Inhibition Of Vascular Endothelial Growth Factor Secretion Research Articles

Red Wine Extract Inhibits VEGF Secretion and Its Signaling Pathway in Retinal ARPE-19 Cells to Potentially Disrupt AMD

Age-related macular degeneration (AMD) is a degenerative disease of the retina where the molecular mechanism involves the production of vascular endothelial growth factor (VEGF), a factor of poor prognosis of the progression of the disease. Previous studies have shown that resveratrol, a polyphenol of grapevines, can prevent VEGF secretion induced by stress from retinal cells. Considering the fundamental role played by VEGF in development and progression of AMD, we investigate the potential effect of red wine extract (RWE) on VEGF secretion and its signaling pathway in human retinal cells ARPE-19. To examine the effect of RWE in ARPE-19, a quantitative and qualitative analysis of the RWE was performed by HPLC MS/MS. We show for the first time that RWE decreased VEGF-A secretion from ARPE-19 cells and its protein expression in concentration-dependent manner. RWE-induced alteration in VEGF-A production is associated with a down of VEGF-receptor 2 (VEGF-R2) protein expression and its phosphorylated intracytoplasmic domain. Subsequently, the activation of kinase pathway is disturbing and RWE prevents the phosphorylation of MEK and ERK 1/2 in human retinal cells ARPE-19. Finally, this study sheds light on the interest that the use of polyphenolic cocktails could represent in a prevention strategy.

Ethyl Acetate Fraction of Teucrium Polium Extract Abolishes Human Umbilical Vein Endothelial Cells (HUVEC) Tubulogenesis in Collagen Bed through Suppression of Cell Proliferation/VEGF Secretion.

Angiogenesis has essential role in growth and metastasis of tumors. Development of therapies aimed to suppress angiogenesis using medicinal plants is one of the effective approaches for prevention/treatment of cancer. The current study was performed to investigate in vitro anti-angiogenic effect of Teucrium Polium (TP) extract and its fractions.The aerial part of Teucrium Polium was powdered and extracted with 50% ethanol. The extract was fractionated in to aqueous (AQ), n-butanol (BU), ethyl acetate (EA) and n-hexane (HE) fractions. Anti-angiogenic effect of TP was examined on human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen matrix. The endothelial cells form capillary-like branches that can be visualized using phase contrast microscope and the number of tube-like structures can be quantified as a measure of in vitro angiogenesis. Furthermore, anti-proliferative and vascular endothelial growth factor(VEGF )suppressive effect of TP as important factors in the process of angiogenesis were assessed using3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)and quantitative ELISA, respectively.Based on our findings, among the TP fractions, EA fraction showed the highest inhibitory activity on angiogenesis. This fraction with IC50: 68 µg/mL, inhibited angiogenesis at 60 µg/mL. The crude extract and other fractions of TP inhibited angiogenesis in a dose-dependent manner at doses higher concentrations than EA fraction, significantly.TP extract and EA fraction were able to inhibit proliferation of HUVEC and inhibited VEGF secretion in a dose dependent manner. The ethyl acetate fraction at 60 µg/ml inhibited VEGF secretion perfectly. Our data indicated that ethyl acetate fraction of Teucrium Polium could be a potential candidate for the prevention of angiogenesis in cancer and other related disorders. However, this suggestion needs more quantitative and in vivo investigations for confirmation.

Apatinib enhances the radiosensitivity of the esophageal cancer cell line KYSE-150 by inducing apoptosis and cell cycle redistribution.

To determine the radiosensitizing effect of apatinib on esophageal cancer cells, and to preliminarily investigate the underlying mechanism, KYSE-150 cells were treated with apatinib, x-ray or apatinib combined with x-ray, and compared with a blank control. It was observed that apatinib significantly inhibited vascular endothelial growth factor (VEGF) secretion and the proliferation of KYSE-150 cells in a dose-dependent manner. As the concentration of apatinib increased, the radiobiological parameters inactivation dose (D0), quasi domain does (Dq) and survival fraction (SF2) of KYSE-150 cells decreased, while the sensitization enhancement ratio SERD0 increased. The rate of apoptosis in cells treated with apatinib and x-ray was markedly higher compared with those of the blank control, x-ray and apatinib alone groups (P<0.05). The proportion of cells in the G2/M phase was significantly increased in the apatinib, x-ray and combination groups compared with the blank control group (P<0.05). Compared with the control and x-ray groups, combination treatment did not significantly alter the expression level of polyADP-ribose polymerase (PARP), although it significantly increased the expression of cleaved-PARP (P<0.05). Moreover, the expression of cell serine/threonine-protein kinase-2 (CHK2) was downregulated (P<0.05), whilst expression of the phosphorylated form, pCHK2, was significantly increased (P<0.05) in the combination group when compared with the control and x-ray groups. In conclusion, the present study suggested that apatinib increases the radiosensitivity of KYSE-150 esophageal cancer cells by inhibiting VEGF secretion and cell proliferation, and promoting apoptosis and cell cycle redistribution.

