The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation

The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.     

CSR1 suppresses tumor growth and metastasis of prostate cancer

Prostate cancer is frequent among men over 45 years of age, but it generally only becomes lethal with metastasis. In this study, we identified a gene called cellular stress response 1 (CSR1) that was frequently down-regulated and methylated in prostate cancer samples. Survival analysis indicated that methylation of the CSR1 promoter, and to a lesser extent down-regulation of CSR1 protein expression, was associated with a high rate of prostate cancer metastasis. Forced expression of CSR1 in prostate cancer cell lines DU145 and PC3 resulted in a two- to threefold decrease in colony formation and a 10-fold reduction in anchorage-independent growth. PC3 cells stably expressing CSR1 had an average threefold decrease in their ability to invade in vitro. Expression of CSR1 in PC3 cell xenografts produced a dramatic reduction (>8-fold) in tumor size, rate of invasion (0 versus 31%), and mortality (13 versus 100%). The present findings suggest that CSR1 is a potent tumor sup-pressor gene.     

EphrinA1 inhibits vascular endothelial growth factor-induced intracellular signaling and suppresses retinal neovascularization and blood-retinal barrier breakdown

The Eph receptor/ephrin system is a recently discovered regulator of vascular development during embryogenesis. Activation of EphA2, one of the Eph receptors, reportedly suppresses cell proliferation and adhesion in a wide range of cell types, including vascular endothelial cells. Vascular endothelial growth factor (VEGF) plays a primary role in both pathological angiogenesis and abnormal vascular leakage in diabetic retinopathy. In the study described herein, we demonstrated that EphA2 stimulation by ephrinA1 in cultured bovine retinal endothelial cells inhibits VEGF-induced VEGFR2 receptor phosphorylation and its downstream signaling cascades, including PKC (protein kinase C)-ERK (extracellular signal-regulated kinase) 1/2 and Akt. This inhibition resulted in the reduction of VEGF-induced angiogenic cell activity, including migration, tube formation, and cellular proliferation. These inhibitory effects were further confirmed in animal models. Intraocular injection of ephrinA1 suppressed ischemic retinal neovascularization in a dose-dependent manner in a mouse model. At a dose of 125 ng/eye, the inhibition was 36.0 +/- 14.9% (P < 0.001). EphrinA1 also inhibited VEGF-induced retinal vascular permeability in a rat model by 46.0 +/- 10.0% (P < 0.05). These findings suggest a novel therapeutic potential for EphA2/ephrinA1 in the treatment of neovascularization and vasopermeability abnormalities in diabetic retinopathy.     

Genistein induces growth arrest and suppresses telomerase activity in brain tumor cells

Genistein, a soy isoflavone, has been reported to exhibit multiple effects, such as inducing cell cycle arrest, triggering apoptosis, inhibiting the activation of NF_KB and inactivating several signaling cascades in human cancer cells. In vivo studies demonstrating antiangiogenesis and antimetastatic effects of genistein have also been reported. Here, we demonstrate that genistein inhibits the growth of glioblastoma multiforme and medulloblastoma cells with different TP53 mutations and radio‐responses by arresting the cells at G2/M phase of the cell cycle. The cell cycle arrest was found to be independent of DNA damage and such an arrest was sustainable for at least 10 days even after drug removal. Annexin V staining revealed absence of apoptotic or necrotic cell populations after genistein treatment. This supports the observation that genistein induces insignificant DNA damage and indicates that the cell cycle arrest triggered does not lead to cell death. Gene and protein expression studies reveal similar changes in the same pathways following treatment in the cell types tested. Genistein was also able to inhibit telomerase activity resulting in telomere shortening. Thus, we demonstrate, for the first time, that genistein induces growth arrest in association with telomerase inhibition in brain tumor cells via the suppression of TR‐ and TERT mRNA. By elucidating the mechanisms of anticancer effects after genistein treatment in brain tumor cells, there will be a premise for the incorporation of genistein dietary sources to complement radiotherapy in brain tumor patients. © 2012 Wiley Periodicals, Inc.     

