OBJECTIVE

Vascular endothelial growth factor (VEGF) expression is prognostic in melanoma, and the activity of VEGF is mediated in part through the receptor tyrosine kinase Flk-1. A Phase II study of SU5416, a preferential inhibitor of Flk-1, was carried out in patients with metastatic melanoma to determine clinical response, tolerability, and changes in tumor vascular perfusion.

METHODS

Patients with documented progressive disease and </=1 prior therapy were eligible. Central nervous system metastases were allowed if stable off medication. SU5416 (145 mg/m(2)) was administered via a central catheter twice weekly for 8 weeks. Premedication with dexamethasone, diphenhydramine, and a H(2) blocker was required because of the Cremophor vehicle. Tumor <em>vascular</em> perfusion was assessed before treatment and during week 8 by dynamic contrast magnetic resonance imaging, and plasma was analyzed for VEGF.

RESULTS

Thirty-one patients were enrolled. Two-thirds had received prior therapy, 21 had visceral metastasis, and 14 had an elevated lactate dehydrogenase. Mean absolute lymphocyte counts were decreased (P = 0.002), and glucose levels were increased (P = 0.001) posttherapy, presumably because of steroid premedication. Four vascular adverse events were observed. Of 26 evaluable patients, 1 experienced a partial response, 1 had stable disease, and 5 had a mixed response. Dynamic contrast magnetic resonance imaging in 5 evaluable patients showed decreased tumor perfusion at week 8 (P = 0.024), and plasma VEGF levels were elevated compared with pretherapy (P = 0.008).

CONCLUSIONS

SU5146 appears to be relatively well tolerated in this population. Although the modest clinical activity and potential effects on tumor vascularity may support additional exploration of VEGF as a target in melanoma, effects from steroid premedication limit further investigation of this agent.

BACKGROUND

Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are of adverse prognostic significance in patients with myeloproliferative disorders (MPD), including agnogenic myeloid metaplasia (AMM), chronic myeloid leukemia in blastic phase (CML-BP), and chronic myelomonocytic leukemia (CMML). VEGF is a soluble, circulating, angiogenic molecule that acts through receptor tyrosine kinases (RTK), including VEGF receptor 2 (VEGFR-2). SU5416 is a small-molecule RTK inhibitor (RTKI) that targets VEGFR-2, c-kit, and fms-related tyrosine kinase Flk2.

METHODS

Adult patients with advanced CMML, AMM, CML-BP, or other BCR-ABL negative MPD were entered on a multicenter, Phase II study.

RESULTS

Thirty-two patients (19 patients with BCR-ABL negative MPD, 6 patients with CMML, 4 patients with CML-BP, and 3 patients with AMM) with a median age of 66 years (range, 29-85 years) received SU5416 145 mg/m(2) twice weekly intravenously for a median of three 4-week cycles (maximum, 12 cycles). Drug-related Grade 3-4 toxicities included acute abdominal pain (13%), bone pain (9%), infusion-related dyspnea (9%) or headache (6%), fatigue (6%), diarrhea (3%), and catheter site reactions (3%). Eleven patients (34%) did not receive a second cycle of therapy (6 patients had progressive disease, 3 because of adverse events; 2 patients withdrew due to lack of response). One patient with AMM achieved a partial response. Eight patients received more than 6 months of therapy.

CONCLUSIONS

SU5416 had minimal clinical activity in patients with MPD. Long-term administration of a twice-weekly, hyperosmolar, intravenous solution containing polyoxyl 35 castor oil was difficult. More tolerable RTKI may be worthy of further investigation in patients with MPD.

Elevated levels of cyclin A1 expression have been implicated in acute myeloid leukemia and in male germ cell tumors. However, a role of cyclin A1 in tumorigenesis of prostate cancer has not been reported. In the present study, expression of cyclin A1 in patients with prostate cancer and a role of cyclin A1 in mediating expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) were investigated. Cyclin A1 was highly expressed in aggressive tumors and was significantly correlated with VEGF expression in 96 patients with prostate cancer. Treatment of LNCaP cells with R1881, a synthetic androgen resulted in increased cyclin A1 expression. Induction of cyclin A1 expression in LNCaP cells led to an increase in VEGF expression and this effect was manifested upon the R1881 treatment. Cyclin A1 failed to mediate VEGF activation in DU-<em>145</em> cells lacking a functional Rb and an androgen receptor (AR). Although AR expression was induced into DU-<em>145</em> cells, cyclin A1 was unable to mediate VEGF expression. However, induced coexpression of cyclin A1, Rb and AR in DU-<em>145</em> cells in the presence of R1881 greatly promoted VEGF promoter activity. This suggests that cyclin A1 mediates VEGF expression in cooperation with Rb- and androgen-dependent pathways in prostate cancer.

