Treatment for malignant pleural effusion of human lung adenocarcinoma by inhibition of vascular endothelial growth factor receptor tyrosine kinase phosphorylation.

Malignant pleural effusion (PE) is associated with advanced human lung cancer. We found recently, using a nude mouse model, that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is responsible for PE induced by non-small cell human lung carcinoma cells. The purpose of this study was to determine the therapeutic potential of a VEGF/VPF receptor tyrosine kinase phosphorylation inhibitor, PTK 787, against PE formed by human lung adenocarcinoma (PC14PE6) cells. PTK 787 did not affect the in vitro proliferation of PC14PE6 cells, whereas it specifically inhibited proliferation of human dermal microvascular endothelial cells stimulated by VEGF/VPF. A specific platelet-derived growth factor receptor tyrosine kinase inhibitor, CGP57148 (used as a control because PTK 787 also inhibits platelet-derived growth factor receptor tyrosine kinases), had no effect on proliferation of PC14PE6 or human dermal microvascular endothelial cells. i.v. injection of PC14PE6 cells into nude mice produced lung lesions and a large volume of PE containing a high level of VEGF/VPF. Oral treatment with CGP57148 had no effect on PE or lung metastasis. In contrast, oral treatment with PTK 787 significantly reduced the formation of PE but not the number of lung lesions. Furthermore, treatment with PTK 787 significantly suppressed vascular hyperpermeability of PE-bearing mice but did not affect the VEGF/VPF level in PE or expression of VEGF/VPF protein and mRNA in the lung tumors of PC14PE6 cells in vivo. These findings indicate that PTK 787 reduced PE formation mainly by inhibiting vascular permeability, suggesting that this VEGF/VPF receptor tyrosine kinase inhibitor could be useful for the control of malignant PE.     

In vivo neutralization of vascular endothelial growth factor (VEGF) vascular permeability factor (VPF) inhibits ovarian carcinoma-associated ascites formation and tumor growth

Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) is emerging as an important growth factor in a variety of tumor types. As a potent endothelial cell mitogen and vascular permeabilizing agent, VEGF/VPF has the unique functional capacity to mediate the component events of solid tumor neovascularization and ascites tumor growth. In the present series of investigations, our experimental hypothesis was that VEGF/VPF is a critical mediator of ovarian carcinoma-associated ascites formation and solid tumor growth. Athymic nude mice xenotransplanted with human ovarian carcinoma cell lines received either a preimmune rabbit serum or VEGF/VPF antiserum. Compared with the control group receiving the preimmune serum, the antiserum-treated animals displayed a 10- and 12-fold reduction in ascites accumulation and solid tumor growth, respectively. The administration of a neutralizing antiserum to VEGF/VPF conferred a modest survival advantage to animals harboring intraperitoneal tumors. These data demonstrate the significance of VEGF/VPF in the pathogenesis of ovarian carcinoma and suggest that interventions targeting this growth factor and/or its receptor may be of therapeutic value in the management of ovarian cancer.     

Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8

We determined whether blockade of nuclear factor (NF)-kappaB/relA activity in human ovarian cancer cells can suppress angiogenesis and growth in an orthotopic nude mouse model. The human ovarian cancer cells SKOV3ip.1 and HEY-A8 were transfected with a mutated IkappaBalpha (IkappaBalphaM), ie., resistant to phosphorylation and degradation, and hence blocks NF-kappaB activity. NF-kappaB signaling blockade significantly inhibited in vitro and in vivo expression of two major proangiogenic molecules, vascular endothelial growth factor and interleukin 8, in cultured cells and in cells implanted into the peritoneal cavity of nude mice. The decreased expression of vascular endothelial growth factor and interleukin 8 directly correlated with decreased tumorigenicity, decreased vascularization of lesions, decreased formation of malignant ascites, and prolonged survival of mice. These findings suggest that inhibition of NF-kappaB/relA activity in ovarian cancer cells can suppress angiogenesis and progressive growth.     

Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer

We determined whether concurrent blockage of vascular endothelial growth factor (VEGF) receptor and epidermal growth factor (EGF) receptor signaling by two novel tyrosine kinase inhibitors, PTK 787 and PKI 166, respectively, can inhibit angiogenesis and, hence, the growth and metastasis of human pancreatic carcinoma in nude mice. Highly metastatic human pancreatic carcinoma L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice began receiving oral doses of PTK 787 and PKI 166 three times weekly. Some groups of mice also received i.p. injections of gemcitabine twice a week. The mice were necropsied when the control mice became moribund. Treatment with PTK 787 and PKI 166, with gemcitabine alone, or with the combination of PTK 787, PKI 166, and gemcitabine produced 69, 50, and 97% reduction in the volume of pancreatic tumors, respectively. Administration of protein tyrosine kinase inhibitors and gemcitabine also significantly decreased the incidence of lymph node and liver metastasis. The therapeutic efficacy directly correlated with a decrease in circulating proangiogenic molecules (VEGF, interleukin-8), a decrease in microvessel density, a decrease in proliferating cell nuclear antigen staining, and an increase in apoptosis of tumor cells and endothelial cells. Therapies produced by combining gemcitabine with either PKI 166 or PTK 787 were similar to those produced by combining gemcitabine with both PKI 166 and PTK 787. These results suggest that blockade of either epidermal growth factor receptor or VEGF receptor signaling combined with chemotherapy provides an effective approach to the therapy of pancreatic cancer.     

Expression of vascular endothelial growth factor and E-cadherin in human ovarian cancer: association with ascites fluid accumulation and peritoneal dissemination in mouse ascites model.

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E-Cadherin is an adhesion molecule that is important for cell-to-cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E-cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC-2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT-PCR methods. AMOC-2 expressed E-cadherin, but not VEGF. HNOA expressed VEGF without E-cadherin expression. HTBOA expressed both VEGF and E-cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC-2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E-cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.     

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.     

Both antiangiogenesis- and angiogenesis-independent effects are responsible for hepatocellular carcinoma growth arrest by tyrosine kinase inhibitor PTK787/ZK222584

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis of hepatocellular carcinoma. Inhibition of VEGF receptors could theoretically reduce angiogenesis and tumor growth in hepatocellular carcinoma, but this remains to be proven with an experimental study. This study examined the angiogenesis-dependent and angiogenesis-independent activities of PTK787/ZK222584 (PTK787), a tyrosine kinase inhibitor of VEGF receptors, in nude mice bearing human hepatocellular carcinoma xenografts. The in vitro effects of PTK787 on proliferation, apoptosis, and cell cycle distribution in human hepatocellular carcinoma cell lines were also studied. Oral administration of PTK787 resulted in a significant reduction in tumor volume and microvessel formation of hepatocellular carcinoma xenografts in nude mice. PTK787 inhibited tumor cell proliferation in a dose-dependent manner and also induced tumor cells to undergo apoptosis both in vivo and in vitro. The proapoptotic response was associated with down-regulation of Bcl-2 and Bcl-x(L) expression and induction of cleavage of caspase-3. In addition, PTK787 induced growth arrest in hepatocellular carcinoma cells, which was associated with G1 arrest and partial G2-M block. This effect correlated with an increase in p21(WAF1/ CIP1) (p21) and p27KIP1 (p27) protein expression. In conclusion, this study showed that PTK787 is a potent inhibitor of tumor growth in hepatocellular carcinoma by both antiangiogenic effect and direct effects on tumor cell proliferation and apoptosis. Our data suggest that blockage of VEGF receptors may provide an effective therapeutic approach for human hepatocellular carcinoma.     

Vascular endothelial growth factor/vascular permeability factor in the pathogenesis of primary effusion lymphomas

Primary effusion lymphomas (PEL), rare lymphomas associated with Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) infection, present as malignant lymphomatous effusions in body cavities. We have recently found that PEL effusions contain high levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). VEGF/VPF, an important regulator of tumor-angiogenesis in vivo, exerts its effects acting through the receptors KDR/Flk-1 and Flt-1 on the endothelial cell membrane. In vitro, the PEL cell lines BC-1, BCP-1 and BCBL-1 produce high levels of VEGF. RT-PCR analysis of RNA from the PEL cell lines amplified the three VEGF/VPF secreted isoforms, VEGF/VPF(121), VEGF/VPF(145) and VEGF/VPF(165). Two of the PEL cell lines express the VEGF/VPF receptor Flt-1, but VEGF did not stimulate proliferation in these cells. SCID/beige mice inoculated intraperitoneally with BCBL-1 cells developed effusion lymphoma of human cell origin with prominent bloody ascites. In contrast, none of the mice treated with a neutralizing anti-human VEGF/VPF antibody developed ascites and effusion lymphoma. Although the precise mechanisms by which VEGF/VPF can promote vascular permeability are not fully understood, VEGF/VPF stimulation of vascular leakage may be critical to the pathogenesis of PEL.     

