Synthesis and Biological Evaluation of Indole‐2‐carbohydrazide Derivatives as Anticancer Agents with Anti‐angiogenic and Antiproliferative Activities

A novel series of indole‐2‐carbohydrazide derivatives were synthesized, characterized, and evaluated for their antiproliferative activities against two cancer cell lines, HCT116 and SW480, and a normal human fetal lung fibroblast cell line, MRC‐5. Among this series, compound 24 f displayed potent cytotoxic activities in vitro against HCT116 and SW480 cell lines with GI₅₀ values of 8.1 and 7.9 μm , respectively, and was inactive against MRC‐5 cells. The newly synthesized compounds were also evaluated for anti‐angiogenesis capabilities by chick chorioallantoic membrane, human umbilical vein endothelial cell (HUVEC) migration, and endothelial microtubule formation assays. Moreover, the effects of 24 f on the vascular endothelial growth factor receptor‐2 and the signaling pathway in HUVECs indicated that this compound inhibits VEGFR‐2 and its downstream related proteins. These results indicate that compound 24 f , as well as the other derivatives, are promising inhibitors of angiogenesis.     

Discovery of Potent Vascular Endothelial Growth Factor Receptor‐2 Inhibitors

Substantial evidence over the last decades has implicated uncontrolled angiogenesis with various pathological states, including cancer. Vascular endothelial growth factor (VEGF) plays a critical role in its regulation. Because the tyrosine kinase VEGF receptor‐2 (VEGFR‐2) is the major mediator of the mitogenic, angiogenic, and permeability‐enhancing effects of VEGF, it has become one of the most profound anti‐angiogenesis targets. Inspired by the anthranilamide class of VEGFR‐2 inhibitors, we performed a computational analysis of some potent representative members, using docking and molecular dynamics calculations. Based on the observations drawn from introducing the effect of the receptor's flexibility in implicit aqueous environment, we designed, synthesized, and characterized several new analogues of related scaffolds with modifications in their steric and electronic characteristics. In vitro evaluation of these compounds revealed several novel VEGFR‐2 inhibitors that are less cytotoxic and more potent than the parent compounds.     

4-Acetylantroquinonol B Suppresses Prostate Cancer Growth and Angiogenesis via a VEGF/PI3K/ERK/mTOR-Dependent Signaling Pathway in Subcutaneous Xenograft and In Vivo Angiogenesis Models

Prostate cancer is a major cause of cancer-related mortality in men in developed countries. The compound, 4-acetylantroquinonol B (4AAQB), is isolated from Antrodia cinnamomea (commonly known as Niu-Chang-Chih), which has been shown to inhibit cancer growth. However, the anticancer activity of 4AAQB has not previously been examined in prostate cancer. This study aimed to investigate the effect of 4AAQB on cancer and angiogenesis, as well as to explore its mechanism of action. Human prostate cancer cells (PC3) and human umbilical vein endothelial cells (HUVEC) were used in cell viability, cell migration, and cell cycle functional assays to evaluate the anticancer and antiangiogenic efficacy of 4AAQB in vitro. The effects of 4AAQB in vivo were determined using xenograft and angiogenesis models. The signaling events downstream of 4AAQB were also examined. The 4AAQB compound inhibited PC3 cell growth and migration, and reduced in vivo cancer growth, as shown in a subcutaneous xenograft model. Furthermore, 4AAQB inhibited HUVEC migration, tube formation, and aortic ring sprouting; it also reduced neovascularization in a Matrigel implant angiogenesis assay in vivo. The 4AAQB compound also decreased metastasis in the PC3 prostate cancer model in vivo. Serum or vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2), phosphoinositide 3-kinase (PI3K)/Ak strain transforming (Akt), and extracellular signal-regulated kinase ½ (ERK ½) phosphorylation were attenuated by 4AAQB in both PC3 and HUVEC. In conclusion, 4AAQB is a potential candidate for prostate cancer therapy.     

