and were further incubated for 48 h. 3 H-thymidine was added to each well (1 μci /well) and incubation was continued for additional 18 h. After completing incubation, the plates were centrifuged and the culture supernatant was removed, the cells were washed three times with PBS and then treated with ice cold PCA for 15 min. The resulting precipitate was dissolved in 0.5 N NaOH and was added to the scintillation fluid and kept overnight in the dark. The radioactivity was counted using a Rack Beta liquid scintillation counter Determination of the effect of Vernolide-A on clonogenic survival by Soft agar assay B16F-10 melanoma cells were exposed to different doses of radiation (0.5, 1, 2, 3, 4, and 5 Gy). Cells were cultured in soft agar (0.3%) in a 24-well culture plate (500 cells/well) in the presence and absence of Vernolide-A (0.1 μg/ml) and were incubated for 6 days. Colonies were then fixed, stained with crystal violet, and counted. Experiments were repeated twice. The surviving fraction (SF) was estimated as follows: 137

8 SF = Number of colonies formed after treatment Number of cells seeded X plating efficiency Determination of the effect of Vernolide-A on vascular endothelial growth factor (VEGF) level (in vitro) B16F-10 cells were grown in complete DMEM to subconfluent monolayers in a 24- well culture plate. After incubation, culture medium was replaced (after washing cells twice with sterile PBS) by serum-free DMEM. In the first set, cells were incubated with or without Vernolide-A (0.1 μg/ml) and the second set of cells were irradiated at 1 Gy with or without Vernolide-A (0.1 μg/ml), which was added just before ionizing radiation. Conditioned medium was collected 24, 48, and 72 hours after irradiation, centrifuged, and used for Matrigel invasion assays or stored frozen at C until analysis of VEGF content. VEGF was measured by ELISA kits (R & D system, USA) according to the manufacturer s instructions. Each sample was analyzed in duplicate and experiments were repeated twice Determination of the effect of Vernolide-A on collagen matrix invasion The invasion assay was carried out in modified Boyden chambers as described by Albini et al. (1987). The lower compartment of the chamber was filled with conditioned medium with maximum VEGF level from the above experiment and a polycarbonate filter coated with 25 μg Type I collagen was placed above this. B16F- 10 melanoma cells were irradiated with 1 Gy and were then seeded (10 5 cells/150 μl DMEM) on to the upper chamber in the presence and absence of Vernolide-A (0.1 μg/ml) and incubated at 37 C in 5% CO 2 atmosphere for 10 h. After incubation, the filters were removed, fixed with methanol and stained with crystal violet. Cells migrating to the lower surface of the polycarbonate filters were counted under a microscope. The results were expressed as percentage inhibition of invasion Determination of the effect of Vernolide-A on MMP activation by gelatin zymography SDS-PAGE was performed with 5% gelatin incorporated in the separating gel (Billings et al., 1991). Conditioned medium with maximum VEGF level from the above experiment was used for zymographic analysis. Fifty microlitres of sample 138

9 (equivalent to 100 μg protein) was activated with 5 μl trypsin solution (75 μg/ml) in the presence and absence of Vernolide-A (0.1 μg/ml) in 0.1 M Tris HCl, 10 mm CaCl 2 buffer (ph-8.0) and incubated for 1 h at room temperature. Samples were mixed with an equal volume of 2X sample buffer and loaded on to 11% polyacrylamide gels containing 5% gelatin. Electrophoresis was carried out at 4 C with constant current of 2 ma/tube until the tracking dye reached the periphery. The gels were then washed with 2% Triton X-100 in 0.1 M Tris HCl, 10 mm CaCl 2 at 37 C for 18 h followed by staining with Gelcode Blue stain reagent for 2 h. Gels were destained to visualize the clear area against the dark background Determination of the effect of Vernolide-A on gene expressions of VEGF, HIF-1α and VEGF receptor (Flk-1) B16F-10 melanoma cells and HUVEC cells ( cells) were suspended in conditioned medium from the above experiment (with maximum VEGF). Cells were seeded in 96-well titre plate and incubated for 24 h at 37 C in 5% CO 2 atmosphere. Cells were incubated into hypoxia (5% CO 2 and 95% N 2 ) in an Incubator Chamber (Billups-Rothenberg, Del Mar, CA) in the presence and absence of Vernolide-A (0.1 μg/ml) for 4 h at 37 C. cdna was synthesised using cell to cdna kit (Ambion Inc.). Cells were washed with phosphate buffered saline and heated in cell lysis buffer (provided in the kit) to release the RNA into the solution followed by a heating step to inactivate endogenous RNases. The genomic DNA was further degraded by treating with DNase followed by inactivation of DNase by heating at 70 C. Reverse transcription was performed at 42 C for 50 min using Moloney murine leukemia virus RT (supplied along with the kit). Gene expression analysis was done by PCR. B16F-10 cell lysate was used for the amplification of mouse VEGF and HIF-1α whereas Flk-1 was amplified using HUVEC cells. The cycling conditions used were as follows: 1 min at 94 C, 1 min at 58 C and 1 min at 72 C for 40 cycles, followed by a 10-minute extension at 72 C. Amplified samples were subjected to electrophoresis in an agarose gel (1.5%) containing 0.5μg/mL ethidium bromide and photographed under UV light. 139

