Modulation Of Vascular Endothelial Growth Factor Expression Research Articles

Analysis of microRNA expression in rat kidneys after VEGF inhibitor treatment under different degrees of hypoxia.

Previously, we found that the incidence of kidney injury in patients with chronic hypoxia was related to the partial pressure of arterial oxygen. However, at oxygen concentrations that contribute to kidney injury, the changes in the relationship between microRNAs (miRNAs) and the hypoxia-inducible factor-1α (HIF-1α)-vascular endothelial growth factor (VEGF) axis and the key miRNAs involved in this process have not been elucidated. Therefore, we elucidated the relationship between VEGF and kidney injury at different oxygen concentrations and the mechanisms mediated by miRNAs. Sprague-Dawley rats were exposed to normobaric hypoxia and categorized into six groups based on the concentration of the oxygen inhaled and injection of the angiogenesis inhibitor bevacizumab, a humanized anti-VEGF monoclonal antibody. Renal tissue samples were processed to determine pathological and morphological changes and HIF-1α, VEGF, and miRNA expression. We performed a clustering analysis of high-risk pathways and key hub genes. The results were validated using two Gene Expression Omnibus datasets (GSE94717 and GSE30718). As inhaled oxygen concentration decreased, destructive changes in the kidney tissues became more severe. Although the kidney possesses a self-protective mechanism under an intermediate degree of hypoxia (10% O2), bevacizumab injections disrupted this mechanism, and VEGF expression was associated with the ability of the kidney to repair itself. rno-miR-124-3p was identified as a crucial miRNA; a key gene target, Mapk14, was identified during this process. VEGF plays an important role in kidney protection from injury under different hypoxia levels. Specific miRNAs and their target genes may serve as biomarkers that provide new insights into kidney injury treatment.NEW & NOTEWORTHY Renal tolerance to hypoxic environments is limited, and the degree of hypoxia does not show a linear relationship with angiogenesis. VEGF plays an important role in the kidney's self-protective mechanism under different levels of hypoxia. miR-124-3p may be particularly important in kidney repair, and it may modulate VEGF expression through the miR-124-3p/Mapk14 signaling pathway. These microRNAs may serve as biomarkers that provide new insights into kidney injury treatment.

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

Modulation of VEGF Expression and Oxidative Stress Response by Iodine Deficiency in Irradiated Cancerous and Non-Cancerous Breast Cells

Breast cancer remains a major concern and its physiopathology is influenced by iodine deficiency (ID) and radiation exposure. Since radiation and ID can separately induce oxidative stress (OS) and microvascular responses in breast, their combination could additively increase these responses. Therefore, ID was induced in MCF7 and MCF12A breast cell lines by medium change. Cells were then X-irradiated with doses of 0.05, 0.1, or 3 Gy. In MCF12A cells, both ID and radiation (0.1 and 3 Gy) increased OS and vascular endothelial growth factor (VEGF) expression, with an additive effect when the highest dose was combined with ID. However, in MCF7 cells no additive effect was observed. VEGF mRNA up-regulation was reactive oxygen species (ROS)-dependent, involving radiation-induced mitochondrial ROS. Results on total VEGF mRNA hold true for the pro-angiogenic isoform VEGF165 mRNA, but the treatments did not modulate the anti-angiogenic isoform VEGF165b. Radiation-induced antioxidant response was differentially regulated upon ID in both cell lines. Thus, radiation response is modulated according to iodine status and cell type and can lead to additive effects on ROS and VEGF. As these are often involved in cancer initiation and progression, we believe that iodine status should be taken into account in radiation prevention policies.

VEGF Promoter Polymorphism Confers an Increased Risk of Pulmonary Arterial Hypertension in a Chinese Population.

