Inhibition Of VEGF Expression Research Articles

Polyethylene glycol modified protamine-chlorin e6 conjugate nanoparticles for enhanced cancer photodynamic therapy

Photodynamic therapy attracts great attention in cancer treatment due to its minimal invasiveness, good tolerance and specific cancer targeting. However, most of tumors are hypoxic and the tumors will become more hypoxic after photodynamic therapy, which might cause the poor prognosis by inducing tumor angiogenesis. In the present work, polyethylene glycol modified protamine-chlorin e6 conjugate (PEG-protamine-Ce6) was synthesized for enhanced cancer photodynamic therapy. PEG-protamine-Ce6 was synthesized by conjugating protamine with methoxypolyethylene glycol acetaldehyde and Ce6 sequentially. The in vitro studies show that PEG-protamine-Ce6 self-assembles into nanoparticles in aqueous solution with uniform size (∼200 nm), positive zeta potential and spherical morphology. The in vitro cell studies show that PEG-protamine-Ce6 nanoparticles exhibit enhanced cellular uptake amount, intracellular ROS level elevation upon laser irradiation, cell apoptosis and photocytotoxicity as compared to Ce6. The in vivo studies show that PEG-protamine-Ce6 nanoparticles exhibit enhanced antitumor effect as compared to Ce6 upon laser irradiation. PEG-protamine-Ce6 with laser irradiation not only inhibits tumor cell proliferation, but also inhibits tumor angiogenesis by suppression of VEGF expression, whereas Ce6 with laser irradiation promotes tumor angiogenesis by enhancing VEGF expression. PEG-protamine-Ce6 nanoparticles developed in this work offer a novel nanomedicine for enhancing cancer photodynamic therapy.

Leech extract inhibits VEGF expression in WERI-RB-1 cells possibly via VEGF/PI3K/AKT signal pathway

Leech extract inhibits VEGF expression in WERI-RB-1 cells possibly via VEGF/PI3K/AKT signal pathway

HIF-2α-targeted interventional chemoembolization multifunctional microspheres for effective elimination of hepatocellular carcinoma

Transcatheter arterial chemoembolization (TACE) is widely used for the treatment of advanced hepatocellular carcinoma (HCC). However, the long-term hypoxic microenvironment caused by TACE seriously affects the therapeutic effect of TACE. HIF-2α plays a crucial role on the chronic hypoxia process, which might be an ideal target for TACE therapy. Herein, a multifunctional polyvinyl alcohol (PVA)/hyaluronic acid (HA)-based microsphere (PT/DOX-MS) co-loaded with doxorubicin (DOX) and PT-2385, an effective HIF-2α inhibitor, was developed for enhanced TACE treatment efficacy. In vitro and in vivo studies revealed that PT/DOX-MS had a superior ability to treat HCC by blocking the tumor cells in G2/M phase, prompting cell apoptosis, and inhibiting tumor angiogenesis. The antitumor mechanisms of PT/DOX-MS were possibly due to that the introduction of PT-2385 could effectively inhibit the expression level of HIF-2α in hypoxic HCC cells, thereby down-regulating the expression levels of Cyclin D1, VEGF and TGF-α. In addition, the combination of DOX and PT-2385 could jointly inhibit VEGF expression, which was another reason accounting for the combined anti-cancer effect of PT/DOX-MS. Overall, our study demonstrated that PT/DOX-MS is a promising embolic agent for enhanced HCC treatment via the combined effect of hypoxia microenvironment improvement, chemotherapy, and embolization.