Novel Drugs with High Efficacy against Tumor Angiogenesis

Angiogenesis is involved in physiological and pathological processes in the body. Tumor angiogenesis is a key factor associated with tumor growth, progression, and metastasis. Therefore, there is great interest in developing antiangiogenic strategies. Hypoxia is the basic initiating factor of tumor angiogenesis, which leads to the increase of vascular endothelial growth factor (VEGF), angiopoietin (Ang), hypoxia-inducible factor (HIF-1), etc. in hypoxic cells. The pathways of VEGF and Ang are considered to be critical steps in tumor angiogenesis. A number of antiangiogenic drugs targeting VEGF/VEGFR (VEGF receptor) or ANG/Tie2, or both, are currently being used for cancer treatment, or are still in various stages of clinical development or preclinical evaluation. This article aims to review the mechanisms of angiogenesis and tumor angiogenesis and to focus on new drugs and strategies for the treatment of antiangiogenesis. However, antitumor angiogenic drugs alone may not be sufficient to eradicate tumors. The molecular chaperone heat shock protein 90 (HSP90) is considered a promising molecular target. The VEGFR system and its downstream signaling molecules depend on the function of HSP90. This article also briefly introduces the role of HSP90 in angiogenesis and some HSP90 inhibitors.     

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five G_i protein-coupled receptors (SST_1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST₁ and SST₅ receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST₁ and SST₅ receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.     

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling

Background: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib^®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. Methods: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. Results: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. Conclusions: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.     

可溶性血管内皮生长因子受体2片段的表达及其对血管内皮细胞增殖的影响

目的探讨可溶性血管内皮生长因子受体2(sKDR)抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞(HUVEC)总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜(CAM)试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子(VEGF)刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。     

The CXCL2/IL8/CXCR2 Pathway Is Relevant for Brain Tumor Malignancy and Endothelial Cell Function

We aimed to evaluate the angiogenic capacity of CXCL2 and IL8 affecting human endothelial cells to clarify their potential role in glioblastoma (GBM) angiogenesis. Human GBM samples and controls were stained for proangiogenic factors. Survival curves and molecule correlations were obtained from the TCGA (The Cancer Genome Atlas) database. Moreover, proliferative, migratory and angiogenic activity of peripheral (HUVEC) and brain specific (HBMEC) primary human endothelial cells were investigated including blockage of CXCR2 signaling with SB225502. Gene expression analyses of angiogenic molecules from endothelial cells were performed. Overexpression of VEGF and CXCL2 was observed in GBM patients and associated with a survival disadvantage. Molecules of the VEGF pathway correlated but no relation for CXCR1/2 and CXCL2/IL8 was found. Interestingly, receptors of endothelial cells were not induced by addition of proangiogenic factors in vitro. Proliferation and migration of HUVEC were increased by VEGF, CXCL2 as well as IL8. Their sprouting was enhanced through VEGF and CXCL2, while IL8 showed no effect. In contrast, brain endothelial cells reacted to all proangiogenic molecules. Additionally, treatment with a CXCR2 antagonist led to reduced chemokinesis and sprouting of endothelial cells. We demonstrate the impact of CXCR2 signaling on endothelial cells supporting an impact of this pathway in angiogenesis of glioblastoma.     

Multifunction Sr doped microporous coating on pure magnesium of antibacterial,osteogenic and angiogenic activities