10 Statistical analysis Values are expressed as mean±s.d. The statistical analysis was done using one-way ANOVA followed by Dunnett's test RESULTS Effect of Vernolide-A on Solid tumour reduction Effect of Vernolide-A on solid tumour reduction is presented in Figure 6.1. Tumour volume was significantly (p<0.05) reduced by the administration of Vernolide-A in all the groups. Mice carrying B16F-10 cells alone (Tumour control) had the tumour volume 1.45 ± 0.2 cm 3 on 15 th day, which was significantly (p<0.05) reduced by the administration of Vernolide-A on the same day (0.68 ± 0.06 cm 3 ). Radiation treatment also significantly decreased the tumour volume. Maximum reduction in the tumour volume was observed when the animals were treated with Vernolide-A along with radiation. Tumour bearing mice exposed to 6 Gy radiation decreased the tumour volume to 0.42 ± 0.05 cm 3 on 15 th day, which was further reduced to 0.38 ± 0.04 cm 3 by the administration of Vernolide-A on the same day Effect of Vernolide-A on vascular endothelial growth factor level (in vivo) Serum VEGF level during tumour progression, radiation and Vernolide-A treatment is given in the Figure 6.2. Serum VEGF level was elevated during tumour progression. Administration of Vernolide-A significantly (p<0.05) reduced the serum VEGF compared to untreated tumour control. Mice carrying B16F-10 cell alone had the VEGF level 2835 ± 62 pg/ml on 15 th day, which was significantly (p<0.05) reduced by the treatment of Vernolide-A on the same day (1142 ± 53 pg/ml). Radiation exposure drastically enhanced the serum VEGF level. Tumour bearing animals exposed to 6 Gy radiation showed a maximum VEGF level on 15 th day (3421 ± 168 pg/ml), which was significantly reduced by (1741 ± 68 pg/ml) the Vernolide-A treatment Effect of Vernolide-A on vascular density Figure 6.3 shows the immunohistochemistry of tumour tissue for CD 31 to analyse the vascular density. Immunohistochemistry studies revealed an increase in CD 31 endothelial cell marker, suggesting a hypoxia mediated activation of the proangiogenic pathways. Data shows that radiation increased the severity of hypoxia 140

11 Figure 6.1 Effect of Vernolide-A on solid tumour reduction C57BL/6 mice were induced solid tumour by injecting 1x10 6 B16F-10 cells, subcutaneously on the hind limbs. After the 8 th day of tumour induction the animals were exposed to different doses of radiation. The tumour volume was measured using vernier calipers at 24 th hour, 7 th day and 15 th day after radiation exposure. Values are mean ± SD. *p<0.05, compared with the respective control.

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13 Figure 6.2 Effect of Vernolide-A on vascular endothelial growth factor level (in vivo) C57BL/6 mice were induced solid tumour by injecting 1x10 6 B16F-10 cells, subcutaneously on the hind limbs. After the 8 th day of tumour induction the animals were exposed to different doses of radiation. Blood was collected by tail vein bleeding at 24 th hour, 7 th day and 15 th day after radiation exposure; serum was separated and used for the estimation of VEGF level using ELISA kits. Values are mean ± SD. *p<0.05, compared with the respective control.