Anti-neovascularization effects of DMBT in age-related macular degeneration by inhibition of VEGF secretion through ROS-dependent signaling pathway.

Choroidal neovascularization (CNV) is the hallmark of late-staged wet age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is a key component in the development and progression of wet AMD. DMBT, 6,6'-bis(2,3-dimethoxybenzoyl)-α,α-D-trehalose, had been proved that it could suppress tumor angiogenesis and metastasis by inhibiting production of VEGF. But the effects of DMBT on CNV were not known. This study was to investigate effects and mechanisms of DMBT on CNV in vitro and in vivo. Results showed that DMBT could inhibit migration and tube formation of RF/6A cells under ARPE-19 hypoxia conditioned medium. DMBT could reduce lesion area in laser-induced CNV model mice. ELISA and Western blotting assay showed that DMBT markedly inhibited secretion of VEGF in vitro and in vivo. Furthermore, DMBT restrained ROS level under hypoxia via suppressing Nrf2/HO-1 pathway. DMBT effectively suppressed hypoxia-induced the up-regulation of p-Akt, p-NF-κB, and HIF-1α. These results suggest that DMBT can inhibit CNV by down-regulation of VEGF in retina through Akt/NF-κB/HIF-1α and ERK/Nrf2/HO-1/HIF-1α pathway. DMBT might be a promising lead molecule for anti-CNV and serve as a therapeutic agent to inhibit CNV.

Abstract A127: The identification and development of selective natural product inhibitors of hypoxia inducible factor-2α for the treatment of renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant accumulation of the hypoxia inducible factor-alpha subunit (HIF-α), which contributes to tumor growth, angiogenesis, and metastasis. Recent studies suggest that this normoxic stabilization of HIF-1α is not sufficient to reproduce tumorigenesis, and HIF-1α is thought to act as a tumor suppressor in ccRCC. Conversely, HIF-2α acts as an oncogene, turning on transcription of a large number of HIF-regulated genes, and upregulation of HIF-2α is often associated with poor prognosis. To this end, two HIF-2α selective inhibitors are currently in clinical trials for RCC. To isolate additional HIF-2α selective compounds for use as research tools and drug development leads, a cell-based high-throughput screen (HTS) of the NCI Natural Products Repository was performed. Stably transformed clones of the human ccRCC-derived cell line 786-0 expressing a luciferase reporter construct downstream of the human vascular endothelial growth factor (VEGF) gene promoter were used to identify natural product samples with inhibitory activity and minimal cellular toxicity. HTS leads were chromatographically separated into component structures yielding ~40 pure compounds with micromolar or submicromolar IC50 values, &amp;gt;80% inhibition, and &amp;lt;10% cell toxicity. These hits were then screened for their ability to inhibit VEGF-A protein secretion by VHL- and HIF1A-negative 786-0 cells, and several compounds showed &amp;gt;90% inhibition of VEGF secretion at micromolar doses under normoxic conditions. Patterns of mRNA expression of HIF-1α, HIF-2α, and several HIF target genes in cell lines engineered to express either HIF alone or both were studied to help identify HIF-2α-selective inhibitors. Several lead compounds reduced VEGF transcription and secretion, and downregulated HIF-2α target genes. To further characterize the HTS leads, ICM-Pro protein/small-molecule docking software was used to dock the ligands of interest to high-resolution HIF-2α and HIF-1α 3D structures. The HIF-2α selective inhibitor currently undergoing clinical investigation, PT2385, has been found to bind directly to a hydrophobic pocket of the HIF-2α:ARNT dimerization. Using the PT2385 binding mode as a benchmark, docking studies of the HTS leads were performed and the results have been linked to empirical data in an effort to explain selectivity for HIF-2α over HIF-1α, reveal key favorable interactions between the protein and ligand, define mode of action, and aid in future drug development studies. Citation Format: Molly M. Lee, Daniel C. Rabe, Tawnya C. McKee, Girma M. Woldemichael, James R. Vasselli, James B. McMahon, W. Marston Linehan, Donald P. Bottaro. The identification and development of selective natural product inhibitors of hypoxia inducible factor-2α for the treatment of renal cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A127.