In vivo inhibition of tumor angiogenesis by a soluble VEGFR-2 fragment

The interaction of vessel endothelial cell growth factor (VEGF) and its receptors (flt-1, FLK-1/KDR) regulates tumor angiogenesis. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for antitumor angiogenesis biological therapy. Our study extracted sFLK-1 fragment from embryo mouse liver using RT-PCR, recombined it to retrovirus vector, and transfected it to tumor cell lines (S180 and B16) by the liposome mediated method, then we observed the biological behavior of transgenic cells in vivo. The results are: (1) Fragment (1034 bp) was extracted from E9, E11 embryo mouse liver tissue, which was identified by sequence analysis. (2) This fragment was cloned to retrovirus vector (PLXSN vector), which was further transfected to tumor cells lines (S180 and B16). SDS-PAGE indicated the suspension of transgenic cells present sVEGFR-2(sFLK-1) fragment; Western blot identified it. (3) In vivo study showed that the weight and size of tumor in the group of transgenic cells were smaller than in control groups. Microvessel density (MVD) and FLK-1 expression were obviously different between transgenic and control groups, but there were no differences in VEGF expression between transgenic and control groups. In short, the isolated soluble VEGFR2 fragment transfected to tumor cells can be secreted to extracellular suspension and can inhibit tumor angiogenesis in vivo.     

Hepatocyte growth factor suppresses tumor cell apoptosis in nasopharyngeal carcinoma by upregulating Bcl-2 protein expression

Hepatocyte growth factor (HGF) is a multifunctional cytokine, but cell apoptosis related to HGF in nasopharyngeal carcinoma (NPC) and the potential mechanisms involved have not yet been identified. In this study, we aimed at determining whether HGF is a potent inhibitor of cell apoptosis in NPC, and tried to find out which antiapoptotic or proapoptotic protein is involved in this process.     

The significance of VEGF-C/VEGFR-2 interaction in the neovascularization and prognosis of nephroblastoma (Wilms' tumour)

AIM: To investigate the immunohistochemical expression of vascular endothelial growth factor (VEGF)-C and VEGFR-2 in nephroblastoma tissue and correlate their presence with the survival rate of children diagnosed with stage III Wilms' tumour. METHODS AND RESULTS: The material included nephroblastoma tissue obtained from 25 children hospitalized in the Department of Paediatric Oncology, Haematology and Transplantology between 1997 and 2003. VEGF-C and VEGFR-2 expression was evaluated by immunohistochemical assay. VEGF-C was expressed in all cells of the blastemal component and in 30% of tumour cells in the stromal part. It was absent from epithelial elements. VEGFR-2 expression was spread over the surface of numerous stromal cells as well as all the epithelial cells forming dysplastic tubules. The blastemal component of Wilms' tumour was VEGFR-2-negative. VEGF-C-immunopositive stromal cells were situated in the closest proximity to VEGF-C-immunonegative but VEGFR-2-immunoreactive tubules. VEGF-C expression was of prognostic value for both clinical progression (P = 0.0005) and tumour-related death (P = 0.0365). CONCLUSIONS: VEGF-C expression in Wilms' tumour constitutes a potent unfavourable risk factor and may direct future antiangiogenic treatment strategies. The proximity of VEGF-C and VEGFR-2 in the stromal and epithelial components of nephroblastoma could be the neoplastic equivalent of the binary VEGF-C function observed in epithelial and endothelial morphogenesis.     

