Molecular signatures of angiogenesis inhibitors: a single-embryo untargeted metabolomics approach in zebrafish.

Angiogenesis is a key process in embryonic development, a disruption of this process can lead to severe developmental defects, such as limb malformations. The identification of molecular perturbations representative of antiangiogenesis in zebrafish embryo (ZFE) may guide the assessment of developmental toxicity from an endpoint- to a mechanism-based approach, thereby improving the extrapolation of findings to humans. Thus, the aim of the study was to discover molecular changes characteristic of antiangiogenesis and developmental toxicity. We exposed ZFEs to two antiangiogenic drugs (SU4312, sorafenib) and two developmental toxicants (methotrexate, rotenone) with putative antiangiogenic action. Molecular changes were measured by performing untargeted metabolomics in single embryos. The metabolome response was accompanied by the occurrence of morphological alterations. Two distinct metabolic effect patterns were observed. The first pattern comprised common effects of two specific angiogenesis inhibitors and the known teratogen methotrexate, strongly suggesting a shared mode of action of antiangiogenesis and developmental toxicity. The second pattern involved joint effects of methotrexate and rotenone, likely related to disturbances in energy metabolism. The metabolites of the first pattern, such as phosphatidylserines, pterines, retinol, or coenzyme Q precursors, represented potential links to antiangiogenesis and related developmental toxicity. The metabolic effect pattern can contribute to biomarker identification for a mechanism-based toxicological testing.

Botulinum toxin type A in multimodal management of age-related macular degeneration and related diseases.

Age related macular degeneration (AMD) is the major cause of visual loss in the aging population in the Western world. In past decade, intra ocular injections of anti-vascular endothelial growth factor (anti-VEGF) pharmaceuticals have revolutionized therapy for exudative (edematous-wet) AMD and become standard practice for the near term. However repeated intra-ocular injections are required for years and long terms results have been limited. The pathogenesis of this condition is multifactorial involving genetic, ischemic, inflammatory factors leading to neovascularization, edema and retinal pigment epithelial scaring resulting in photoreceptor destruction. Based on coincidental observation in reduction in AMD related macular edema on ocular coherence tomography (OCT) in a BoNT A treated patient with facial movement disease, BoNT-A at conventional doses targeting the para orbital area was added to therapeutic regiment in a small number of patients with exudative macular degeneration or related diseases. Measurements of edema and choriocapillaris using Spectral Doman (OCT) and Ocular Coherence Angiography (OCT-A) and Snellen visual acuity were made over the evaluation period. 15 eyes in 14 patients averaged 361 µm central sub foveal edema (CSFT) pre injection and average of 266 µm (CSFT) post injection over an average of 21 months and 5.7 cycles using BoNT A alone at conventional doses (n = 86 post injection measurements, paired t-test p < 0.001 two tailed). Visions at baseline in patients with 20/40 or worse averaged 20/100- pre injection improved to an average of 20/40- in the post injection period (n = 49 measurements p < 0.002 paired t-test). The previous data was added to a group of 12 more severely afflicted patients receiving anti VEGF (aflibercept or bevacizumab) (total 27 patients). With this 27-patient group, patients were followed for an average of 20 months and receiving average of 6 cycles at conventional doses. Improvement in exudative edema and vision were noted with pre injection baseline CSFT average 399.5, post injection average 267, n = 303 post measurement, independent t-test P < 0.0001.). Snellen vision 20/128 baseline average improved to average of 20/60- during post injection period (n = 157 post injection measurements, p < 0.0001 paired t-test to baseline). No substantial adverse effects were noted. Cyclic effects were noted corresponding to duration of action of BoNT-A on a number of patients. The above data is preliminary and is skewed toward early leakage for all conditions. BoNT A may have a role in the treatment of aged related macular degeneration. Controlled studies are needed with careful staging and baseline stratifications for multi-modal management paradigms. The findings are discussed relative to known botulinum toxin type A pharmacology and AMD pathogenesis.

A systematic comparison of anti-angiogenesis efficacy and cardiotoxicity of receptor tyrosine kinase inhibitors in zebrafish model.