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15 Figure 6.3 Effect of Vernolide-A on vascular density Thin tumour sections (5 μm) after deparaffinization and rehydration were treated with monoclonal rat anti-mouse CD31 IgG antibody and further incubated with HRP conjugated rabbit anti-mouse IgG secondary antibody. DAB was used as the chromagen and methyl green as the counterstain. Arrow represents CD 31 positive cells (endothelial cells). a) Tumour alone, b) Tumour + Radiation (6 Gy), c) Tumour + Radiation (4 Gy), d) Tumour + Radiation (2 Gy), e) Tumour + Vernolide-A, f) Tumour + Radiation (6 Gy) + Vernolide-A, g) Tumour + Radiation (4 Gy) + Vernolide-A, h) Tumour + Radiation (2 Gy) + Vernolide-A.

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17 in tumour. Vascular density was significantly reduced by the treatment of Vernolide- A. We could see maximum vascular density in the tumour section from the tumour bearing mice exposed to 6 Gy radiation whereas microvessels were markedly reduced by the Vernolide-A treatment Effect of Vernolide-A on the expressions of VEGF and HIF-1α genes Gene expression of VEGF and HIF-1α is given in the Figure 6.4. We could see the upregulated expression of these genes in the untreated control. Tumour bearing mice exposed to 6 Gy radiation showed more significant expression, compared to other control groups. Vernolide-A administration down regulated the expression of VEGF and HIF-1α in the tumour bearing mice Effect of Vernolide-A on cell proliferation Figure 6.5 shows the effect of radiation and Vernolide-A on the inhibition of B16F- 10 cell proliferation. Cells proliferation was analyzed by 3 H-thymidine incorporation assay. Cell proliferation was markedly decreased by the increase of radiation dose. Proliferation was further decreased when the cells were treated with Vernolide-A along with radiation Effect of Vernolide-A on clonogenic survival Soft agar assay shows the effect of radiation on the rate of cell survival of tumour cells (Figure 6.6). The number of cell colonies was reduced by the radiation exposure. Survival fraction was decreased with the increase of radiation dose. B16F- 10 cells treated with Vernolide-A along with radiation further decreased the survival fraction supporting the cell proliferation data Effect of Vernolide-A on vascular endothelial growth factor level (in vitro) Figure 6.7 shows the effect of radiation on in vitro VEGF level. Our data indicate that radiation could elevate the VEGF level. B16F-10 cells after 48 h of radiation showed maximum VEGF level ( ± 121), which was significantly (p<0.05) decreased by the treatment of Vernolide-A (935.7 ± 82) Effect of Vernolide-A on collagen matrix invasion Effect of Vernolide-A on the invasion of irradiated B16F-10 cells is presented in the Figure 6.8. Conditioned medium after 48 h of irradiation from the previous 141

18 Figure 6.4 Effect of Vernolide-A on gene expressions of VEGF and HIF-1α Total RNA was isolated from the tumour, and cdna was synthesized using Moloney murine leukemia virus reverse transcriptase. Amplification was performed using specific primers of VEGF and HIF-1α. a) VEGF b) HIF-1α Lane 1: Molecular weight marker, Lane 2: Tumour control, Lane 3: Tumour + Vernolide-A, Lane 4: Tumour + Radiation (6 Gy), Lane 5: Tumour + Radiation (6 Gy) + Vernolide-A, Lane 6: Tumour + Radiation (4 Gy), Lane 7: Tumour + Radiation (4 Gy) + Vernolide-A, Lane 8: Tumour + Radiation (2 Gy), Lane 9: Tumour + Radiation (2 Gy) + Vernolide-A, Lane 10: GAPDH.

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20 Figure 6.5 Effect of Vernolide-A on cell proliferation B16F-10 melanoma cells ( cells/well) were grown in 96 well flat bottom plate. After 24 h, the cells were exposed to different doses of radiation (0.5, 1, 2, 3, 4, and 5 Gy) in the presence and absence of vernolide-a (0.1 μg/ml) and were further incubated for 48 h. 3 H-thymidine was added to each well (1 μ Ci /well) and incubation was continued for 18 h. Cells were lysed and radioactivity was counted by using Rack Beta liquid scintillation counter. Values are mean ± S.D.

