VEGFR2 Phosphorylation Research Articles

Apatinib Combined With Radiotherapy Enhances Antitumor Effects in an In Vivo Nasopharyngeal Carcinoma Model.

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are highly expressed in nasopharyngeal carcinoma; therefore, blocking the binding of VEGF and VEGFR may be a potential way to treat nasopharyngeal carcinoma. Apatinib inhibits tumor angiogenesis. Previous studies have suggested that treatment with apatinib has an antitumor effect on nasopharyngeal carcinoma. This study will investigate the effect of apatinib combined with radiotherapy. In this study, nude mice injected with CNE-2 nasopharyngeal carcinoma cells were randomly divided into 6 groups. Therapeutic effects were assessed by evaluating tumor inhibition rate, phosphorylation of VEGFR-2, CD31, partial oxygen pressure, and tumor metabolism. We found that the tumor inhibition of mice in the treated groups was better compared to that of the control group. In mice treated with apatinib alone, angiogenesis was prevented, and the tumor tissue partial oxygen pressure was reduced, thereby achieving an antitumor effect. Moreover, the tumor inhibitory effect of combined treatment was stronger than treatment with either apatinib or radiotherapy alone. Compared with monotherapy treatment, combined treatment better resisted angiogenesis. Apatinib combined with radiotherapy to treat nasopharyngeal carcinoma has synergistic effects, which may be related to enhanced antiangiogenesis.

Alternagin-C (ALT-C), a disintegrin-like protein, attenuates alpha2beta1 integrin and VEGF receptor 2 signaling resulting in angiogenesis inhibition

Angiogenesis, a crucial process in tumor progression, is mainly regulated by vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. Studies have shown the interaction between α2β1 integrin, a collagen receptor, and VEGFR2 in VEGF-driven angiogenesis in vitro and in vivo. Alternagin-C (ALT-C), an ECD-disintegrin-like protein from Bothrops alternatus snake venom, has high affinity for α2β1 integrin and shows antiangiogenic activity in concentrations higher than 100 nM. Despite previous results, its mechanism of action on angiogenic signaling pathways has not been addressed. Here we evaluate the antiangiogenic activity of ALT-C in human umbilical vein endothelial cells (HUVECs) associated or not with VEGF, as well as its interference in the α2β1/VEGFR2 crosstalk. ALT-C (1000 nM) affected actin cytoskeleton, decreased the number of cell filopodia, and strongly inhibited HUVEC tube formation, adhesion to type I collagen and cell migration. Down-regulation of α2β1/VEGFR2 crosstalk by ALT-C decreased the protein content and phosphorylation of VEGFR2 and β1 integrin subunit, inhibited ERK 1/2 and PI3K signaling and regulated FAK/Src and paxillin pathways. Furthermore, ALT-C increased the content of the autophagic markers LC3B and Beclin-1 in the presence of VEGF, which is associated with decreased angiogenesis. In conclusion, we suggest that ALT-C, after binding to α2β1 integrin, inhibits VEGF/VEGFR2 signaling, which results in impaired angiogenesis. These results demonstrate that ALT-C may be a potential candidate for the development of antiangiogenic therapies for tumor and metastasis treatment and help to understand the complexity and fundamental role of integrin inhibition in the tumor microenvironment.

Isolinderalactone suppresses human glioblastoma growth and angiogenic activity in 3D microfluidic chip and in vivo mouse models.

Glioblastoma multiforme (GBM) is a lethal and highly vascular type of brain tumor. We previously reported that isolinderalactone enhances GBM apoptosis in vitro and in vivo, but its role in tumor angiogenesis is unknown. Here, we investigated the anti-angiogenic activity of isolinderalactone and its mechanisms. In a human GBM xenograft mouse model, isolinderalactone significantly reduced tumor growth and vessels. Isolinderalactone decreased the expression of vascular endothelial growth factor (VEGF) mRNA, protein, and VEGF secretion in hypoxic U-87 GBM cells and also in xenograft GMB tissue. In addition, we demonstrated that isolinderalactone significantly inhibited the proliferation, migration, and capillary-like tube formation of human brain microvascular endothelial cells (HBMECs) in the presence of VEGF. We also found that isolinderalactone decreased sprout diameter and length in a 3D microfluidic chip, and strongly reduced VEGF-triggered angiogenesis in vivo Matrigel plug assay. Isolinderalactone downregulated hypoxia-inducible factor-1α (HIF-1α) and HIF-2α proteins, decreased luciferase activity driven by the VEGF promoter in U-87cells under hypoxic conditions, and suppressed VEGF-driven phosphorylation of VEGFR2 in HBMECs. Taken together, our results suggest that isolinderalactone is a promising candidate for GBM treatment through tumor angiogenesis inhibition.

