Abstract
Purpose: To assess the role of phosphatidylinositol-3 kinase (PI3K) inhibition in vascular permeability, angiogenesis, and vascular remodeling in tumor vessels and peritoneal lining in an athymic mouse model of i.p. human ovarian carcinoma.
Experimental Design: Mice were inoculated i.p. with cells from the human ovarian cancer cell line, OVCAR-3. Fourteen days after inoculation, mice were treated with or without the PI3K inhibitor LY294002, 3 days weekly for 4 weeks. At the end of the experiment, some mice were anesthetized and injected via the tail vein with FITC-labeled lycopersicon lectin and perfused through the aorta before sacrifice. The peritoneal wall and tumor from all mice were removed and embedded in 10% agarose. Tumor sections were visualized by fluorescence microscopy.
Results: Ascites in the LY294002-treated group (0.69 ± 0.27 mL) was reduced by 72.4% compared with the control group (2.5 ± 1.2 mL). Tumor burden in the LY294002-treated group (0.62 ± 0.32 g) was reduced by 47.3% compared with the control group (1.18 ± 0.41 g). LY294002 inhibited peritoneal and tumor vascularization resulting in numerous leaky, irregular, tortuous vessels in scant, straight, relatively impermeable vessels.
Conclusions: The data indicate that LY294002 inhibits ascites formation in our mouse model of human ovarian cancer by inhibiting tumor and peritoneal neovascularization as well as vascular permeability. The data also show that LY294002 directly inhibits vascular endothelial growth factor (VEGF) protein expression and release from ovarian carcinoma and suggest that LY294002 blocks the VEGF signaling pathway involved in angiogenesis and vascular permeability.
- phosphatidylinostol 3-kinase
- vascular permeability
- Gynecological cancers: ovarian
- Cell signaling
- Angiogenic factors and receptors
The predominant factor in malignant ascites formation associated with ovarian cancer is increased permeability of the tumor and peritoneal microvasculature (1). Marked neovascularization of the parietal peritoneum has been reported in patients with malignant ascites (2), and the amount of ascites parallels the extent of neovascularization (2). Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, plays an important role in the ascites formation associated with ovarian cancer. However, the regulation of VEGF release and the signaling pathways involved in the VEGF-mediated increase in vascular permeability remain to be elucidated definitively.
VEGF signals, in part, through phosphatidylinositol 3-kinase (PI3K) and its downstream signaling cascade, including protein kinase B/Akt (3). Recent studies indicate that inhibition of PI3K signaling interferes with angiogenesis, and PI3K signaling also mediates VEGF expression in endothelial cells (4). In turn, VEGF increases the permeability of the endothelial cell monolayer by activation of the protein kinase B/Akt pathway (5). VEGF signals hyperpermeability through PI3K/Akt in vivo in the microcirculation (6). VEGF derived from human ovarian cancer increases vascular permeability in vitro1 and formation of ascites in vivo (5). Our previous studies of ovarian carcinoma in our athymic mouse model inoculated with human OVCAR-3 cells indicated that the PI3K inhibitor LY294002 not only inhibits tumor growth but also ascites formation (7, 8). We hypothesized that LY294002 inhibits vascular permeability and ascites formation by inhibition of VEGF release from ovarian cancer cells, and that VEGF derived from ovarian cancer increases ascites by activation of the PI3K/Akt pathway. To test these hypotheses, we assessed the effects of PI3K inhibition on vascular permeability, angiogenesis, and vascular remodeling in tumor vessels and peritoneal lining vessels in our athymic mouse model of i.p. ovarian carcinoma.
Materials and Methods
Materials. LY294002, a PI3K inhibitor, was obtained from Eli Lilly (Indianapolis, IN). The human OVCAR-3 cell line was kindly provided by Dr. T. Hamilton (Fox Chase Cancer Center, Philadelphia, PA). Athymic immunodeficient mice were obtained from Simonsen Laboratories (Gilroy, CA). All cell culture reagents were obtained from the Cell Culture Facility, University of California, San Francisco. Fluorescein lycopersicon esculentum lectin was obtained from Vector Laboratory (Burlingame, CA). Rabbit anti-human VEGF polyclonal antibody was obtained from Chemicon (Temecula, CA). Cy3-conjugated secondary polyclonal antibody was obtained from Jackson ImmunoResearch (West Grove, PA).