Pathological angiogenesis is fundamental to progression of cancerous tumors and blinding eye diseases. Anti-angiogenic receptor tyrosine kinase inhibitors (TKIs) are in broad use for the treatment of these diseases. With more and more TKIs available, it is a challenge to make an optimal choice. It remains unclear whether TKIs demonstrate similar anti-angiogenesis activities in different tissues. Many TKIs have shown varying degrees of toxic effects that should also be considered in clinical use. This study investigates the anti-angiogenic effects of 13 FDA-approved TKIs on the intersegmental vessels (ISVs), subintestinal vessels (SIVs) and retinal vasculature in zebrafish embryos. The results show that vascular endothelial growth factor receptor TKIs (VEGFR-TKIs) exhibit anti-angiogenic abilities similarly on ISVs and SIVs, and their efficacy is consistent with their IC50 values against VEGFR2. In addition, VEGFR-TKIs selectively induces the apoptosis of endothelial cells in immature vessels. Among all TKIs tested, axitinib demonstrates a strong inhibition on retinal neovascularization at a low dose that do not strongly affect ISVs and SIVs, supporting its potential application for retinal diseases. Zebrafish embryos demonstrate cardiotoxicity after VEGFR-TKIs treatment, and ponatinib and sorafenib show a narrow therapeutic window, suggesting that these two drugs may need to be dosed more carefully in patients. We propose that zebrafish is an ideal model for studying in vivo antiangiogenic efficacy and cardiotoxicity of TKIs.

Antitumor and antiangiogenic effects of Tonantzitlolone B, an uncommon diterpene from Stillingia loranthacea.

Natural products have played a pivotal role for the discovery of anticancer drugs. Tonantzitlolones are flexibilan-type diterpenes rare in nature; therefore, few reports have shown antiviral and cytotoxic activities. This study aimed to investigate the in vivo antitumor action of Tonantzitlolone B (TNZ-B) and its toxicity. Toxicity was evaluated in mice (acute and micronucleus assays). Antitumor activity of TNZ-B (1.5 or 3 mg/kg intraperitoneally - i.p.) was assessed in Ehrlich ascites carcinoma model. Angiogenesis and reactive oxygen species (ROS) and nitric oxide (NO) production were also investigated, in addition to toxicological effects after 7-day treatment. The LD50 (lethal dose 50%) was estimated at around 25 mg/kg (i.p.), and no genotoxicity was recorded. TNZ-B reduced the Ehrlich tumor's volume and total viable cancer cell count (p < 0.001 for both). Additionally, TNZ-B reduced peritumoral microvessel density (p < 0.01), suggesting antiangiogenic action. Moreover, a decrease was observed on ROS (p < 0.05) and nitric oxide (p < 0.001) levels. No significant clinical findings were observed in the analysis of biochemical, hematological, and histological (liver and kidney) parameters. In conclusion, TNZ-B exerts antitumor and antiangiogenic effects by reducing ROS and NO levels and has weak in vivo dose-repeated toxicity. These data contribute to elucidate the antitumor action of TNZ-B and point the way for further studies with this natural compound as an anticancer drug.

Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate.

Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure.

BACKGROUND: Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH. METHODS: We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5). RESULTS: We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV. CONCLUSIONS: Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.

Sorafenib not only impairs endothelium-dependent relaxation but also promotes vasoconstriction through the upregulation of vasoconstrictive endothelin type B receptors.

As a VEGF-targeting agent, sorafenib has been used to treat a number of solid tumors but can easily lead to adverse vascular effects. To elucidate the underlying mechanism, rat mesenteric arteries were subjected to organ cultured in the presence of different concentrations of sorafenib (0, 3, 6 and 9 mg/L) with or without inhibitors (U0126, 10-5 M; SB203580, 10-5 M; SP200126, 10-5 M) of MAPK kinases, and then acetylcholine- or sodium nitroprusside-induced vasodilation and sarafotoxin 6c-induced vasoconstriction were monitored by a sensitive myograph. The NO synthetases, the nitrite levels, the endothelial marker CD31,the ETB and ETA receptors and the phosphorylation of MAPK kinases were studied. Next, rats were orally administrated by sorafenib for 4 weeks (7.5 and 15 mg/kg/day), and their blood pressure, plasma ET-1, the ETB and ETA receptors and the phosphorylation of MAPK kinases in the mesenteric arteries were investigated. The results showed that sorafenib impairs endothelium-dependent vasodilation due to decreased NO levels and the low expression of eNOS and iNOS. Weak staining for CD31 indicated that sorafenib induced endothelial damage. Moreover, sorafenib caused the upregulation of vasoconstrictive ETB receptors, the enhancement of ETB receptor-mediated vasoconstriction and the activation of JNK/MAPK. Blocking the JNK, ERK1/2 and p38/MAPK signaling pathways by using the inhibitors significantly abolished ETB receptor-mediated vasoconstriction. Furthermore, it was observed that the oral administration of sorafenib caused an increase in blood pressure and plasma ET-1, upregulation of the ETB receptor and the activation of JNK in the mesenteric arteries. In conclusion, sorafenib not only impairs endothelium-dependent vasodilatation but also enhances ETB receptor-mediated vasoconstriction, which may be the causal factors for hypertension and other adverse vascular effects in patients treated with sorafenib.