Secretion-mediated STAT3 activation promotes self-renewal of glioma stem-like cells during hypoxia

High-grade gliomas (HGGs) include the most common and the most aggressive primary brain tumor of adults and children. Despite multimodality treatment, most high-grade gliomas eventually recur and are ultimately incurable. Several studies suggest that the initiation, progression, and recurrence of gliomas are driven, at least partly, by cancer stem-like cells. A defining characteristic of these cancer stem-like cells is their capacity to self-renew. We have identified a hypoxia-induced pathway that utilizes the Hypoxia Inducible Factor 1α (HIF-1α) transcription factor and the JAK1/2-STAT3 (Janus Kinase 1/2 - Signal Transducer and Activator of Transcription 3) axis to enhance the self-renewal of glioma stem-like cells. Hypoxia is a commonly found pathologic feature of HGGs. Under hypoxic conditions, HIF-1α levels are greatly increased in glioma stem-like cells. Increased HIF-1α activates the JAK1/2-STAT3 axis and enhances tumor stem-like cell self-renewal. Our data further demonstrate the importance of Vascular Endothelial Growth Factor (VEGF) secretion for this pathway of hypoxia-mediated self-renewal. Brefeldin A and EHT-1864, agents that significantly inhibit VEGF secretion, decreased stem cell self-renewal, inhibited tumor growth, and increased the survival of mice allografted with S100β-v-erbB/p53−/− glioma stem-like cells. These agents also inhibit the expression of a hypoxia gene expression signature that is associated with decreased survival of HGG patients. These findings suggest that targeting the secretion of extracellular, autocrine/paracrine mediators of glioma stem-like cell self-renewal could potentially contribute to the treatment of HGGs.

Tanshinone IIA Inhibits VEGF Secretion and HIF-1α Expression in Cultured Human Retinal Pigment Epithelial Cells under Hypoxia

ABSTRACTPurpose: The current work intends to study the activity of tanshinone IIA on secretion of vascular endothelial growth factor (VEGF) and expression of hypoxia inducible factor 1α (HIF-1α) in human retinal pigment epithelial cells (ARPE-19 cells) under hypoxic condition.Methods: The cytotoxicity of tanshinone IIA was tested in ARPE-19 cells by MTT assay. ARPE-19 cells were incubated with different concentrations of cobalt chloride (100, 150, and 200 µM) for 12 h, and levels of expressed HIF-1α and secreted VEGF were quantified through Western blot and ELISA, respectively. Further, ARPE-19 cells were pretreated for 1 h with different concentrations of tanshinone IIA (5, 10, 15, and 18 µM). After 1 h, the cells were subjected to hypoxic condition using 150 µM cobalt chloride for 12 h in the presence and absence of tanshinone IIA. The cells were then harvested, and the secreted VEGF and expressed HIF-1α was studied.Results: Tanshinone IIA at concentrations of 5, 10, 15, and 18 μM did not show cytotoxicity in ARPE-19 cells. Chemical hypoxia induced by cobalt chloride caused a significant increase in VEGF level in a dose-dependent manner, and HIF-1α expression peaked at 150 µM. Based on the data, cobalt chloride concentration was maintained at 150 μM for further studies. Tanshinone IIA decreased the level of HIF-1α and VEGF secretion in a dose-dependent manner under hypoxic condition.Conclusion: Tanshinone IIA could be explored as a new potential candidate for treating wet AMD.

Melatonin inhibits angiogenesis in SH-SY5Y human neuroblastoma cells by downregulation of VEGF.