PurposeEvidence on the contribution of genes to the hereditary predisposition to pulmonary arterial hypertension (PAH) is limited.Materials and MethodsIn this study, we hypothesized that single nucleotide variants in vascular endothelial growth factor (VEGF) gene may alter gene function and expression and may be associated with PAH risk. Five putatively functional loci (rs699947C>A and rs833061T>C in the promoter, rs3025040C>T, rs10434G>A and rs3025053G>A in the 3'-UTR) in the VEGF gene were genotyped and analyzed in a retrospective study of 587 patients with PAH and 736 healthy subjects from southern China.ResultsWe found that the rs833061T>C polymorphism was significantly associated with PAH risk, while the other single nucleotide polymorphisms were not. Compared to carriers with TT genotype, those with rs833061C variant genotype (CT/CC) had an increased risk of PAH (odds ratio=1.47, 95% confidence interval=1.18–1.83, p=0.001). Functional assays indicated that CT/CC variant genotype had significantly higher mRNA levels of VEGF in peripheral blood mononuclear cells than TT genotype (p=0.021). Luciferase reporter assay indicated that having a C allele conferred a significantly higher transcription activity than that with a T allele.ConclusionOur findings suggest that the functional polymorphism rs833061T>C in VEGF gene promoter modulates VEGF expression and may be a valuable biomarker for predicting PAH susceptibility.

Enhancement of radiosensitivity in human esophageal carcinoma cells by fenofibrate and its potential mechanism.

Fenofibrate is a specific agonist of PPARα, and is characterized by relatively low systemic toxicity. Recent studies have revealed that fenofibrate suppresses the growth of several cancer lines in vitro, but the exact relation between fenofibrate and irradiation has not been explored. The purpose of this study was to investigate the radiosensitivity enhancement effects of fenofibrate combined with radiation on the human esophageal carcinoma cell lines Eca-109 and TE1, and the potential mechanism underlying these effects. The Eca-109 and TE1 cell lines were tested by the CCK-8 assay for cell proliferation. The multitarget click model was used to delineate the survival curve and radiosensitivity was determined after cells were treated with fenofibrate and/or x-ray radiation. Flow cytometry was used to examine the effect of fenofibrate and radiation on the cell cycle. The expression of vascular endothelial growth factor (VEGF) protein was detected by Western blot analysis. When given alone, fenofibrate had a time- and concentration-dependent cytotoxic effect on cells. The dose-enhancement ratio for combined fenofibrate and radiation increased markedly compared with fenofibrate alone. Further, the ratio of cells in the G2/M phase after fenofibrate and radiation was higher than that after fenofibrate or irradiation alone. The expression of VEGF protein was suppressed after treatment with fenofibrate alone or fenofibrate plus radiation. Fenofibrate can enhance the radiosensitivity of human esophageal carcinoma cells by increasing G2/M phase arrest. Modulation of VEGF expression could contribute in vivo to a favorable interaction.

Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

C-myc but not Hif-1α-dependent downregulation of VEGF influences the proliferation and differentiation of HL-60 cells induced by ATRA

Vascular endothelial growth factor (VEGF) plays an important role in solid tumor growth, progression and metastasis as well as in the proliferation and differentiation of hematological malignancies. However, the molecular mechanism that modulates VEGF expression and secretion in leukemia cells has not yet to be elucidated. The purpose of the present study was to investigate the role of the signal pathway in modulating the expression of VEGF in HL-60 cells. Specific siRNAs targeting VEGF were transfected into HL-60 cells and the VEGF expression was measured with reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. The cell proliferation of HL-60 cells was detected by the cell counting kit-8 (CCK-8) assay and the differentiation of HL-60 cells induced by all-trans-retinoic acid (ATRA) was detected by the RT-PCR assay and flow cytometry assay for CD11b. The upstream transcription factors that were related to VEGF expression such as P53, SP-1, c-jun, VHL, cox-2, c-myc and stat3 were detected by RT-PCR assay. In addition, the chromatin immunoprecipitation (ChIP) assay was used to reveal the role of c-myc by binding the target gene VEGF. The results demonstrated the hypoxia-inducible factor1α-related signaling pathway, not the same as in solid tumors, might not play a key role in modulating VEGF expression. c-myc contributes to the modulation of VEGF expression by targeting the promoter of VEGF, which was indicated by the ChIP assay. In conclusion, our data demonstrate that VEGF plays an important role in the differentiation and proliferation of HL-60 cells; c-myc-dependent downregulation of VEGF induced by ATRA contributes to the differentiation of HL-60 cells.