Evaluation of curcumin-loaded polymeric nanocapsules with different coatings in chick embryo model: influence on angiogenesis, teratogenesis and oxidative stress.

BACKGROUND: Curcumin (CUR) is a bioactive compound with several proven pharmacological properties. However, the major limitation for therapeutic use of CUR is its low bioavailability. In this sense, an alternative to this question is the use of polymeric nanocapsules (NC) as drug/nutraceutical delivery systems. Thus, the aim of current study was to assess the effect of CUR-loaded NC and their different coatings in chick embryo model, evaluating angiogenic, teratogenic and oxidative stress parameters. METHODS: The physicochemical characterization of unloaded and loaded NC with different coatings: (U-NC (P80), U-NC (PEG), U-NC (EUD), U-NC (CS), CUR-NC (P80), CUR-NC (PEG), CUR-NC (EUD) and CUR-NC (CS)) were performed. After 9 days of incubation, eggs were treated (10 mL/kg eggs; via injection) with NC (unloaded and loaded with CUR) and CUR-solution. In sequence, hen's egg test-chorioallantoic membrane (HET-CAM), angiogenic assay, external abnormalities, weight of embryos and oxidative stress markers (TBARS, NPSH, ROS and CAT) were analyzed. RESULTS: CUR-NC (P80, PEG, EUD and CS) treatments caused antiangiogenic and non-teratogenic effects in chick embryo model. Still, CUR-NC (P80), CUR-NC (PEG), CUR-NC (EUD) and CUR-NC (CS) did not alter markers of oxidative stress (TBARS, NPSH, CAT) studied. Only CUR-NC (EUD) caused increase in ROS levels. CONCLUSION: Wherefore, these findings of present study represent a advance in research of drug/nutraceutical delivery systems.

Dasatinib and PD-L1 inhibitors provoke toxicity and inhibit angiogenesis in the embryo.

Dasatinib is a targeted cancer therapy, while programmed death ligand 1 (PD-L1) inhibitors are a form of immune checkpoint therapy used to treat various types of cancers. Several studies showed the potential efficacy of these drugs in the management of triple-negative breast cancer- an aggressive subtype of breast cancer, which can develop during pregnancy. Nevertheless, side effects of Dasatinib (DA) and PD-L1 drugs during pregnancy, especially in the early stages of embryogenesis are not explored yet. The aim of this study is to assess the individual and combined toxicity of DA and PD-L1 inhibitors during the early stages of embryogenesis and to evaluate their effect(s) on angiogenesis using the chorioallantoic membrane (CAM) model of the embryo. Our results show that embryos die at greater rates after exposure to DA and PD-L1 inhibitors as compared to their matched controls. Moreover, treatment with these drugs significantly inhibits angiogenesis of the CAM. To further elucidate key regulator genes of embryotoxicity induced by the actions of PD-L1 and DA, an RT-PCR analysis was performed for seven target genes that regulate cell proliferation, angiogenesis, and survival (ATF3, FOXA2, MAPRE2, RIPK1, INHBA, SERPINA4, and VEGFC). Our data revealed that these genes are significantly deregulated in the brain, heart, and liver tissues of exposed embryos, compared to matched control tissues. Nevertheless, further studies are necessary to evaluate the effects of these anti breast cancer drugs and elucidate their role during pregnancy.

Efficacy of the thromboxane receptor antagonist NTP42 alone, or in combination with sildenafil, in the sugen/hypoxia-induced model of pulmonary arterial hypertension.

