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Vascular Endothelial Growth Factor Immunoneutralization Plus Paclitaxel Markedly Reduces Tumor Burden and Ascites in Athymic Mouse Model of Ovarian Cancer

      Ovarian cancer is characterized by rapid growth of solid intraperitoneal tumors and production of large volumes of ascites. Our previous studies of intraperitoneal ovarian carcinoma in an athymic mouse model demonstrated that a monoclonal antibody (mAb) to human vascular endothelial growth factor (VEGF) could prevent ascites formation. Although ascites was almost completely inhibited, tumor burden was variably reduced. To develop more effective therapy, we assessed the combination of a human VEGF mAb plus paclitaxel. Four groups of female athymic nude mice were inoculated intraperitoneally with OVCAR3 cells. Two weeks after inoculation, one group was treated with a human VEGF mAb intraperitoneally twice weekly plus paclitaxel intraperitoneally three times weekly for 6 weeks. The second group was treated with VEGF mAb alone. The third group was treated with paclitaxel alone. The remaining group was treated with vehicle only. Tumor burden in the VEGF mAb plus paclitaxel and paclitaxel alone groups was reduced by 83.3% and 85.7% and 58.5% and 59.5%, respectively, in two separate experiments, compared to controls. VEGF mAb alone caused no significant decrease in tumor burden, nor did treatment of mice inoculated intraperitoneally with HEY-A8 cells, a non-VEGF-secreting ovarian cell line. Virtually no ascites developed in the combined treatment group or the group treated with VEGF mAb alone. Paclitaxel alone reduced ascites slightly, but not significantly. Morphological studies demonstrated that VEGF immunoneutralization enhanced paclitaxel-induced apoptosis in these human ovarian cancers. Thus, combination therapy with inhibitors of VEGF plus paclitaxel may be an effective way to markedly reduce tumor growth and ascites in ovarian carcinoma.
      Ovarian cancer is characterized by rapid growth and spread of solid intraperitoneal tumors and, in some patients, the formation of large volumes of ascites. It is the major cause of death from gynecological malignancy and is the fifth most common cause of death from cancer in American women. Despite improved methods of surgery and chemotherapy, the mortality rates in women with advanced, recurrent, or persistent ovarian cancer have remained largely unchanged for the last 4 decades.
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      Cancer of the ovary.
      Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein, specific for endothelial cells, which stimulates angiogenesis. It also possesses potent vascular permeability-enhancing activity
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      and is also known as vascular permeability factor (VPF). VEGF/VPF induces ascites accumulation, at least in part, by increasing the permeability of diaphragmatic and tumor-associated vasculature.
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      Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease.
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      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
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      Inhibition of malignant ascites and growth of human ovarian carcinoma by oral administration of a potent inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.
      Our previous studies in a model of intraperitoneal ovarian carcinoma in athymic mice inoculated with SKOV3 cells demonstrated that a monoclonal antibody (mAb) to human VEGF can prevent ascites.
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      We also showed that administration of a VEGF mAb could reverse pre-existing ascites in mice inoculated with cells derived from an OVCAR3 cancer cell line, in which ascites develops earlier in the course of the disease than with the SKOV3 cell line.

      Hu L, Huang Z, Ferrara N, Jaffe RB: Reversal of pre-existing ovarian carcinoma-associated ascites with a human monoclonal antibody to vascular endothelial growth factor. Abstracts, 81st Annual Meeting Endocrine Society, San Diego, CA, June 12–15, 1999