Strontium (Sr) ions were added to porous magnesium (Mg) oxide with silicon and fluorine by microarc oxidation (MAO) to improve its osteogenic and pro-angiogenic properties. First, pure Mg was oxidized by MAO, and Sr was added by electrolysis. The surface of the resulting Sr coating was characterized by SEM, EDS, and EDS mapping. The release of Sr ions was monitored by ICP-OES. The antibacterial property of the coating was assessed against Staphylococcus aureus. The effect of Sr coating on osteogenesis was tested in MC3T3-E1 cell line by performing cell adhesion and proliferation tests, alkaline phosphatase (ALP) activity detection, cell morphology characterization, alizarin red staining, and osteogenic-related gene expression analysis. Finally, HUVECs cells were used to test the effect of Sr coating on angiogenesis through cell migration and tube formation assays, VEGF quantification, chicken embryo chorioallantoic membranes (CAM) test, and angiogenic-related gene expression analysis. The results showed that Sr coating had a hierarchical microstructure with a microporous structure evenly covered with nano-grains and that the Sr elements from the coating were released slowly and continuously. Sr coating had effective antibacterial properties and promoted cell adhesion, proliferation, ALP release, calcium nodule formation, and upregulated osteogenic gene expression. Moreover, the coating could promote migration, tube formation, VEGF expression, and angiogenic gene upregulation in endothelial cells. Sr coating also enhanced angiogenesis of CAM. This study supports that Sr coating on Mg- MAO enhances osteogenesis and angiogenesis.     

Resveratrol,an Inhibitor Binding to VEGF,Restores the Pathology of Abnormal Angiogenesis in Retinopathy of Prematurity (ROP) in Mice: Application by Intravitreal and Topical Instillation

Retinopathy of prematurity (ROP) is a severe eye disease leading to blindness. Abnormal vessel formation is the pathological hallmark of neovascular ROP. In forming vessels, vascular endothelial growth factor (VEGF) is an important stimulator. The current anti-ROP therapy has focused on bevacizumab, a monoclonal antibody against VEGF, and pazopanib, a tyrosine kinase inhibitor on the VEGF receptor (VEGFR). Several lines of evidence have proposed that natural compounds may be more effective and safer for anti-VEGF function. Resveratrol, a common natural compound, binds to VEGF and blocks its interaction with VEGFR, thereafter suppressing angiogenesis. Here, we evaluate the efficacy of intravitreal injection, or topical instillation (eye drops), of resveratrol into the eyes of mice suffering from oxygen-induced retinopathy, i.e., developing ROP. The treatment of resveratrol significantly relieved the degree of vascular distortion, permeability and hyperplasia; the efficacy could be revealed by both methods of resveratrol application. In parallel, the treatments of resveratrol inhibited the retinal expressions of VEGF, VEGFR and CD31. Moreover, the applied resveratrol significantly relieved the damage caused by oxygen radicals through upregulating the level of superoxide dismutase (SOD) and downregulating the level of malondialdehyde (MDA) in the retina. Taken together, the potential therapeutic benefit of resveratrol in pro-angiogenic diseases, including retinopathy, can be considered.     

A Critical Overview of Targeted Therapies for Vestibular Schwannoma

Vestibular schwannoma (VS) is a benign tumor that originates from Schwann cells in the vestibular component. Surgical treatment for VS has gradually declined over the past few decades, especially for small tumors. Gamma knife radiosurgery has become an accepted treatment for VS, with a high rate of tumor control. For neurofibromatosis type 2 (NF2)-associated VS resistant to radiotherapy, vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR)-targeted therapy (e.g., bevacizumab) may become the first-line therapy. Recently, a clinical trial using a VEGFR1/2 peptide vaccine was also conducted in patients with progressive NF2-associated schwannomas, which was the first immunotherapeutic approach for NF2 patients. Targeted therapies for the gene product of SH3PXD2A-HTRA1 fusion may be effective for sporadic VS. Several protein kinase inhibitors could be supportive to prevent tumor progression because merlin inhibits signaling by tyrosine receptor kinases and the activation of downstream pathways, including the Ras/Raf/MEK/ERK and PI3K/Akt/mTORC1 pathways. Tumor-microenvironment-targeted therapy may be supportive for the mainstays of management. The tumor-associated macrophage is the major component of immunosuppressive cells in schwannomas. Here, we present a critical overview of targeted therapies for VS. Multimodal therapy is required to manage patients with refractory VS.     

Rho GTPases in Retinal Vascular Diseases

The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell–cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.     

SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate–SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate–SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.     