Fifty years of research on Tinospora cordifolia: From botanical plant to functional ingredient in foods

Consumer demand, combined with industry interest, highlights the need to develop foods that have health promoting characteristics beyond basic nutrition. In this regard, Tinospora cordifolia ( giloy ) (TC), has paved the way in the food industry because of multi-purpose utilization for health care management. Being reservoir of bioactive components, TC has been exploited since ages for its therapeutic properties. Its’ consumption by people in the crude form has limited its utility for disease treatment and management. The greatest approach to avoid any disease is to prevent it thus, application of TC preparations in the development of functional foods would definitely address this issue and broaden the horizon of food market. However, this is relatively a nascent area and there is an urgent need to investigate the mechanisms and interactions between diverse food matrices and TC bioactive components. This review provides comprehensive information on TC and its health-related properties, elucidating the physiological role in human body and focusing its utilization in food industry. Literature is based on the findings of several researches on its protective role against various patho-physiological diseases. The potential of this medicinal plant as a therapeutic agent against SARS-COV-2 virus has also been reviewed. Research needs of food scientists dealing with development of functional foods using TC has also been catered under this review. About 45 active constituents have been isolated from TC and include alkaloids, glycosides, terpenoids, steroids, polysaccharides, and are mainly responsible for its biological roles. These components provide the defense against diseases by several mechanisms viz., modulation of host immune responses via TLR4 dependent pathway, macrophage activation, inhibiting VEGF expression, activating enzyme system generating cyclic AMP, translocation of nuclear transcription factor etc. TC has also been explored in development of functional foods and edible packaging films as antioxidant, antimicrobial, lipase inhibitor and bioactive ingredient. Extensive research is required to investigate the suitable techniques for the stability of TC preparations in food matrix. • T. cordifolia (TC) is well-recognized plant as a reservoir of therapeutic properties. • Alkaloids and terpenoids are the most active components from TC. • The review describes the potential application of TC in wide variety of foods. • Foods incorporated with TC can improve storage stability and functionality. • Future challenges include stabilization of TC preparation in food matrix and their interactions.

MicroRNA-15b-5p shuttled by mesenchymal stem cell-derived extracellular vesicles protects podocytes from diabetic nephropathy via downregulation of VEGF/PDK4 axis.

Diabetic nephropathy (DN) is a severe complication of diabetes lethal for end-stage renal disease, with less treatment methodologies and uncertain pathogenesis. In the current study, we determined the role of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) containing microRNA (miR)-15b-5p in DN. After extraction and identification of MSC-derived EVs, mouse podocyte line MPC5 was selected to establish an in vitro high-glucose (HG) cell model, where expression of miR-15b-5p, pyruvate dehydrogenase kinase 4 (PDK4) and VEGFA expression in tissues and cells were determined. The loss- and gain- function assays were conducted to determine the roles of miR-15b-5p, PDK4 and VEGFA. MPC5 cells were then co-cultured with MSC-derived EVs and their biological behaviors were detected by Western blot, CCK-8 assay, and flow cytometry. The binding relationship between miR-15b-5p and PDK43 by dual luciferase reporter gene assay. The expression of miR-15b-5p was downregulated in podocytes under HG environment, but highly expressed in mouse MSCs-derived EVs. EVs-derived miR-15b-5p could protect MPC5 cell apoptosis and inflammation. miR-15b-5p inhibited the expression of PDK4 by directly bound to the 3'UTR region of PDK4 gene. miR-15b-5p inhibits VEGF expression by binding to PDK4. Inhibition of PDK4 decreased VEGFA expression and reduced apoptosis and inflammation. Collectively, miR-15b-5p shuttled by MSC-derived EV can play protective roles in HG-induced mouse podocyte injury, possibly by targeting PDK4 and decreasing the VEGFA expression.