      Although ascites was almost completely inhibited, tumor burden was variably reduced.
      In an effort to develop more effective forms of therapy for ovarian carcinoma, we sought to develop VEGF mAb-based combination therapy. In the past few years, several chemotherapeutic agents, including paclitaxel (Taxol), cis-platin, and etoposide have demonstrated some efficacy in the treatment of ovarian carcinoma.
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      Paclitaxel: the first of the taxanes, an important new class of antitumor agents.
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      Phase II study of high-dose cisplatin, etoposide, and cyclophosphamide for refractory ovarian cancer.
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      Adjuvant therapy of ovarian germ cell tumors with cisplatin, etoposide, and bleomycin: a trial of the Gynecologic Oncology Group.
      However, resistance to these agents usually supervenes. Paclitaxel, a naturally-occurring diterpenoid originally isolated from the Pacific yew, is the first representative of a class of anti-tumor agents that is now widely used in the treatment of many forms of cancer, including those of the ovary, breast, and lung.
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      Inhibition of malignant ascites and growth of human ovarian carcinoma by oral administration of a potent inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.
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      Phase II trial of paclitaxel, an active drug in the treatment of metastatic breast cancer.
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      Paclitaxel promotes assembly of microtubules, inhibits tubulin disassembly, and blocks cell cycling at the G2/M stage.
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      Coupling cell division and cell death to microtubule dynamics.
      Paclitaxel also inhibits DNA synthesis,
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      releases tumor necrosis factor-α,
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      New natural products in cancer chemotherapy.
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      Paclitaxel increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.
      and causes apoptotic cell death in a variety of cancer cell types.
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      Paclitaxel: the first of the taxanes, an important new class of antitumor agents.
      Because of its unique mechanism of action and its wide spectrum of activity,
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      and because of recent studies in which paclitaxel was used in combination with other agents in other types of neoplasms,
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      Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma.
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      • Dinney CP
      Treatment of human metastatic transitional cell carcinoma of the bladder in a murine model with the anti-vascular endothelial growth factor receptor monoclonal antibody DC101 and paclitaxel.
      we elected to test paclitaxel combined with the mAb to VEGF/VPF in our athymic mouse model
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      to assess the extent of inhibition of tumor burden as well as ascites. We explored the possible effects of the interaction between immunoneutralization of VEGF and paclitaxel by assessing tumor burden and ascites volume before, during, and after treatment. In addition, we assessed the effects of treatment on apoptosis. To further assess the central role of VEGF in these processes, we also studied the effects of the VEGF mAb plus paclitaxel in a non-VEGF-expressing ovarian cancer cell line.

      Materials and Methods

      Materials

      Paclitaxel was obtained from Sigma Chemical Co. (St. Louis, MO). A mouse mAb (A4.6.1) directed against human VEGF was used to neutralize VEGF activity in vivo. Characterization of this antibody, including its specificity for human VEGF and its ability to inhibit VEGF activity in vitro and in vivo, as well as its ability to block binding of VEGF to its receptors in vivo, has been described previously.
      • Kim KJ
      • Li B
      • Winer J
      • Armanini M
      • Gillett N
      • Phillips HS
      • Ferrara N
      Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.
      All cell culture reagents were obtained from the Cell Culture Facility, University of California, San Francisco (UCSF).

      Experimental Animals

      Five- to seven-week-old female athymic immunodeficient mice (Simonsen Laboratories, Gilroy, CA) were delivered to the UCSF Laboratory Animal Resource Center, 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 Research, UCSF.

      Experimental Design

      Experiment 1

      Four groups of female athymic nude mice (5 to 7 weeks of age) were inoculated intraperitoneally with OVCAR3 cells (n = 18). Two weeks after inoculation, one group (n = 5) was treated with the human VEGF mAb plus paclitaxel for 6 weeks. The second group of mice (n = 5) was treated with VEGF mAb alone. The third group (n = 4) was treated with paclitaxel alone. The remainder (n = 4) were treated with the same volume of vehicle (phosphate-buffered saline). The human VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly as in our previous studies.
      • Kraft A
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      Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease.
      The dose of paclitaxel (20 μg/g body weight), was based on previous studies.
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      • Matsuzaki SW
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      Antitumor activity of paclitaxel against human breast carcinoma xenografts serially transplanted into nude mice.
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      Antitumor activity of paclitaxel (NSC-125973) in human ovarian carcinomas growing in the peritoneal cavity of nude mice.
      Administration was twice weekly in the first week and increased to three times weekly for the last 5 weeks. There was no apparent toxicity.