Vascular endothelial growth factor (VEGF) produced from tumor cells plays a crucial role in the pathogenesis and neovascularization of neuroblastoma. Inhibition of VEGF secretion by tumor cells, as well as VEGF-regulated signaling in endothelial cells, are important to reduce the angiogenesis and growth of neuroblastoma. Since melatonin has anti-angiogenic effects in tumor cell lines, the aim of the present study was to study melatonin modulation of the pro-angiogenic effects of VEGF in neuroblastoma cells (SH-SY5Y). We used co-cultures of SH-SY5Y and endothelial cells. VEGF expression and protein levels were analyzed by quantitative RT-PCR and ELISA, respectively. Endothelial cell migration was assessed by wound-healing assay and endothelial angiogenesis by a tube formation assay. Melatonin inhibited the pro-angiogenic effects of SH-SY5Y cells. The conditioned medium collected from the neuroblastoma cells was angiogenically active and stimulated proliferation, migration and tube formation in endothelial cells. This effect was significantly counteracted by the addition of either anti-VEGF or melatonin. Melatonin inhibited VEGF expression and secretion in SH-SY5Y cells, decreasing the levels of VEGF available for endothelial cells. Melatonin has anti-angiogenic effects at different steps of the angiogenic process in SH-SY5Y neuroblastoma cells, through the downregulation of VEGF.

Dopamine agonist inhibits vascular endothelial growth factor protein production and secretion in granulosa cells.

BackgroundDopamine receptor 2 agonists (D2-ags) inhibit vascular endothelial growth factor (VEGF) secretion in luteinized granulosa cells (LGCs) both in vitro and in vivo. However, the mechanism of D2 regulation of the VEGF/VEGF Receptor 2 (VEGFR-2) pathway remains to be elucidated. We sought to determine the effects of D2 signaling on VEGF transcription and translation in LGCs, with the expectation of identifying potential D2-ag-based therapies for ovarian hyperstimulation syndrome (OHSS).FindingsLGCs from egg donors were cultured with chorionic gonadotropin (hCG) in the presence of Actinomycin-D (ActD) or Brefeldin-A (BFA) to evaluate the effects of a D2-ag, cabergoline (Cb2), on VEGF secretion. The contribution of the conventional Gi/Go, Gz and AKT/β-Arrestin pathways in the VEGF regulation was assessed by adding pertussis toxin (PTX), phorbol 12-myristate 13-acetate (PMA), or wortmannin (WT). While Cb2 inhibited VEGF secretion by interfering with VEGF peptide translation and secretion, inhibition of conventional D2 transduction pathways did not reverse Cb2-mediated inhibition of VEGF secretion.ConclusionsThe effects of D2-ag on VEGF translation and secretion are mediated by D2 signaling pathways that have yet to be described. We found that D2-ag inhibits VEGF secretion at the post-transcriptional level, suggesting that D2-ag treatment should be combined with therapies that inhibit VEGF transcription, such as the employment of LH or GnRH for triggering ovulation, to improve the efficacy of OHSS prevention.

Inhibitory effect of thalidomide combined with interferon on the proliferation of Kasumi-1 cells

目的探讨沙利度胺联合干扰素（IFN）对急性髓系白血病细胞系Kasumi-1细胞的增殖抑制作用及其机制。方法沙利度胺、IFN以及两药联合处理Kasumi-1细胞，采用CCK-8法检测细胞增殖抑制率，流式细胞术检测细胞凋亡率，ELISA法检测细胞培养上清血管内皮生长因子（VEGF）水平，Western blot法检测凋亡相关蛋白的表达。结果在50~500 µg/ml范围内沙利度胺可抑制Kasumi-1细胞增殖，并呈浓度依赖性[24 h的IC50值为（451.13±6.92）µg/ml、48 h的IC50值为（362.50±14.52）µg/ml]；在500~5 000 U/ml范围内，IFN浓度依赖性地抑制Kasumi-1细胞增殖[24 h的IC50值为（2 209±127）U/ml、48 h的IC50值为（1 393±63) U/ml]。350 µg/ml沙利度胺和1 400 U/ml IFN作用48 h, Kasumi-1细胞的凋亡率分别为（14.68±2.61）％和（21.71±0.71）%，与对照组[（0.89±0.38）%]相比，差异有统计学意义（P值均<0.01）；两药联合作用后Kasumi-1细胞的增殖抑制率为（88.50±2.40）%，凋亡率为（41.95±3.41）%，与对照组和各单药组比较，差异均有统计学意义（P值均<0.01）。沙利度胺、IFN单药及两药联合组VEGF水平分别为（141.11±3.70）、（119.90±2.00）和（94.61±5.46) ng/L，两药联合组明显低于单药组（P值均<0.05）。沙利度胺、IFN单药作用Kasumi-1细胞48 h能不同程度下调Bcl-2的表达，上调p-P38（同时P38表达量下降）、Bax、细胞色素C、活化型（cleaved）Caspase-3、8、9的表达，与对照组比较差异均有统计学意义（P值均<0.05）；沙利度胺联合IFN该作用进一步加强（P值均<0.05）。结论沙利度胺和IFN可协同抑制Kasumi-1细胞增殖、诱导其凋亡，其可能是通过线粒体和死亡受体途径，并活化P38信号通路诱导细胞凋亡和抑制Kasumi-1细胞VEGF的自分泌来实现。

Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking.

Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.

Effect of 20 (S)-ginsenoside Rg3 on the proliferation inhibition and secretion of vascular endothelial growth factor of multiple myeloma cell line U266

To explore the effect of 20 (S)-ginsenoside Rg3 [20 (S)-Rg3] on the proliferation inhibition and secretion of vascular endothelial growth factor (VEGF) of multiple myeloma (MM) cell line U266. The proliferation inhibition rate of U266 cells after treatment with different doses of 20 (S)-Rg3 was detected by MTT method, the cell cycle and apoptosis by flow cytometry, the expression of apoptosis related proteins of caspase-3, 8 and 9 by Western blot, VEGF concentration in the culture supernatant by ELISA. It showed that 20 (S)-Rg3 could inhibit the proliferation of U266 in a dose-dependent manner (P<0.05) with IC50 of (71.07 ± 2.63)μmol/L and (44.06 ± 3.98) μmol/L at 24 h and 48 h, respectively. VEGF concentration in the culture supernatant showed a dosedependent reduction (P<0.05), decreased from (419.93 ± 36.76) pg/106 cells in the control group to (314.82 ± 27.05) pg/106 cells in 80 μmol/L 20 (S)-Rg3 treated group by ELISA assay. Flow cytometry with Annexin-V/PI double staining revealed that 20(S)-Rg3 may induce U266 cells apoptosis in a concentration-dependent manner from (0.51 ± 0.05)% at control group to (8.32 ± 0.83)%, (10.72 ± 1.29)% and (15.27 ± 2.26)% at 20, 40 and 80 μmol/L treatment groups, respectively (P<0.05). Flow cytometry with PI staining showed that the ratio of cells in G0/G1 phase increased from (49.11 ± 1.71)% to (52.72 ± 7.75)%, (60.29 ± 5.76)% and (61.81 ± 3.46)%, respectively (P<0.05). Western blot analysis indicated that the expression of caspase-3, 8 and 9 declined, and that of cleaved-caspase-3, 8 and 9 significantly increased (P<0.05) with 20 (S)-Rg3 concentration increased. 20(S)-Rg3 can inhibit the proliferation of U266 cells by cell cycle arrest in G1 phase and induce cell apoptosis by increasing the expressions of cleaved-caspase-3, -8 and -9. It can also inhibit VEGF secretion of U266 cells, which makes it a potential agent for multiple myeloma therapy.

Dopamine receptor 2 activation inhibits ovarian vascular endothelial growth factor secretion in vitro: implications for treatment of ovarian hyperstimulation syndrome with dopamine receptor 2 agonists

To ascertain whether vascular endothelial growth factor (VEGF) secretion by luteinized granulosa cells (GCs) is modulated by the dopaminergic system in a dose-dependent fashion and how this is related to the differential efficacy of dopamine receptor 2 (D2)-agonists (D2-ag) in preventing ovarian hyperstimulation syndrome (OHSS). The relationship between the dopaminergic system and VEGF secretion in luteinized GCs was evaluated. Archived human ovaries were immunostained to characterize D2 expression. University affiliated infertility center. Premenopausal women and egg donors. Luteinized GCs were cultured with the D2-ag cabergoline. Human ovarian sections were immunostained for D2. The VEGF was measured by ELISA and D2 expression was evaluated by In-Cell ELISA. The D2 expression throughout the luteal phase was characterized by immunohistochemistry. The VEGF secretion was decreased by the D2-ag in a dose-dependent fashion. The efficiency of this process was correlated with the amount of D2 expressed by luteinized GCs. A decrease in D2 expression in ovarian sections was observed during the late luteal phase. The efficacy of D2-ags in preventing OHSS might rely on their capacity to inhibit VEGF secretion by luteinized GCs. Because this capacity is dose-dependent, increasing the intraovarian concentration of D2-ags should be explored as a means of increasing the efficacy of these drugs in preventing OHSS.

