Inhibition of adipose tissue angiogenesis prevents rebound weight gain after caloric restriction in mice fed a high-fat diet.

AIMS: We investigated whether modulation of white adipose tissue (WAT) vasculature regulates rebound weight gain (RWG) after caloric restriction (CR) in mice fed a high-fat diet (HFD). MAIN METHODS: We compared changes in energy balance, hypothalamic neuropeptide gene expression, and characteristics of WAT by RT-qPCR, ELISA, immunohistochemistry, and adipose-derived stromal vascular fraction spheroid sprouting assay in obese mice fed a HFD ad libitum (HFD-AL), mice under 40 % CR for 3 or 4 weeks, mice fed HFD-AL for 3 days after CR (CRAL), and CRAL mice treated with TNP-470, an angiogenic inhibitor. KEY FINDINGS: WAT angiogenic genes were expressed at low levels, but WAT vascular density was maintained in the CR group compared to that in the HFD-AL group. The CRAL group showed RWG, fat regain, and hyperphagia with higher expression of angiogenic genes and reduced pericyte coverage of the endothelium in WAT on day 3 after CR compared to the CR group, indicating rapidly increased angiogenic activity after CR. Administration of TNP-470 suppressed RWG, fat regain, and hyperphagia only after CR compared to the CRAL group. Changes in circulating leptin levels and hypothalamic neuropeptide gene expression were correlated with changes in weight and fat mass, suggesting that TNP-470 suppressed hyperphagia independently of the hypothalamic melanocortin system. Additionally, TNP-470 increased gene expression related to thermogenesis, fuel utilization, and browning in brown adipose tissue (BAT) and WAT, indicating TNP-470-induced increase in thermogenesis. SIGNIFICANCE: Modulation of the WAT vasculature attenuates RWG after CR by suppressing hyperphagia and increasing BAT thermogenesis and WAT browning.

Methionine aminopeptidase­2 is a pivotal regulator of vasculogenic mimicry.

Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase­2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP­470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2­knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re­expression of wild­type MetAP2 restored VM in the MetAP2­KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP­470 as potent VM­suppressing agents.

The methionine aminopeptidase 2 inhibitor, TNP-470, enhances the antidiabetic properties of sitagliptin in mice by upregulating xenin.

The therapeutic mechanism of action of methionine aminopeptidase 2 (MetAP2) inhibitors for obesity-diabetes has not yet been fully defined. Xenin, a K-cell derived peptide hormone, possesses an N-terminal Met amino acid residue. Thus, elevated xenin levels could represent a potential pharmacological mechanism of MetAP2 inhibitors, since long-acting xenin analogues have been shown to improve obesity-diabetes. The present study has assessed the ability of the MetAP2 inhibitor, TNP-470, to augment the antidiabetic utility of the incretin-enhancer drug, sitagliptin, in high fat fed (HFF) mice. TNP-470 (1 mg/kg) and sitagliptin (25 mg/kg) were administered once-daily alone, or in combination, to diabetic HFF mice (n = 10) for 18 days. Individual therapy with TNP-470 or sitagliptin resulted in numerous metabolic benefits including reduced blood glucose, increased circulating and pancreatic insulin and improved glucose tolerance, insulin sensitivity, pyruvate tolerance and overall pancreatic islet architecture. Further assessment of metabolic rate revealed that all treatments reduced respiratory exchange ratio and increased locomotor activity. All sitagliptin treated mice also exhibited increased energy expenditure. In addition, treatment with TNP-470 alone, or in combination with sitagliptin, reduced food intake and body weight, as well as elevating plasma and intestinal xenin. Importantly, combined sitagliptin and TNP-470 therapy was associated with further significant benefits beyond that observed by either treatment alone. This included more rapid restoration of normoglycaemia, superior glucose tolerance, increased circulating GIP concentrations and an enhanced pancreatic beta:alpha cell ratio. In conclusion, these data demonstrate that TNP-470 increases plasma and intestinal xenin levels, and augments the antidiabetic advantages of sitagliptin.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis.

Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma.

CONTEXT: Resistance of cancer cells to chemotherapeutic drugs is a major pitfall of the failure of chemotherapy treatment for cholangiocarcinoma (CCA). A new therapeutic strategy that can improve treatment efficacy is mandatory for CCA patients. Our previous findings demonstrated the overexpression of methionine aminopeptidase-2 (MetAP2) in CCA patients. In addition, supplementation of TNP-470, a MetAP2 inhibitor, significantly inhibited the growth and metastatic activities of CCA cell lines. However, the molecular mechanism of antitumor activity of TNP-470 and the synergistic antitumor activity of TNP-470 combined with chemotherapeutic drugs are still unknown. AIMS: The aim of this study is to evaluate the molecular mechanism of anticancer activity and the potential use of TNP-470 as a chemosensitizing agent in CCA cell lines. MATERIALS AND METHODS: Cell cycle and apoptosis of CCA cell lines were evaluated using flow cytometry with propidium iodide staining. Expression of apoptosis regulatory proteins was measured by Western blotting. The chemosensitizing effect of TNP-470 was determined using combination index. RESULTS: TNP-470 inhibited the growth of CCA cells via induction of apoptosis through activation of the p38-phosphorylation and up- and down-regulation of Bax and Bcl-xL, respectively. Furthermore, TNP-470 significantly enhanced the antitumor activity of 5-fluorouracil, cisplatin, doxorubicin, and gemcitabine. CONCLUSIONS: The present results show that TNP-470 could be a potential therapeutic or adjuvant agent for CCA.

Systems Modeling of Developmental Vascular Toxicity.

The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.

Comparative evaluation of anti-angiogenic effects of noscapine derivatives.

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumor progression. Synthetic derivatives of anti-cancer compound, noscapine (an opium alkaloid) such as Cl-noscapine, Br-noscapine and Folate-noscapine along with two of the reference compounds, TNP-470 and paclitaxel were examined for anti-angiogenic activities by using human umbilical vein endothelial cells (HUVECs). The noscapine derivatives showed anti-angiogenic activity albeit at high concentration compared to the reference compounds. All the tested compounds inhibited angiogenesis in a dose-dependent manner; the drug concentration causing 50% inhibition of cell survival was 11.87 µM for Cl-noscapine, 6.9 µM for Br-noscapine and 6.79 µM for folate-noscapine. Besides, all the noscapine derivatives significantly inhibited cord formation (IC50 for Cl-noscapine is 50.76 µM, for Br-noscapine is 90.08 µM and for folate-noscapine is 18.44 µM) as well as migration and invasion (IC50 value of Cl-noscapine is 28.01 µM, for Br-noscapine is 19.78 µM and for folate-noscapine is 10.76 µM) of endothelial cells. Based on these results, we speculated that the inhibitory effects on human endothelial cell proliferation of noscapine derivatives might be important for anti-angiogenesis.

Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Embryonic vascular disruption adverse outcomes: Linking high-throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High-throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Advanced Chemoembolization by Anti-angiogenic Calcium-Phosphate Ceramic Microspheres Targeting the Vascular Heterogeneity of Cancer Xenografts.