Abstract 108: Inhibiting methionine aminopeptidase 2 prevents liver fibrosis and hepatocellular carcinoma

Abstract Background: The methionine aminopeptidase 2 (MetAP2) pathway, a key regulator of angiogenesis progress, has been implicated in common pathologic conditions including diabetes, non-alcoholic steatohepatitis (NASH) and cancer. MetAP2 inhibition suppresses cancer cell proliferation and xenograft tumor growth. In this study we investigated the hypothesis that MetAP2 inhibition can prevent liver fibrosis and hepatocellular carcinoma (HCC) induced with diethylnitrosamine (DEN) in rat models by inhibiting neo-vascularization. Methods: Male Wistar rats received weekly intraperitoneal injections of DEN at 50 mg/kg for 18 weeks. After 8 weeks, rats were randomly assigned to treatment with one of two MetAP2 inhibitors ZGN1345 or ZGN1136 versus control vehicle (n = 10 per group): 1) water (control); or 2) ZGN-1345 (3 mg/kg) by daily gavage; 3) 0.15% DMSO (control); or 4) ZGN-1136 (0.3 mg/kg) by daily subcutaneous injection. All rats were sacrificed at 19 weeks. Serum chemistries were performed to evaluate the liver function. Liver fibrosis was assessed with hydroxyproline measurement, Sirius Red and H&E staining with review by a liver pathologist. Liver tumor nodules were recorded and analyzed with IHC staining. Results: ZGN1345 treatment improved ALT and AST on DEN-induced cirrhosis in rat models. Both inhibitors improved the liver fibrosis, including lowering collagen deposition (hydroxyproline measurement and amount of collagen measured with Sirius Red staining) and improving the Ishak fibrosis score. Both inhibitors reduced the number of tumor nodules and suppressed PCNA expression in the liver. More importantly, both inhibitors suppressed neo-vascularization and angiogenesis by inhibiting VEGF expression. Conclusions: MetAP2 inhibitors ZGN1345 and ZGN1136 effectively prevent DEN-induced liver fibrosis and HCC in rat models, potentially by inhibiting neo-vascularization. These data suggest that inhibition of MetAP2 may represent a new prevention strategy for HCC. Table. Effect of ZGN1345 and ZGN1136 on rat liver fibrosis and HCC.GroupsASTALTHydroxyprolineCollagen (%)Fibrosis score rateNodule numbers (>5 mm)PCNA expressionVEGF expressionWater (Control), PO100.0 ± 35.5100.0 ± 29.5100.0 ± 39.913.1 ± 3.8F4:25.0%,F3:37.5%,F2:37.5%27.4 ± 14.2100.0± 36.4100.0 ± 44.6ZGN-1345, PO45.5 ± 11.1 ***44.7 ± 14.3 ***36.9 ± 10.5 ***4.7 ± 2.2 ***F2:11.1%,F1:77.8%,F0:11.1%5.7 ± 3.9 ***40.1 ± 13.3 ***21.9 ± 9.8 ***0.15% DMSO (Control), SC100.0 ± 28.1100.0 ± 21.1100.0 ± 63.99.2 ± 3.5F3:42.9%,F2:42.9%,F1:14.3%41.1 ± 40.2100.0 ± 22.8100.0 ± 29.4ZGN-1136, SC89.1 ± 11.3107.1 ± 37.242.9 ± 8.6 *2.9 ± 1.7 **F2:14.3%,F1:42.9%,F0:42.9%4.1 ± 2.8 *29.9 ± 9.4 ***52.3 ± 21.6 *** Citation Format: YONGTAO WANG, Mozhdeh Sojoodi, Guoliang Qiao, Zenan Lin, Stephen C. Barrett, Lawrence Zukerberg, Michael Lanuti, Motaz Qadan, Kenneth K. Tanabe. Inhibiting methionine aminopeptidase 2 prevents liver fibrosis and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 108.

Nanodelivery of Resveratrol-Loaded PLGA Nanoparticles for Age-Related Macular Degeneration.

