Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications.

BACKGROUND: We investigated the expression of CXCR4, CCR7, estrogen receptor (ER), progesterone receptor (PR) and HER2-neu in human metastatic breast cancers to determine whether these biological biomarkers were preferentially expressed in any organ-specific metastases. MATERIALS AND METHODS: CXCR4, CCR7, ER, PR and HER2-neu expression levels were evaluated by immunohistochemical staining using paraffin-embedded tissue sections of metastatic breast cancers (n = 41) obtained by either diagnostic biopsy or surgical resection. RESULTS: The metastatic sites included the following: bone (n = 15), brain (n = 14), lung (n = 6), liver (n = 2), and omental metastases (n = 2). CXCR4 was expressed in 41% of cases, CCR7 expression was demonstrated in 10%, and HER2-neu overexpression was present in 27%. CXCR4 was more likely to be expressed in bone metastases than visceral metastases (67% versus 26%, P = 0.020). Visceral sites demonstrated a lower rate of CXCR4 positivity (33% and 23%, respectively, for lung and brain metastases). Similarly, CCR7 was more likely to be found in bone metastases than visceral sites (27% versus 0%, P = 0.037). CONCLUSIONS: These results indicate that CXCR4 can contribute to the homing of breast cancer cells to the bone. This finding might have important clinical implications since patients with metastatic bone disease may achieve the highest benefit from a CXCR4-targeted therapy.

Epidermal hyperplasia overlying human melanoma correlates with tumour depth and angiogenesis.

The aim of this study was to determine whether epidermal hyperplasia overlying cutaneous human melanoma is associated with increased tumour angiogenesis, tumour growth and the potential for metastasis. Forty-two surgical specimens of cutaneous human melanoma of different depths, each containing epidermis present in the tumour-free margin, were analysed by immunohistochemistry for the expression of the pro-angiogenic molecules basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) and the anti-angiogenic molecule interferon-beta (IFN-beta). The epidermis overlying intermediate and thick (1.0-10.0 mm), but not thin (0.5-1.0 mm), melanoma specimens was hyperplastic. Although the expression level of bFGF, VEGF and IL-8 in the epidermis directly overlying the tumour was similar to that in the distant epidermis, the expression of IFN-beta was significantly decreased in keratinocytes overlying intermediate and thick, but not thin, melanomas. The microvessel density was also increased in intermediate and thick specimens. Human melanoma cells were injected subcutaneously into nude mice. The resulting tumours were used to determine the association between overlying epidermal hyperplasia and neoplastic angiogenesis. Similar to human autochthonous melanomas, epidermal hyperplasia was found only over lesions produced by metastatic cells. Although there was no change in the expression of the pro-angiogenic molecules, the expression of IFN-beta was significantly decreased in the hyperplastic epidermis. Conditioned medium collected from cultures of the metastatic cell line induced in vitro proliferation of mouse keratinocytes, whereas conditioned medium collected from cultures of the non-metastatic cell line did not. Collectively, the data demonstrate that metastatic melanoma cells induce keratinocyte proliferation, leading to decreased expression of the negative regulator of angiogenesis, IFN-beta, and hence to increased angiogenesis.

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells.

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.

Angiopoietin-1 inhibits tumour growth and ascites formation in a murine model of peritoneal carcinomatosis.

Angiopoietin-1 is an important regulator of endothelial cell survival. Angiopoietin-1 also reduces vascular permeability mediated by vascular endothelial growth factor. The effects of angiopoietin-1 on tumour growth and angiogenesis are controversial. We hypothesised that angiopoietin-1 would decrease tumour growth and ascites formation in peritoneal carcinomatosis. Human colon cancer cells (KM12L4) were transfected with vector (pcDNA) alone (control) or vector containing angiopoietin-1 and injected into the peritoneal cavities of mice. After 30 days, the following parameters were measured: number of peritoneal nodules, ascites volume, and diameter of the largest tumour. Effects of angiopoietin-1 on vascular permeability were investigated using an intradermal Miles assay with conditioned media from transfected cells. Seven of the nine mice in the pcDNA group developed ascites (1.3+/-0.5 ml (mean+/-s.e.m.)), whereas no ascites was detectable in the angiopoietin-1 group (0 out of 10) (P<0.01). Number of peritoneal metastases (P<0.05), tumour volume, (P<0.05), vessel counts (P<0.01), and tumour cell proliferation (P<0.01) were significantly reduced in angiopoietin-1-expressing tumours. Conditioned medium from angiopoietin-1-transfected cells decreased vascular permeability more than did conditioned medium from control cells (P<0.05). Our results suggest that angiopoietin-1 is an important mediator of angiogenesis and vascular permeability and thus could theoretically serve as an anti-neoplastic agent for patients with carcinomatosis from colorectal cancer.

Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model.

We hypothesised that the combination of anti-angiogenic and anti-epidermal growth factor (EFG)-receptor (R) therapies would more effectively inhibit gastric cancer growth than single-agent therapy. TMK-1 gastric cancer cells were injected into the gastric wall of nude mice to generate tumours. After 4 days, mice were randomly assigned to the following groups: control, DC101 ([vascular endothelial growth factor (VEGF)-receptor (R)-2 antibody], C225 (EGF-R antibody), or a combination of DC101 and C225. The combination therapy significantly inhibited gastric tumour growth compared with the control group, whereas the decrease in tumour growth in mice treated with DC101 or C225 alone did not reach statistical significance. All mice administered DC101 demonstrated decreased tumour vascularity and increased endothelial cell apoptosis. C225 alone did not affect angiogenesis, but inhibited tumour cell proliferation. The combination therapy led to a further decrease in tumour cell proliferation. The combination of anti-VEGF-R and anti-EGF-R therapies was effective in inhibiting gastric cancer growth. These findings support the hypothesis that inhibiting multiple biological pathways that mediate tumour growth may be an effective therapeutic strategy.

In vivo intracellular signaling as a marker of antiangiogenic activity.

Alterations in endothelial cell (EC) signaling could serve as a marker of effective antiangiogenic therapy. We determined the effect of an antiangiogenic tyrosine kinase inhibitor, SU6668, on tumor EC signaling in liver metastases in mice. In vitro immunofluorescence verified that pretreatment of ECs with SU6668 before exposure to VEGF decreased in vitro phosphorylation of Erk and Akt. Using double-fluorescence immunohistochemistry, phosphorylated Erk and Akt were constitutively expressed in ECs in liver metastases in untreated mice, but SU6668 blocked activation of these signaling intermediates. Determining the activation status of the Erk and Akt signaling pathways in tumor ECs may serve as a surrogate marker for the effectiveness of antiangiogenic regimens.

Differential expression of angiopoietin-1 and angiopoietin-2 in colon carcinoma. A possible mechanism for the initiation of angiogenesis.

BACKGROUND: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are important regulators of endothelial cell (EC) survival. Current models suggest that an increase in Ang-2 expression in ECs leads to the initiation of angiogenesis. The authors hypothesized that the imbalance of Ang-1 and Ang-2 activities in colon carcinoma leads to a net gain in Ang-2 function. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses and immunofluorescent double-staining were performed to examine human colon carcinoma cell lines, surgical specimens, normal mucosa, and liver metastases for the expression of Ang-1 and Ang-2. RESULTS: RT-PCR analyses revealed that 7 of 18 colon carcinoma cell lines expressed Ang-1, and 14 of 18 colon carcinoma cell lines expressed Ang-2 (P < 0.05). Of the surgical specimens from patients with colon carcinoma, 6 of 11 specimens expressed Ang-1, and 11 of 11 specimens expressed Ang-2 (P < 0.05). However, Ang-1 and Ang-2 were expressed with relative equal frequency in normal mucosa (P = 0.62). Immunofluorescent staining (n = 20 specimens) revealed the presence of Ang-2 protein in normal mucosa and tumor epithelium, but Ang-1 was expressed only in normal mucosa. A similar pattern was found for hepatic colorectal metastases. Double staining for Ang-1 or Ang-2 and cytokeratin-22 (an epithelial marker) demonstrated that Ang-1 was produced by uninvolved, normal colonic epithelium, whereas Ang-2 was produced by normal and malignant colonic epithelium. CONCLUSIONS: In patients with colon carcinoma, Ang-2 is expressed ubiquitously in tumor epithelium, whereas expression of Ang-1 in tumor epithelium is rare. The net gain of Ang-2 activity is possibly an initiating factor for tumor angiogenesis.

Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors.

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.

An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration.

Abnormal angiogenesis accompanies many pathological conditions including cancer, inflammation, and eye diseases. Proliferative retinopathy because of retinal neovascularization is a leading cause of blindness in developed countries. Another major cause of irreversible vision loss is retinitis pigmentosa, a group of diseases characterized by progressive photoreceptor cell degeneration. Interestingly, anecdotal evidence has long suggested that proliferative diabetic retinopathy is rarely associated clinically with retinitis pigmentosa. Here we show that neonatal mice with classic inherited retinal degeneration (Pdeb(rd1)/Pdeb(rd1)) fail to mount reactive retinal neovascularization in a mouse model of oxygen-induced proliferative retinopathy. We also present a comparable human paradigm: spontaneous regression of retinal neovascularization associated with long-standing diabetes mellitus occurs when retinitis pigmentosa becomes clinically evident. Both mouse and human data indicate that reactive retinal neovascularization either fails to develop or regresses when the number of photoreceptor cells is markedly reduced. Our findings support the hypothesis that a functional mechanism underlying this anti-angiogenic state is failure of the predicted up-regulation of vascular endothelial growth factor, although other growth factors may also be involved. Preventive and therapeutic strategies against both proliferative and degenerative retinopathies may emerge from this work.

Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis.

Since the NF-kappaB/relA transcription factor is constitutively activated in human prostate cancer cells, we determined whether blocking NF-kappaB/relA activity in human prostate cancer cells affected their angiogenesis, growth, and metastasis in an orthotopic nude mouse model. Highly metastatic PC-3M human prostate cancer cells were transfected with a mutated IkappaBalpha (IkappaBalphaM), which blocks NF-kappaB activity. Parental (PC-3M), control vector-transfected (PC-3M-Neo), and IkappaBalphaM-transfected (PC-3M-IkappaBalphaM) cells were injected into the prostate gland of nude mice. PC-3M and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastasis, whereas PC-3M-IkappaBalphaM cells produced slow growing tumors with low metastatic potential. NF-kappaB signaling blockade significantly inhibited in vitro and in vivo expression of three major proangiogenic molecules, VEGF, IL-8, and MMP-9, and hence decreased neoplastic angiogenesis. Inhibition of NF-kappaB activity in PC-3M cells also resulted in the downregulation of MMP-9 mRNA and collagenase activity, resulting in decreased invasion through Matrigel. Collectively, these data suggest that blockade of NF-kappaB activity in PC-3M cells inhibits angiogenesis, invasion, and metastasis.

Suppression of angiogenesis and therapy of human colon cancer liver metastasis by systemic administration of interferon-alpha.

The purpose of this study was to determine whether systemic administration of interferon-alpha (IFN-alpha) can inhibit liver metastasis produced in nude mice by human colon cancer cells. KM12L4 (IFN-alpha-sensitive) or KM12L4 IFN(R) (IFN-alpha-resistant) cells were injected into the spleen of nude mice. Seven days later, the mice were treated with subcutaneous (s.c.) injections of IFN-alpha (70,000 units/week) at different dosing schedules (1, 2, or 7 times/week). Significant inhibition of tumor growth, vascularization and expression of basic fibroblast growth factor (bFGF) or matrix metalloproteinase-9 (MMP-9) mRNA and protein occurred in mice given daily injections of IFN-alpha. Kinetic analysis of therapy showed that daily s.c. administrations of 10,000 units of IFN-alpha induced apoptosis in liver metastasis-associated endothelial cells, followed by inhibition of tumor cell division and apoptosis of tumor cells. These data suggest that the antiangiogenic activity of IFN-alpha-2a depends on frequent administration of the optimal biologic dose.

Expression of interleukin-8 in human metastatic endometrial carcinoma cells and its regulation by inflammatory cytokines.