Inhibition of p38 MAPK Reduces Expression of Vascular Endothelial Growth Factor in Allergic Airway Disease

The p38 mitogen-activated protein kinase (MAPK) appears to play an important role in various pathophysiological responses and has been suggested to be involved in many processes considered critical to the inflammatory response and tissue remodeling. Bronchial asthma is a chronic inflammatory disorder of the airway accompanied by increased vascular permeability. Vascular endothelial growth factor (VEGF) is a potent stimulator of bronchial inflammation, airway remodeling, and physiologic dysregulation that augments antigen sensitization and T-helper type 2 cell (Th2)-mediated inflammation in allergic airway diseases. However, there are little data on the relationship between p38 MAPK signaling and VEGF expression in allergic airway disease. This study aimed to investigate the role of p38 MAPK on the pathogenesis of allergic airway disease, more specifically in VEGF expression. Using ovalbumin (OVA)-inhaled mice and a selective p38 MAPK inhibitor, SB 239063, the involvement of p38 MAPK in allergen-induced VEGF expression in the airway was evaluated. The increases of phosphorylation of p38 MAPK, VEGF protein expression, and vascular permeability in the lung after OVA inhalation were decreased substantially by the administration of SB 239063. In addition, SB 239063 significantly reduced the increase of Th2 cytokines and OVA-specific IgE. The inhibition of p38 MAPK or VEGF signaling prevented and also decreased the increases in the number of inflammatory cells and airway hyperresponsiveness in OVA-induced allergic airway disease. These results indicate that inhibition of p38 MAPK may attenuate allergen-induced airway inflammation and vascular leakage through modulation of VEGF expression in mice.

P4-08-02: Estradiol (E2) Mediated Secretion of Vascular Endothelial Growth Factor (VEGF) and Stimulation of T-Regulatory Cells (T-Regs): Their Role for the Worse Prognosis of Breast Cancer (BC) in Premenopause.

Abstract Background: E2 plays a key role in all the phases of human reproduction, including promotion of uterine basal membrane erosion, embryo implantation, placental villi development, and pregnancy maintenance. E2 mediates all these functions through the induction of VEGF secretion and stimulation of T-Regs with increased tolerance for the eterologous embryo. BC might utilize the same pathways for its growth and for disease progression: Modulation of VEGF expression in BC cells through transcriptional activation and regulation of the peripheral development of CD4+CD25+ T-Regs cells. Both VEGF and T-Regs are important prognostic factors: In fact high levels of VEGF expression are associated with significantly worse survival in breast cancer patients, even in sub-groups expected to have a better prognosis and T-Regs quantification in breast tumors is valuable for assessing disease prognosis and progression. However few clinical studies investigated the association between E2, VEGF and T-Regs in humans. Objective of this study was to evaluate whether E2 suppression with an LH-RH analogue was able to down regulate VEGF expression, and decrease T-Regs in premenopausal patients with high-risk estrogen receptor positive (ER+) and negative (ER-) early BC. Material and Methods: From 04–2003 to 10–2008, 100 premenopausal early BC patients were entered into the study. At baseline, in the follicular phase of the menstrual cycle, after surgery, plasma E2, VEGF and T-Regs were measured. Measurements were repeated every six months. Treatment: LH-RH analogue for 5 years, chemotherapy tailored to the biological characteristics of each patient, radiation therapy, and 5-year hormonal therapy in ER+ tumors. Primary end-point was the evaluation of VEGF and T-Regs. Secondary end points were progression-free survival (PFS) and overall survival (OS). Results: Median age was 43 years (range 26–45). Median number of positive axillary nodes was 3.3. Eighty-three patients were ER+, 17 were ER - and progesterone receptor negative (PGR-), 10 were triple negative, 20 were Herb-2 positive. Median KI-67 was 33% (range 15%-100%). A statistically significant decrease of E2,VEGF and T-Regs was observed after 1 and 5 years, in ER+ and ER- patients: In particular, VEGF (P<0.001) and T-Regs (P<0.001) decreased in 95% of patients that were disease-free. No unexpected toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogues administration. After a median follow-up of 55 months (range 30–95), 5-year PFS and OS rate were 95% and 100%, respectively. Discussion: E2 deprivation with an LH-RH analogue is able to decrease plasma VEGF and T-Regs levels in premenopausal high risk ER+ and ER- BC patients. These data show how E2, through VEGF and T-Regs modulation, may be responsible for the worst prognosis that is observed in premenopausal BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-08-02.