Experimental animals. Two groups of athymic immunodeficient mice (Simonsen Laboratories) were delivered to the University of California, San Francisco Animal Care Facility, housed in isolated conditions, fed autoclaved standard pellets and water, and allowed to adapt to their new environment. All protocols involving immunodeficient mice were approved by the Committee on Animal Care, University of California, San Francisco.
Methods. To prepare cells for inoculation, they were collected from ascites fluid of athymic mice inoculated with OVCAR-3 cells. Ascites fluid was collected and placed in a 4°C refrigerator for 1 to 2 hours. The supernatant was discarded and the cells were diluted with RPMI 1640 supplemented with 2.0 g/L glucose and 0.3 g/L l-glutamine that had been prewarmed in a 37°C incubator. Athymic mice (5-7 weeks) were inoculated i.p. with OVCAR-3 cells (n = 20; 2 × 106 cells per mouse in 500 μL RPMI 1640). Abdominal circumference and body weight were measured twice weekly. At the end of the experiment, mice underwent euthanasia with CO2. The volume of ascites was measured, tumor tissue was excised, weighed, and fixed in 4% paraformaldehyde (pH 7.4) at 4°C for 24 hours and embedded in paraffin. Paraffin sections (5 μm) were used for histochemical analysis.
Experimental design. Two groups of athymic nude mice (5-7 weeks) were inoculated (i.p.) with OVCAR-3 cells (n = 20; 2 × 106 cells per mouse in 500 μL RPMI 1640). Fourteen days after inoculation, one group of mice was treated with LY294002 (100 μg/g body weight) i.p. thrice weekly, as in our previous studies (8). Another group of mice was treated with the same volume of vehicle. Abdominal circumference and body weight were measured twice weekly.
Tumor vessel imaging. At the end of the experiment, mice were anesthetized and injected i.v. with 100 μL FITC-labeled lycopersicon lectin. Ten minutes later, mice were perfused through the ascending aorta with 4 % paraformaldehyde in PBS for 2 minutes. Tumors were removed and placed in fixative for 2 hours followed by immersion in 30% sucrose. Tissues were embedded in 10% agarose and cut with a Vibratome or cryostat (100-μm thickness). Blood vessels were visualized by their uptake of fluorescent lycopersicon lectin.
Immunofluorescence and confocal microscopy. The ovarian cancer tissue from the mice inoculated with OVCAR-3 and injected with FITC-labeled lycopersicon lectin was washed extensively in PBS. Before immunostaining, the tissues were pretreated with 3% horse serum in PBS plus 0.05% Tween 20 for 1 hour to improve penetration and block nonspecific antibody absorption. The tissues were rinsed in PBS and incubated with 1:100 rabbit anti-VEGF antibody for 16 hours (overnight) on an orbital shaker at 4°C. Tissues were thoroughly washed in PBS before application of 1:200 anti-rabbit IgG Cy3-conjugated antibody diluted in PBS containing 0.05% Tween 20 for 1 hour at room temperature and finally washed again in PBS. Immunostained tissues were examined using a Zeiss Axiophot 200M fluorescence microscope and a Zeiss LSM 510 confocal microscope. The confocal images were processed using Adobe PhotoShop and stored on a CD-R (Memorex, Cerritos, CA). All images shown are individual middle sections of projected Z series.
Light microscopy and analysis. Tissue sections of ovarian cancer from OVCAR-3-inoculated mice treated with LY294002 and injected with FITC-labeled lycopersicon lectin were examined with a Leica DMRB or Leica Ortholux II photomicroscope at low and high magnifications. Images were collected with a Photonics DEI-470 CCD camera and a RasterOps 24XLTV frame grabber, imported directly into Adobe PhotoShop, and stored on a CD-R. Photomicrographic plates were composed from the original data in Photoshop, without alteration or manipulation, and annotated with rub-on letters and symbols.
To determine whether LY294002 affects VEGF release from nude mice inoculated with OVCAR-3 cells, at the end of experiment, blood samples and ascites were collected from the mice with and without LY294002 treatment. VEGF protein concentrations in serum and ascites were determined using ELISA (9).
Statistics. Results are presented as means ± SE. Data were analyzed using one-way ANOVA followed by unpaired Student's t test for comparison between groups. Differences between groups were considered statistically significant at P < 0.05. Experiments were done in duplicate.