Age-related macular degeneration, precisely neovascular form, is the leading cause of vision loss and the key treatment includes intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. A method to increase local concentration of drug at posterior segment of the eye and to reduce the frequency of intravitreal injections is an unmet need. Resveratrol, a naturally occurring antioxidant and anti-inflammatory polyphenol, was loaded in PLGA polymeric nanoparticles to study their sustained release property and effectiveness in reducing expression of VEGF protein in vitro. Nanoparticles were characterized using FTIR, DSC, size, encapsulation efficiency, TEM, and in vitro drug release studies. Using MTT assay, the cytotoxicity of formulation was evaluated on ARPE-19 cells. The cellular uptake and VEGF expression levels were also evaluated in in vitro settings. The optimized formulation had a particle size of 102.7nm with - 47.30mV of zeta potential. Entrapment efficiency was found to be 65.21%. The cell viability results suggested compatibility of developed formulation. Cellular uptake and VEGF expression levels for the formulated nanoparticles specified that the developed formulation showed potential cellular uptake and had displayed anti-angiogenic property by inhibiting VEGF expression in vitro. The results showed successful development of resveratrol-loaded nanoparticles which may be used for neovascular AMD treatment alone or in combination with anti-VEGF agents.

YAP promotes endothelial barrier repair by repressing STAT3/VEGF signaling

AimsEndothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. Main methodsA trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. Key findingsYAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SignificanceElucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.

Anti-PD-1 antibody combined with chemotherapy suppresses the growth of mesothelioma by reducing myeloid-derived suppressor cells

BackgroundThe combination of anti-PD-1/PD-L1 antibody with chemotherapy has been approved for the first-line therapy of lung cancer. However, the effects against malignant mesothelioma (MPM) and the immunological mechanisms by which chemotherapy enhances the effect of targeting PD-1/PD-L1 in MPM are poorly understood. Materials and MethodsWe utilized syngeneic mouse models of MPM and lung cancer and assessed the therapeutic effects of anti-PD-1 antibody and its combination with cisplatin (CDDP) and pemetrexed (PEM). An immunological analysis of tumor-infiltrating cells was performed with immunohistochemistry. ResultsWe observed significant therapeutic effects of anti-PD-1 antibody against MPM. Although the effect was associated with CD8+ and CD4+ T cells in tumors, the number of Foxp3+ cells was not reduced but rather increased. Consequently, combination with CDDP/PEM significantly enhanced the antitumor effects of anti-PD-1 antibody by decreasing numbers of intratumoral myeloid-derived suppressor cells (MDSCs) and vessels probably through suppression of VEGF expression by CDDP + PEM. ConclusionsThe combination of anti-PD-1 antibody with CDDP + PEM may be a promising therapy for MPM via inhibiting the accumulation of MDSCs and vessels in tumors.

CircTLK1 Promotes the Proliferation and Metastasis of Renal Cell Carcinoma by Sponging miR-136-5p

Background: Circular RNAs (circRNAs), a novel type of ncRNAs, are covalently linked to make up circular configuration via a loop structure. Accumulating evidence indicate that circRNAs are potential biomarkers and key regulators in the development and progression of tumors. However, the precise role of circRNAs in renal cell carcinoma (RCC) remains unknown. Methods: Though circRNA high-throughput sequencing from RCC cell lines, we determined circRNA TLK1 (circTLK1) as a novel candidate circRNA derived from TLK1 gene. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of mRNAs, circRNAs and miRNAs in RCC tissues and cells. Loss-of function experiments were executed to detect the biological roles of circTLK1 on the phenotypes of RCC cells in vitro and in vivo. RNA-FISH, RNA-pull down, dual-luciferase reporter, western blot and immunohistochemistry assay were used to investigate the molecular mechanisms underlying the functions of circTLK1. Findings: CircTLK1 is overexpressed in RCC and positively corelated with distant metastasis and unfavorable prognosis. Silence of circTLK1 significantly inhibits the proliferation, migration and invasion of RCC cells in vitro and in vivo. Mechanistically, circTLK1 is mainly distributed in the cytoplasm and positively regulates CBX4 expression by sponging miR-136-5p. In addition, forced expression of CBX4 reverses the phenotypes inhibition of RCC cells induced by circTLK1 suppression. Moreover, CBX4 expression is positively corelated with VEGF expression in renal cancer tissues. Knockdown of CBX4 significantly inhibited VEGF expression in RCC cells. Interpretation: Collectively, our findings demonstrate that circTLK1 plays a critical role in RCC progression by sponging miR-136-5p to increase CBX4 expression. CircTLK1 may act as a diagnostic biomarker and therapeutic target for RCC. Funding Statement: This study was supported by grants from Guangdong Key Laboratories (2017B030314074) and the Shenzhen Project of Science and Technology (JCYJ20170413100245260). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The present study was approved the Institutional Review Board of Peking University Shenzhen Hospital. In vivo assay was approved by the animal management committee of Perking University Shenzhen Hospital, and all experimental procedures and animal care were in line with the institutional ethics guidelines for animal experiments.