Abstract P2-09-16: Pre-clinical evaluation of novel anti-angiogenic agents as breast cancer therapeutics

Abstract Background There is a need to develop novel drugs that will improve survival in breast cancer patients. Angiogenesis is essential for breast tumour progression. To date, the most promising approach to inhibit angiogenesis in breast cancer patients has been the drug bevacizumab which targets the pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF). Recently, there has been controversy regarding the efficacy of bevacizumab for breast cancer treatment. In clinical trials, bevacizumab failed to establish an overall-survival benefit and was associated with serious toxicities. This resulted in the FDA revoking approval for the drug for the first line treatment of advanced breast cancer. Due to this uncertainty surrounding the efficacy of bevacizumab for the treatment of breast cancer it is clear that there is a clinical need for more effective novel anti-angiogenic drugs with better toxicity profiles for the treatment of breast cancer. Aim This study aimed to identify novel small-molecule anti-angiogenic agents with therapeutic potential in human breast cancer. Methods Compounds with physiochemical properties consistent with drug-like compounds were screened for anti-angiogenic activity by high-throughput screening involving zebrafish larvae. Human breast tumour explants were treated with the lead compound and secretion of angiogenic factors was assessed by ELISA. Results We have identified a novel small-molecule agent ‘SMG1’ that significantly inhibited inter-segmental blood-vessel development in zebrafish and showed no toxicity. Treatment of breast tumour explants with SMG1 significantly inhibited secretion of the potent pro-angiogenic cytokine VEGF (p = 0.01). Furthermore, SMG1 inhibited VEGF secretion more than the standard targeted breast cancer therapies tamoxifen and Herceptin® which have been reported to inhibit angiogenesis. Conclusion Continuing pre-clinical work will determine if SMG1 has potential as a therapeutic agent for human breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-16.

Metronomic chemotherapy combined with dendritic cell vaccine inhibits VEGF secretion

背景与目的肺癌生存期有限，需要探索一种新颖的治疗模式。本研究应用免疫治疗联合小剂量化疗的方法，观察联合治疗的抑瘤效应。方法C57/BL6J小鼠负荷3LL肿瘤，按照不同的治疗模式：对照组、小剂量持续化疗组、树突状细胞（dendritic cells, DCs）生物免疫治疗组和小剂量化疗联合DC生物免疫治疗组；观察不同治疗模式下小鼠的生存时间；应用微渗析分析联合液相芯片分析瘤体内bFGF和VEGF的分泌。结果接受小剂量化疗联合DC生物免疫治疗组的小鼠中位生存时间为（27.6±3.2）天，与对照组（13.5±2.7）天、生物免疫治疗组（13.1±2.3）天、小剂量持续化疗组（11.8±3.0）天间比较，差异具有统计学意义（P < 0.05）。联合治疗组瘤体内在48 h、72 h内VEGF的分泌下降，较其余治疗组的差异具有统计学意义（P < 0.05）。结论小剂量化疗联合DC生物免疫治疗能抑制肿瘤内VEGF的分泌。

Urotensin II Stimulates Vascular Endothelial Growth Factor Secretion From Adventitial Fibroblasts in Synergy With Angiotensin II

The adventitia plays an important role in and is considered to be the initiating site for vascular remodeling. Urotensin II (UII) and angiotensin II (Ang II) are the two most important vascular peptides involved in vascular remodeling in the adventitia. Nevertheless, little is known about their effect on the expression of vascular endothelial growth factor (VEGF). It was hypothesized that both UII and Ang II could induce VEGF expression in adventitial fibroblasts and VEGF may play a role in cell proliferation and collagen I synthesis induced by UII or Ang II. Growth-arrested adventitial fibroblasts were incubated in serum-free medium with UII and/or Ang II and inhibitors of the mitogen-activated protein kinase (MAPK) pathway or VEGF-neutralizing antibodies. The VEGF expression was evaluated using enzyme-linked immunosorbent assay (ELISA), while the proliferation and collagen I synthesis were detected using methyl thiazol tetrazolium (MTT) assay and ELISA. It was found that: (1) both UII and Ang II could stimulate VEGF expression in adventitial fibroblasts and they had a synergistic effect; (2) MAPK pathway inhibitors could inhibit VEGF secretion induced by UII and/or Ang II; and (3) VEGF-neutralizing antibodies could inhibit UII/Ang II-induced cell proliferation and collagen synthesis in adventitial fibroblasts. Induction of VEGF expression may be a new mechanism involved in vascular remodeling for UII and Ang II.