In the present study, we analyzed the expression of a multifunctional cytokine, interleukin-8 (IL-8), in metastatic endometrial carcinoma cells. Our data demonstrate that human serous papillary endometrial adenocarcinoma (SPEC) and human endometrial adenocarcinoma (HEC) cells expressed steady-state IL-8-specific mRNA transcript and secreted IL-8 protein. The levels of IL-8 mRNA in SPEC-2 cells established from stage IV serous papillary adenocarcinoma were three-fold higher as compared to endometrial adenocarcinoma cells, HEC-1 A, established from stage IA endometrial cancer. Further, we observed higher levels of IL-8 mRNA and protein expression in the metastatic variants of SPEC-2 and HEC-1A cells as compared to the parent cell lines, demonstrating that IL-8 expression was associated with metastatic potential. Further, the treatment of endometrial carcinoma cells with inflammatory cytokines, IL-1beta and tumor necrosis factor-alpha (TNF-alpha), demonstrated that IL-1beta and TNF-alpha induced IL-8 expression in endometrial cancer cells. IL-1beta was a more potent inducer of IL-8 expression than TNF-alpha in our studies. These data demonstrate that constitutive and induced IL-8 expression in endometrial carcinoma cells might be an important regulatory mechanism of tumor growth and metastasis.

The effects of angiopoietin-1 and -2 on tumor growth and angiogenesis in human colon cancer.

Angiopoietin (Ang) 1 and Ang-2 are important regulators of endothelial cell survival. Current models suggest that an increase in Ang-2 expression in endothelial cells leads to initiation of angiogenesis. We stably transfected HT29 colon cancer cells with cDNA constructs for Ang-1 or -2 or with vector alone, injected the cells s.c. into nude mice, and assessed tumor growth. Immunohistochemical analyses confirmed sustained increases of Ang-1 and -2 in the tumors. The tumors produced by the Ang-2-transfected cells were larger than the tumors produced in the other groups; those tumors also had higher vessel counts and proliferative indices than tumors in the other groups. Tumors produced by the Ang-1 transfectants had fewer vessels and lower tumor cell proliferative indices than tumors in the other groups. These data suggest that imbalances between Ang-1 and -2 that result in a net gain of Ang-2 activity lead to enhanced tumor angiogenesis and growth.

Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms.

Redundant mechanisms mediate colon cancer angiogenesis. Targeting multiple angiogenic factors simultaneously may improve survival of mice with colon cancer metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate liver metastases and received administration of either vehicle alone or a tyrosine kinase inhibitor for vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). Mice were sacrificed when they became moribund as determined by a blinded observer. In a parallel experiment, groups of mice were sacrificed at earlier time points to better define the kinetics of the effect of SU6668 on angiogenic parameters over time. SU6668 increased median survival by 58% (P < 0.001) and led to a progressive increase in tumor cell and endothelial cell apoptosis that increased over time. In addition, pericyte vessel coverage and tumor vascularity were significantly decreased in mice treated with SU6668. Based on current knowledge of endothelial cell survival, these data suggest that SU6668 may prevent tumor endothelial cell survival directly (vascular endothelial growth factor) and indirectly (pericyte coverage) by affecting endothelial cell survival mechanisms.

EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells.

Catechins are key components of teas that have antiproliferative properties. We investigated the effects of green tea catechins on intracellular signalling and VEGF induction in vitro in serum-deprived HT29 human colon cancer cells and in vivo on the growth of HT29 cells in nude mice. In the in vitro studies, (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea extract, inhibited Erk-1 and Erk-2 activation in a dose-dependent manner. However, other tea catechins such as (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) did not affect Erk-1 or 2 activation at a concentration of 30 microM. EGCG also inhibited the increase of VEGF expression and promoter activity induced by serum starvation. In the in vivo studies, athymic BALB/c nude mice were inoculated subcutaneously with HT29 cells and treated with daily intraperitoneal injections of EC (negative control) or EGCG at 1.5 mg day(-1)mouse(-1)starting 2 days after tumour cell inoculation. Treatment with EGCG inhibited tumour growth (58%), microvessel density (30%), and tumour cell proliferation (27%) and increased tumour cell apoptosis (1.9-fold) and endothelial cell apoptosis (3-fold) relative to the control condition (P< 0.05 for all comparisons). EGCG may exert at least part of its anticancer effect by inhibiting angiogenesis through blocking the induction of VEGF.

Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis.

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.

Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells.

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

Induction of angiogenesis by hyperplastic colonic mucosa adjacent to colon cancer.

We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-alpha than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-beta inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-beta than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis.

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.