Results
Tumor burden and ascites. The data obtained from quantification of tumor burden and ascites were similar to those in our previous study (7). Ascites in the LY294002-treated group (0.69 ± 0.27 mL) was reduced by 72.4% compared with the control group (2.5 ± 1.2 mL). Tumor burden in the LY294002-treated group (0.62 ± 0.32 g) was reduced by 47.3% compared with the control group (1.18 ± 0.41 g).
Peritoneal lining vessels. Vascular architecture in the peritoneal lining of OVCAR-3-inoculated nude mice is shown in Fig. 1A . Vessels of the peritoneal lining of normal (non–OVCAR-3 inoculated) mice have many intact, uniform vessels oriented parallel to the peritoneal surface (a). Vessels of the peritoneal lining from OVCAR-3-inoculated mice (b) are dense compared with those of the controls (a) and are irregular in shape with multiple loops. Extravasation of FITC-lectin from the vessels of the peritoneal lining is apparent. In contrast, the vessels of the peritoneal lining from OVCAR-3-inoculated mice treated with LY294002 have low vascular density compared with the nontreated group (b), and the vessels are straighter and less tortuous. The amount of extravasated FITC-lectin is also decreased compared with the untreated mice.
Fluorescence microscopy and immunocytochemistry in peritoneal and tumor vessels in mice inoculated with OVCAR-3 human ovarian cancer cells and treated with or without LY294002. A, fluorescence microscopy of peritoneal blood vessel distribution, shape, and permeability in mice inoculated with OVCAR-3 human ovarian cancer cells with and without LY294002 treatment. Mice were anesthetized and injected via the tail vein with FITC-labeled lycopercicon lectin followed by perfusion through the aorta with paraformaldehyde. a, vessels in nude mouse not inoculated with OVCAR-3 cells. b, vessels in nude mouse inoculated with OVCAR-3 cells without treatment. c, vessels in nude mouse inoculated with OVCAR-3 cells and, 2 weeks later, treated with LY294002 for 4 weeks. B, fluorescence microscopy of tumor blood vessel distribution, shape, and permeability in mice inoculated with OVCAR-3 human ovarian cancer cells with and without LY294002 treatment. Procedures are identical to those in (A). a, vessels in nude mouse inoculated with OVCAR-3 cells. b, vessels in nude mouse inoculated with OVCAR-3 cells and treated with LY294002. C, immunostaining of VEGF in tumor tissue from mice inoculated with OVCAR-3 human ovarian cancer cells with and without LY294002 treatment (confocal microscopy). a, VEGF immunostaining in tumor tissue from control mouse. b, VEGF immunostaining in tumor tissue from LY294002-treated mouse.
Tumor vessels. The vascular architecture in tumor tissue from OVCAR-3-inoculated mice is shown in Fig. 1B. Tumor vessels from the untreated mice have a branching pattern with multiple loops and an extensive microvascular network comprised of numerous tortuous irregular vessels. Extravasation of FITC-lectin from the vessels is apparent (q). In contrast, the tumor vessels from the LY294002-treated group have straight vessels without loops (b).
Vascular endothelial growth factor protein expression. To explore whether PI3K mediates angiogenesis in ovarian cancer, we used an anti-rabbit VEGF antibody to perform immunoassay on the tumor tissue from the mice inoculated with OVCAR-3 cells with and without LY294002 treatment and injected i.v. with 100 μL FITC-labeled lycopersicon lectin. Figure 1C shows that VEGF staining is extensive in the control tumor tissue. In contrast, VEGF staining is reduced in the tumor tissue from the LY294002-treated mice. The extent of vascularization parallels the expression of VEGF.
Vascular endothelial growth factor concentrations in serum and ascites. Results of the study of control of VEGF release by treatment with LY294002 are shown in Fig. 2A . The mean VEGF serum concentrations in the LY294002-treated group (54.72 ± 2.58 pg/mL) were significantly lower (58.28%, P < 0.001) than those of the controls (131.18 ± 23.47 pg/mL). The VEGF concentrations in ascites (3,967 ± 964 pg/mL) in the LY294002-treated group were markedly reduced (67.02 %, P < 0.001) compared with the control group (12,033 ± 322 pg/mL).
VEGF release detected by ELISA from nude mice inoculated with OVCAR-3 cells with and without treatment with LY294002. A, VEGF concentrations in serum. B, VEGF concentration in ascites.
