Thienopyrimidine-based agents bearing diphenylurea: Design, synthesis, and evaluation of antiproliferative and antiangiogenic activity.

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.

Etiology of OHSS and use of dopamine agonists

This study was designed to evaluate the antiangiogenic properties of emodin and its ability to inhibit tyrosine-kinase-mediated phosphorylation of vascular endothelial growth factor (VEGF) receptors in colon cancer cells. The effects of emodin on VEGF-receptor (VEGFR) phosphorylation were determined by assaying the tyrosine kinase activity and by Western blot analysis. The antiproliferative and proapoptotic activities of emodin were evaluated by soft agar colony formation, flow cytometric analysis of cell cycle, and by apoptotic assay. Emodin causes a dose-dependent inhibition of VEGFR phosphorylation in colon cancer cells. Treatment with 40 muM of emodin decreased the relative activity of VEGFR-1 to 22.4%, when compared to the control group (assigned a value of 100%); VEGFR-2 and -3 showed a similar reduction in relative activity at 58.5% and 31.6%, respectively (p < 0.01, in each case). Treatment with emodin reduced VEGFR phosphorylation, as evidenced by Western blot analysis. Flow cytometric analysis showed that, upon treatment with emodin, the HCT116 cell cycle was blocked at the G2/M phase. Emodin also increased the apoptosis of HCT116 cells in a dose-dependent manner; treatment with 40 muM emodin increased the apoptotic rate from 8.1% +/- 2.7% in the control group to 27.8% +/- 10.9% in the treated group (p < 0.01). These studies demonstrate that emodin may inhibit cancer-cell growth by blocking VEGFR signaling and indicate that emodin can be used as a potential inhibitor for tumor angiogenesis.

Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway

PARP-1, a DNA base repair enzyme, is activated by DNA breaks induced by oxidative (ROS) and nitrosative (RNS) stress. By consuming NAD(+), PARP-1 activation can lead to ATP depletion and cell death. Studies suggest that inhibiting PARP-1 activity can attenuate pathologies associated with vascular smooth muscle and endothelial dysfunction. PARP-1 inhibition can also activate the prosurvival serine/threonine kinase, Akt. Vascular endothelial growth factor (VEGF) regulates endothelial cell survival via Akt activation downstream of VEGF receptor 2 (VEGFR2) activation. Here we investigated the hypothesis that PARP-1 inhibition protects human umbilical vein endothelial cells (HUVECs) from ROS- and RNS-induced cell death by limiting NAD(+) depletion and by activating a prosurvival signaling pathway via VEGFR2 phosphorylation. We activated PARP-1 in HUVECs by treatment with hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)). Both depleted HUVECs of NAD(+) and ATP, processes that were limited by the PARP-1 inhibitor, PJ34. ONOO(-) and H(2)O(2)-induced cell death and apoptosis were attenuated in cells treated with PJ34 or PARP-1 siRNA. PARP-1 inhibition increased Akt, BAD, and VEGFR2 phosphorylation in HUVECs and in PJ34-treated rabbit aortas. The VEGFR2-specific tyrosine kinase inhibitor SU1498 decreased PARP-1 inhibition-mediated phosphorylation of VEGFR2 and Akt, and also reversed survival effects of PJ34. Finally, PARP-1 inhibition protected cells from death induced by serum starvation, evidence for a role in cell survival independent of energy protection. PARP-1 inhibition prevents ROS- and RNS-induced HUVEC death by maintaining cellular energy in the form of NAD(+) and ATP, and also by activating a survival pathway via VEGFR2, Akt, and BAD phosphorylation.

The extracellular region (EC) of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) contains seven immunoglobulin-like (Ig-like) domains that are required for specific ligand binding and receptor dimerization. Studies of domain 4-7 deletions and substitutions provided insights into the interaction between receptors in the absence of VEGF. In this study, we investigated the effect of domain 4 in ligand-independent VEGFR-2 dimerization and activation in human vascular endothelial cells and human breast cancer cells. To confirm the role of domain 4 in ligand-independent receptor dimerization and activation, two VEGFR-2 fragments with and without domain 4, KFP1 and KFP2, were generated by recombinant DNA technology. We measured the affinity of KFP1 and KFP2 with VEGFR-2, and the roles of KFP1 and FKP2 in dimerization and phosphorylation of VEGFR-2. We also evaluated the effect of KFP1 and FKP2 on cell proliferation and migration in HUVECs and in human breast cancer cells. We showed that KFP1 did not affect the interaction of VEGFR-2 and VEGF but bound VEGFR-2 in the absence of VEGF. Furthermore, cross-linking and cross-linking immunoblotting demonstrated that KFP1 could form a complex with VEGFR-2, which resulted in VEGFR-2 dimerization in the absence of VEGF. Importantly, we found that the KDR fragment with domain 4 induced phosphorylation of VEGFR-2, as well as phosphorylation of downstream receptor kinases in HUVECs and VEGFR-2-positive breast cancer cells. Consistent with these results, this ligand-independent activation of VEGFR-2 also promoted downstream signaling and cell proliferation and migration. The domain 4 of VEGFR-2 plays an important role in the interaction between VEGFR receptors in the absence of VEGF.