PBX3 Promotes Tumor Growth and Angiogenesis via Activation of AT1R/VEGFR2 Pathway in Papillary Thyroid Carcinoma

PBX3 (Pre-B-cell leukemia homeobox 3) had been considered to be a multifunctional oncogene which involved in tumor growth, invasion, and metastasis in leukemia and some solid tumors. However, the contribution of PBX3 to papillary thyroid carcinoma (PTC) remains unclear. In this study, we found that PBX3 expression was significantly upregulated in PTC tissues compared to adjacent normal tissues, and high levels of PBX3 were correlated with tumor size, lymphatic metastasis, TMN stage, and poor prognosis of PTC patients. Overexpression of PBX3 in PTC cell lines promoted cell proliferation. Consistently, knockdown of PBX3 by shRNA induced cell cycle arrest at G0/G1 phase, and inhibited angiogenesis and tumor growth in vitro and in vivo. Furthermore, PBX3 promoted PTC cell proliferation and angiogenesis through activation of AT1R/VEGFR2 pathway while overexpression of AT1R and treatment with VEGFA reversed PBX3-shRNA-induced decreased phosphorylation of VEGFR2 and its downstream (ERK1/2, AKT and Src). It demonstrated that PBX3 could be used as a potential prognostic biomarker and therapeutic target for PTC.

Low Dose of Penfluridol Inhibits VEGF-Induced Angiogenesis.

Metastasis is considered a major burden in cancer, being responsible for more than 90% of cancer-related deaths. Tumor angiogenesis is one of the main processes that lead to tumor metastasis. Penfluridol is a classic and commonly used antipsychotic drug, which has a great ability to cross the blood–brain barrier. Recent studies have revealed that penfluridol has significant anti-cancer activity in diverse tumors, such as metastatic breast cancer and glioblastoma. Here, we aim to identify the effect of low doses of penfluridol on tumor microenvironment and compare it with its effect on tumor cells. Although low concentration of penfluridol was not toxic for endothelial cells, it blocked angiogenesis in vitro and in vivo. In vitro, penfluridol inhibited VEGF-induced primary endothelial cell migration and tube formation, and in vivo, it blocked VEGF- and FGF-induced angiogenesis in the matrigel plug assay. VEGF-induced VEGFR2 phosphorylation and the downstream p38 and ERK signaling pathways were not affected in endothelial cells, although VEGF-induced Src and Akt activation were abrogated by penfluridol treatment. When cancer cells were treated with the same low concentration of penfluridol, basal Src activation levels were mildly impaired, thus impacting their cell migration and wound healing efficiency. The potential of cancer-induced paracrine effect on endothelial cells was explored, although that did not seem to be a player for angiogenesis. Overall, our data demonstrates that low penfluridol levels, similar to the ones clinically used for anti-psychotic conditions, suppress angiogenic efficiency in the tumor microenvironment.

Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice.

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

Tumor necrosis factor receptor-2 signaling pathways promote survival of cancer stem-like CD133+ cells in clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC) contains cancer stem‐like cells (CSCs) that express CD133 (ccRCC‐CD133+). CSCs are rarely in cell cycle and, as nonproliferating cells, resist most chemotherapeutic agents. Previously, we reported that tumor necrosis factor receptor‐2 (TNFR2) signaling promotes the cell cycle entry of ccRCC‐CD133+CSCs, rendering them susceptible to cell‐cycle‐dependent chemotherapeutics. Here, we describe a TNFR2‐activated signaling pathway in ccRCC‐CD133+CSCs that is required for cell survival. Wild‐type (wt)TNF or R2TNF but not R1TNF (TNF muteins that selectively bind to TNFR2 and TNFR1) induces phosphorylation of signal transducer and activator of transcription 3 (STAT3) on serine727 but not tyrosine705, resulting in pSTAT3Ser727 translocation to and colocalization with TNFR2 in mitochondria. R2TNF signaling activates a kinase cascade involving the phosphorylation of VEGFR2, PI‐3K, Akt, and mTORC. Inhibition of any of the kinases or siRNA knockdown of TNFR2 or STAT3 promotes cell death associated with mitochondrial morphological changes, cytochrome c release, generation of reactive oxygen species, and TUNEL+cells expressing phosphorylated mixed lineage kinase‐like (MLKL). Pretreatment with necrostatin‐1 is more protective than z‐VAD.fmk, suggesting that most death is necroptotic and TNFR2 signaling promotes cell survival by preventing mitochondrial‐mediated necroptosis. These data suggest that a TNFR2 selective agonist may offer a potential therapeutic strategy for ccRCC.