Estrogen and vascular endothelial growth factor (VEGF): Their role in breast cancer (BC) carcinogenesis and disease progression in premenopause.

540 Background: Estrogen has a key role in BC development and there is evidence in vitro, that it modulates VEGF expression in BC cells through transcriptional activation. However few clinical studies investigated the association between VEGF and estrogens in humans. Objective of this study was to evaluate wether estrogen suppression with an LH-RH analogue was able to down regulate VEGF expression, and thus decrease its plasma concentration, in premenopausal patients with high-risk estrogen receptor positive (ER+) and negative (ER−) early BC. Methods: From 04-2003 to 10-2008, 100 premenopausal early BC patients were entered into the study. At baseline, after surgery, plasma VEGF and sex hormones were measured. Measurements were repeated every six months. Treatment: LH-RH analogue for 5 years, chemotherapy tailored to the biological characteristics of each patient, radiation therapy, and 5-year hormonal therapy in ER+ tumors. Primary end-point was the evaluation of VEGF. Secondary end points were progression-free survival (PFS) and overall survival (OS). Results: Median age was 43 years (range 26-45). Mean number of positive axillary nodes was 3.3. Eighty-three patients were ER+, 17 were ER − and progesterone receptor (PGR) -. Median KI-67 was 33% (range 15%−100%). Twenty patients were Herb−2 positive. A statistically significant decrease of VEGF was observed after 1 and 5 years, both in ER+ and ER− patients: In particular, VEGF decreased (p<0.001) in 94% of patients that were disease-free, while in 6% of patients with disease relapse, it increased with respect to baseline values. No unexpected toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogues administration. After a median follow-up of 50 months (range 24-90), 5-year disease free survival and overall survival rate were 94% and 100%, respectively. Conclusions: Estrogen deprivation with an LH-RH analogue is able to decrease plasma VEGF levels in premenopausal high risk ER+ and ER- BC patients. These data show how estrogens, through VEGF modulation, may be responsible for the worst prognosis that is observed in premenopausal BC patients.

Abstract 5055: The role of estrogens and vascular endothelial growth factor (VEGF) in premenopausal breast cancer carginogenesis and disease progression

Abstract Background. Estrogen is important in breast cancer development and there is evidence in vitro, that it modulates VEGF expression in breast cancer cells through transcriptional activation. However few clinical studies have investigated the association between VEGF and estrogens in humans. Objective of this study was to evaluate weather estrogen suppression with an LH-RH analogue was able to downregulate VEGF expression, and thus decrease its plasma concentration, in premenopausal patients with high-risk early breast cancer. Patients and methods: From April 2003 to October 2008 we conducted this study on 100 premenopausal early breast cancer patients. At baseline, after surgery, before any type of therapy, plasma VEGF and sex hormones (estradiol, progesterone, FSH, LH) were evaluated. Evaluation was repeated each year. Patients received an LH-RH analogue for 5 years, a chemotherapy that was tailored to the biological characteristics of each patient, radiation therapy, and hormonal therapy in estrogen receptor positive (ER+) tumors. Primary end point was the evaluation of VEGF. Secondary end points were progression-free survival (PFS) and overall survival (OS). Results: Median age was 43 years (range 26-45); mean number of positive axillary nodes was 3.3. (Range 0-25). Fifty-three patients were ER+ and progesterone receptor positive (PGR+), 17 were ER negative (−) and PGR-, 30 were ER+ and PGR-, Median KI-67 was 33% (range 15%-100%). Twenty patients were Herb-2 positive by FISH. A statistically significant decrease of VEGF was observed after 1 year. At 5 years and during the study period, plasma VEGF continued to decrease (p&amp;lt;0.001) in 94% of patients that were disease-free, while in 6% of patients with disease relapse, VEGF increased with respect to baseline values. No unexpected toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogues administration. After a median follow-up of 50 months (range 24-90), 8-year disease free survival and overall survival rated were 94% and 100%, respectively. Conclusion: Estrogen deprivation with an LH-RH analogue is able to decrease plasma VEGF levels in premenopausal high risk breast cancer patients. These data show how estrogens, through VEGF modulation, may be responsible for the worst prognosis that is observed in premenopausal breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5055. doi:10.1158/1538-7445.AM2011-5055