Rosiglitazone, a PPAR-γ agonist, inhibits VEGF secretion by peripheral blood mononuclear cells and ROS production by human leukocytes

To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes. PBMCs and leukocytes were obtained from venous blood samples collected from 20 healthy individuals. VEGF secretion was evaluated using a commercial ELISA kit, while ROS production was determined using a luminol-dependent chemiluminescence assay. Rosiglitazone and calphostin C (a protein kinase C inhibitor) inhibited VEGF secretion by PBMCs by 63.7 and 62.3%, respectively. Both agents reduced ROS production in non-stimulated human leukocytes and down-regulated the enhanced generation of ROS in leukocytes that had been stimulated with the PKC activator phorbol 12,13-dibutyrate. The results support the involvement of PKC as a direct, and/or NADPH-oxidase as an indirect, target for the action of rosiglitazone on VEGF secretion by PBMCs and ROS production in human leukocytes.

Differential Effect of Intraperitoneal Albendazole and Paclitaxel on Ascites Formation and Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Cell-Bearing Athymic Nude Mice

The purposes of our study were to evaluate the effect of intraperitoneal albendazole on tumor growth, ascites formation, and vascular endothelial growth factor (VEGF) mRNA expression, and to assess the synergistic effect of paclitaxel in OVCAR-3-bearing nude mice. In all, 4 groups of mice were injected intraperitoneally with weekly albendazole (450 mg/kg per week), paclitaxel (30 mg/kg per week), albendazole plus paclitaxel, or normal saline for 4 weeks. Ascitic fluid accumulation (2.47, 2.65, 2.88, and 5.90 mL, respectively) and in ascitic VEGF levels were significantly reduced in the 3 treatment groups compared to the control group (170.83, 229.16, 267, and 1625 pg/mL, respectively). However, complete tumor suppression was more prominent in the paclitaxel group, and VEGF mRNA expression was more strongly inhibited in the albendazole group (P < .05). No synergistic effect of albendazole and paclitaxel was observed. We demonstrated a differential effect of albendazole and paclitaxel in a xenograft model of ovarian carcinoma; albendazole suppressed ascites formation by inhibiting VEGF secretion, and paclitaxel exerted its effects by direct cytotoxicity.

Evidences for the Existence of a Low Dopaminergic Tone in Polycystic Ovarian Syndrome: Implications for OHSS Development and Treatment

The dopamine/dopamine receptor 2 (D2/Drd2) pathway modulates vascular endothelial growth factor (VEGF)-dependent vascular permeability and angiogenesis in the ovary. Deregulation of the VEGF/VEGF receptor (VEGFR)-2 pathway leading to increased risk of ovarian hyperstimulation syndrome has been described in the ovary of patients suffering from polycystic ovarian syndrome (PCOS). The objective of the study was to ascertain whether deregulation of the VEGF/VEGFR-2 might a least be partially due to abnormalities of the D2/Drd2 pathway in PCOS women. Dated, archived ovaries from PCOs and control group patients as well as human chorionic gonadotropin-stimulated luteinized granulosa cells form PCOS and non-PCOS oocyte patients were used. The study was conducted at a private research center. PCOS and nonpolycystic ovarian patients and oocyte patients participated in the study. Human ovarian sections were stained against the Drd2 antibody. Human chorionic gonadotropin-stimulated luteinized granulosa cells (LGC) were cultured in the presence/absence and the Drd2 agonist cabergoline. Drd2 and vascularized stained area in the theca layer of antral (< 8 mm) and luteinized follicles was quantified. VEGF, D2, and its related metabolites were measured in the supernatant of cultured LGC by ELISA and HPLC, respectively. VEGFR-2 and Drd2 expressed by LGC was quantified through an In-Cell ELISA. Decreased Drd2 expression and increased vascularization in the theca layer of antral and luteinized follicles of PCOS ovaries was observed. A lower dopamine production and reduced efficacy of cabergoline in inhibiting VEGF secretion was uncovered in LGC from PCOS. Decreased dopaminergic tone as well as deregulated Drd2 signaling might explain higher VEGF and vascularization leading to increased ovarian hyperstimulation syndrome risk in PCOS.

Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo-D-arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo-D-arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.