      Experiment 2

      The design of experiment 2 was similar to that of experiment 1 except that paclitaxel was administrated three times weekly for 6 weeks, while paclitaxel was administrated twice weekly in the first week and increased to three times weekly for the last 5 weeks in experiment 1.
      Four groups of female athymic nude mice (5 to 7 weeks of age) were inoculated intraperitoneally with OVCAR3 cells (n = 49). Two weeks after inoculation, one group of mice (n = 12) was treated with the human VEGF mAb (5 μg/g body weight) twice weekly plus paclitaxel (20 μg/g body weight) three times weekly for 6 weeks. The second group of mice (n = 13) was treated with VEGF mAb alone. The third group (n = 12) was treated with paclitaxel alone. The remainder (n = 12) was treated with the same volume of vehicle.
      As an additional control, we inoculated nude mice (n = 20) with HEY-A8 ovarian cancer cells, which do not express VEGF mRNA and/or secrete the protein.
      • Yoneda J
      • Kuniyasu H
      • Crispens MA
      • Price JE
      • Bucana CD
      • Fidler IJ
      Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice.
      The protocol for HEY-A8-inoculated mice was the same as for OVCAR3 inoculated mice except that mice inoculated with HEY-A8 were treated for 4 weeks because they developed advanced disease earlier than the mice inoculated with OVCAR3, and did not survive longer than 4 weeks of treatment.

      Methods

      To prepare cells for inoculation, they were collected from the ascites fluid of athymic mice inoculated with the OVCAR3 line. Ascites fluid was collected and placed in a 4°C refrigerator for 1 to 2 hours. The supernatant was then discarded. The cells were diluted with medium RPMI 1640 supplemented with 2.0 g/L of glucose and 0.3 g/L of l-glutamine, which had been prewarmed in a 37°C incubator. Athymic nude mice (5 to 7 weeks of age) were inoculated intraperitoneally with OVCAR3 cells (n = 49; 2 × 106 cells per mouse in 500 μl of RPMI 1640). In addition, 20 athymic nude mice (5 to 7 weeks of age) were inoculated intraperitoneally with HEY-A8 cells (2 × 106 cells per mouse in 500 μl of 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, 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.

      Light Microscopy and Analysis

      Tumor tissue sections from OVCAR3-inoculated mice treated with VEGF mAb plus paclitaxel 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 4.0, and stored on a ZIP external 100 MB drive (Iomega). Photomicrographic plates were composed from the original data in Photoshop, without alteration or manipulation, and annotated with rub-on letters and symbols.

      Assessment of Apoptosis

      Paraffin sections (5 μm) of cancer tissue from OVCAR3 cell-inoculated mice treated with VEGF mAb plus paclitaxel were used to assess apoptosis. DNA labeling with digoxigenin deoxy-UTP and terminal transferase, followed by immunocytochemical staining with peroxidase-coupled anti-digoxigenin antibody and diaminobenzidine, was performed with the reagents supplied in the Apoptag kit (Intergen, Purchase, NY) according to the manufacturer's instructions, except that Tris was substituted for phosphate in the wash buffer. After light counterstaining with hematoxylin, nuclei that stained brown were scored as positive for apoptosis and those that stained blue were scored as negative. At least five ×300 microscopic fields were scored, and the apoptotic index was calculated as the percentage of cells that were scored positive.

      Statistics

      Results are presented as means ± SE. Data were analyzed using one-way analysis of variance followed by unpaired Student's t-test for comparison between groups. Differences between groups were considered statistically significant at P < 0.05. Experiments were performed in duplicate.

      Results

      We examined the potential interactions occurring between the angiogenesis inhibitor VEGF mAb (A.4.6.1) and paclitaxel, given singly and in combination, in the control of ovarian tumor growth and ascites formation, to assess whether these interactions increase the therapeutic effects of each agent individually.
      At postmortem examination, tumors were found on the surface of the peritoneum, intestines, mesentery, and uterus in both treatment and control groups. Eighty-seven percent, 58%, and 20% of the mice in the control, VEGF antibody-treated and paclitaxel-treated groups, respectively, had tumors on the diaphragm and in the hilus of the liver. However, these tumors were not found in the combined VEGF mAb plus paclitaxel treatment groups. There were numerous small bead-like tumors in the VEGF mAb plus paclitaxel-treated mice, which are not commonly found in untreated control mice.