Tumor Promoting Effect of BMP Signaling in Endometrial Cancer

The effects of bone morphogenetic proteins (BMPs), members of the transforming growth factor-β (TGF-β) family, in endometrial cancer (EC) have yet to be determined. In this study, we analyzed the TCGA and MSK-IMPACT datasets and investigated the effects of BMP2 and of TWSG1, a BMP antagonist, on Ishikawa EC cells. Frequent ACVR1 mutations and high mRNA expressions of BMP ligands and receptors were observed in EC patients of the TCGA and MSK-IMPACT datasets. Ishikawa cells secreted higher amounts of BMP2 compared with ovarian cancer cell lines. Exogenous BMP2 stimulation enhanced EC cell sphere formation via c-KIT induction. BMP2 also induced EMT of EC cells, and promoted migration by induction of SLUG. The BMP receptor kinase inhibitor LDN193189 augmented the growth inhibitory effects of carboplatin. Analyses of mRNAs of several BMP antagonists revealed that TWSG1 mRNA was abundantly expressed in Ishikawa cells. TWSG1 suppressed BMP7-induced, but not BMP2-induced, EC cell sphere formation and migration. Our results suggest that BMP signaling promotes EC tumorigenesis, and that TWSG1 antagonizes BMP7 in EC. BMP signaling inhibitors, in combination with chemotherapy, might be useful in the treatment of EC patients.     

Vasostatin Inhibits VEGF-Induced Endothelial Cell Proliferation,Tube Formation and Induces Cell Apoptosis under Oxygen Deprivation

Anti-angiogenesis treatment has been a promising new form of cancer therapy. Endothelial cells are critical for vascular homeostasis and play important roles in angiogenesis, vascular and tissue remodeling. Vasostatin, the 180 amino acid N-terminal fragment of the calreticulin protein, is reported to be a potent endogenous inhibitor of angiogenesis, suppressing tumor growth. However, the mechanism of these effects has not been sufficiently investigated. This study was performed to investigate the possible mechanism of vasostatin effects on primary cultured human umbilical vein endothelial cells (HUVEC). We found that vasostatin could inhibit the cell viability of HUVEC and induce cell apoptosis through mitochondrial pathways via activation of caspase-3 under oxygen deprivation conditions. Meanwhile, vasostatin also inhibited vascular endothelial growth factor-induced proliferation and tube formation of HUVEC. The possible mechanism of vasostatin-inhibited proliferation of HUVEC could be through down-regulation of endothelial nitric oxide synthase. These findings suggest that vasostatin could regulate endothelial cell function and might be used in anti-angiogenesis treatment.     

Anti-Angiogenic Properties of BDDPM,a Bromophenol from Marine Red Alga Rhodomela confervoides,with Multi Receptor Tyrosine Kinase Inhibition Effects

Bis-(2,3-dibromo-4,5-dihydroxy-phenyl)-methane (BDDPM) is a bromophenol first isolated from Rhodomelaceae confervoides. Our previous studies showed that BDDPM exerts PTP1B-inhibiting activity and anti-cancer activity against a wide range of tumor cells while it also showed lower cytotoxicity against normal cells. In the present study, we found that BDDPM exhibits significant activities toward angiogenesis in vitro. BDDPM inhibits multiple angiogenesis processes, including endothelial cell sprouting, migration, proliferation, and tube formation. Further kinase assays investigations found that BDDPM is a potent selective, but multi-target, receptor tyrosine kinase (RTKs) inhibitor. BDDPM (10 μM) inhibits the activities of fibroblast growth factor receptor 2 and 3 (FGFR2, 3), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor α (PDGFRα) (inhibition rate: 57.7%, 78.6%, 78.5% and 71.1%, respectively). Moreover, BDDPM also decreases the phosphorylation of protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS), as well as nitric oxide (NO) production in a dose dependent manner. These results indicate that BDDPM can be exploited as an anti-angiogenic drug, or as a lead compound for the development of novel multi-target RTKs inhibitors.     

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.