NTP42 is a novel antagonist of the thromboxane A2 receptor (TP) in development for the treatment of pulmonary arterial hypertension (PAH). Recent studies demonstrated that NTP42 and TP antagonism have a role in alleviating PAH pathophysiology. However, the efficacy of NTP42 when used in combination with existing PAH therapies has not yet been investigated. Herein, the Sugen 5416/hypoxia (SuHx)-induced PAH model was employed to evaluate the efficacy of NTP42 when used alone or in dual-therapy with Sildenafil, a PAH standard-of-care. PAH was induced in rats by injection of Sugen 5416 and exposure to hypoxia for 21 days. Thereafter, animals were treated orally twice-daily for 28 days with either vehicle, NTP42 (0.05 mg/kg), Sildenafil (50 mg/kg), or NTP42+Sildenafil (0.05 mg/kg + 50 mg/kg, respectively). While Sildenafil or NTP42 mono-therapy led to non-significant reductions in the SuHx-induced rises in mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RSVP), combined use of NTP42+Sildenafil significantly reduced these increases in mPAP and RVSP. Detailed histologic analyses of pulmonary vessel remodelling, right ventricular hypertrophy and fibrosis demonstrated that while NTP42 and Sildenafil in mono-therapy resulted in significant benefits, NTP42+Sildenafil in dual-therapy showed an even greater benefit over either drug used alone. In summary, combined use of NTP42+Sildenafil in dual-therapy confers an even greater benefit in treating or offsetting key aetiologies underlying PAH. These findings corroborate earlier preclinical findings suggesting that, through antagonism of TP signalling, NTP42 attenuates PAH pathophysiology, positioning it as a novel therapeutic for use alone or in combination therapy regimens.

Endothelial HIF-2α as a Key Endogenous Mediator Preventing Emphysema.

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema.Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice.Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression.Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α-knockout mice developed more severe emphysema, whereas EC Hif-2α-overexpressing mice were protected. EC Hif-2α-knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression.Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

Longitudinal Toxicity over Time (ToxT) analysis to evaluate tolerability: a case study of lenalidomide in the CALGB 50401 (Alliance) trial.

Evaluation of tolerability is increasingly relevant for patients with haematological malignancies treated with chronically administered therapies. Adverse events from these agents might affect the ability of patients to tolerate treatment over time. Conventional toxicity tables that include the incidence of high-grade adverse events, defined by the Common Terminology Criteria for Adverse Events, do not provide information on the time profile of these adverse events or reflect the continuous, lower grade symptomatic toxicities that are particularly relevant to treatment tolerability for patients living with indolent disease. Modern approaches to the evaluation and reporting of toxicity that capture the tolerability of treatment to the patient are imperative. In this Viewpoint, we present a focused, pilot, and longitudinal Toxicity over Time analysis of adverse events from lenalidomide and lenalidomide with rituximab in patients with follicular lymphoma treated in the CALGB 50401 (Alliance; NCT00238238) trial to define the trajectory of adverse events and quantify the burden of continuous, low-grade events. Toxicity over Time analyses provided clinically relevant descriptions of neutropenia and fatigue trajectories caused by lenalidomide that were not identified by standard analysis of the maximum grade events defined by the Common Terminology Criteria for Adverse Events. Systematic, rigorous incorporation of patient-reported outcomes in clinical trials will be crucial to our understanding of the tolerability of chronically administered therapies in patients with haematological malignancies.

A novel piperine analogue exerts in vivo antitumor effect by inducing oxidative, antiangiogenic and immunomodulatory actions.

Structural diversity characterizes natural products as prototypes for design of lead compounds. The aim of this study was to synthetize, and to evaluate the toxicity and antitumor action of a new piperine analogue, the butyl 4-(4-nitrobenzoate)-piperinoate (DE-07). Toxicity was evaluated against zebrafish, and in mice (acute and micronucleus assays). To evaluate the DE-07 antitumor activity Ehrlich ascites carcinoma model was used in mice. Angiogenesis, Reactive Oxygen Species (ROS) production and cytokines levels were investigated. Ninety-six hours exposure to DE-07 did not cause morphological or developmental changes in zebrafish embryos and larvae, with estimated LC50 (lethal concentration 50%) higher than 100 µg/mL. On the acute toxicity assay in mice, LD50 (lethal dose 50%) was estimated at around 1000 mg/kg, intraperitoneally (i.p.). DE-07 (300 mg/kg, i.p.) did not induce increase in the number of micronucleated erythrocytes in mice, suggesting no genotoxicity. On Ehrlich tumor model, DE-07 (12.5, 25 or 50 mg/kg, i.p.) induced a significant decrease on cell viability. In addition, there was an increase on ROS production and a decrease in peritumoral microvessels density. Moreover, DE-07 induced an increase of cytokines levels involved in oxidative stress and antiangiogenic effect (IL-1ß, TNF-α and IL-4). No significant clinical toxicological effects were recorded in Ehrlich tumor transplanted animals. These data provide evidence that DE-07 presents low toxicity, and antitumor effect via oxidative and antiangiogenic actions by inducing modulation of inflammatory response in the tumor microenvironment.