Discussion
Here, we show that the PI3K inhibitor LY294002 not only inhibits tumor burden but also reduces ascites formation in athymic mice inoculated with human OVCAR-3 cells, consistent with our previous studies (7, 8). Furthermore, we explored possible mechanisms leading to inhibition of ascites formation, as well as the role of PI3K in neovascularization and its correlation with VEGF protein expression and release from the OVCAR-3 cell–inoculated mice. We provide direct evidence that LY294002 inhibits both tumor and peritoneal neovascularization and reduces vascular permeability. A key finding is that inhibition of ascites formation by blocking the PI3K signaling pathway is, at least in part, due to inhibition of tumor and peritoneal neovascularization and down-regulation of VEGF protein expression and release from ovarian cancer cells.
PI3K is involved in the signaling pathways of many cancers (10) and is implicated as an oncogene in ovarian cancer (11). The gene encoding PI3KCA has increased copy number in > 40% of primary ovarian cancer and ovarian cancer cell lines (11). Recent studies indicate that PI3KCA overexpression induces increased angiogenesis in ovarian carcinoma by up-regulating VEGF via hypoxia-inducible factor-1α (12). VEGF is essential for vasculogenesis and angiogenesis, and its expression is regulated by hypoxia, which induces binding of hypoxia-inducible factor-1α to the hypoxia response element in the VEGF gene promoter region (13–15).
The interaction between VEGF and PI3K in vascular permeability and ascites formation has not been completely documented previously. A recent study showed that PI3K signaling mediates angiogenesis and the expression of VEGF in endothelial cells (4). LY294002 has been reported to inhibit VEGF expression in both endothelial and cancer cells (4, 16). PI3K and Akt may stimulate angiogenesis through the activation of VEGF (17), whereas activated PI3K and Akt both increase the levels of VEGF mRNA in a chicken chorioallantoic membrane assay (4). Overexpression of PIK3CA positively correlated with the expression of VEGF mRNA and protein levels in ovarian carcinoma (12). In the present study, immunostaining indicated that LY294002 inhibited VEGF protein expression in ovarian cancer tissue, and VEGF ELISA showed that VEGF protein levels in either serum or ascites was reduced in OVCAR-3 cell–inoculated mice treated with LY294002 compared with controls.
Additionally, PI3K and Akt are sensitive to VEGF (12). The protein kinase Akt exerts one of its functions as a downstream signal of VEGF to promote survival in endothelial cells (18). The function of VEGF in vascular permeability is of importance because angiogenesis is accompanied by an increase in vascular permeability and may be required for vessel sprouting (19). Akt is one of the major downstream effectors for PI3K. Akt signaling is essential for VEGF induction of vascular permeability (20). A previous study indicated that VEGF increases permeability of the endothelial cell monolayer by activation of Akt (5). VEGF activates Akt and causes phosphorylation of eNOS, a downstream target of Akt in human umbilical vascular endothelial cells, increasing permeability (20). VEGF also signals hyperpermeability through PI3K/Akt in the microcirculation in vivo (6). Our study shows that inhibition of PI3K by LY294002 significantly reduces ascites formation and suggests that PI3K/Akt is involved in the signaling pathway leading to VEGF-stimulated hyperpermeability.
As noted, ascites formation is largely dependent on vascular permeability and neovascularization. The present study shows that inhibition of neovascularization and vascular permeability by LY294002 occurs not only in tumor tissues but also in peritoneal lining tissues. These results indicate that malignant ascites formation originates not only from tumor vessels but also from the vasculature of the peritoneal lining. The hyperpermeability of peritoneal lining microvessels was found to increase in parallel with hyperpermeability of tumor microvessels. The close correlation of VEGF concentrations in ascites with the inhibition of hyperpermeable tumor and peritoneal microvessels by LY294002 supports the notion that PI3K may act as an angiogenesis regulatory gene (21) and that activated PI3K and Akt are strong inducers of neovascularization and endothelial cell proliferation (22).
Taken together, the present study indicates that LY294002 inhibits ascites formation in a human ovarian cancer mouse model by inhibiting tumor and peritoneal neovascularization and vascular permeability. We also show that LY294002 directly inhibits VEGF protein expression and release from ovarian carcinoma, indicating that LY294002 blocks the VEGF signaling pathway in angiogenesis and vascular permeability.
Footnotes
↵1 Hu L., Ferrara N., and Jaffe R.B., unpublished.
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Grant support: National Cancer Institute grant P50-CA 83609 and Helene Jaffe Ovarian Cancer Fund.
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Accepted March 24, 2005.
- Received January 31, 2005.
- Revision received March 16, 2005.
References
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