Tranexamic Acid Inhibits Angiogenesis and Melanogenesis in Vitro by Targeting VEGF Receptors.

Melasma is a common but complex skin condition concerning cosmetic problems. Tranexamic acid (TA) has been proved to be effective in treatment of melasma with still unclear mechanisms. Here, we show that VEGF165 enhanced the expression of VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2 and NRP-1) in human umbilical vein endothelial cells (HUVECs), which was attenuated by TA. VEGF165 also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in HUVECs, which was again abolished by TA. TA further showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting cell proliferation, migration, invasion and tube formation of HUVECs induced by VEGF165, suggesting that TA could inhibit angiogenesis by targeting VEGFRs in HUVECs. In addition, VEGF165 enhanced the expression of VEGFRs and promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human melanocytes, which were also attenuated by TA. Furthermore, TA showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting tyrosinase activity, melanin production and even melanogenic proteins induced by VEGF165, suggesting that TA could reduce melanogenesis via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that TA can inhibit angiogenesis and melanogenesis in vitro at least in part by targeting VEGFRs, which may offer a new understanding of the pathogenesis of melasma as well as the molecular mechanism for TA in treatment of the disease.

Activation of VEGF receptors in response to UVB promotes cell proliferation and melanogenesis of normal human melanocytes

VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and signaling via VEGFRs extends beyond the classical roles in blood vessel formation. We previously showed VEGFRs were also expressed in epidermal keratinocytes and activation of VEGFR-2 by ultraviolet B (UVB) was involved in the pro-survival mechanism. Here, we show that both VEGF165 and UVB enhanced the expression of VEGFRs (including VEGFR-1, VEGFR-2 and NRP-1) in normal human melanocytes, and increased expression of VEGFRs by UVB was mediated through hypoxia and oxidative stress. Also, VEGF165 and UVB promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, and UVB-induced phosphorylation of VEGFR-1 and VEGFR-2 required PKA but not P38 MAPK. In addition, UVB and VEGF165 contributed to the over-expression of melanogenic proteins in melanocytes, which could be reduced by neutralization of VEGFR-1 and/or VEGFR-2. UVB, but not VEGF165 promoted cell proliferation, while neutralization of VEGFR-1 and/or VEGFR-2 abolished this effect. UVB showed stronger than VEGF165 in promoting tyrosinase activity and melanin production, while neutralization of VEGFR-2 was stronger in reducing these effects than that of VEGFR-1. Furthermore, tranexamic acid (TA) decreased tyrosinase activity and melanin production via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that VEGFRs are functionally involved in UVB-induced melanogenesis, and TA can inhibit melanogenesis at least in part by targeting VEGFRs in melanocytes.

YY1 Promotes Endothelial Cell-Dependent Tumor Angiogenesis in Hepatocellular Carcinoma by Transcriptionally Activating VEGFA.

Hepatocellular carcinoma (HCC) is a typical hypervascular solid tumor that requires neoangiogenesis for growth. The vascular endothelial growth factor (VEGF) is the most potent proangiogenic factor in neovascularization. The multifunctional Yin-Yang 1 (YY1) is involved in the regulation of tumor malignancy of HCC. However, the relationship between YY1 and endothelial cell-dependent tumor angiogenesis in HCC remains unclear. In this study, we observed that YY1 is positively correlated with microvessel density (MVD) and poor prognosis in HCC tissues. We further found that YY1 promotes the transcriptional activity of VEGFA by binding its promoter in HCC. The secreted VEGFA from HCC cells activates phosphorylation of VEGFR2 to promotes tube formation, cell migration, and invasion of vascular endothelial cells in vitro, and promotes tumor growth and angiogenesis in vivo. In addition, upregulation of YY1 enhanced resistance of bevacizumab in HCC cells. These results indicate that YY1 plays essential roles in HCC angiogenesis and resistance of bevacizumab by inducing VEGFA transcription and that YY1 may represent a potential molecular target for antiangiogenic therapy during HCC progression.

VEGFR2-Specific Ligands Based on Affibody Molecules Demonstrate Agonistic Effects when Tetrameric in the Soluble Form or Immobilized via Spider Silk.

Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-ZVEGFR2) was formatted into a tetrameric construct (tetra-ZVEGFR2) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-ZVEGFR2 induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-ZVEGFR2. In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-ZVEGFR2-silk and tetra-ZVEGFR2-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-ZVEGFR2-silk and tetra-ZVEGFR2-silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-ZVEGFR2-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended.

Distinct downstream signaling and the roles of VEGF and PlGF in high glucose-mediated injuries of human retinal endothelial cells in culture

Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) plays a crucial role in breakdown of the blood-retinal barrier due to hyperpermeability in diabetic retinopathy (DR). However, the distinct signaling driven by VEGF and PlGF in the pathogenesis of DR remains unclear. In this study, we investigated VEGF- and PlGF- related signaling pathways and their roles in cultured human microvascular retinal endothelial cells (hRECs) under high glucose conditions (HG; 25 mM). Apoptotic cell death was evaluated, and FITC conjugated bovine serum albumin across monolayer hRECs served as an index of permeability. Western blots were used to assess the protein levels of VEGF and PlGF, as well as the phosphorylation of p38MAPK, STAT1 and Erk1/2. Knockdown of VEGF and PlGF was performed by using siRNA. Following HG treatment, increases of VEGF and PlGF as well as PKC activity were detected in hRECs. Increased phosphorylations of p38MAPKThr180/Thr182, STAT1Ser727, and Erk1/2Tyr202/Tyr185 as well as VEGFR1Tyr1213 and VEGFR2Tyr1175 were also detected in HG-treated hRECs. Inhibition of PKC activity by Go 6976 prevented HG-induced increases of phosphor-Erk1/2 and nitric oxide synthase (NOS1) expressions as well as hyperpermeability, whereas inhibition of p38MAPK pathway by SB203580 selectively suppressed activation of STAT1 and decreased apoptotic cell death under HG conditions. Moreover, VEGF knockdown predominantly inhibited activation of VEGFR2, and phosphorylation of p38MAPK and STAT1, as well as apoptotic cell death in HG-treated hRECs. Nevertheless, PlGF knockdown mainly suppressed phosphorylation of VEGFR1, PKC, and Erk1/2, as well as NOS1 expressions and hyperpermeability. Taken together, we provide evidence demonstrating that HG-induced elevation of PlGF is responsible for hyperpermeability mainly through increasing activation of PKC-Erk1/2-NOS axis via VEGFR1, while HG-induced elevation of VEGF is associated with induction of apoptotic cell death mainly through increasing activation of p38MAPK/STAT1 signaling via VEGFR2.

Apatinib-loaded nanoparticles inhibit tumor growth and angiogenesis in a model of melanoma

Anti-angiogenic drugs are an effective therapeutic method for the treatment of melanomas. Apatinib is a small-molecule tyrosine kinase inhibitor, which has potent inhibitory activity on tumor angiogenesis. Due to the low water solubility and stability of Apatinib, we aimed to design and develop poly (lactic-co-glycolic acid) (PLGA) and Poloxamer 407 nanoparticles to encapsulate Apatinib (Apa/p NPs) to improve the efficacy of application in melanoma treatment. The size and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro proliferation assays were used to assess the capacity of Apa/p NPs to suppress the growth of B16 cells. Furthermore, we constructed melanoma models using C57BL/6 mice, and preliminary evaluation of the effect and mechanism of Apa/p NPs on tumor inhibition was performed in vivo. The results showed that the size of Apa/p NPs averaged 136 ± 0.27 nm and the nanoparticles were evenly dispersed. Moreover, Apa/p NPs significantly inhibited the growth of B16 cells and melanoma tumors, compared with the naked drug treatment and control groups. The protein levels of VEGFR-2, phosphorylated (p)-VEGFR-2 and p-ERK1/2 in tumor tissues were inhibited by Apa/p NP treatment, as detected by Western blot. The results of this study suggested that Apa/p NPs could inhibit the growth of melanoma tumors by inhibiting the phosphorylation and expression of VEGFR-2 and downstream ERK1/2, providing a theoretical basis for the clinical application of Apatinib in the treatment of melanoma.

A novel 2-aminobenzimidazole-based compound Jzu 17 exhibits anti-angiogenesis effects by targeting VEGFR-2 signalling.

Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.

Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo.

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5–2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5–4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism.

Diverse bioactive substances derived from marine organisms have been attracting growing attention. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. Being identified as a sarcoplasmic calcium-binding protein, ASP-3 exhibited strong inhibitory effects on the proliferation of Human hepatocellular carcinoma (HepG2) cells with an IC50 value of 171.18 ± 18.59 μg/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. More importantly, the therapeutic potential of ASP-3 as an anti-angiogenesis agent was further confirmed by an in vitro model using VEGF-induced tube formation assay of human umbilical vein endothelial cells (HUVECs), as well as an in vivo model using transgenic zebrafish model. Taken together, the ASP-3 provides a good framework for the development of even more potent anticancer proteins and provides important weapon for cancer treatment using novel approaches such as gene therapy.