The purpose of the present study was to develop an advanced method of anti-angiogenic chemoembolization to target morphological vascular heterogeneity in tumors and further the therapeutic efficacy of cancer treatment. This new chemoembolization approach was designed using resorbable calcium-phosphate ceramic microspheres (CPMs), in a mixture of three different sizes, which were loaded with an anti-angiogenic agent to target the tumor vasculature in highly angiogenic solid tumors in humans in vivo. The human uterine carcinosarcoma cell line, FU-MMT-3, was used in this study because the tumor is highly aggressive and exhibits a poor response to radiotherapy and chemotherapeutic agents that are in current use. CPMs loaded with TNP-470, an anti-angiogenic agent, were injected into FU-MMT-3 xenografts in nude mice three times per week for 8 weeks. The treatment with TNP-470-loaded CPMs of three different diameters achieved a greater suppression of tumor growth in comparison to treatment with single-size TNP-470-loaded CPMs alone, and the control. Severe loss of body weight was not observed in any mice treated with any size of TNP-470-loaded CPMs. These results suggest that treatment with a mixture of differently-sized anti-angiogenic CPMs might be more effective than treatment with CPMs of a single size. This advanced chemoembolization method, which incorporated an anti-angiogenic agent to target the morphological vascular heterogeneity of tumors may contribute to effective treatment of locally advanced or recurrent solid tumors.

Specific cell targeting with APRPG conjugated PEG-PLGA nanoparticles for treating ovarian cancer.

Good biocompatibility, specific tumor targeting, effective drug loading capacity and persistence in the circulation in vivo are imperative prerequisites for the antitumor efficiency of nanoparticles and their further clinical application. In this study, APRPG (Ala-Pro-Arg-Pro-Gly) peptide-modified poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) nanoparticles (NP-APRPG) encapsulating inhibitors of angiogenesis (TNP-470) (TNP-470-NP-APRPG) were fabricated. TNP-470-NP-APRPG was designed to feature maleimide-PEG-PLA and mPEG-PLA as carrier materials, the APRPG peptide for targeting angiogenesis, PEG for prolonging circulation in vivo and PLA for loading TNP-470. TNP-470-NP-APRPG was confirmed to be approximately 130 nm in size with negative ζ-potential (-14.3 mV), narrow distribution (PDI = 0.27) and spherical morphology according to dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. In addition, X-ray photoelectron spectra (XPS) analyses confirmed 7.73% APRPG grafting on the TNP-470-NP. In vitro, TNP-470-NP-APRPG exhibited effective inhibition of proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Similar findings were observed for the retardation of tumor growth in SKOV3 ovarian cancer-bearing mice, suggesting the significant inhibition of angiogenesis and antitumor efficiency of TNP-470-NP-APRPG. Moreover, no obvious toxic drug responses were observed. Further evidence obtained from the immunohistochemical examination demonstrated that the tumor growth inhibition was closely correlated with the high rate of apoptosis among endothelial cells and the effective blockade of endothelial cell proliferation. These results demonstrate that NP-APRPG is a promising carrier for delivering TNP-470 to treat ovarian cancer and that this approach has the potential to achieve broad tumor coverage in the clinic.

The effect of TNP-470 treatment on ascitic-fluid type hepatocarcinoma H22 / 肿瘤研究与临床

Objective To study the effect of TNP-470 treatment on ascitic hepatocarcinoma H22 and potential mechanisms. Methods The experimental approaches employed in the study included in vitro assays of cell proliferation and migration, and in vivo ascitic hepatocarcinoma mouse model. Results In vitro, TNP-470 inhibited cell proliferation of both H22 and human microvascular endothelial cell HMEC. Furthermore, TNP-470 inhibited HMEC migrations. In vivo, TNP-470 treatment significantly improved the life quality of the tumor-loaded mice, including lessen the ascitic effasion, decreased tumor cell viability, lessen the eye blindness, and prolonged the life-span. Conclusion These observations demonstrate that TNP-470 would be an effective drug for treatment of ascitic-fluid type hepatocarcinoma mice.