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22 Figure 6.6 Effect of Vernolide-A on clonogenic survival B16F-10 melanoma cells were exposed to different doses of radiation (0.5, 1, 2, 3, 4, and 5 Gy). Cells were cultured in soft agar in the presence and absence of vernolide-a (0.1 μg/ml) and were incubated for 6 days. Colonies were then fixed, stained with crystal violet, and counted. The surviving fraction (SF) was estimated. Values are mean ± S.D.

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24 Figure 6.7 Effect of Vernolide-A on vascular endothelial growth factor level (in vitro) B16F-10 cells were grown in complete DMEM and incubated with or without vernolide- A (0.1 μg/ml) and also the cells were irradiated at 1 Gy with or without Vernolide-A (0.1 μg/ml), which was added just before ionizing radiation. Conditioned medium was collected 24, 48, and 72 hours after irradiation, centrifuged, and analysed VEGF level by ELISA kit. Values are mean ± SD. *p<0.05, compared with the respective control.

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26 Figure 6.8 Effect of Vernolide-A on collagen matrix invasion B16F-10 cells (10 5 cells/150 μl DMEM) were irradiated with 1 Gy and were then seeded on to the upper chamber of Boyden chamber in the presence and absence of Vernolide-A (0.1 μg/ml) for 10 h at 37 0 C. Membrane was removed, fixed, stained and the cells that had migrated in the test and control were counted. Experiments were performed in triplicate and the results are expressed as percentage inhibition of invasion. a) Untreated control, b) Vernolide-A (0.1 μg/ml) treated

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28 experiment was used for the invasion assay. Irradiated control cells showed the maximum invasion through polycarbonate filter membrane. Vernolide-A treatment significantly inhibited the cell invasion by 82.5% Effect of Vernolide-A on MMP activation Vernolide-A inhibited the activation of matrix metalloproteinases produced by the irradiated B16F-10 melanoma cells as shown in Figure 6.9. Conditioned medium with maximum VEGF level from the irradiated B16F-10 cells from the previous experiment was used as the source of matrix metalloproteinase s. Conditioned medium after trypsin activation showed digested clear areas at 92 and 72 kd which was identical to MMP-9 and MMP-2 activity. Gels loaded with conditioned medium, without trypsin activation, did not show any clear areas, indicating the inactive form of the enzyme collagenase. Trypsin activated conditioned medium loaded gels; after incubation with 10 mm EDTA did not show clear areas which indicate that the enzyme responsible for degradation is metalloproteinase. When conditioned medium was treated with Vernolide-A during trypsin activation, no clear bands were observed indicating that Vernolide-A inhibited the activation of procollagenase to active collagenase Effect of Vernolide-A on the expression of VEGF, HIF-1α and VEGF receptor (Flk-1) genes. VEGF, HIF-1α and Flk-1 genes were clearly expressed in hypoxia exposed untreated cells (Figure 6.10). Expression of all these genes were down regulated or suppressed by the treatment of Vernolide-A DISCUSSION Tumour hypoxia is associated with resistance to radiation therapy and with increased invasion and metastatic potential (Postovit et al., 2004). This is an important phenomenon in the tumour microenvironment. Hypoxic tumours are more aggressive and resistant to anti-neoplastic treatments. HIF-1α plays a major role in the response of tumours to hypoxia, and it is mainly responsible for the angiogenic switch. HIF- 1α contributes to tumour aggressiveness, invasiveness and resistance to radiotherapy and chemotherapy (Diaz-onzalez and Russell, 2005). Hypoxic cells are 2-to 3-fold more resistant to radiation than well-oxygenated cells because the biological effect 142

29 Figure 6. 9 Effect of Vernolide-A on MMP activation a) Conditioned medium from untreated irradiated B16F-10 cells without trypsin activation. b) Conditioned medium from untreated irradiated B16F-10 cells after trypsin activation. c) Conditioned medium from untreated irradiated B16F-10 cells after trypsin activation+edta. d) Conditioned medium from irradiated B16F-10 melanoma cells (0.1µg/ml Vernolide-A) after trypsin activation.

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31 Figure 6.10 Effect of Vernolide-A on the expression of VEGF, HIF-1α and VEGF receptor (Flk-1) genes Cells were exposed to hypoxia in the presence and absence of Vernolide-A (0.1 μg/ml) for 4 h at 37 C. cdna was synthesized and amplified with appropriate primer for VEGF, HIF-1α and Flk-1. Lane 1: Molecular weight marker, Lane 2: VEGF of control cells Lane 3: VEGF of treated cells Lane 4: HIF-1α of control cells Lane 5: HIF-1α of treated cells Lane 6: Flk-1 of control cells Lane 7: Flk-1 of treated cells Lane 8: GAPDH.