Respiratory distress syndrome (RDS) secondary to preterm birth and surfactant deficiency is characterized by severe hypoxemia, lung injury, and impaired production of nitric oxide (NO) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). Since hypoxia-inducible <em>factors</em> (HIFs) mediate the effects of both NO and VEGF in part through regulation by prolyl-hydroxylase-containing domains (PHDs) in the presence of oxygen, we hypothesized that HIF-1alpha and -2alpha in the lung are decreased following severe RDS in preterm neonatal lambs. To test this hypothesis, fetal lambs were delivered at preterm gestation (115-day gestation, term = <em>145</em> days; n = 4) and mechanically ventilated for 4 h. Lambs developed respiratory failure characterized by severe hypoxemia despite treatment with mechanical ventilation with high inspired oxygen concentrations. Lung samples were compared with nonventilated control animals at preterm (115-day gestation; n = 3) and term gestation (142-day gestation; n = 3). We found that HIF-1alpha protein expression decreased (P < 0.05) and PHD-2 expression increased (P < 0.005) at birth in normal term animals before air breathing. Compared with age-matched controls, HIF-1alpha protein and HIF-2alpha protein expression decreased by 80% and 55%, respectively (P < 0.005 for each) in preterm lambs with RDS. Furthermore, VEGF mRNA was decreased by 40%, and PHD-2 protein expression doubled in RDS lambs. We conclude that pulmonary expression of HIF-1alpha, HIF-2alpha, and the downstream target of their regulation, VEGF mRNA, is impaired following RDS in neonatal lambs. We speculate that early disruption of HIF and VEGF expression after preterm birth and RDS may contribute to long-term abnormalities in lung <em>growth</em>, leading to bronchopulmonary dysplasia.

Microdialysate fluid from <em>145</em> severely injured NSICU-patients, 88 with subarachnoidal haemorrage (SAH), and 57 with traumatic brain injury (TBI), was collected by microdialysis during the first 7 days following impact, and levels of the neurotrophins fibroblast <em>growth</em> <em>factor</em>-2 (FGF2) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) were analysed. The study illustrates both similarities and differences in the reaction patterns of the 2 inflammatory proteins. The highest concentrations of both FGF2 and VEGF were measured on Day 2 (mean (+/- SE) values being 47.1 +/- 15.33 and 116.9 +/- 41.85 pg/ml, respectively, in the pooled patient material). The VEGF concentration was significantly higher in TBI-patients, while the FGF2 showed a tendency to be higher in SAH-patients. This is the first report presenting in some detail the human cerebral response of FGF2 and VEGF following SAH and TBI. Apart from increasing the understanding of the post-impact inflammatory response of the human brain, the study identifies potential threshold values for these chemokines that may serve as monitoring indicators in the NSICU.

Differentiation antagonizing non-protein coding RNA (DANCR) is a newly identified oncogenic long noncoding RNA found in various cancers. However, the functional role of DANCR in tumor angiogenesis and the underlying mechanisms are still unclear. The expression of DANCR was determined in ovarian malignant tissues and cell lines. The functional role of DANCR in tumor angiogenesis was revealed by the following methods: CD31 staining of ovarian tumor tissues, matrigel-plug assay tissues, HUVEC-related tube formation assay, and invasion assay. Enzyme-linked immunosorbent assay, Western blotting, luciferase assay, and rescue experiments were used to investigate the underlying mechanisms of DANCR-regulating angiogenesis. DANCR was upregulated in ovarian malignant tissues and ovarian cancer cells. Knockdown of DANCR efficiently impaired ovarian tumor <em>growth</em> through inhibition of tumor angiogenesis. Furthermore, the conditional culture medium from DANCR-knockdown ovarian cells significantly inhibited tube formation and invasion of HUVEC in vitro. Mechanistic investigation indicated that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A, VEGF) plays a crucial role during DANCR inhibition of tumor angiogenesis in ovarian cancer. Further results demonstrated that miR-<em>145</em> is the direct binding target of DANCR during regulation of VEGF expression and tumor angiogenesis in ovarian cancer cells. Collectively, DANCR plays a promotional role in tumor angiogenesis in ovarian cancer through regulation of miR-<em>145</em>/VEGF axis. Therefore, DANCR may be a novel therapy target for ovarian cancer.