      Experiment 1

      The results of this initial study of treatment of VEGF mAb and paclitaxel, singly and in combination, are shown in Figure 1A. The mean tumor burden in the combined VEGF mAb plus paclitaxel-treated group was 1.47 ± 0.31 g (n = 4). (At the beginning of the experiment, there were five mice in this group. One mouse had leakage of ascites fluid because the peritoneum was accidentally torn with the injection needle 2 weeks before the end of the experiment; this mouse was not included in the analysis.)
      Figure thumbnail gr1
      Figure 1Effects of VEGF mAb plus paclitaxel on tumor burden (A) and ascites formation (B) in mice inoculated with OVCAR3 cells. Experiment 1. Four groups of athymic immunodeficient mice were used. OVCAR3 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatment was initiated 2 weeks after inoculation. Treatment groups consisted of control (vehicle alone), VEGF mAb alone, VEGF mAb plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 6 weeks. Administration of paclitaxel (20 μg/g body weight) was twice weekly in the first week and increased to three times weekly for the last 5 weeks. At autopsy, ascites fluid was quantified and tumors were excised and weighed (n = 15). Data are expressed as mean ± SE **. P < 0.01 versus control; ++, P < 0.01 versus paclitaxel.
      The tumor burden in the group treated with paclitaxel alone (3.64 ± 0.42 g) was significantly reduced compared to that of the controls (8.78 ± 1.32 g) (n = 2). (At the beginning of the experiment, there were four mice in the control group. However, two died 10 and 22 days, respectively, before the end of the experiment, most likely as a result of the marked tumor burden and ascites.) The tumor burdens in the anti-VEGF plus paclitaxel and paclitaxel alone groups were reduced by 83.3% and 58.5%, respectively, compared to the control group. VEGF mAb alone (7.79 ± 1.33 g) (n = 4) had no significant effect on tumor burden compared to the control group.
      Figure 1B shows the results of the initial study of the control of ascites formation by the VEGF mAb and paclitaxel treatment, singly and in combination. The mean volume of ascites in the control group was 3.15 ml. In contrast, virtually no ascites developed in the VEGF mAb plus paclitaxel-treated group nor in the group that was treated with VEGF mAb alone. Paclitaxel alone reduced ascites formation slightly, but not significantly.

      Experiment 2

      As in experiment 1, tumor burden in both the combined VEGF mAb plus paclitaxel (n = 12) and paclitaxel alone (n = 12) treated groups was significantly reduced compared to the control group (Figure 2A). The tumor burden of the VEGF mAb plus paclitaxel group (n = 12) and the group treated with paclitaxel alone (n = 12) was reduced by 85.7% (0.529 ± 0.12 g) and 59.5% (150 ± 0.21 g), respectively, compared to the control group (3.71 ± 0.37 g) (n = 12). There was no significant difference in tumor burden between the group treated with VEGF mAb alone (n = 13) and the control group.
      Figure thumbnail gr2
      Figure 2Effects of VEGF mAb plus paclitaxel on tumor burden (A) and ascites formation (B) in mice inoculated with OVCAR3 cells. Experiment 2. Four groups of athymic immunodeficient mice were used. The OVCAR3 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatment was initiated 2 weeks after inoculation. Treatment groups consist of control (vehicle alone), VEGF mAb alone, VEGF antibody plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 6 weeks. Administration of paclitaxel (20 μg/g body weight) was three times weekly for 6 weeks. At autopsy, ascites fluid was quantified and tumors were excised and weighed (n = 49). Data are expressed as mean ± SE. **, P < 0.01 versus control; ++, P < 0.01 versus paclitaxel.
      Changes in ascites volume (Figure 2B) also were similar to the initial study. The volume of ascites in mice that did not receive VEGF mAb (control), was 3.6 ml, whereas ascites in the VEGF mAb alone or VEGF mAb plus paclitaxel-treated groups was barely detectable. Again, paclitaxel alone slightly, but not significantly, reduced ascites formation.
      As an additional control we inoculated nude mice with HEY-A8 ovarian cancer cells, which do not express VEGF mRNA and do not secrete VEGF.
      • Yoneda J
      • Kuniyasu H
      • Crispens MA
      • Price JE
      • Bucana CD
      • Fidler IJ
      Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice.
      We found that the mice inoculated with HEY-A8 did not produce ascites, and the VEGF mAb did not enhance the inhibitory effect of paclitaxel alone on tumor growth (Figure 3). The tumor burden in the group treated with paclitaxel alone (0.704 ± 0.09 g) (n = 5) was significantly reduced (44.3%) compared to the controls (1.26 ± 0.32 g) (n = 5). There were no significant differences in tumor burden between the group treated with paclitaxel alone and VEGF-mAb plus paclitaxel (0.678 ± 0.13 g) (n = 5) in the mice inoculated with HEY-A8. VEGF alone (1.346 ± 0.18 g) (n = 5) had no effect on tumor burden compared to the control group.
      Figure thumbnail gr3
      Figure 3Effects of VEGF mAb plus paclitaxel on tumor growth in mice inoculated with Hey-A8 cells. Four groups of athymic immunodeficient mice were used. Hey-A8 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatments were started 2 weeks after inoculation. Treatment groups consisted of control (vehicle alone), VEGF mAb alone, VEGF mAb plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 4 weeks. Paclitaxel (20 μg/g body weight) was administered three times weekly for 4 weeks. At autopsy, tumors were excised and weighed (n = 20). Data are expressed as mean ± SE. *, P < 0.05 versus control.