NOX2 in autoimmunity,tumor growth and metastasis

Myeloid cell NADPH oxidase isoform 2 (NOX2) generates reactive oxygen species (ROS) that participate in defense against microbial pathogens. Humans with compromised NOX2-mediated ROS formation develop chronic granulomatous disease characterized by recurrent bacterial and fungal infections. Additionally, impaired NOX2 function entails hyperactive lymphocytes and autoimmunity in humans and in murine models. The impact of NOX2 and ROS on cancer development is only partly explored. Recent research published in the Journal of Pathology showed that genetic depletion of any of the NOX2 subunits Cyba, Cybb, Ncf1, Ncf2 and Ncf4 reduced the formation of lung metastases following intravenous injection of murine tumor cells. These findings, together with the role of NOX2 in maintaining self-tolerance, imply that NOX2 is a targetable immune checkpoint in cancer. In particular, the possibility of modulating NOX2 to improve lymphocyte-mediated control of metastatic cells merits further investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

High levels of vascular endothelial growth factor and its receptors (VEGFR-1, VEGFR-2, neuropilin-1) are associated with worse outcome in breast cancer

Vascular endothelial growth factor has been shown to be up-regulated in breast cancers. Vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, are the principal mediators of its effects. Together with VEGFR-1 and VEGFR-2, neuropilin-1 may act as a coreceptor for vascular endothelial growth factor. Although vascular endothelial growth factor exerts important effects on endothelial cells, VEGFRs are likely present on tumor cells as well. We used AQUA to analyze tumor-specific expression of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 on a large cohort of breast cancer tissue microarray. Two-fold redundant arrays were constructed from 642 cases of primary breast adenocarcinomas. Automated image analysis with AQUA (Automated Quantitative Analysis) was then performed to determine a quantitative expression score. Scores from redundant arrays were normalized and averaged. Kaplan-Meier survival analysis showed that high levels of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 were all significantly associated with survival (Miller Siegmeund corrected P = .0020, .0160, and .0320, respectively). In addition, vascular endothelial growth factor and neuropilin-1 retained a significant association with survival independent of other standard prognostic factors. Vascular endothelial growth factor, VEGFR-1 and -2, and neuropilin-1 are expressed to varying degrees in primary breast cancers and have prognostic significance. Further study of the functional significance of this finding is warranted as well as the prognostic value of these biomarkers in other tumor microenvironment-specific compartments (eg, vessels).     

Immunolocalization of VEGF, VEGFR-1 and VEGFR-2 in lung tissues after acute hemorrhage in rats

In treatment of hypovolemia it is important to reestablish normal tissue hemodynamics after fluid resuscitation. Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) have been identified as important in many physiological and pathological processes. In this study, we aimed to investigate the histo-physiological effects of VEGF, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1) in resuscitation with different plasma substitutes on lung tissues after acute hemorrhage in rats. Male Sprague-Dawley rats (n=25) were used in this study. The left femoral vein and artery were cannulated for the administration of volume expanders and for direct measurement of mean arterial blood pressure (MAP) (Power-Lab) and heart rate (HR). Fifteen rats were bled (5 ml/10 min) and infused (5 ml/5 min) with one of three randomly selected fluids: (a) dextran-70 (Macrodex); (b) gelatin (Gelofusine); or (c) physiological saline (PS, 0.9% isotonic saline) solutions. Five rats were bled and none were infused (hypovolemia group) and five rats were untreated as the control group. At the end of the experiment, rats were sacrificed and lung tissues were removed for routine processing and paraffin wax embedding. Sections of tissue were stained with hematoxylin and eosin (H&E) and selected blocks were then prepared for indirect immunohistochemical labeling for anti-VEGF, anti-VEGFR-1 and anti-VEGFR-2 primary antibodies. It was observed that both MAP and HR decreased parallel to blood withdrawn in this time interval. The MAP and HR were restored in the following periods. In the control rats, positive immunoreactivity of VEGF and its receptors (VEGFR-1 and VEGFR-2) were detected in respiratory epithelial cells, respiratory and vascular smooth muscle cells, alveolar cells and endothelial cells. While strong immunoreactivities of VEGF and VEGFR-1 were observed in the hypovolemia group, only moderate immunoreactivity of VEGFR-2 was seen in this group. Moderately strong immunolabeling of VEGF and VEGFR-1 were observed in the dextran-70, gelatin and PS resuscitated groups, whereas only weak immunolabeling of VEGFR-2 was observed in these groups. In summary, the vascular protecting effects of these factors were observed with fluid resuscitation, contributing to the pathophysiological changes seen in hypovolemia.     