The endothelium is involved in the pathogenesis of inflammatory bowel disease (IBD). So far knowledge of the precise role of soluble adhesion molecules and angiogenic <em>factors</em> at different periods of activity in IBD is scarce or contradictory. Our goal in this study was to determine the serum levels of adhesion molecules and angiogenic <em>factors</em> in IBD patients at different periods of disease activity--clinical remission, biochemical evidence of inflammation, and clinical evidence of activity. We used a cross-sectional study design consisting of 218 patients (<em>145</em> with Crohn's disease [CD] and 73 with ulcerative colitis [UC]) and 115 randomly assymptomatic blood donors. To assess disease activity, Harvey and Bradshaw's and Truelove-Witts' indexes were used. Circulating plasma sE-selectin (sE-S), sP-selectin (sP-S), human soluble <em>vascular</em> cell adhesion molecule-1 (sVCAM-1), and human soluble intercellular adhesion molecule-1 (sICAM-1) and serum levels of human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), angiogenin (ANG), and placenta <em>growth</em> <em>factor</em> (P/GF) were measured with ELISAs. The amount of mRNA VEGF in blood mononuclear cells was also evaluated. In inactive CD patients, serum levels of sP-S, sE-S, sVCAM, and sICAM were significantly lower (P < 0.05) than in controls. In active CD patients, only the sE-S values were higher than in controls. In UC patients, sP-S and sVCAM levels were significantly lower than those in controls. Considering <em>growth</em> <em>factors</em>, CD patients in remission had levels of ANG and VEGF lower than those found in controls. The VEGF RNAm in blood mononuclear cells was similar among all CD activity groups. In conclusion, in UC patients the serum levels of VEGF, ANG, and P/GF were similar to those in controls. The serum levels of adhesion molecules and angiogenic <em>factors</em> were low in IBD patients in periods of remission. Low levels of angiogenic <em>factors</em> in inactive CD patients suggest dysfunction of the angiogenic process and wound repair.

Treatment of metastatic renal cell carcinoma (mRCC) typically entails mechanistically distinct agents across the first- and second-line setting. Activity of these agents may be predicated on selective pressure that modulates RCC biology. Circulating tumor DNA (ctDNA) is a platform to noninvasively ascertain temporal changes in genomic profile.

To assess the ctDNA profile in a large cohort of mRCC patients, and to assess changes across patients receiving first-line and later lines of therapy.

We obtained the ctDNA profile in mRCC patients who received ctDNA profiling as part of routine clinical care at progression using a 73-gene Clinical Laboratory Improvement Amendments-certified ctDNA platform.

Genomic alterations (GAs) were pooled for the entire cohort. A comparison of first- and postfirst-line was performed with grouping based on conventional practice patterns (first-line regimens included sunitinib, pazopanib, and bevacizumab, and postfirst-line regimens included everolimus, axitinib, cabozantinib, and nivolumab).

ctDNA clinical results from a nationwide cohort of 220 consecutive patients with mRCC were assessed (<em>145</em> men, 75 women; median age: 63 yr, interquartile range: 57-70). GAs were detected in 78.6% of patients. The most frequent GAs in the overall cohort included TP53 (35%), VHL (23%), EGFR (17%), NF1 (16%), and ARID1A (12%). Thirty-eight and 64 patients were coded as receiving first-line and later line agents, respectively. The highest disparity in GA frequencies in postfirst-line versus first-line were in TP53 (49% vs 24%), VHL (29% vs 18%), NF1 (20% vs 3%), EGFR (15% vs 8%), and PIK3CA (17% vs 8%) while ARID1A was equivalent (13% vs 11%). Restricting the analysis to later lines versus first-line <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> inhibitors, these differences were even more prominent, particularly for TP53 (64% vs 31%) and NF1 (29% vs 4%).

In the largest assessment of ctDNA-detected GAs prevalence in mRCC to date, the majority of patients demonstrated clinically and biologically relevant GAs. Increasing p53 and mechanistic target of rapamycin pathway (eg, NF1, PIK3CA) alterations in postfirst-line patients with first-line vascular endothelial growth factor-directed therapy may underlie mechanisms of resistance. Routine ctDNA assessment during the clinical course of mRCC patients may have therapeutic implications.

Collection of circulating tumor DNA is feasible in patients with metastatic renal cell carcinoma, and analysis of a large cohort demonstrates significant changes in circulating tumor DNA profile across patients' clinical course which may have therapeutic implications.