Effect of Silencing Information Regulator on Rat Cerebral Venous Thrombosis Through Regulating Vascular Endothelial Growth Factor

Cerebral venous thrombosis (CVST) can cause severe dysfunction and even death. Silencing information regulator (SIRT1) involves in neurodegenerative diseases. However, whether SIRT1 participates in CVST is unclear. SD rats were divided into 3 groups, control group; CVST group and SIRT1 group (transfected with AAV-SIRT1 plasmid) followed by analysis of brain tissue SIRT1 and VEGF expression by Real time PCR, neurological deficit scores and brain tissue water content. Brain vascular endothelial cells (bVECs) were cultured and divided into NC group, SIRT1 group and si-SIRT1 group followed by analysis of cell proliferation by MTT assay, Caspase 3 activity, SIRT1 expression by Real time PCR and Western blot and VEGF expression and secretion by Real time PCR and ELISA. SIRT1 expression was decreased and VEGF expression was increased, along with increased score of neurological deficit and water content of brain tissue in CVST rats. Transfection of AAV-SIRT1 plasmid up-regulated SIRT1 expression in CVST rats, inhibited VEGF expression, improved neurological deficit score and brain tissue water content (P < 0.05). Overexpression of SIRT1 in bVECs significantly decreased cell proliferation, elevated Caspase 3 activity, and decreased VEGF expression and secretion, compared to NC group (P < 0.05). Knockdown of SIRT1 expression in bVECs reversed the above changes (P < 0.05). SIRT1 expression is decreased in CVST rats and up-regulation of SIRT1 can inhibit VEGF expression and improve neurological function. SIRT1 can inhibit the proliferation of bVECs and regulate cerebral venous thrombosis by regulating VEGF.

The effects of cooking oil fumes-derived PM2.5 on blood vessel formation through ROS-mediated NLRP3 inflammasome pathway in human umbilical vein endothelial cells

BackgroundCooking oil fumes (COFs), a main pollutant in kitchen air, is a major risk to human health. In our previous research, exposure to COFs-derived PM2.5 could cause umbilical vascular endothelial dysfunction, leading to decreased fetal weight. Here, to test the role of ROS-mediated NLRP3 inflammasome pathway in blood vessel formation of human umbilical vein endothelial cells (HUVECs) caused by COFs-derived PM2.5, the cells were exposed to COFs-derived PM2.5 at different concentrations with and without N-acetyl-L-cysteine (NAC). MethodsMTT assay was used to determine HUVECs viability. Intracellular ROS and mitochondrial ROS levels were assessed with DCFH-DA and MitoSOX™ assay. The levels of proteins and mRNA involved in NLRP3 inflammasome signaling pathway and VEGF were measured by western blot and real-time PCR (RT-PCR). Tube formation in HUVECs was detected by tube formation assay. ResultsThe results revealed that COFs-derived PM2.5 exposure reduced HUVECs viability, increased the intracellular and mitochondrial ROS levels in cells, and up-regulated the levels of proteins and mRNA involved in NLRP3 inflammasome signaling pathway. However, the protein and mRNA expression of VEGF were reduced with the increasing exposure concentrations. In addition, COFs-derived PM2.5 also affected the tube formation. However, co-incubation with NAC effectively rescued the damages caused by COFs-derived PM2.5 exposure. ConclusionsThis study proved that COFs-derived PM2.5 could significantly reduce HUVECs viability, induce the overproduction of ROS, lead to inflammation and inhibit VEGF expression, thus affect angiogenesis of HUVECs in vitro. It was revealed that the impact caused by COFs-derived PM2.5 on blood vessel formation through a ROS-mediated NLRP3 inflammasome pathway.

