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.

In Vivo Imaging of Methionine Aminopeptidase II for Prostate Cancer Risk Stratification.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

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.

The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis.

During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.

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.

TNP-470 skews DC differentiation to Th1-stimulatory phenotypes and can serve as a novel adjuvant in a cancer vaccine.

Fumagillin is an antiangiogenic and antineoplastic fungal natural product, and TNP-470 is one of its most potent analogs. Decades of studies revealed that TNP-470 has potent anticancer activities via destruction of neovasculature. In stark contrast, TNP-470 has been reported to suppress lymphocyte proliferation, thereby limiting its clinical potentials. In an attempt to investigate whether the similar or opposite immunomodulatory effect of TNP-470 could act on myeloid cells, we found that TNP-470 potentiates the immunogenicity of dendritic cells (DCs) toward a phenotype with T helper cell type 1 (Th1)-stimulatory features. Using DC vaccine on a murine melanoma cancer model, the TNP-470-treated DC vaccine could significantly induce tumor-specific immunogenicity and substantially enhance tumor eradication when compared with vehicle-treated DC vaccine in a prophylactic setting. Enhanced tumor-specific immunogenicity and delayed tumor progression were observed in a therapeutic setting upon the TNP-470-treated DC vaccine. Our data showed that TNP-470 potentiates Toll-like receptor signaling, including NF-κB activation, in DCs to transcriptionally activate interleukin-12 production, thus inducing a Th1-immune response. Our current study uncovers a novel immune function of TNP-470 in DCs and redefines its role as a novel class of small molecule immune adjuvant in DC-based cancer vaccine given potentiation of DC immunogenicity is a major roadblock in DC vaccine development. Our study not only provides a novel adjuvant for ex vivo-cultured patient-specific DC vaccines for cancer treatment but also discovers the distinct immunostimulatory function of TNP-470 in DCs of myeloid lineage that differs from its immunosuppressive function in lymphoid cells.

Evolving multidimensional pharmacological approaches to CNV therapy in AMD.

PURPOSE: The leading cause of severe visual loss world-wide is age-related macular degeneration. Although anti-Vascular Endothelial Growth Factor agents have significantly led to the initial pharmacologic reversal of vision loss in many cases of exudative macular degeneration, there still has been recurrence of choroidal neovascularization, and/or the onset of chorioretinal atrophy with fibrosis. MATERIALS AND METHODS: In this review we discuss the status of anti- Vascular Endothelial Growth Factor in age-related macular degeneration and describe different studies focused on new potential therapeutic targets beyond anti- Vascular Endothelial Growth Factor. RESULTS: Further investigations have elicited that Vascular Endothelial Growth Factor is only one of many angiogenic, and pro-inflammatory factors that bring about the growth and leakage of active choroidal neovascularization. Various new multifaceted strategies, including inhibitors to down-stream targets of endothelial cell division, such as TNP-470, may lead to a more permanent inactivation of choroidal neovascularization. CONCLUSIONS: Based on the accumulated results in the treatment of age-related macular degeneration, it is hoped that the appropriate combination of anti-Vascular Endothelial Growth Factor agents with longer-acting and multidimensional pharmaceuticals, such as Methionine Aminopeptidase-2 inhibitors, will more effectively control choroidal neovascularization, prevent atrophy and fibrosis, and reduce the burden of frequent intraocular injections in age-related macular degeneration.

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.

Earliest phases of chondrogenesis are dependent upon angiogenesis during ectopic bone formation in mice.

Endochondral ossification is the process where cartilage forms prior to ossification and in which new bone forms during both fracture healing and ectopic bone formation. Transitioning to ossification is a highly coordinated process between hypertrophic chondrocytes, vascular endothelial cells, osteoblasts and osteoclasts. A critical biological process that is central to the interactions of these various cell types is angiogenesis. Although it is well established that angiogenesis is crucial for fracture repair, less is known pertaining to the role of angiogenesis in ectopic bone formation. Furthermore, fracture repair models are complicated by extensive trauma, subsequent inflammatory responses and concurrent repair processes in multiple tissues. In order to more definitively characterize the relationship between angiogenesis and postnatal endochondral ossification, a model of ectopic bone formation was used. Human demineralized bone matrix (DBM) was implanted in immune-deficient mice (rag null (B6.129S7-Rag1tm1/MOM/J)) to induce ectopic bone. Inhibition of angiogenesis with either a small molecule (TNP-470) or a targeted biological (Vascular Endothelial Growth Factor Receptor type 2 [VEGFR2] blocking antibody) prevented ectopic bone formation by 83% and 77%, respectively. Most striking was that the progression of chondrogenesis was halted during very early phases of chondrocyte differentiation between condensation and prehypertrophy (TNP-470) or the proliferative phase (VEGFR2 blockade) prior to hypertrophy, while osteoclast recruitment and resorption were almost completely inhibited. Our results demonstrate angiogenesis plays a developmental role in endochondral bone formation at a much earlier phase of chondrogenesis than suggested by prior findings.