Discovery of a chiral fluorinated azetidin-2-one as a tubulin polymerisation inhibitor with potent antitumour efficacy.

Inhibition of tubulin polymerisation with small molecules has been clinically validated as a promising therapy for multiple solid tumours. Herein, a series of chiral azetidin-2-ones were asymmetrically synthesised and biologically evaluated for antitumour activities. Among them, a chiral fluorinated azetidin-2-one, 18, was found to exhibit the most potent activities against five cancer cell lines, including a drug-resistant cell line, with IC50 values ranging from 1.0 to 3.6 nM. Further mechanistic studies revealed that the compound 18 worked by disrupting tubulin polymerisation, blocking the cell cycle in the G2/M phase, inducing cellular apoptosis, and suppressing angiogenesis. Additionally, 18 exhibited higher human-microsomal metabolic stability and aqueous solubility compared to those of combretastatin A-4. Finally, 18 was also found to effectively inhibit tumour growth in a xenograft mice model with low toxicity and thus might be a promising lead for further clinical development.

Curcumin attenuates bevacizumab-induced toxicity via suppressing oxidative stress and preventing mitochondrial dysfunction in heart mitochondria.

Heart failure was subsequently noted in 2-4% of patients on bevacizumab (BEV). Whereas mitochondria play an important role in myocardial tissue homeostasis, deterioration in mitochondrial function will eventually lead to cardiomyocyte cell death and consequently cardiovascular dysfunction. Therefore, the aim of our study is to search the effects of BEV on isolated rat heart mitochondria and cardiomyocytes, and survey the effect of curcumin as a mitochondrial protective and cardioprotective agent. Rat heart mitochondria and cardiomyocytes were isolated from adult rat heart ventricular. By using biochemical and flow cytometry evaluations, the parameters of mitochondrial toxicity including succinate dehydrogenase (SDH) activity, mitochondrial swelling, mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) formation and lipid peroxidation (LP), and cellular assays such as cytotoxicity and MMP collapse were evaluated. Results revealed that BEV (up to 50 µg/ml) induced a concentration- and time-dependent rise in mitochondrial ROS formation, MMP collapse, mitochondrial swelling, LP, and inhibition of SDH in rat heart mitochondria. Our results showed that curcumin (10-100 µM) significantly ameliorated BEV-induced mitochondrial toxicities. Also, our results in cellular assays confirmed amelioration effect of curcumin against BEV toxicity. These results indicate that the cardiotoxic effects of BEV are associated with mitochondrial dysfunction and ROS formation, which finally ends in MMP collapse and mitochondrial swelling as the "point of no return" in the cascade of events leading to apoptosis. Also, results of this study suggest that probably the combination therapy of BEV and curcumin could decrease mitochondrial effects of this drug.

Developmental toxicity of fipronil in early development of zebrafish (Danio rerio) larvae: Disrupted vascular formation with angiogenic failure and inhibited neurogenesis.