Monocarboxylate transporter1 is an independent prognostic factor in esophageal squamous cell carcinoma.

The monocarboxylate transporter 1 (MCT1) has been reported to have significant prognostic value in several solid tumors. The present study aimed to explore its clinical significance in esophageal squamous cell carcinoma (ESCC). After acquiring and analyzing MCT1 (solute carrier family 16 member; SLC16A1) mRNA expression in The Cancer Genome Atlas (TCGA) database, the prognostic potential of MCT1 was assessed by immunohistochemistry (IHC). The impact of the knockdown of MCT1 by shRNA was evaluated using Cell Counting Kit‑8 (CCK‑8) and colony formation assays to determine whether MCT1 suppression affected the proliferation and survival of ESCC cells. MCT1 expression was found to correlate with T stage (P=0.005), N stage (P=0.036) and TNM stage (P=0.035). Kaplan‑Meier survival analysis showed that patients in a high‑MCT1 group had a lower overall survival (OS) (P<0.001) and lower progression‑free survival (PFS) (P<0.001). The results of univariate and multivariate Cox regression analyses demonstrated that MCT1 is an independent prognostic factor for OS (P=0.001 and 0.01) and PFS (P=0.001 and 0.012). Downregulation of MCT1 suppressed proliferation and survival of ESCC cells in vitro. The proliferation rate and colony numbers were decreased in the sh‑MCT1 groups (all P<0.05). Downregulation of MCT1 suppressed VEGF expression (all P<0.05). MCT1 may act as a biomarker for ESCC to identify patients with poor outcomes.

Quadruplex-forming oligonucleotide targeted to the VEGF promoter inhibits growth of non-small cell lung cancer cells.

BackgroundVascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression.ResultsOur experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death.ConclusionOur data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression.

The effects of dietary polyunsaturated fatty acids on miR-126 promoter DNA methylation status and VEGF protein expression in the colorectal cancer cells

BackgroundThere is increasing evidence indicating an aberrant expression of miRNAs in colorectal cancer (CRC) development. Growing evidence has suggested that polyunsaturated fatty acids (PUFAs) could modulate the remodeling of the epigenome. No study has yet been published to examine the direct effect of PUFA on the promoter methylation of miRNAs. This study aimed to examine the potential clinical application of PUFA on the promoter DNA methylation of miR-126 and its angiogenic target molecule (VEGF) in the CRC cells.MethodsWe investigated the direct effect of 100 μM EPA, DHA, and LA for 24 h on promoter methylation status of miR-126 in a panel of five CRC cell lines (HCT116, HT29/219, Caco2, SW742, and LS180) by methylation-specific PCR (MSP). We also quantified the miR-126 and VEGF transcript expression levels in five CRC cell lines affected by PUFA by real-time PCR. Moreover, we analyzed the protein expression level of VEGF, as a target of miR-126, by western blotting assay.ResultsMSP analysis showed extensive DNA methylation of the miR-126 promoter in all five CRC cell lines, and among all three PUFAs, only DHA completely demethylated the promoter of miR-126 in HCT116 and Caco2 cell lines. We found that only DHA significantly induces the expression level of miR-126 in HCT116 and Caco2 cell lines, respectively, by 20.1-fold and 1.68-fold (p < 0.05). Our finding indicates that the downregulation of VEGF protein level is also effectively observed only in DHA-treated HCT116 and Caco2 cells compared to control cells (p < 0.05).ConclusionsOur results provide evidence that n-3 PUFAs are able to modulate cellular miR-126 DNA methylation and inhibit VEGF expression level in a cell-type specific manner in colorectal cancer cells. DHA always showed higher efficacy than EPA and LA in our experiment. Overall, our results suggest a potential clinical application of n-3 PUFAs as anti-angiogenic agents in CRC therapy.