We investigated native Japanese subjects whether C702T, C936T and G1612A polymorphisms in the 3' untranslated region (3'-UTR) of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) gene are associated with the risk of renal cell carcinoma (RCC). Genomic DNAs from <em>145</em> RCC patients and <em>145</em> healthy controls were examined by polymerase chain reaction-based restriction fragment length polymorphism. Variant allele frequencies of C702T, C936T and G1612A were 0.00, 0.20 and 0.13 in the controls, respectively. The C702T and G1612A allele frequencies were significantly different between the Japanese population and the Caucasian population reported elsewhere. For each of C936T and G1612A polymorphisms, there was no statistically significant difference in the distribution of genotype frequencies between the cases and controls. Odds ratios and 95% confidence intervals computed by logistic regression analyses were not statistically significant. Stratification for the RCC cases according to pathological cell subtype, grade or stage failed to reveal any significant heterogeneity with respect to the genotype of each VEGF polymorphism. We revealed that there are significant ethnic differences in the C702T and G1612A allele frequencies, but suggested that C702T, C936T and G1612A polymorphisms in the 3'-UTR of VEGF gene are not associated with the risk of RCC, at least in Japanese population.

BACKGROUND

Antagonists of growth hormone releasing hormone (GHRH) as well as antagonists of bombesin/gastrin releasing peptide (BN/GRP) inhibit the growth of various malignancies (cancers) including prostate cancer.

METHODS

We investigated the effects of GHRH antagonists MZ-J-7-118 and RC-J-29-18, BN/GRP antagonists RC-3940-II and RC-3940-Et and the combination of MZ-J-7-118 and RC-3940-II on the growth of PC-3 and DU-145 human androgen independent prostate cancers xenografted s.c. into nude mice. To elucidate the mechanisms of action of these analogs, growth factors like IGF-II (insulin-like growth factor-II), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor receptor/human epidermal growth factor receptor (EGF-R/HER) family were measured in tumors as well as IGF-I in serum.

RESULTS

Antagonists of GHRH and BN/GRP alone or in combination significantly inhibited growth of PC-3 and DU-145 tumors, the greatest inhibition of tumor volume being achieved by combination of MZ-J-7-118 (5 microg/day) and RC-3940-II (10 microg/day). BN/GRP and GHRH antagonists and their combination also decreased the expression of VEGF significantly in PC-3 and non-significantly in DU-145, as measured by radioimmunoassay for VEGF protein and RT-PCR for mRNA levels of VEGF. GHRH and BN/GRP antagonists reduced bFGF concentrations and the maximal binding capacity of EGF receptors, and their mRNA levels in PC-3 and DU-145 tumors. mRNA levels for HER-2 and -3 were also diminished in PC-3 tumors by GHRH and BN/GRP antagonists. No changes in HER-4 were found after treatment. Serum IGF-I and tumoral IGF-II levels were not affected by the analogs.

CONCLUSIONS

BN/GRP and GHRH antagonists inhibit growth of PC-3 and DU-145 prostate cancers by suppressing the expression of tumoral growth factors such as VEGF and bFGF as well as the receptors for EGF and related HER-2 and -3. Additive effects on tumor inhibition (TI) in vivo, but not on VEGF, bFGF, or members of the EGF/HER receptor family, can be achieved by the joint administration of both classes of analogs.

BACKGROUND

Tumors commonly outgrow their blood supply, thereby creating hypoxic conditions, which induce apoptosis and increase expression of angiogenic growth factors. The bcl-2 oncogene inhibits apoptosis induced by a variety of stimuli, including hypoxia. On the basis of bcl-2's role in regulating apoptosis in response to hypoxia, we hypothesized that this oncogene might affect other responses to hypoxia, such as the expression of angiogenic growth factors.

METHODS

Three prostate carcinoma cell lines, PC3, LNCaP, and DU-145, were stably transfected with a bcl-2 complementary DNA (cDNA), and transfectants were analyzed in vitro for the expression of angiogenic factors after exposure to either normoxic (19% O(2)) or hypoxic (1% O(2)) conditions. The in vivo angiogenic potential of the transfected cells was determined by analyzing vessel density in xenografts derived from them and by measuring the ability of these xenografts to induce neovascularization when implanted in mouse corneal micropockets. Statistical tests were two-sided.