转入WIF-1或5-氮杂-2’-脱氧胞苷去甲基化抑制骨肉瘤模型鼠的肿瘤生长

BACKGROUND:WIF-1 is a tumor suppressor gene. Promoter hypermethylation causes WIF-1 down- regulation in most tumors. DNA methylation inhibitor can lead to gene demethylation and restore its expression. OBJECTIVE:To observe the differences of tumor pathology and, WIF-1 mRNA and protein changes using WIF-1 or 5-aza-2'-deoxycytidine demethylation in animal models of osteosarcoma. METHODS:Murine osteosarcoma models were established and divided into three groups. In the control group, no treatment was given. In the 5-aza-2'-deoxycytidine group, an appropriate amount of 5-aza-2'-deoxycytidine was injected in each mouse daily. In the WIF-1 group, an appropriate amount of Wnt/β-catenin signal transduction pathway inhibitor WIF-1 was injected in each mouse daily. Seven days after medication, the weight of nude mouse was weighed every 7 days. Short tumor diameter (a) and the long diameter (b) were measured. The relative tumor volume was calculated. The relative growth rate of tumor was calculated at 7, 14, 21, 28 and 56 days. Four nude mice from ach group were sacrificed by pulling the neck at 7, 14, 21, 28 and 56 days after medication. Tumor tissues were stripped and the weight of them was weighed. Pathological analysis of the tumor was conducted. The expression of WIF-1 protein and WIF-1 mRNA was detected in osteosarcoma at 56 days after medication in the three groups. RESULTS AND CONCLUSION: (1) Compared with the medication and control groups, the weight of nude mice was increased at 7, 14, 21, 28 and 56 days in the treatment group. No significant difference was found between the medication and control groups. (2) The tumor size was significantly smaller in the medication group than in the control group. WIF-1 mRNA and WIF-1 protein expression was increased in the medication group compared with the control group to different degrees. (3) Results suggested that WIF-1 gene promoter methylation is one of the mechanisms of the development of osteosarcoma. Use of WIF-1 or 5-aza-2'-deoxycytidine demethylation can inhibit tumor growth in animal models of osteosarcoma.     

Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006)

Activation of the Raf-MEK-ERK signal transduction pathway in endothelial cells is required for angiogenesis. Raf is the kinase most efficiently inhibited by the multikinase inhibitor sorafenib, which has shown activity against certain human cancers in clinical trials. To understand the mechanisms underlying this activity, we studied how it controlled growth of K1735 murine melanomas. Therapy caused massive regional tumor cell death accompanied by severe tumor hypoxia, decreased microvessel density, increased percentage of pericyte-covered vessels, and increased caliber and decreased arborization of vessels. These signs of K1735 angiogenesis inhibition, along with its ability to inhibit Matrigel neovascularization, showed that sorafenib is an effective anti-angiogenic agent. Extracellular signal-regulated kinase (ERK) activation in tumor endothelial cells, revealed by immunostaining for phospho-ERK and CD34, was inhibited, whereas AKT activation, revealed by phospho-AKT immunostaining, was not inhibited in K1735 and two other tumor types treated with sorafenib. Treatment decreased endothelial but not tumor cell proliferation and increased both endothelial cell and tumor cell apoptosis. These data indicate that sorafenib's anti-tumor efficacy may be primarily attributable to angiogenesis inhibition resulting from its inhibition of Raf-MEK-ERK signaling in endothelial cells. Assessing endothelial cell ERK activation in tumor bio-psies may provide mechanistic insights into and allow monitoring of sorafenib's activity in patients in clinical trials.     