      Apoptosis

      Figure 4 illustrates apoptotic cells in cancer tissue from OVCAR3-inoculated mice. The brown nuclei (indicated by arrows) indicate cells that underwent apoptosis. Approximately 30% of OVCAR3 cells in the paclitaxel-treated group were apoptotic, whereas more than 50% in the slides from the VEGF mAb plus paclitaxel group were necrotic and apoptotic, with cytoplasmic debris and calcification. There were no significant differences between the control group and the group treated with the VEGF mAb alone.
      Figure thumbnail gr4
      Figure 4Histological appearance and apoptosis in tumor tissue from OVCAR3-inoculated athymic mice with and without paclitaxel or VEGF mAb plus paclitaxel treatment. A: Representative section of tumor from control group. Tumor cells have large, atypical nuclei, prominent nucleoli, a moderate amount of cytoplasm, grow in sheets, and undergo mitosis. B: Section of tumor from VEGF mAb plus paclitaxel treatment group. Focus of necrosis and apoptosis with cytoplasmic debris and calcification. A few clusters of viable tumor cells surround this central focus (bottom right). Similar foci were scattered throughout tumors removed from the treated animals. The brown-stained cells (arrows) indicate apoptosis. C: Section of tumor from VEGF mAb-treated group shows tumor cells with large, atypical nuclei. Some cells are swollen, whereas in others the cytoplasm is dense and decreased in amount. No evidence of apoptosis was detected. D: Section of tumor from paclitaxel-treated group shows degenerative changes, including decreased nuclear size, hyperchromasia, and smudging of the nuclear chromatin. A few cells are swollen, whereas in others the cytoplasm is dense and decreased in amount. Arrows indicate apoptotic cells. Original magnifications, ×300.