RESULTS

When exposed to hypoxic conditions, prostate carcinoma cells overexpressing bcl-2 expressed statistically significantly higher levels of vascular endothelial growth factor (VEGF), an angiogenic factor, than control-transfected cells (P = .001 for PC3, P = .04 for DU-145 after 48 hours). This effect of bcl-2 was independent of its antiapoptotic activity because increased expression of VEGF was detected in PC3 cells overexpressing bcl-2 even though PC3 cells are inherently resistant to hypoxia-induced apoptosis. In vivo, xenograft tumors derived from the bcl-2-overexpressing prostate carcinoma cell lines displayed increased angiogenic potential and grew more aggressively than tumors derived from the control cell lines (P =.03 for PC3). Treatment of bcl-2-overexpressing and control tumors with the antiangiogenic drug TNP-470 neutralized the aggressive angiogenesis in bcl-2-overexpressing tumors (P = .04 for PC3, P = .004 for DU-145) and the moderate angiogenesis in control tumors (P = .01 for PC3, P = .05 for DU-145), resulting in similar growth rates for both tumors.

CONCLUSIONS

bcl-2 may play a dual role in tumorigenesis by suppressing apoptosis and by stimulating angiogenesis.

Increasing evidence points towards a prothrombotic state in atherosclerosis and its manifestations, such as peripheral artery disease (PAD), which is associated with thrombosis-related complications, such as acute limb ischaemia, graft thrombosis and stroke. We hypothesized that the increased risk of thrombogenesis in PAD may be related to abnormal angiogenesis and, thus, an increased risk of future <em>vascular</em> disease. To test this hypothesis, we measured plasma levels of tissue <em>factor</em> (TF) and related levels to indices of angiogenesis, that is <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its soluble receptor sFlt-1. We studied 234 patients (<em>145</em> males; mean age 68.6+/-10 years) with proven PAD (ankle brachial pressure index <0.8) and compared them with 50 healthy controls. Levels of VEGF ( P =0.001) and TF ( P =0.043) were increased in patients compared with controls. There were significant correlations between VEGF and TF levels in both patients (Spearman r =0.351, P <0.001) and healthy controls (Spearman r =0.335, P =0.017). Amongst PAD patients, levels of VEGF were related to gender, with women having higher levels than men. There was no difference in the levels of sFlt-1 between the patients and controls, or between the subgroups of patients. There were however significant correlations between the levels of sFlt-1 and TF (Spearman r =0.268, P <0.001) and between sFlt-1 and VEGF (Spearman r =0.499, P <0.001). In conclusion, patients suffering from proven PAD have higher plasma levels of TF and VEGF compared with controls, with a significant correlation between the two. This suggests a link between the hypercoagulable state in PAD and the process of angiogenesis.

Maspin is a mammary serine protease inhibitor or serpin with tumor suppressive and antiangiogenic activity that inhibits tumor motility, invasion and metastasis, at least by its actions on cell membrane and extracellular matrix (ECM) proteins. Previous studies documented that the quinazoline-derived alpha1-adrenoceptor antagonist doxazosin affects the attachment and migration of prostate cancer cells. In this study, we investigated the effect of maspin overexpression on the apoptotic/antiadhesion response of prostate cancer cells to doxazosin. The response of maspin-overexpressing clones of human prostate cancer cells DU-<em>145</em> to doxazosin was evaluated by determining cell viability, apoptosis and cell proliferation on the basis of the trypan blue exclusion assay/methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, Hoechst staining and caspase-3 activation, and [(3)H]thymidine incorporation assay. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), transforming <em>growth</em> <em>factor</em> betaRII (TGFbetaRII), Smad4 (a TGFbeta intracellular effector) and bax expression was evaluated at the mRNA and protein level using reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The effect of doxazosin on cell attachment of maspin-expressing prostate cancer cells was evaluated on collagen- and fibronectin-coated plates. Cell migration was assessed using the wounding assay. In response to tumor necrosis <em>factor</em>-related apoptosis-inducing ligand, DU-<em>145</em>-maspin expressing cells undergo apoptosis, via poly(ADP-ribose) polymerasecleavage and caspase-3 activation. DU-<em>145</em>-maspin cells exhibited higher sensitivity to doxazosin and an earlier temporal activation of caspase-3. The number of apoptotic cells detected in response to doxazosin was significantly higher compared to the neo control (P<0.0001). Doxazosin resulted in dramatic downregulation of the 189 isoform of VEGF in maspin transfectants, while a fivefold induction of Smad4 mRNA expression was detected in those cells after 24 h of treatment. Maspin overexpression in prostate cancer cells resulted in an increased ability to attach to ECM-coated plates, and doxazosin treatment considerably antagonized this effect by decreasing the attachment potential to collagen and fibronectin. The present study supports the ability of maspin to enhance the apoptotic threshold of prostate cancer cells to the quinazoline-based alpha1-adrenoceptor antagonist doxazosin. These findings may have therapeutic significance in the development of antiangiogenic targeting by doxazosin and derivative agents for advanced prostate cancer.