Pentoxifylline suppresses interleukin-2-mediated activation of immature human natural killer cells by inhibiting endogenous tumor necrosis factor-alpha secretion

We recently reported that immature human peripheral blood-derived natural killer (NK) cells, the free NK subset, can be activated by interleukin-2 (IL-2) to become killer cells and to undergo proliferation. Activation by IL-2 is dependent on endogenous secretion of tumor necrosis factor-alpha (TNF-) by the free cells. Because pentoxifylline (PTX) inhibits TNF- synthesis and secretion in monocytes, we hypothesized that PTX may also inhibit TNF- secretion by NK cells and thus would inhibit IL-2-mediated activation of free cells. The free NK cells were separated from purified NK cells by flow cytometry and cell sorting of non-target binding cells. IL-2-mediated secretion of TNF- by the free cells was inhibited by PTX. In the presence of PTX, IL-2-mediated activation of free cells into cytotoxic function, proliferation, and recruitment of binder and killer cells was markedly inhibited. Also, PTX inhibited IL-2-triggered upregulation of the expression of CD69, CD25, ICAM-1, and p75TNF-R on the cell surface. These findings demonstrate that PTX has a marked suppression on IL-2-mediated activation of immature free NK cells and that the suppression is due, in large part, to PTX-mediated inhibition of endogenous TNF- secretion. The implication of these findings in the clinical use of PTX for therapy is discussed.     

The aspartic protease napsin A suppresses tumor growth independent of its catalytic activity

Members of the aspartic protease family have been implicated in cancer progression. The aspartic protease napsin A is expressed in type II cells of the lung, where it is involved in the processing of surfactant protein B (SP-B). Napsin A is also expressed in kidney, where its function is unknown. Here, we examined napsin A mRNA expression in human kidney tissues using in situ hybridization. Whereas strong napsin A mRNA expression was observed in kidney proximal tubules, expression was detected in only one of 29 renal cell carcinomas. This result is consistent with previous observations of loss of napsin A expression in high-grade lung adenocarcinomas. We re-expressed napsin A in the tumorigenic HEK293 kidney cell line and examined the phenotype of stably transfected cells. Napsin A-expressing HEK293 cells showed an altered phenotype characterized by formation of cyst-like structures in three-dimensional collagen cultures. Napsin A-expressing cells also showed reduced capacity for anchorage-independent growth and formed tumors in SCID mice with a lower efficiency and slower onset compared to vector-transfected control cells. Mutation of one of the aspartic acid residues in the napsin A catalytic site inactivated enzymatic activity, but did not influence the ability to suppress colony formation in soft agar and tumor formation. The mutation of the catalytic site did not affect processing, glycosylation or intracellular localization of napsin A. These data show that napsin A inhibits tumor growth of HEK293 cells by a mechanism independent of its catalytic activity.     

1,6-O,O-Diacetylbritannilactone suppresses activation of mast cell and airway hyper-responsiveness

Mast cells play critical roles in allergic disorders such as atopic dermatitis and allergic asthma. The aim of this study was to investigate the anti-inflammatory and anti-asthmatic activities of 1,6-O,O-diacetylbritannilactone (OODBL) isolated from Inula japonica Thunb. (I. japonica) in a murine asthma model and bone marrow-derived mast cells (BMMCs). In an ovalbumin-induced asthma model, OODBL administration attenuated the airway hyper-responsiveness induced by aerosolized methacholine and serum IgE level in asthmatic mice. In vitro system, we found that OODBL reduced leukotriene C₄ production and degranulation through the suppression of cytosolic phospholipase A₂ phosphorylation and phospholipase Cγ-mediated Ca²⁺ influx in IgE/antigen-stimulated BMMCs. Taken together, OODBL may have therapeutic potential in the treatment of allergic diseases such as asthma.