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis. TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLCγ1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5. Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is a critical protein involved in tumor progression, making it an attractive target for cancer therapy. This study aimed to synthesize and evaluate novel thieno[2,3-d]pyrimidine analogues as potential anticancer VEGFR-2 inhibitors. The thieno[2,3-d]pyrimidine analogues were synthesized following the pharmacophoric features of VEGFR-2 inhibitors. The anticancer potential was assessed against PC3 and HepG2 cell lines. The VEGFR-2 inhibition was evaluated through IC50 determination. Cell cycle analysis and apoptosis assays were performed to elucidate the mechanisms of action. Molecular docking, molecular dynamics simulations, MM-GBSA, and PLIP studies were conducted to investigate the binding affinities and interactions with VEGFR-2. Additionally, in silico ADMET studies were performed. Compound 8b demonstrated significant anti-proliferative activities with IC50 values of 16.35 μM and 8.24 μM against PC3 and HepG2 cell lines, respectively, surpassing sorafenib and exhibiting enhanced selectivity indices. Furthermore, compound 8b showed an IC50 value of 73 nM for VEGFR-2 inhibition. Cell cycle analysis revealed G2-M phase arrest, while apoptosis assays demonstrated increased apoptosis in HepG2 cells. Molecular docking and dynamic simulations confirmed the binding affinity and interaction of compound 8b with VEGFR-2, supported by MMGBSA and PLIP studies. In silico ADMET studies indicated the drug development potential of the synthesized thieno[2,3-d]pyrimidines. The study highlights compound 8b as a promising VEGFR-2 inhibitor with potent anti-proliferative activities. Its mechanism of action involves cell cycle arrest and induction of apoptosis. Further, molecular docking and dynamic simulations support the strong binding affinity of compound 8b to VEGFR-2.

Vascular endothelial cadherin (VEC) inhibits the signaling of the vascular endothelial growth factor receptor (VEGFR), which may contribute to contact-mediated growth inhibition, a phenomenon that is lost in cancerous cells. Lampugnani et al. show that VEC sequesters VEGFR at the plasma membrane, preventing internalization. When sequestered at the plasma membrane at the site of cell-cell junctions, VEC promotes the dephosphorylation of VEGFR through the recruitment of the phosphatase DEP-1 to the complex. The authors studied VEGFR endocytosis and signaling in human umbilical vein endothelial cells (HUVECs) and VEC-null or VEC-positive cultured cell lines. In HUVECs, VEGF-stimulated internalization of VEGFR was decreased in confluent cultures compared with that observed in sparse cultures. A similar lower amount of internalization was observed in VEC-positive cultured cells compared with that in VEC-null cells. Internalized VEGFR was active, and the VEC-null cells showed greater abundance of tyrosine-phosphorylated VEGFR and activated phospholipase C-γ on internal membranes than did the VEC-positive cells. Internalization was clathrin mediated, and inhibition of clathrin-mediated endocytosis in the VEC-null cells returned VEGFR signaling--measured as tyrosine phosphorylation of mitogen-activated protein kinases (MAPKs) p44 and p42--to levels similar to those in VEC-positive cells. VEC was not endocytosed with VEGFR, suggesting that VEGFR activity was inhibited at the plasma membrane. VEC-positive cells in which DEP-1 was knocked down with silencing RNA showed enhanced VEGFR internalization and VEGFR phosphorylation and MAPK activation. Thus, VEC appears to stabilize VEGFR at the cell surface by stimulating VEGFR dephosphorylation at sites of cell-cell contact through the recruitment of DEP-1. Signaling by VEGFR after internalization appears to be an important mechanism of signal transduction by this growth factor receptor, and blocking this internalization is one mechanism by which activity can be controlled. M. G. Lampugnani, F. Orsenigo, M. C. Gagliani, C. Tacchetti, E. Dejana, Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J. Cell Biol. 174 , 593-604 (2006). [Abstract] [Full Text]