Fipronil has been widely used in agriculture to prevent aggressive insects from damaging agricultural products. Fipronil residues circulate in the environment and they have been detected in non-targeted organisms in aquatic environments. To study the effect of fipronil toxicity on environmental health, 6 h post fertilization (hpf) zebrafish embryos were treated with fipronil for 72 h. LC50 value was obtained by applying varying concentrations of fipronil to zebrafish embryos for 72 h. As zebrafish embryos are useful vertebrate models for studying developmental and genetic findings in toxicology research, they were exposed to fipronil to study detailed elucidating mechanisms with hazardous end points of toxicity. Cell cycle arrest-related apoptosis supported pathological alterations, such as increased mortality, shortened body length, and reduced hatchability. Furthermore, observed heart defects, including edema and irregular heartbeat were caused due to abnormal blood circulation. In transgenic zebrafish models (fli1:eGFP and olig2:dsRED), disrupted blood vessel formations were indicated by eGFP+ endothelial cells. Moreover, neurogenic defects were observed by studying dsRED+ motor neurons and oligodendrocytes. This study demonstrates fipronil accumulation in aquatic environment and its ability to impair essential processes, such as angiogenesis and neurogenesis during early developmental stage of zebrafish, along with general developmental toxicity.

Design and synthesis of novel pyridazinoquinazoline derivatives as potent VEGFR-2 inhibitors: In vitro and in vivo study.

Worldwide, Hepatocellular Carcinoma (HCC) endures to be a prominent cause of cancer death. Treatment of HCC follows multiple therapies which are not entirely applicable for treatment of all patients. HCC usually arises contextual to chronic liver diseases and is often discovered at later stages which makes treatment options more complex. The present study aimed at design, synthesis & evaluation of new pyridazinoquinazoline derivatives as potential nontoxic anti-hepatocellular carcinoma (HCC) agents, through inhibition of Vascular endothelial growth factor -2 (VEGFR-2). Novel Pyridazino[3, 4, 5-de]quinazoline derivatives (2-6) were designed & synthesized. Their structures were confirmed via spectral and microanalytical data. They were tested for their in vitro VEGFR-2 inhibition & anticancer activity against human liver cancer cell line (HEPG-2). Molecular docking was investigated into VEGFR-2 site. In vivo studies of VEGRF-2 inhibition and the anti-apoptotic effect of the new compounds were determined in liver of irradiated rats. Toxicity of synthesized compounds was also assessed. The results showed that compounds 3-6 have significant antitumor activity and proved to be non-toxic. The ethoxy aniline derivative 6, exhibited the highest activity both in vitro and in vivo compared to the reference drug used, sorafenib. Compound 6 could be considered a promising nontoxic anti HCC agent and this could be partially attributed to its VEGFR-2 inhibition. Future preclinical investigation would be carried out to confirm the specific and exact mechanism of action of these derivatives especially compound 6 as an effective pharmaceutical agent after full toxicological and pharmacological assessment.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as novel FAK inhibitors with antitumor and anti-angiogenesis activities.

A series of 2,4-diarylaminopyrimidine derivatives containing dithiocarbamate moiety were designed by molecular hybridization strategy and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most of these compounds exhibit significant antiproliferative activities on human cancer cell lines expressing high levels of FAK at nanomolar concentrations. The compound 14z was identified as the most potent FAK inhibitor among these candidates. 14z has excellent anti-proliferative effect with IC50 values from 0.001 µM to 0.06 µM on HCT116, PC-3, U87-MG and MCF-7 cell lines and relatively less cytotoxicity to a nonmalignant cell line MCF-10A compared with MCF-7 cells (SI value > 10). 14z also exhibits significant FAK inhibitory activity (IC50 = 0.07 nM). In addition, compound 14z causes cell cycle arrest at G2/M and prompted apoptosis in both HCT116 and MCF-7 cells in a dose-dependent manner. Further studies show that compound 14z inhibits migration of MCF-7 and has anti-angiogenesis effect on HUVEC cells.

Discovery of chalcone-modified estradiol analogs as antitumour agents that Inhibit tumour angiogenesis and epithelial to mesenchymal transition.

Angiogenesis plays an essential role in tumourigenesis and tumour progression, and anti-angiogenesis therapies have shown promising antitumour effects in solid tumours. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been regarded as a potential antitumour agent mainly targeting angiogenesis. Here we synthesized a novel series of chalcones based on 2-methoxyestradiol and evaluated their potential activities against tumours. Compound 11e was demonstrated to have potent antiangiogenic activity. Further studies showed that 11e suppressed tumour growth in human breast cancer (MCF-7) xenograft models without obvious side effects. Evaluation of the mechanism revealed that 11e targeted the epithelial to mesenchymal transition (EMT) process in MCF-7 cells and inhibited HUVEC migration and then contributed to hindrance of angiogenesis. Thus, 11e may be a promising antitumour agent with excellent efficacy and low toxicity.