Experiments were designed to investigate the expression and regulation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3-5 post-LH surge), mid (MCL; Day 6-8 post-LH surge), mid-late (MLCL; Days 10-12 post-LH surge), late (LCL; Days 14-16 post-LH surge), and very late (Days 17- 18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, <em>145</em>, and 121. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and 121 sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P < 0.05) from ECL to MLCL but then declined (P < 0.05) by LCL. Although VEGF 121 mRNA levels were limited in ECL, they increased significantly in MCL (P < 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two- to fourfold higher for VEGF 165 versus VEGF 121. Also, VEGF 165 levels were higher (P < 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P < 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P < 0.05) VEGF levels, whereas LH exposure increased (P < 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.

Geldanamycin (GA), a benzoquinone ansamycin, is a naturally occurring inhibitor of heat shock protein (Hsp90), which regulates the transcription activity of hypoxia-inducible <em>factor</em> 1 (HIF-1alpha). Under hypoxia, HIF-1alpha is activated in tumor cells, and induces the transcription of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), which is the prime regulator for angiogenesis. VEGF promotes the formation of new blood vessels by stimulating <em>endothelial</em> cell division and migration. This eventually forms a <em>vascular</em> network that allows for tumor <em>growth</em> and metastasis. In this study, we used GA to inhibit HIF-1alpha transcription function. Human prostate cancer DU-<em>145</em> cells were incubated in a hypoxic chamber at 1% O(2) and 37 degrees C for different durations. Both mRNA and protein levels of HIF-1alpha and VEGF were upregulated under hypoxic conditions. We demonstrated that GA treatment of hypoxic DU-<em>145</em> cells abolished the induction of HIF-1alpha protein in a time-dependent manner and decreased VEGF mRNA and its protein levels. The transient transfection of DU-<em>145</em> cells with luciferase reporter gene construct (5HRE/hCMVmp-luc) showed that the transcriptional activity of HIF-1alpha was significantly induced in response to hypoxia, but inhibited by GA. In addition, using conditioned medium from GA-treated hypoxic cells led to a significant decrease in cell invasion in comparison with using conditioned medium from nontreated hypoxic cells. These data provide evidence for the important role of GA in inhibition of angiogenesis and also invasion mediated by HIF-1alpha in prostate cancer cells.

The splice forms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) differ in biological properties such as the receptor types that they recognize and their interaction with heparan sulfate proteoglycans. We have identified a new VEGF mRNA splice form encoding a VEGF species containing 162 amino acids (VEGF(162)) in human A431 ovarian carcinoma cells. This novel mRNA contains the peptides encoded by exons 1-5, 6A, 6B, and 8 of the VEGF gene. Recombinant VEGF(162) is biologically active. It induces proliferation of <em>endothelial</em> cells in vitro and angiogenesis in vivo as determined by the alginate bead assay. VEGF(162) binds less efficiently than VEGF(<em>145</em>) but more efficiently than VEGF(165) to a natural basement membrane produced by corneal <em>endothelial</em> cells. VEGF(138), an artificial VEGF form that contains exon 6B but lacks exons 6A and 7, did not bind to this basement membrane at all, indicating that exon 6B probably interferes with the interaction of exon 6A with heparin and heparan sulfate proteoglycans.

We report a case of a patient with probable von Hippel-Lindau (VHL) syndrome and metastatic renal cell cancer (RCC) who had a complete radiological and metabolic response to SU5416 (semaxanib). The patient was enrolled on a clinical study examining the efficacy of SU5416 in patients with metastatic cancer. Treatment with SU5416 was given at a dose of <em>145</em> mg/m2 intravenously twice-weekly for 11 doses. The patient achieved an early metabolic response on an F-18 fluorodeoxyglucose (FDG) Positron Emission Tomographic (PET) scan within 2 weeks of therapy. Subsequent computerized tomography (CT) and PET scans (9 and 12 months after treatment, respectively) confirmed ongoing complete radiological and metabolic response. He remains tumor-free 18 months after treatment. This is the first documented report of metastatic RCC in the setting of presumed VHL syndrome responding to treatment with SU5416. While <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) inhibitors have been shown to produce a modest response in sporadic metastatic RCC, further studies utilizing VEGF inhibitors in patients with VHL syndrome and RCC warrants exploration.