Multimerin-2 maintains vascular stability and permeability

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2−/− (Mmrn2−/−) mice. Although Mmrn2−/− mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2−/− mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2−/− vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2−/− mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Fruquintinib and its use in the treatment of metastatic colorectal cancer.

Fruquintinib is a potent, highly selective and orally active inhibitor of VEGFR1, 2, 3 tyrosine kinases. It inhibits VEGF-induced VEGFR2 phosphorylation, endothelial cell proliferation and tubule formation. Currently, it has been approved for the treatment of metastatic colorectal cancer in patients who have failed at least two prior systemic antineoplastic therapies in China. However, it is not approved outside China, and there is another similar small molecular VEGFR multitarget drug approved in China, USA, Europe, etc. Here, we summarize the mechanism characteristics and clinical development of fruquintinib supporting its use in the treatment of metastastic colorectal cancer as well as explorations in other tumor types.

Impact of tivozanib on patient outcomes in treatment of advanced renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common type of kidney malignancy, and the clear-cell subtype represents the majority of RCCs. RCC is a heterogeneous disease in terms of genetic and histological features which determine the behavior of the disease. The von Hippel–Lindau (VHL) is a tumor suppressor gene and mutations of this gene are seen in 95% of clear-cell RCCs. Inactivation of VHL causes the accumulation of hypoxia-inducible factor-1 (HIF-1), and in turn, accumulation of HIF-1 induces overexpression of vascular endothelial growth factor (VEGF); the increase in VEGF expression makes RCC a highly vascularized tumor, and forms the rationale for antiVEGF treatment. In the past decade, improvement in the survival of RCC patients has been observed due to new effective therapies, such as antiVEGF and mammalian target of rapamycin (mTOR) targeting agents and immune checkpoint inhibitors. The majority of VEGF targeted agents are not just selective to VEGF receptors, but usually also have inhibitory effects on other kinases, such as c-KIT and FLT3. Tivozanib is an extremely potent and selective tyrosine kinase inhibitor (TKI) of VEGFR-1, 2, and 3, with a relatively long half-life, that is approved by the European Commission for the treatment of advanced/metastatic RCC. Tivozanib, at very low serum concentration can inhibit phosphorylation of VEGFR −1, −2, and −3 tyrosine kinase activity. This article summarizes the clinical data on tivozanib in the treatment of advanced/metastatic RCC.

The strigolactone analog GR-24 inhibits angiogenesis in vivo and in vitro by a mechanism involving cytoskeletal reorganization and VEGFR2 signalling

Phytohormones have shown great potential as natural anticancer compounds, being interesting in cancer prevention and therapy. Strigolactones are a class of plant hormones involved in the inhibition of root branching and sprouting. The antiproliferative capacity of the synthetic strigolactone analog GR-24 has been described against breast cancer cell lines in vitro. In this study, we show for the first time that GR-24 is a potent antiangiogenic compound in vivo and in vitro. In the in vivo tests, GR-24 shows a great inhibitory effect on vasculature formation in the chicken chorioallantoic membrane and in two different zebrafish models. Our in vitro results show that GR-24 inhibits the growth of endothelial cells and different cancer cell lines with a micromolar range of half inhibitory concentration (IC50) values. In addition, GR-24 inhibits key steps of the angiogenic process in vitro, such as tubulogenesis, invasion, extracellular matrix remodeling capacity, migration and adhesion of endothelial cells at non-cytotoxic concentrations. Our data point to an effect of GR-24 on cytoskeleton organization in endothelial cells, in addition to a decrease in focal adhesion kinase (FAK) presence in these cells. All these data, together with the observed increase in surface expression of vascular endothelial-cadherin (VE-cadherin) and platelet and endothelial cell adhesion molecule 1 (PECAM-1), suggest that GR-24 prevents angiogenesis by maintaining the quiescent phenotype in endothelial cells. The proposed mechanism of action underlying the antiangiogenic activity of GR-24 involves the inhibition of VEGFR2 phosphorylation, and the downstream reduction in activation of FAK, a key regulator protein implicated in angiogenesis. Our results suggest that GR-24 may be a promising new compound for antiangiogenic therapy of cancer and other angiogenesis-dependent diseases.