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33 of radiation is greatly influenced by the presence or absence of molecular oxygen at the time of irradiation (Hall, 1994; Gray et al., 1953). Many studies demonstrated that the growth of solid tumours and their metastasis are depending on angiogenesis, which is regarded as a critical event in tumour development (Bergers and Benjamin, 2003). Overexpression of HIF-1α has correlation with vascular density in tumours indicating that HIF-1α is a key initiator of angiogenic activity (Giatromanolaki et al., 2001; Sivridis et al., 2002). The overexpression of HIF-1α in human cancers is correlated with poor patient prognosis and poor response to radiotherapy. HIF-1α is therefore considered to be a potential therapeutic target and HIF-1α blockade is pursued as a promising strategy for cancer therapy in combination with radiation (Palayoor et al., 2008). Administration of Vernolide-A along with radiation significantly reduced the tumour volume compared to untreated radiation control of tumour bearing mice. Vernolide-A treatment along with the radiation also decreased the cell proliferation and survival fraction of B16F-10 cells. VEGF, a 40- to 45-kDa disulphide-linked homodimeric glycoprotein, is a potent endothelial cell mitogen that has been associated with increased angiogenesis in malignancies. VEGF is produced by tumour cells, and its binding with VEGF receptor Flk-1, which is expressed on vascular endothelial cells, leads to the proliferation and migration of endothelial cells (Ferrara, 2004; Liu et al., 2005). Recent studies showed that the expression of VEGF and its receptors was upregulated in human HCC tissue (Mise et al., 1996; Yamaguchi et al., 2000). It is stimulated by hypoxia, growth factors, cytokines and oncogenes. HIF-1α contributes to the angiogenic switch by binding to DNA at hypoxia response elements and activating transcription of the VEGF gene. VEGF expression has been shown to possess a prognostic value in predicting metastasis of various malignant solid tumours. Several clinicopathologic studies have shown a direct association between VEGF expression and increased microvascular density (MVD) demonstrating intratumoural angiogenesis (Ferrara et al., 2003). Serum VEGF level was elevated during tumour progression. Vernolide-A significantly reduced the elevated serum VEGF level in tumour bearing animals. Ionizing radiation also drastically enhanced the serum VEGF level, which was found to be decreased by the administration of Vernolide-A. Gene expressions of VEGF, HIF-1α, and VEGF receptor, Flk-1 were found to be downregulated by the treatment of Vernolide-A. CD31 (PECAM-1) is a 143

34 transmembrane glycoprotein that is highly expressed in endothelium. Its localization at the endothelial cell junctions suggests an important role in transendothelial cellular migration (Zocchi et al., 1996). Immunohistochemical analysis showed comparitively less number of tumour vasculature in Vernolide-A treated tumour bearing animals. Matrix metalloproteinases (MMP) are zinc-dependent proteolytic endopeptidases (Matrisian, 1992) involved in cancer progression. They stimulate cancer cell growth, migration, invasion, and metastasis (Egeblad and Werb, 2002). MMPs are secreted in a latent (pro) form and activated by proteolytic removal of the NH 2 -terminal propeptide (Belotti et al., 2003). MMP-2 (gelatinase-a), and MMP-9 (gelatinase-b) are important in the proteolytic degradation of extracellular matrix (ECM) and their expression and activation is found to be increased after radiation (Speake et al., 2005). Our study clearly demonstrated that Vernolide-A treatment significantly inhibited the activation of MMP-2 and MMP-9 in B16F-10 cells. It also decreased the B16F-10 cells invasion through polycarbonate filter membrane, supporting the above observations. Our results indicate that Vernolide-A administration significantly reduced the tumour volume in experimental mice. Vernolide-A treatment inhibited the tumour cell invasion, MMP activation, VEGF level, and gene expressions of HIF-1α and VEGF induced by ionizing radiation. The results of the above experiments strongly suggest that Vernolide-A inhibited the radiation induced tumour invasion by regulating HIF-1α, MMP-2, MMP-9 and VEGF. 144