      Discussion

      Recent studies indicate that the combination of anti-angiogenic agents and conventional chemotherapeutic agents can significantly inhibit tumor growth and metastasis.
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      The present data are consistent with these studies and demonstrate that the combination of the human VEGF mAb plus paclitaxel, which markedly inhibited tumor growth, was extremely effective in the treatment of ovarian carcinoma in that it virtually eliminated ascites and reduced tumor burden by >85.7%. Thus, the effect of paclitaxel on ovarian cancer was markedly enhanced by combination with the VEGF mAb. The anti-metastatic, anti-tumor, and anti-ascites effects of the VEGF mAb plus paclitaxel were markedly greater than those of paclitaxel alone.
      The mechanism by which this combination therapy exerts its effect is not clear. A recent study demonstrates that survivin, an inhibitor of apoptosis, plays a role in the maintenance of microtubules within the mitotic spindle and appears to be overexpressed in common cancers but not in corresponding normal adult tissues.
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      We have demonstrated that the PI3K inhibitor, LY294002, enhances the effects of paclitaxel on tumor growth and ascites formation and decreases development of resistance to paclitaxel.
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      We also have demonstrated that OVCAR3 cells release high levels of VEGF,
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      • Ferrara N
      • Jaffe RB
      Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells.
      which can result in overexpression of survivin. Our use of the VEGF mAb to neutralize VEGF could, in turn, reduce the expression of survivin and decrease the drug resistance that develops with paclitaxel alone.
      Paclitaxel inhibits microtubule depolymerization and blockade of cell division in the G2/M phase of the cell cycle.
      • Sorger PK
      • Dobles M
      • Tournebize R
      • Hyman AA
      Coupling cell division and cell death to microtubule dynamics.
      In addition, paclitaxel can inhibit angiogenesis by suppressing VEGF expression.
      • Lau DH
      • Xue L
      • Young LJ
      • Burke PA
      • Cheung AT
      Paclitaxel (Taxol): an inhibitor of angiogenesis in a highly vascularized transgenic breast cancer.
      Thus, tumor growth might be affected not only by direct cytotoxicity but also by inhibition of new vessel formation, and the VEGF mAb could enhance the anti-angiogenic effects of paclitaxel, as well as decreasing development of drug resistance to paclitaxel.
      • Hu L
      • Hofmann J
      • Lu Y
      • Mills GB
      • Jaffe RB
      Inhibition of phosphatidylinositol 3′-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models.
      On the other hand, the inhibition of angiogenesis might have led to the death of tumor cells most distal to the established vasculature, thereby decreasing tumor volume and facilitating access of the cytotoxic agents throughout the tumor tissue.
      • Satoh H
      • Ishikawa H
      • Fujimoto M
      • Fujiwara M
      • Yamashita YT
      • Yazawa T
      • Ohtsuka M
      • Hasegawa S
      • Kamma H
      Combined effects of TNP-470 and paclitaxel in human non-small cell lung cancer cell lines.
      When we administered combination therapy to mice that had been inoculated with HEY-A8 ovarian cancer cells, which do not express VEGF mRNA and do not secrete VEGF,
      • Yoneda J
      • Kuniyasu H
      • Crispens MA
      • Price JE
      • Bucana CD
      • Fidler IJ
      Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice.
      we found that the mice did not produce ascites and that the VEGF mAb did not enhance the inhibitory effect of paclitaxel on tumor growth.
      The present study indicates that, similar to our previous experiment,
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      the human VEGF mAb markedly inhibits the ascites that develops after intraperitoneal inoculation with OVCAR3 cells. The function-blocking human VEGF mAb, A.4.6.1, neutralized the tumor-derived VEGF activity, blocking access of VEGF to its receptors, specifically inhibiting the activity of tumor-derived VEGF, as it is specific for human VEGF.
      • Kim KJ
      • Li B
      • Winer J
      • Armanini M
      • Gillett N
      • Phillips HS
      • Ferrara N
      Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.
      The mechanisms by which the VEGF mAb inhibits ascites formation also are not completely understood. Ascites is linked to peritoneal, as well as tumor microvascular hyperpermeability,
      • Kubota T
      • Matsuzaki SW
      • Hoshiya Y
      • Watanabe M
      • Kitajima M
      • Asanuma F
      • Yamada Y
      • Koh JI
      Antitumor activity of paclitaxel against human breast carcinoma xenografts serially transplanted into nude mice.
      and several studies have implicated VEGF in ascites formation by increasing vascular permeability.
      • Kraft A
      • Weindel K
      • Ochs A
      • Marth C
      • Zmija J
      • Schumacher P
      • Unger C
      • Marme D
      • Gastl G
      Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease.
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      Tumor secretion of VEGF is essential for ascites accumulation.
      • Kraft A
      • Weindel K
      • Ochs A
      • Marth C
      • Zmija J
      • Schumacher P
      • Unger C
      • Marme D
      • Gastl G
      Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease.
      • Luo JC
      • Toyoda M
      • Shibuya M
      Differential inhibition of fluid accumulation and tumor growth in two mouse ascites tumors by an antivascular endothelial growth factor/permeability factor neutralizing antibody.