Abstract 5307: VEGF downregulation by HSP90 inhibitor NVP-AUY922 visualized with 89Zr-bevacizumab PET in triple negative breast cancer (TNBC)

Abstract Zirconium-89 (89Zr) labeled bevacizumab visualizes tumor vascular endothelial growth factor (VEGF) excretion in vivo, and can therefore serve as a potential biomarker for targeted drugs affecting VEGF levels. TNBC is more dependent upon alternative routes for their maintenance, such as angiogenesis, than hormone receptor or HER2 positive breast cancer. HSP90 inhibitors are known to impact the angiogenesis pathway via degradation of HSP90 client proteins such as HIF1α and VEGFRs, and in vitro studies have shown that breast cancer cells (including TNBC) are sensitive to these compounds. The aim of this study was to image modulation of VEGF expression in response to HSP90 inhibition with NVP-AUY922, a highly potent non-geldamycin HSP90 inhibitor, in a mouse model with a TNBC xenograft. Methods: Three groups of animals were used in this study, a control (n = 6), treated (n = 7) and placebo (n = 6) group. Twenty-two days after inoculation of TNBC cell line MDA MB-231, all mice received co-injection of 89Zr-bevacizumab and Indium-111 (111In) labeled IgG as aspecific control. VEGF-PET was performed 144 h after tracer injection. Ex vivo biodistribution studies were performed in the control group after the first scan week, for the treatment and placebo group after the second scan week, which was after 3 weeks of treatment. The control group received no treatment, the treatment and placebo group received respectively 50 mg/kg NVP-AUY922 or vehicle every-third-day (q3d) administrated intraperitoneally. Treatment effect was verified with histological response. Results: VEGF-PET showed a decreased 89Zr-bevacizumab tumor uptake of 44% (P = 0.0021) after 3 weeks NVP-AUY922 treatment compared to the baseline scan. Placebo treated mice showed no significant difference in bevacizumab uptake. These in vivo imaging data were supported by ex vivo biodistribution data. IgG tumor uptake was in all groups consistent, indicating that there was no influence on tumor perfusion due to the anti-angiogenic treatment. VEGF downregulation in the tumors was shown by immunohistochemistry of VEGF-A. Conclusion: NPV-AUY922 downregulates VEGF in a TNBC mouse model. VEGF-PET using 89Zr-bevacizumab can be used in vivo to monitor downregulation of VEGF excretion in response to HSP90 inhibition, and can therefore potentially serve as biomarker for early HSP90 inhibition effect in TNBC. Supported by a grant of the Dutch Cancer Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5307. doi:10.1158/1538-7445.AM2011-5307

Reduced Choroidal Neovascular Membrane Formation in Cyclooxygenase-2 Null Mice

To assess the degree of laser-induced choroidal neovascular membrane formation in wild-type (WT) and COX-2 null mice and to measure vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α levels in the retina and choroid. Four laser burns were placed in each eye of WT and COX-2 null mice to induce choroidal neovascularization. Fluorescein angiography (FA) was performed at 14 days, and retinal pigment epithelium-choroid-sclera (choroidal) flat mounts were prepared. The retina and choroid were isolated from WT and COX-2 null mice at 24, 72, and 168 hours after laser photocoagulation and from unlasered eyes and were tested for VEGF, IL-1β, and TNF-α. COX-2 null mice demonstrated 58% (P = 0.001) and 48% (P = 0.001) reductions in CNV formation on FA and choroidal flat mounts, respectively, compared with WT mice. For unlasered mice, mean VEGF concentrations in the retina and choroid were 1.2 ± 0.42 pg/mg protein for WT but only 0.42 ± 0.2 pg/mg protein for COX-2 null mice (P < 0.05). After laser photocoagulation, WT mice showed significantly greater VEGF and IL-β expression in the retina and choroid by 168 hours (P < 0.05) and 72 hours (P < 0.05), respectively, compared with COX-2 null mice. COX-2 null mice exhibited significantly less choroidal neovascular membrane formation associated with reduced expression of VEGF. The results of this study suggest that COX-2 modulates VEGF expression in CNV and implicates a potential therapeutic role for nonsteroidal anti-inflammatory drugs.