Albendazole: a potent inhibitor of vascular endothelial growth factor and malignant ascites formation in OVCAR-3 tumor-bearing nude mice.

PURPOSE: Angiogenesis and vessel hyperpermeability are the two factors leading to the formation of ascites. Vascular endothelial growth factor (VEGF) plays a pivotal role in malignant ascites formation. We have recently shown that albendazole inhibits peritoneal growth of human colorectal cancer cells (HT-29). The present study was designed to find out if albendazole can suppress ascites formation in ascites-producing peritoneal carcinomatosis. EXPERIMENTAL DESIGN: Female nude mice bearing peritoneal tumors of human ovarian cancer cells (OVCAR-3) were treated with albendazole. Following i.p. inoculation and ascites development, mice were given i.p. albendazole (150 mg/kg) or the vehicle x 3 weekly for 4 weeks. RESULTS: Whereas vehicle-treated mice developed overt ascites requiring repeated aspiration, ascites formation in the albendazole-treated mice was markedly suppressed. As a result of this, 7 of 10 mice from the control group had to be euthanized before the course of treatment was over. Suppressed ascites production and reduced tumor vascularity observed was a result of dramatic reduction in tumor VEGF production as revealed by profoundly lower VEGF ascites fluid and plasma levels. In vitro, incubation of SKOV-3 cells with various concentrations of albendazole led to significant dose-dependent inhibition of VEGF secretion. Examination of floating tumor cells collected from the peritoneal wash revealed profound down-regulation of VEGF mRNA in albendazole-treated mice. CONCLUSIONS: These findings suggest for the first time that in nude mice bearing OVCAR-3 peritoneal tumors, by inhibiting VEGF production, albendazole abolishes tumor angiogenesis and ascites formation.     

Expression of Vascular Endothelial Growth Factor and E-Cadherin in Human Ovarian Cancer: Association with Ascites Fluid Accumulation and Peritoneal Dissemination in Mouse Ascites Model

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E-Cadherin is an adhesion molecule that is important for cell-to-cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E-cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC-2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT-PCR methods. AMOC-2 expressed E-cadherin, but not VEGF. HNOA expressed VEGF without E-cadherin expression. HTBOA expressed both VEGF and E-cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC-2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E-cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.     

Enhanced susceptibility of irradiated tumor vessels to vascular endothelial growth factor receptor tyrosine kinase inhibition

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.     

Vascular Endothelial Growth Factor/Vascular Permeability Factor in the Pathogenesis of Primary Effusion Lymphomas

Primary effusion lymphomas (PEL), rare lymphomas associated with Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) infection, present as malignant lymphomatous effusions in body cavities. We have recently found that PEL effusions contain high levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). VEGF/VPF, an important regulator of tumor-angiogenesis in vivo, exerts its effects acting through the receptors KDR/Flk-1 and Flt-1 on the endothelial cell membrane. In vitro, the PEL cell lines BC-1, BCP-1 and BCBL-1 produce high levels of VEGF. RT-PCR analysis of RNA from the PEL cell lines amplified the three VEGF/VPF secreted isoforms, VEGF/VPF₁₂₁, VEGF/VPF₁₄₅ and VEGF/VPF₁₆₅. Two of the PEL cell lines express the VEGF/VPF receptor Flt-1, but VEGF did not stimulate proliferation in these cells. SCID/beige mice inoculated intraperitoneally with BCBL-1 cells developed effusion lymphoma of human cell origin with prominent bloody ascites. In contrast, none of the mice treated with a neutralizing anti-human VEGF/VPF antibody developed ascites and effusion lymphoma. Although the precise mechanisms by which VEGF/VPF can promote vascular permeability are not fully understood, VEGF/VPF stimulation of vascular leakage may be critical to the pathogenesis of PEL.     

Depleted dopamine in gastric cancer tissues: dopamine treatment retards growth of gastric cancer by inhibiting angiogenesis.