Parthenolide inhibits tumor-promoting effects of nicotine in lung cancer by inducing P53 - dependent apoptosis and inhibiting VEGF expression

The correlation between cigarette smoking and the onset of non-small cell lung cancer is well documented. Enhanced proliferation, angiogenesis induction, and resistance to apoptosis were reported as direct results associated with exposure to nicotine (the active ingredient of cigarettes). Parthenolide is a sesquiterpene lactone with anticancer activity against different cancer types. In this study, we tested the ability of parthenolide to inhibit the proliferating effect of nicotine in lung cancer cell lines. MTT assay was used to measure cell survival of A549 and H526 cells treated with nicotine, parthenolide, and their combination. Angiogenesis inhibition was measured using VEGF detection kit and apoptosis induction was evaluated by measuring caspase-3 activity. Real time PCR assay was used to detect the change in expression of several genes associated with cell proliferation and apoptosis (CASP3, CASP7, CASP8, CASP9, P53, GADD45, BAX, BIM, Bcl-2, TOPO I, and TOPO II). Parthenolide inhibited lung cancer cells in a concentration-dependent manner and decreased the proliferation stimulating effect of nicotine. Caspase-3 activity and VEGF assays evidenced an apoptosis-inducing and VEGF- inhibiting effects of parthenolide. The real time PCR assay demonstrated that parthenolide down-regulated the expression of Bcl-2 and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which indicates an activation of P53- dependent apoptosis pathway in response to parthenolide. Furthermore, this pathway remained active in the presence of nicotine suggesting the ability of parthenolide to exclude the anti-apoptotic effect of nicotine. Our results indicate that parthenolide inhibits nicotine proliferating effect on lung cancer. The anticancer effect of parthenolide is mediated by angiogenesis inhibition and activation of P53- dependent apoptosis. Parthenolide is a promising natural product for inhibiting and treating nicotine-associated lung cancer. However, further studied on more lung cancer cell lines and on protein level are needed to fully understand its mechanisms of action.

Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells

PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo−/− mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome—3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo−/− mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).

Peptides derived from the integrin \u03b2 cytoplasmic tails inhibit angiogenesis

BackgroundIntegrins are essential regulators of angiogenesis. However, the antiangiogenic potential of peptides derived from the integrin cytoplasmic tails (CT) remains mostly undetermined.MethodsHere we designed a panel of membrane-penetrating peptides (termed as mβCTPs), each comprising a C-terminal NxxY motif from one of the conserved integrin β CTs, and evaluated their antiangiogenic ability using both in vitro and in vivo approaches.ResultsWe found that mβ3CTP, mβ5CTP and mβ6CTP, derived respectively from the integrin β3, β5 and β6 CTs, but not others, exhibit antiangiogenic ability. Interestingly, we observed that the integrin β3, β5 and β6 CTs but not others are able to interact with β3-endonexin. In addition, the antiangiogenic core in mβ3CTP is identical to a previously identified β3-endonexin binding region in the integrin β3 CT, indicating that the antiangiogenic mβCTPs may function via their binding to β3-endonexin. Consistently, knockdown of endogenous β3-endonexin in HUVECs significantly suppresses tube formation, suggesting that β3-endonexin is proangiogenic. However, neither treatment with the antiangiogenic mβCTPs nor knockdown of endogenous β3-endonexin affects integrin-mediated HUVEC adhesion and migration, indicating that their antiangiogenic effect may not rely on directly regulating integrin activity. Importantly, both treatment with the antiangiogenic mβCTPs and knockdown of endogenous β3-endonexin in HUVECs inhibit VEGF expression and cell proliferation, thereby providing mechanistic explanations for the functional consequences.ConclusionOur results suggest that the antiangiogenic mβCTPs can interact with β3-endonexin in vascular endothelial cells and suppress its function in regulating VEGF expression and cell proliferation, thus disclosing a unique pathway that may be useful for developing novel antiangiogenic strategies.