      • Shibuya M
      • Luo JC
      • Toyoda M
      • Yamaguchi S
      Involvement of VEGF and its receptors in ascites tumor formation.
      Anti-VEGF targeting agents block ascites formation
      • Xu L
      • Yoneda J
      • Herrera C
      • Wood J
      • Killion JJ
      • Fidler IJ
      Inhibition of malignant ascites and growth of human ovarian carcinoma by oral administration of a potent inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.
      • Luo JC
      • Toyoda M
      • Shibuya M
      Differential inhibition of fluid accumulation and tumor growth in two mouse ascites tumors by an antivascular endothelial growth factor/permeability factor neutralizing antibody.
      • Verheul HM
      • Hoekman K
      • Jorna AS
      • Smit EF
      • Pinedo HM
      Targeting vascular endothelial growth factor blockade: ascites and pleural effusion formation.
      and immunoneutralization of VEGF alone in our nude mouse model inhibits ascites.
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      In contrast, inoculation with HEY-A8 cells, which do not express VEGF, was not associated with ascites formation. We have also demonstrated that VEGF release from OVCAR-3 cells modulates endothelial cell monolayer permeability in a dual-chamber permeability assay (L Hu, N Ferrara, and RB Jaffe, submitted). In that study, we used HEY-A8 and OCC1 cells as controls, as neither cell line expresses VEGF, and found that neither the HEY-A8 nor OCC1 cells induced significant increases in monolayer permeability.
      Angiogenesis plays a pivotal role in the progression of cancer by permitting tumor growth and facilitating metastasis. The present studies, similar to our previous study using the SKOV3 cell line,
      • Mesiano S
      • Ferrara N
      • Jaffe RB
      Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization.
      indicate that blocking tumor-derived VEGF activity with A.4.6.1 alone does not markedly inhibit intraperitoneal human ovarian tumor growth, although it did decrease subcutaneous SKOV3 cell growth during treatment.
      Interestingly, in VEGF mAb-treated mice there were numerous small bead-like tumors, which are not commonly found in untreated control mice. This observation suggests that the inhibition of angiogenesis by the human VEGF antibody, which only inhibits tumor-derived VEGF, led to the inhibition of new vessel development, thereby limiting growth of tumors; cell death occurred in the cells most distant from the established vasculature. Because endogenous VEGF and/or other angiogenic factors may support some tumor growth and spread, tumors near established vasculature remain small and bead-like. Further, Xu and colleagues
      • Xu L
      • Xie K
      • Mukaida N
      • Matsushima K
      • Fidler IJ
      Hypoxia-induced elevation in interleukin-8 expression by human ovarian carcinoma cells.
      have suggested that although VEGF/VPF may be the major substance involved in the ascites associated with ovarian carcinoma, interleukin-8, which also is angiogenic, may play a major role in solid tumor growth of ovarian and other
      • Kitadai Y
      • Takahashi Y
      • Haruma K
      • Naka K
      • Sumii K
      • Yokozaki H
      • Yasui W
      • Mukaida N
      • Ohmoto Y
      • Kajiyama G
      • Fidler IJ
      • Tahara E
      Transfection of interleukin-8 increases angiogenesis and tumorigenesis of human gastric carcinoma cells in nude mice.
      • Singh RK
      • Gutman M
      • Radinsky R
      • Bucana CD
      • Fidler IJ
      Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice.
      tumors. A recent report indicated that IL-8 reduced tumorigenicity of human ovarian cancer.
      • Lee F
      • Hellendall RP
      • Wang Y
      • Haskill JS
      • Mukaida N
      • Matsushima K
      • Ting JP
      IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration.
      VEGF likely is not the only angiogenic factor that can be involved in the maintenance and growth of intraperitoneal carcinomatosis.
      Our morphological observations, in agreement with previous studies,
      • Costa A
      • Villa R
      • Zaffaroni N
      • Santi S
      • Bearzatto A
      • Silvestrini R
      Paclitaxel induces apoptosis in various cancer cell lines: comparison among different methodological approaches.
      • Al-alami O
      • Sammons J
      • Martin JH
      • Hassan HT
      Divergent effect of paclitaxel on proliferation, apoptosis and nitric oxide production in MHH225 CD34 positive and U937 CD34 negative human leukaemia cells.
      • Frankel A
      • Buckman R
      • Kerbel RS
      Abrogation of paclitaxel-induced G2-M arrest and apoptosis in human ovarian cancer cells grown as multicellular tumor spheroids.
      indicate that paclitaxel induces nuclear pyknosis and fragmentation as well as reduced cytoplasmic volume in tumor cells, indicating apoptosis. Using digoxigenin-UTP and terminal transferase labeling with immunocytochemistry, we have also demonstrated that the VEGF mAb enhanced paclitaxel-induced OVCAR3 cell apoptosis in vivo. More than 50% of the cells from the VEGF mAb plus paclitaxel-treated groups underwent necrosis, perhaps because of apoptosis that subsequently can lead to necrosis.
      • Sandoval D
      • Gukovskaya A
      • Reavey P
      • Gukovsky S
      • Sisk A
      • Braquet P
      The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis.
      In summary, our studies suggest that combination therapy with an anti-angiogenic agent, such as the VEGF mAb plus paclitaxel, administered intraperitoneally or via another route, may be an effective way to markedly inhibit tumor growth and ascites in ovarian cancer.

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