VEGF signaling through activation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an endothelial cell-specific receptor tyrosine kinase is indispensable for developmental angiogenesis and cancer progression. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of anti-angiogenic therapies. As part of this strategy inducing apoptosis in cancer cells could make the therapeutic agents very effective. The in vitro anti-angiogenic activity of water soluble form of JFD named as JFD-WS was examined by ECMatrixTM gel assay with human umbilical vein endothelial cells (HUVEC) using 0.01-10µM concentrations at different time intervals (0, 4 and 8 hrs). A complete inhibition of in vitro angiogenesis was observed at 8 hrs after treating with 10µM concentration of JFD-WS. Furthermore, the VEGFR2 phosphorylation was inhibited by 38.65% in JFD-WS (1.0µM) treated HUVEC cells. Besides the in vitro anti-angiogenic activity, JFD-WS is also proven as a potent anti-cancer drug in this study by using GI-101A (human breast adenocarcinoma) xenograft implanted athymic nude mice. In our in vivo experiments, the intraperitoneal (i.p.) injection of JFD-WS (at a dose of 100 mg/kg body weight) was able to significantly inhibit the tumor growth compared to the control group. At the end of the treatment period nearly 35.19% inhibition of tumor growth was observed in JFD-WS treated animals along with prolongation of survival. The tumor inhibition was lot more effective when JFD-WS was combined with Taxol (10 mg/kg). Eventually, the serum levels of MUC1 proved a significant regression of the tumor burden in the experimental animals. In addition to the anti-angiogenic ability, the anti-tumor activity of JFD-WS seems to be further enhanced due to induction of apoptosis signals in the xenograft tumor implanted animals. The protein expression levels of key apoptotic signaling molecules such as Bax, Bcl2, cytochrome c, Apaf-1 and cleaved caspase-3 and p53 were analyzed in the tumor samples that were extracted from experimental animals. Interestingly, a significant increase in the expression of pro-apoptotic proteins p53 (3 fold), Bax (10.6 fold) and Apaf-1 (2.2 fold) were observed in JFD-WS treated animals. In support of the induction of apoptotic signals, the expression of anti-apoptotic protein Bcl2 was decreased by 1.8 fold. Consequently, the release of cytochrome c and the cleavage of caspase 3 found in the cytosolic fraction were significantly higher in JFD-WS treated group as compared with the untreated controls. Finally, the pharmacokinetics (PK) property of JFD-WS was analyzed by measuring the concentration in plasma and urine samples of Balb/c mice at different time intervals (Plasma: 2.5, 5, 10, 15, 30, 60, 120, 1440 min; and Urine: 15, 30, 60, 120, 1440 min), using a HPLC method. Our experiments showed that JFD-WS can reach peak plasma concentration after 15 mins and a maximum elimination in urine was observed after 30 mins of i.p. injection. The compound was undetectable in both plasma and urine after 24 hrs. Moreover, a two compartment model of absorption, distribution and elimination for JFD-WS, with the half-life (t1/2) of around 33 mins is suspected. Our results from both in vivo and in vitro experiments confirm the anti-angiogenic and pro-apoptotic effects of JFD-WS in xenograft tumor implanted athymic nude mice. Furthermore, our data clearly indicate that, JFD-WS is highly effective in causing tumor inhibition with minimal toxicity. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Manasa Subbarao, Janelle Alvarez, Meera Bhalani, Appu Rathinavelu. The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A05.

Human hepatocellular carcinoma is one of the most common recurrent malignancies since there is no effective therapy for it. Silibinin, a widely used drug and supplement for various liver disorders, demonstrated anti-cancer effects on human hepatocellular carcinoma, human prostate adenocarcinoma cells, human breast carcinoma cells, human ectocervical carcinoma cells, and human colon cancer cells. Considering the anti-hepatotoxic activity of silibinin and its strong preventive and anti-cancer efficacy against various epithelial cancers, we investigated the efficacy of silibinin against human HCC and HUVEC cell lines. Silibinin effects on the growth and mode of cell death of these two cell lines are presented in this paper. HepG2 and HUVEC cells were incubated with different doses of silibinin (12.5, 25, 50, 100, 150 and 200 μg/mL) at 24, 48, and 72 h. Cytotoxicity was assessed using MTT and Trypan blue assays. Mode of cell death induced by silibinin was investigated using LDH assay and acridine orange/PI double dye staining. The results showed that silibinin has dose-dependent inhibitory effect on the viability of HepG2 and HUVEC cells. However, Silibinin causes a more continuous dose-dependent cytotoxicity in HepG2 cells compared to the HUVEC cells in which some degrees of resistance is apparent at the beginning. The mode of cell death looks also different in these two cell lines with HepG2 cells being more in favor of apoptosis while necrosis is more evident for the HUVEC cells.

Increased placental angiogenesis in late and early onset pre-eclampsia is associated with differential activation of vascular endothelial growth factor receptor 2

Soluble and Immobilized Vegf Induce Distinct Patterns of VEGFR2 Phosphorylation Mediated by Intracellular Trafficking

Vascular Repair After Menstruation Involves Regulation of Vascular Endothelial Growth Factor-Receptor Phosphorylation by sFLT-1