Antiangiogenic cytokines as potential new therapeutic targets for resveratrol in diabetic retinopathy.

Diabetes mellitus (DM) affects >350 million people worldwide. With many complications that can reduce the patient’s quality of life, vision loss is one of the most debilitating disorders it can cause. Active research in the field of diabetes includes microvascular complications in diabetic retinopathy (DR). Disturbances in the balance of pro-angiogenesis and anti-angiogenesis factors can lead to the progression of DR. The retinal pigment epithelium (RPE) is the outermost layer of the retina, and it is essential in maintaining the visual function. The RPE produces and secretes growth factors as well as protective agents which maintain structural integrity of the retina. Small natural molecules, such as resveratrol, may influence neurotrophic factors of the retina. The pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1) are secreted by RPE cells. These two proteins inhibit angiogenesis and inflammation in RPE cells. An alteration of their production contributes to various eye diseases. There is a critical balance between two important factors secreted on opposite sides of the RPE: at the basal side, vascular endothelial growth factor (VEGF; acts on the choroidal endothelium) and, on the apical side, PEDF (acts on neurons and photoreceptors). Resveratrol inhibits VEGF expression in human adult RPE cells and limits the development of proliferative vitreoretinopathy, by attenuating transforming growth factor-β2-induced wound closure and cell migration. Possible new mechanisms could include PEDF and TSP-1 expression alterations under physiological and pathological conditions. Resveratrol is currently of interest due to its capacity to influence the cell’s secretory activity. Some limitations arise from its low bioavailability. Several drug delivery systems are currently tested, promising to improve tissue concentrations. This article reviews biological pathways involved in the pathogenesis of DR that could be influenced by resveratrol. A study of these pathways could identify new potential targets for the reduction of diabetic complications.

In vitro evaluation of natural thermal mineral waters in human keratinocyte cells: a preliminary study.

We aimed to test the anti-inflammatory and angiogenic properties of two different thermal waters at the cellular level in human keratinocyte cells in the present study. Two different thermal waters, thermo-mineral BJ1 (Bursa, Turkey) and oligomineral BG (Bolu, Turkey), were tested in human keratinocyte (HaCaT) cell line. HaCaT cells were incubated for 3days with thermal waters; RNA isolation was carried out in the treated and untreated cells. The gene expressions of TNFα, IL-1α, and VEGF were measured using the RT-qPCR. The tested thermal waters significantly decreased the expression of IL-1α (BJ1 93% p = 0.0024 and BG 38% p = 0.0303). BJ1 and BG thermal waters downregulated the expression of TNFα (59% p = 0.0001 and 23% p = 0.0238 respectively). Furthermore, BJ1 and BG significantly downregulated the gene expression of VEGF (98% p = 0.0430 and 15% p = 0.0120). The observed decrease in the gene expression of TNFα and IL1α could be interpreted as an anti-inflammatory effect of mineral waters on HaCaT cells. Moreover, the suppressed VEGF expression might be an indicator of the antiangiogenic effect on human keratinocytes. Therefore, we hypothesized that depending on their specific chemical composition such as silica (128mg/L) in BJ1 and hydrogen sulfide (1.2mg/L) in BG, thermal waters suppress pro-inflammatory cytokines and angiogenic growth factor. These preliminary findings might give insight on the underlying mechanisms of the therapeutic benefits observed in some skin diseases such as rosacea and psoriasis.