Tumor angiogenesis is a critical step for the <em>growth</em> and metastasis of solid tumors. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is the most important angiogenic molecule associated with tumor-induced neovascularization. VEGF exerts its activity through binding to its receptor tyrosine kinase, KDR/Flk-1, expressed on the surface of <em>endothelial</em> cells. From the screening of medicinal plants, we have identified 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) from the roots of Paeonia lactiflora that inhibited the binding of VEGF to KDR/Flk-1. PGG efficiently blocked VEGF-induced human umbilical vein <em>endothelial</em> cell proliferation and the <em>growth</em> of immortalized human microvascular <em>endothelial</em> cells, but did not affect the <em>growth</em> of HT1080 human fibrosarcoma and DU-<em>145</em> human prostate carcinoma cells. PGG also blocked VEGF-induced capillary-like tube formation of <em>endothelial</em> cell on Matrigel. Our results suggest that PGG could be a candidate for developing anti-angiogenic agent.

Metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) is a long non‑coding RNA (lncRNA) that has an oncogenic role in some types of cancers, uncluding breast cancer (BC). To investigate the role of MALAT1 in human BC progression, we detected MALAT1 expression levels based on tissue samples from 20 BC cases and 20 healthy controls and found MALAT1 expression levels to be significantly high (P<0.05). Then, we knocked down endogenous MALAT1 in MCF‑7 cells using MALAT1 short hairpin RNA (shRNA). The results revealed that MALAT1 knockdown could significantly inhibit proliferation, migration, and tube formation in vitro. In addition, miR‑<em>145</em> expression inversely changed in BC tissue cases. Furthermore, knockdown of endogenous MALAT1 significantly increased miR‑<em>145</em> levels in MCF‑7 cells. This finding indicated an interaction between MALAT1 and miR‑<em>145</em>. In addition, knockdown of MALAT1 significantly reduced the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> in MCF‑7 cells. This outcome revealed that MALAT1 promoted angiogenesis in BC, which may be related to the expression of miR‑<em>145</em>.

The potential anti-angiogenic activities of water-soluble condensed tannins isolated from black beans were evaluated using HEL 299 normal human fibroblast lung cells, Caco-2 colon, MCF-7 and Hs578T breast, and DU <em>145</em> human prostatic cancer cells. Condensed tannins at 0.24-24 microM did not affect the <em>growth</em> of normal cells, but dose-dependently induced cancer cell death by apoptosis as shown by a concentration-dependent decrease in ATP and cell gross morphology. After 24h exposure to Caco-2, MCF-7, Hs578T, and DU <em>145</em> cancer cells, water-soluble black bean condensed tannins at 24 microM suppressed fetal bovine serum stimulated cell migration, the secretion of matrix metalloproteinase-2 (MMP-2 or gelatinase A), matrix metalloproteinase-9 (MMP-9 or gelatinase B), and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> VEGF(165) receptor expression by the cancer cells in the conditioned media. The potential health enhancing properties of condensed tannins from black beans as inhibitors of angiogenesis is discussed.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a highly specific <em>factor</em> for <em>vascular</em> <em>endothelial</em> cells. Five VEGF-A isoforms (splice variants 121, <em>145</em>, 165, 189 and 206) are generated as a result of alternative splicing from a single VEGF-A gene. These differ in their molecular weights and in biological properties such as their ability to bind to cell-surface heparan sulfate proteoglycans. Deregulated VEGF-A expression contributes to the development of solid tumors by promoting tumor angiogenesis. More specifically, VEGF-A189 expression is related to angiogenesis and prognosis in certain human solid tumors. VEGF-A189 expression is also related to the xenotransplantability of human cancers into immunodeficient mice in vivo. Consequently, inhibition of VEGF-A or VEGF-A189 signaling regulates the development and metastasis of a variety of tumors. This review focuses on recent studies of the mechanisms by which VEGF-A regulates angiogenesis in the cancer stroma and on our recent findings concerning the potential mechanisms of VEGF-A189 expression on tumor <em>growth</em> and metastasis.

BACKGROUND

Vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-1 and Ang-2 are key factors in angiogenic signaling. In this study the expression of these factors was identified in cartilage tumors. As interleukin (IL)-1beta has been found to be an indispensable factor in angiogenic signaling, we further analyzed the effect of IL-1beta on the expression of VEGF-A, Ang-1, and Ang-2 using a previously established cell culture model.