PURPOSE: It has been recently shown that the catecholamine neurotransmitter dopamine (DA) strongly and selectively inhibits vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-induced angiogenesis. Gastric cancer is highly angiogenic and is dependent on VEGF for its growth and progression. Because substantial amounts of DA present in normal stomach tissues has been implicated in several gastric functions, we therefore investigated the role, if any, of this neurotransmitter in the growth and progression of gastric cancer. EXPERIMENTAL DESIGN: Initially, the status of DA and tyrosine hydroxylase, the rate-limiting enzyme required for DA synthesis, were determined in human gastric cancer tissues and in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer tissues of rats. On the basis of our observation of inverse correlation between stomach DA and gastric cancer growth, we determined the effect of pharmacological dose of DA on the angiogenesis and growth of MNNG induced gastric cancer in rats and Hs746T human gastric cancer in nude mice. RESULTS: DA and tyrosine hydroxylase were absent in both human and rat gastric cancer tissues. On the contrary, a low nontoxic pharmacological dose of DA significantly retarded tumor angiogenesis by inhibiting VEGFR-2 phosphorylation in tumor endothelial cells, which expressed DA D(2) receptors. This action of DA was associated with the growth inhibition of both MNNG-induced rat malignant gastric tumors and xenotransplanted human gastric cancer in nude mice. CONCLUSIONS: Our study demonstrates that there is an inverse correlation between endogenous stomach DA and gastric cancer and indicates that DA already in clinical use for other purposes might have a role as an antiangiogenic agent in the treatment of gastric cancer.     

Profound but dysfunctional lymphangiogenesis via vascular endothelial growth factor ligands from CD11b+ macrophages in advanced ovarian cancer

Severe ascites is a hallmark of advanced ovarian cancer (OVCA), yet the underlying mechanism that creates an imbalance between peritoneal vascular leakage and lymphatic drainage is unknown. Here, we identified and characterized peritoneal lymphatic vessels in OVCA mice, a model generated by implantation of human OVCA cells into athymic nude mice. The OVCA mice displayed substantial lymphangiogenesis and lymphatic remodeling, massive infiltration of CD11b(+)/LYVE-1(+) macrophages and disseminated carcinomatosis in the mesentery and diaphragm, and progressive chylous ascites formation. Functional assays indicated that the abnormally abundant lymphatic vessels in the diaphragm were not conductive in peritoneal fluid drainage. Moreover, lipid absorbed from the gut leaked out from the aberrant mesenteric lymphatic vessels. Our results indicate that vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF-A from CD11b(+) macrophages are responsible for producing OVCA-induced dysfunctional lymphangiogenesis, although other cell types contribute to the increased ascites formation. Accordingly, the combined blockade of VEGF-C/D and VEGF-A signaling with soluble VEGF receptor-3 and VEGF-Trap, respectively, markedly inhibited chylous ascites formation. These findings provide additional therapeutic targets to ameliorate chylous ascites formation in patients with advanced OVCA.     

Efficient inhibition of intra-peritoneal tumor growth and dissemination of human ovarian carcinoma cells in nude mice by anti-L1-cell adhesion molecule monoclonal antibody treatment

The L1 cell adhesion molecule is implicated in the control of proliferation, migration, and invasion of several tumor cell types in vitro. Recently, L1 overexpression was found to correlate with tumor progression of ovarian carcinoma, one of the most common causes of cancer-related deaths in gynecologic malignant diseases. To evaluate L1 as a potential target for ovarian cancer therapy, we investigated the effects of anti-L1 monoclonal antibodies (chCE7 and L1-11A) on proliferation and migration of L1-positive human SKOV3ip ovarian carcinoma cells in vitro and the therapeutic efficacy of L1-11A against i.p. SKOV3ip tumor growth in nude mice. In vitro, both anti-L1 antibodies efficiently inhibited the proliferation of SKOV3ip cells as well as other L1-expressing tumor cell lines (renal carcinoma, neuroblastoma, and colon carcinoma). On two cell lines, hyper-cross-linking of L1-11A with a secondary antibody was necessary for significant inhibition of proliferation, indicating that cross-linking of L1 is required for the antiproliferative effect. L1-negative prostate carcinoma cells were not influenced by antibody treatment. Biweekly treatment of ovarian carcinoma-bearing mice with L1-11A led to a dose-dependent and significant reduction of tumor burden (up to -63.5%) and ascites formation (up to -75%). This effect was associated with reduced proliferation within the tumors. L1-directed antibody-based inhibition of peritoneal growth and dissemination of human ovarian carcinoma cells represents important proof-of-principle for the development of a new therapy against one of the leading gynecologic malignant diseases.     

The vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584 inhibits growth and migration of multiple myeloma cells in the bone marrow microenvironment

Our prior studies show that multiple myeloma (MM) cell lines and patient cells express high-affinity vascular endothelial growth factor (VEGF) receptor (VEGFR) Flt-1 but not Flk-1/KDR. Moreover, these studies have shown that VEGF induces proliferation and migration of MM cells, and we have begun to delineate the signaling cascades mediating those sequelae. In this study, we examined the activity of PTK787/ZK 222584 (PTK787), a molecule designed to bind specifically to the tyrosine kinase domain of VEGFR and inhibit angiogenesis. We show that PTK787 acts both directly on MM cells and in the bone marrow microenvironment. Specifically, PTK787 (1-5 micro M) inhibits proliferation of MM cells by 50%, as assayed by [(3)H]thymidine uptake. This effect of PTK787 is dose dependent in both MM cell lines and patient cells that are both sensitive and resistant to conventional therapy. PTK787 enhances the inhibitory effect of dexamethasone on growth of MM cells and can overcome the protective effect of interleukin 6 (IL-6) against dexamethasone-induced apoptosis. PTK787 (1 micro M) also blocks VEGF-induced migration of MM cells across an extracellular matrix. Importantly, PTK787 also inhibits the increased MM cell proliferation and increased IL-6 and VEGF secretion in cultures of MM cells adherent to bone marrow stem cells. These findings therefore demonstrate that PTK787 both acts directly on MM cells and inhibits paracrine IL-6-mediated MM cell growth in the bone marrow milieu. The demonstrated anti-MM activity of PTK787, coupled with its antiangiogenic effects, provides the framework for clinical trials of this agent to overcome drug resistance and improve outcome in MM.     

Effect of VEGF receptor inhibitor PTK787/ZK222584 [correction of ZK222548] combined with ionizing radiation on endothelial cells and tumour growth.

The vascular endothelial growth factor (VEGF) receptor is a major target for anti-angiogenesis-based cancer treatment. Here we report the treatment effect of ionizing radiation in combination with the novel orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on endothelial cell proliferation in vitro and with tumour xenografts in vivo. Combined treatment of human umbilical vein endothelial cells with increasing doses of PTK787/ZK222584 and ionizing radiation abrogated VEGF-dependent proliferation in a dose-dependent way, but inhibition of endothelial cell proliferation was not due to apoptosis induction. In vivo, a combined treatment regimen of PTK787/ZK222584 (4 x 100 mg/kg) during 4 consecutive days in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumour growth delay for radiation-resistant p53-dysfunctional tumour xenografts derived from SW480 colon adenocarcinoma cells while each treatment modality alone had only a minimal effect on tumour size and neovascularization. SW480 tumours from animals that received a combined treatment regimen, displayed not only an extended tumour growth delay but also a significant decrease in the number of microvessels in the tumour xenograft. These results support the model of a cooperative anti-tumoral effect of angiogenesis inhibitor and irradiation and show that the orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 is suitable for combination therapy with irradiation.     

The antitumor and antiangiogenic activity of vascular endothelial growth factor receptor inhibition is potentiated by ErbB1 blockade.

PURPOSE: Receptor tyrosine kinases of the ErbB family play important roles in the control of tumor growth. Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation, enhances vascular permeability, and plays an important role in tumor vascularization. We evaluated the effects of selective VEGF receptor (VEGFR; PTK787/ZK222584) and ErbB (PKI166 and ZD1839) inhibitors on tumor growth and angiogenesis and asked whether additional therapeutic benefit was conferred by combination treatment. EXPERIMENTAL DESIGN: The antitumor activity of each inhibitor alone or in combination was assessed in human cancer models in immunocompromised mice. ErbB receptor expression and activation of downstream signaling pathway was evaluated in both tumor and endothelial cells. RESULTS: Both ErbB inhibitors significantly enhanced the antitumor activity of PTK787/ZK222584. In vitro, ErbB1 inhibition blocked VEGF release by tumor cells and proliferation of both tumor and endothelial cells. In an in vitro angiogenesis assay, epidermal growth factor (EGF) stimulated the release of VEGF by smooth muscle cells resulting in increased angiogenesis, a response blocked by administration of PTK787/ZK222584. Under basal condition, both ZD1839 and PTK787/ZK222584 blocked sprouting, likely via inhibition of an autocrine ErbB1 loop and VEGFR signaling, respectively, in endothelial cells. In conditions of limiting VEGF, EGF plays an important role in endothelial cell proliferation, survival, and sprouting. CONCLUSION: We have shown that activation of ErbB1 triggers a plethora of effects, including direct effects on tumor and endothelial cells and indirect effects mediated via induction of VEGF release. Simultaneous blockade of ErbB1 and VEGFR pathways results in a cooperative antitumor effect, indicating that this combination may represent a valid therapeutic strategy.     