DLC1 Negatively Regulates Angiogenesis in a Paracrine Fashion

The Rho GTPase-activating protein DLC1 is a tumor suppressor that is often deleted in liver cancer and downregulated in other cancers. DLC1 regulates the actin cytoskeleton, cell shape, adhesion, migration, and proliferation through its Rho GTPase-activating protein activity and focal adhesion localization. In this study, we silenced DLC1 in nonmalignant prostate epithelial cells to explore its tumor suppression functions. Small hairpin RNA-mediated silencing of DLC1 was insufficient to promote more aggressive phenotypes associated with tumor cell growth. In contrast, DLC1 silencing promoted pro-angiogenic responses through vascular endothelial growth factor (VEGF) upregulation, accompanied by the accumulation of hypoxia-inducible factor 1α and its nuclear localization. Notably, modulation of VEGF expression by DLC1 was dependent on epidermal growth factor receptor-MAP/ERK kinase-hypoxia-inducible factor 1 signaling but on RhoA pathways. Clinically, VEGF upregulation is a highly significant event in prostate cancers in which DLC1 is downregulated. Thus, our results strongly suggest that loss of DLC1 may serve as a "second hit" in promoting angiogenesis in a paracrine fashion during tumorigenesis.

Antidepressant hyperforin up-regulates VEGF in CNS tumour cells

Vascular endothelial growth factor (VEGF) is a key player in neo-angiogenesis; it sustains the progression of solid neoplasias, brain tumours included. It has recently been demonstrated that the use of antidepressants correlates with increasing VEGF levels in the central nervous system (CNS). In order to elucidate whether the most used natural antidepressant [St. John's wort (SJW) extract] modulates VEGF expression, possible relationship between ≤ μM hyperforin (Hyp, the bioactive component in SJW) and VEGF in CNS tumours has been now examined in medulloblastoma and glioblastoma cells. Real-time PCR and ELISA revealed that under Hyp VEGF expression increased more than three fold in DAOY medulloblastoma cells; while, U87 glioblastoma cells – constitutively expressing high VEGF levels – showed no significant differences. Moreover, Hyp induced endothelial pro-angiogenic behaviour in a multi-parametric Matrigel colonisation assay, and down-modulation of pro-MMP-2 and pro-MMP-9 activities as measured by gelatin zymography. Should these results be confirmed in vivo for this and other types of CNS tumour, the antidepressant use of SJW extracts must be carefully re-considered, in particular for brain tumour patients.

MiR‐20b modulates VEGF expression by targeting HIF‐1α and STAT3 in MCF‐7 breast cancer cells

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR-20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF-7 breast cancer cells under normoxic and hypoxia-mimicking conditions (CoCl(2) exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24 h following CoCl(2) treatment, and VEGF mRNA at 4 h of treatment. In addition, miR-20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre-miR-20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR-20b. Downregulation of VEGF mRNA by miR-20b under a 4 h of CoCl(2) treatment was associated with reduced levels of nuclear HIF-1 alpha subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1 alpha, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl(2) treatment. This effect was inhibited by transfection of cells with pre-miR-20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl(2)-mediated HIF-1 alpha nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF-1 and STAT3 in a miR-20b-dependent manner.

Abstract 5350: Does hepatitis C virus core protein promote liver cancer through androgen-receptor mediated signaling?

Abstract Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinoma (HCC), which is one of the male dominant diseases. Androgen signaling in liver may be related to carcinogenesis. In this study, we investigated whether HCV proteins cross-talk with androgen receptor (AR) signaling pathway for promotion of liver cancer. We have demonstrated that HCV core protein alone or in context with other HCV proteins enhances AR-mediated transcriptional activity, and further augments in the presence of androgen. Subsequent study suggested that HCV core protein activates STAT3, which in turn enhances AR-mediated transcription. This activity was blocked by a pharmacological inhibitor of Jak/Stat signaling pathway, AG490. Vascular endothelial growth factor (VEGF) is a target gene of androgen receptor in liver, and plays an important role in angiogenesis. Therefore, we examined whether HCV infection modulates VEGF expression in hepatocytes. Our results demonstrated that HCV enhances VEGF expression and facilitates tube formation in human coronary microvascular endothelial cells in the presence of AR. We are in the process of evaluating the AR targets in the hepatocytes following HCV infection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5350.