METHODS

Surgical specimens of enchondromas, conventional chondrosarcomas, and dedifferentiated chondrosarcomas were obtained from 72 patients. VEGF-A, Ang-1, and Ang-2 mRNA expression was detected by conventional and quantitative reverse transcription polymerase chain reaction (PCR). VEGF-A expression was also detected by immunohistochemistry or Western blot.

RESULTS

Differential expression of VEGF-A, Ang-1, and Ang-2 was clearly demonstrated in cartilage tumors. VEGF-A expression was positively correlated with the tumor type. Higher VEGF-A expression levels were detected in conventional chondrosarcomas Grades II and III (using a 3-tier grading system) than in dedifferentiated chondrosarcomas (P < .05). A typical pattern of VEGF-A isoforms was identified, including VEGF(121), VEGF(145), VEGF(165), and VEGF(189). Ang-1 presented as a low-level transcript with slightly elevated levels in chondrosarcomas (P < .05). Highly variable Ang-2 expression levels were detected in solitary cases of conventional chondrosarcomas. IL-1beta regulated VEGF-A and Ang-1 expressions in a dose-dependent manner. Whereas low IL-1beta concentrations increased VEGF-A and Ang-1 transcription, high IL-1beta concentrations had the opposite effect. IL-1beta did not activate Ang-2 expression.

CONCLUSIONS

Angiogenic signaling in cartilage tumors is variable and at least partly regulable by IL-1beta. The findings are of therapeutic relevance, either as a desired effect or a side effect in medical treatment.

Angiogenesis plays crucial roles in development and progression of hepatocellular carcinoma (HCC). Placenta <em>growth</em> <em>factor</em> (PLGF), belonging to <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) family, is involved in angiogenesis associated with cancer. Soluble VEGF receptor-1 (sVEGFR1) has been thought to be an intrinsic negative regulator for PLGF. We investigated whether serum PLGF and serum sVEGFR1 is associated with prognosis of HCC. Serum PLGF and sVEGFR1 levels were measured in <em>145</em> patients with HCC using enzyme-linked immunosorbent assay. The levels of these <em>factors</em> and the ratio of PLGF to sVEGFR1 were analyzed in relation with clinical parameters. The higher level of sVEGFR1 and the lower ratio of PLGF/sVEGFR1 were significantly associated with poor survival in HCC. Cox regression analysis revealed that the lower ratio of PLGF/sVEGFR1 independently correlated to prognosis of patients with HCC. The ratio of PLGF/sVEGFR1 was independent prognostic indicator for HCC. The ratio of PLGF/sVEGFR1 should be addressed in anti-angiogenic therapy for HCC.

OBJECTIVE

To examine the effect of loss of cell-cell contacts on the gene expression of vascular endothelial growth factor (VEGF) and other factors in primary culture of human retinal pigment epithelial (RPE) cells with real-time reverse transcription-PCR.

METHODS

The dissociation of postconfluent RPE cells was induced by calcium chelation, low-calcium medium, anti-E-cadherin, and anti-N-cadherin antibodies. Total RNA was isolated from the cultured RPE cells and reverse transcribed to cDNA. VEGF was quantified by real-time PCR with a fluorescence detector. VEGF isoforms were differentially measured by specific exon-spanning primers. Besides VEGF, the gene expression levels of some other growth factors were also examined in calcium-mediated dissociation.

RESULTS

Disruption of cell-cell contacts of RPE cells was induced by calcium chelation and low-calcium medium, but not by anti-E-cadherin and anti-N-cadherin antibodies. Calcium-mediated dissociation of RPE cells significantly increased the gene expression levels of VEGF. The mRNA levels of VEGF increased by 6.3-fold on treatment with EGTA and by 4.7-fold in the low-calcium medium at 6 hours. Splice variants of VEGF showed the differential pattern of gene expression. Whereas the expression of VEGF(121) and VEGF(165) was upregulated on calcium-induced dissociation of RPE cells, that of VEGF(145) and VEGF(189) was unchanged. VEGF(206) was not detected. On calcium-induced dissociation, bFGF, IL-6, matrix metalloproteinase (MMP)-1, and placental growth factor (PlGF) were upregulated, whereas acidic (a)FGF and pigment epithelium-derived factor (PEDF) were both downregulated.

CONCLUSIONS

The results show that loss of intercellular contacts promotes increased gene expression of VEGF and other angiogenic factors in human RPE cells.