Experimental studies of angiocytotoxic therapy against growth and metastasis of pancreatic carcinoma / 中华消化杂志

Objective The anti tumor and anti metastasis effects of angiocytotoxic therapy (TNP 470/Gemcitabine) were investigated using a model of human pancreatic carcinoma by surgical orthotopic implantation (SOI). Methods The SOI model was developed by suturing small pieces of SW1990 tumors into the tail of pancreas in nude mice. Twenty four male mice were randomly divided into control group, G100 group receiving 100 mg/kg Gemcitabine intraperitoneally injection on days 0,3,6 and 9 after transplantation, and T30 group receiving 30 mg/kg TNP 470 subcutaneous injection on alternate days for 8 weeks. Another thirty two male mice were randomly divided into control group, T15 group, G50 group and combination group (TNP 470 30 mg/kg+ Gemcitabine 50 mg/kg). Animals were sacrificed ten weeks after transplantation. Results G100 group had a significant inhibitory effect on tumor growth of pancreatic carcinoma compared to T30 group, while the metastasis of tumor was significantly inhibited by T30 group compared to G100 group. Neither G100 group nor T30 group showed a significant improvement on survival rate. T15 group and G50 group alone had no significant inhibitory effect on the tumor growth and its metastasis. Mean while a significant anti tumor, anti metastatic effect and a significant improvement on the survival rate were observed in combination group. The inhibitory effect of G50 group was enhanced by 2 times with T30, and 2/8 of the tumors bearing animals were cured by the combination therapy. The level of microvessel density in T30 group was significantly lower than that in T15 group and control group ( P

Synergistic Inhibition of TNP-470 and Recombinant Human Endostatin on the Growth of Mice Lung Adenocarcinoma LA795 / 中国肿瘤生物治疗杂志

Objective: To investigate the combined inhibitive effect of TNP-470 and rhES on the growth of lung adenocarcinoma LA795 in T739 mice. Methods: The purified rhES was acquired by using methanol to induce the recombinant pichia pastoris. GS115 and heparin affinity chromatography. The T739 mice inoculated with LA795 cells were randomized into three groups, 10 mice per group, one group was injected with PBS for 14 days, the other two groups were respectively treated with rhES and TNP-470+rhES. To observe the tumor growth in different groups, and the tumor volume was measured with caliper. The microvessel density(MVD) of tumors were measured by using immunohistochemistry. Results: The purified rhES was acquired. In compared with PBS group, the tumor growth of other two groups was inhibited significantly. And the tumor volume of TNP-470+rhES group are smaller than the rhES group (P

Inhibitory Effect of Liver Metastasis of Human Colon Cancer by TNP-470 in Combination with 5-FU / 中国肿瘤生物治疗杂志

Objective: To study the effect of angiogenesis inhibitor TNP-470 combination with 5-FU on liver metastasis of human colon cancer. Methods: Human colon cancer cell line, LOVO cells, were injected intrasplenically into BALB/c nude mice to produce diffuse liver metastases. Mice were randomly divied into four groups; TNP-470 treated group, 5-FU treated group, TNP-470 +5-FU treated group and control group. Animals were sacrificed after 4 weeks, and their livers were processed for histological examination. Liver metastatic rate and tumor foci in liver were counted. Tumor microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined by immunohistochemistry SABC method with image analyse system. Results: TNP-470 in combination with 5-FU and TNP-470 alone display a significant inhibitory effect on liver metastasis compared to the control ( P

Effects of TNP-470 on proliferation, the cell cycle and apoptosis in human colon cancer Lovo cells / 中国药理学通报

AIM To study the effect of TNP 470 on proliferation, cell cycle and apoptosis in the cultured human colon cancer Lovo cells. METHODS The growth inhibition of TNP 470 on Lovo cells was evaluated by an MTT.assay The effect of TNP 470 on cell cycle and apoptosis measured by flow cytometry, and ultrastructural feature of Lovo cells were observed with electromicroscope. RESULTS TNP 470 inhibited the growth of Lovo cells. flow cytometry analysis showed that G 0/G 1 phase rate was increased but S phase rate was decreased. Apoptosis rate of TNP 470 treated group was significant high than that of control and typical chang of apoptosis in Lovo cells was observed. CONCLUSION TNP 470 can inhibit proliferation of Lovo cells, and this inhibition is associated with cell cycle block and apoptosis.