P16 Modulates VEGF Expression via Its Interaction With HIF-1α in Breast Cancer Cells

ABSTRACTThe degree of tumor progression (such as growth, angiogenesis, and metastasis) directly correlates with the expression of vascular endothelial growth factor (VEGF), but inversely correlates with the expression of tumor-suppressor gene p16, therefore we examined whether the restoration of p16 in breast cancer cells would modulate VEGF expression. Adenoviralmediated p16 expression downregulated VEGF gene expression in breast cancer cells, and inhibited breast cancer cell-induced angiogenesis by a dorsal air sac model in mice. Moreover, p16 appears to form a complex with HIF-1a, the transcription factor for the VEGF gene promoter. Taken together, the binding between p16 and HIF-1a protein may alter HIF-1a's ability to transactivate VEGF expression.

ERK5 Contributes to VEGF Alteration in Diabetic Retinopathy

Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.

Immunologic Mechanisms Mediate Benefits of Lutenizing Hormone-Releasing Hormone (LH-RH) in High Risk Premenopausal Breast Cancer Patients (HR-Pre-BC).

Abstract Background: Systematic reviews have found that LH-RH agonists are effective in the treatment of premenopausal women with early breast cancer. In an extended phase II study, 180 premenopausal women with HR-Pre-BC were treated with an LH-RH analogue in order to protect ovaries from the damage of chemotherapy. We hypothesized that the benefits of LH-RH administration could extend, far beyond ovarian protection, to the improvement of immune function and to the decrease of vascular endothelial growth factor (VEGF). In fact estradiol (E2) at physiological doses not only expands T regulatory cells (T-regs) in different tissues but also increases expression of the Foxp3 gene, a hallmark for CD4+CD25+ T-reg cell function, and the interleukin-10 gene as well. In addition, recent studies indicate that E2 modulates VEGF expression in breast cancer cells through transcriptional activation.Methods: From 05-1997 to 05-2007 180 patients with HR-Pre-BC were entered into the study. At baseline, one week before chemotherapy, patients received an LH-RH analogue which was continued for five years. Adjuvant chemotherapy was tailored to the peculiar biologic features of each patient, including cyclophosphamide, methotrexate, and 5-fluorouracil in 24 patients, an anthracycline and taxane based regimens in 156 patients (40 with positive c-erb-2 tumors). Seventeen patients with &amp;gt; 10 positive axillary lymph nodes and 36 patients with triple negative tumors, received a platinum-based consolidation high-dose chemotherapy with or without autologous peripheral blood progenitor cell transplantation, respectively. Radiation therapy was administered to 145 patients. During the 5 years of LH-RH analogue, patients received a strong psychological support, biphosphonates, and after the end of chemotherapy, estrogen receptor positive (ER+) patients received an aromatase inhibitor.Results: Characteristics of patients. The mean patient age was 42 years (range, 26–50 yrs). One hundred-twenty five women were ER+, 55 were estrogen receptor negative (ER-). Total number of positive nodes was 479, with a mean of 2.6 for each patient (range 0-25). UICC stage was II in 114 women and III in 66 women. All patients had their serum E2 suppressed to values &amp;lt; 40 pg/mL. A statistically significant improvement was observed in lymphocyte number (p&amp;lt; 0.005), and a decrease in T-reg number (p&amp;lt; 0.005) and VEGF (p&amp;lt; 0.005) with respect to baseline values, after LH-RH administration. After a median follow-up of 97 months, 10-years overall progression-free survival and overall survival rate were both 83.2% with no statistical significant difference between ER+ and ER- patients.Conclusions: These data show that the administration of an LH-RH analogue improves lymphocyte number, decreases T-regs and VEGF, and seems to improve the expected outcome of HR-Pre-BC patients through immunologic mediated mechanisms. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4092.