Impact of adjuvant inhibition of vascular endothelial growth factor receptor tyrosine kinases on tumor growth delay and local tumor control after fractionated irradiation in human squamous cell carcinomas in nude mice

PURPOSE: Previous experiments have shown that adjuvant inhibition of the vascular endothelial growth factor receptor after fractionated irradiation prolonged tumor growth delay and may also improve local tumor control. To test the latter hypothesis, local tumor control experiments were performed. METHODS AND MATERIALS: Human FaDu and UT-SCC-14 squamous cell carcinomas were studied in nude mice. The vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584 (50 mg/kg body weight b.i.d.) was administered for 75 days after irradiation with 30 fractions within 6 weeks. Tumor growth time and tumor control dose 50% (TCD(50)) were determined and compared to controls (carrier without PTK787/ZK222584). RESULTS: Adjuvant administration of PTK787/ZK222584 significantly prolonged tumor growth time to reach 5 times the volume at start of drug treatment by an average of 11 days (95% confidence interval 0.06;22) in FaDu tumors and 29 days (0.6;58) in UT-SCC-14 tumors. In both tumor models, TCD(50) values were not statistically significantly different between the groups treated with PTK787/ZK222584 compared to controls. CONCLUSIONS: Long-term inhibition of angiogenesis after radiotherapy significantly reduced the growth rate of local recurrences but did not improve local tumor control. This indicates that recurrences after irradiation depend on vascular endothelial growth factor-driven angiogenesis, but surviving tumor cells retain their clonogenic potential during adjuvant antiangiogenic treatment with PTK787/ZK222584.     

Contributions of stromal metalloproteinase-9 to angiogenesis and growth of human ovarian carcinoma in mice

BACKGROUND: The expression level of several matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, in ovarian cancer cells is directly associated with their invasive and metastatic potentials. MMP-9 is also expressed in stromal cells adjacent to the tumor. To investigate the contribution of MMP-9 expression in stromal cells to ovarian tumor growth, we examined angiogenesis and progressive growth of human ovarian cancer cells implanted into mice with and without the MMP-9 gene. METHODS: Human ovarian cancer cells SKOV3.ip1 and HEY-A8 were implanted into the peritoneal cavities of nude mice that lacked the gene for MMP-9 (MMP-9(-/-)) or were wild type for MMP-9 (MMP-9(+/+)) (10 mice of each genotype per cell line). Tumor incidence, tumor size, and volume of ascites fluid were recorded for each mouse at 30 and 45 days after HEY-A8 and SKOV3.ip1 cell injections, respectively. Blood vessel density and macrophage infiltration into the lesions were analyzed in excised tumors by immunohistochemistry and double immunofluorescence. Tumor growth was also studied in MMP-9(-/-) nude mice that had been reconstituted with spleen cells collected from either MMP-9(+/+) or MMP-9(-/-) nude mice. All statistical tests were two-sided. RESULTS: HEY-A8 cells expressed high levels of MMP-9, and SKOV3.ip1 cells expressed low levels. Nevertheless, tumor incidence and growth were statistically significantly lower in MMP-9(-/-) mice than in MMP-9(+/+) mice injected with cells from either line (for tumor size, P =.006 and.042 for HEY-A8 and SKOV3.ip1 cells, respectively). Compared with MMP-9(+/+) mice injected with human ovarian cancer cells, MMP-9(-/-) mice injected with human ovarian cancer cells displayed decreased microvessel density and decreased macrophage infiltration into the lesions. Compared with MMP-9(-/-) mice that received spleen cells (a rich source of macrophages) from MMP-9(-/-) mice, those that received spleen cells from MMP-9(+/+) mice before cancer cell injections displayed increased angiogenesis and tumorigenicity of the cancer cells. The growing tumors contained MMP-9-expressing macrophages. CONCLUSION: Host-derived MMP-9 expression, most likely in tumor-infiltrating macrophages, appears to play a critical role in angiogenesis and progressive growth of human ovarian tumors in mice.