Methionine aminopeptidase 2 is a key regulator of apoptotic like cell death in Leishmania donovani.

We investigate the role of methionine aminopeptidase 2 (MAP2) in miltefosine induced programmed cell death (PCD) in promastigote form of L. donovani. We report that TNP-470, an inhibitor of MAP2, inhibits programmed cell death in miltefosine treated promastigotes. It inhibits the biochemical features of metazoan apoptosis, including caspase3/7 protease like activity, oligonucleosomal DNA fragmentation, collapse of mitochondrial transmembrane potential, and increase in cytosolic pool of calcium ions but did not prevent the cell death and phosphatidyl serine externalization. The data suggests that the MAP2 is involved in the regulation of PCD in parasite. Moreover, TNP-470 shows the leishmanicidal activity (IC50 = 15 µM) and in vitro inhibition of LdMAP2 activity (K i = 13.5 nM). Further studies on MAP2 and identification of death signaling pathways provide valuable information that could be exploited to understand the role of non caspase proteases in PCD of L. donovani.

TNP-470 Reduces Glioblastoma Angiogenesis in Three Dimensional GelMA Microwell Platform.

Tumor angiogenesis is a promising target for cancer treatment, because severing the supply of oxygen and nutrients to tumors halts tumor growth. Unfortunately, many anticancer drugs, including angiogenesis inhibitors, fail in clinical trials, despite showing high efficiency during in vitro and in vivo experiments. This inconsistency from in vitro and in vivo experiments to clinical trials represents a major obstacle in drug development and cancer treatment. Therefore, we set out to demonstrate how our rapid, stable, easy-to-use three-dimensional (3-D) in vitro angiogenesis model can be used to investigate tumor formation and implement drug screening. In this study, we utilized a 3-D in vitro angiogenesis model, based on gelatin methacrylate (GelMA) hydrogel microwells, to mimic the native microenvironment of tumor angiogenesis. Using this model, we were able to quantify the immigration of endothelial cells into a cancer spheroid during the angiogenic process. Next, we tested the anti-angiogenic effect of the angiogenesis inhibitor, TNP-470, on the cancer spheroids in the model. Based on our results, we believe that this novel in vitro system can be widely used for the high-throughput screening of other anti-angiogenic drugs, and could contribute to the development of personalized medicine in the future.

Clinical Trials of Antiangiogenesis Therapy in Recurrent/Persistent and Metastatic Cervical Cancer.

BACKGROUND: Treatment options for women with metastatic, persistent, or recurrent cervical cancer are limited and thus the disease portends a poor prognosis. It is critical to understand the pathophysiology of cervical cancer to better delineate therapeutic targets. The development of antiangiogenic therapies and their subsequent analysis in rigorous therapeutic trials have redefined current management strategies and is an exciting area of current exploration. RESULTS: Translational trials have furthered the understanding of molecular determinants of angiogenesis. Phase II trials have shown promising trends with developing antiangiogenic therapies. A practice-changing phase III trial has recently been published. Given the potential benefits and different toxicity spectrum compared with standard cytotoxic chemotherapy, antiangiogenic options are under active investigation for this vulnerable patient population. Emerging data are promising for other antiangiogenic-directed therapeutics, as well as cervical cancer molecular biomarkers to guide diagnosis and treatment. CONCLUSION: Antiangiogenic therapies have evolved during the past 20 years and remain an exciting area of current exploration. IMPLICATIONS FOR PRACTICE: Understanding of the angiogenic microenvironment has furthered understanding of tumor biology and management. Antiangiogenic therapies show promise for women with advanced cervical cancer. A review of the evolution of these biologic agents shows them to be an effective and tolerable management strategy for many patients in this vulnerable population, with exciting future potential.

Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis.

Angiogenesis has been identified as a crucial process in the development and spread of cancers. There are many regulators of angiogenesis which are not yet fully understood. Methionine aminiopeptidase is a metalloenzyme with two structurally distinct forms in humans, Type-1 (MetAP-1) and Type-2 (MetAP-2). It has been shown that small molecule inhibitors of MetAP-2 suppress endothelial cell proliferation. The initial discovery by Donald Ingber of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation similar to the discovery of penicillin activity by Sir Alexander Fleming. From a drug design perspective, MetAP-2 is an attractive target. Fumagillin and ovalicin, known natural products, bind with IC50 values in low nanomolar concentrations. Crystal structures of the bound complexes provide 3-dimensional coordinates for advanced computational studies. More recent discoveries have shown other biological activities for MetAP-2 inhibition, which has generated new interests in the design of novel inhibitors. Semisynthetic fumagillin derivatives such as AGM-1470 (TNP-470) have been shown to have better drug properties, but have not been very successful in clinical trials. The rationale and development of novel multicyclic analogs of fumagillin are reviewed.

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.

Prostatic angiogenic responses in late life: antiangiogenic therapy influences and relation with the glandular microenvironment in the transgenic adenocarcinoma of mouse prostate (TRAMP) model.

BACKGROUND: Aging is considered one of the main predisposing factors for the development of prostate malignancies. Angiogenesis is fundamental for tumor growth and its inhibition represents a promising therapeutic approach in cancer treatment. Thus, we sought to determine angiogenic responses and the effects of antiangiogenic therapy in the mouse prostate during late life, comparing these findings with the prostatic microenvironment in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. METHODS: Male mice (52 week-old FVB) were submitted to treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c.). Finasteride was administered (20 mg/kg; s.c.), alone or in association to both inhibitors. The dorsolateral prostate was collected for VEGF, HIF-1α, FGF-2 and endostatin immunohistochemical and Western Blotting analyses and for microvessel density (MVD) count. RESULTS: Senescence led to increased MVD and VEGF, HIF-1α and FGF-2 protein levels in the prostatic microenvironment, similarly to what was observed in TRAMP mice prostate. The angiogenic process was impaired in all the treated groups, demonstrating significantly decreased MVD. Antiangiogenic and/or finasteride treatments resulted in decreased VEGF and HIF-1α levels, especially following TNP-470 administration, either alone or associated to SU5416. The combination of these agents resulted in increased endostatin levels, regardless of the presence of finasteride. CONCLUSIONS: Prostatic angiogenesis stimulation during senescence favored the development of neoplastic lesions, considering the pro-angiogenic microenvironment as a common aspect also observed during cancer progression in TRAMP mice. The combined antiangiogenic therapy was more efficient, leading to enhanced imbalance towards angiogenic inhibition in the organ. Finally, finasteride administration might secondarily upregulate the expression of pro-angiogenic factors, pointing to the harmful effects of this therapy.

Endothelial cell-derived non-canonical Wnt ligands control vascular pruning in angiogenesis.

Multiple cell types involved in the regulation of angiogenesis express Wnt ligands. Although ß-catenin dependent and independent Wnt signaling pathways have been shown to control angiogenesis, the contribution of individual cell types to activate these downstream pathways in endothelial cells (ECs) during blood vessel formation is still elusive. To investigate the role of ECs in contributing Wnt ligands for regulation of blood vessel formation, we conditionally deleted the Wnt secretion factor Evi in mouse ECs (Evi-ECKO). Evi-ECKO mice showed decreased microvessel density during physiological and pathological angiogenesis in the postnatal retina and in tumors, respectively. The reduced microvessel density resulted from increased vessel regression accompanied by decreased EC survival and proliferation. Concomitantly, survival-related genes were downregulated and cell cycle arrest- and apoptosis-inducing genes were upregulated. EVI silencing in cultured HUVECs showed similar target gene regulation, supporting a mechanism of EC-derived Wnt ligands in controlling EC function. ECs preferentially expressed non-canonical Wnt ligands and canonical target gene expression was unaffected in Evi-ECKO mice. Furthermore, the reduced vascularization of Matrigel plugs in Evi-ECKO mice could be rescued by introduction of non-canonical Wnt5a. Treatment of mouse pups with the non-canonical Wnt inhibitor TNP470 resulted in increased vessel regression accompanied by decreased EC proliferation, thus mimicking the proliferation-dependent Evi-ECKO remodeling phenotype. Taken together, this study identified EC-derived non-canonical Wnt ligands as regulators of EC survival, proliferation and subsequent vascular pruning during developmental and pathological angiogenesis.

Angiostatic effects of NK cell-derived IFN-γ counteracted by tumour cell Bcl-xL expression.

Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the anti-apoptotic protein Bcl-xL , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressive activity in a murine lymphoma model. Bcl-xL overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-xL protection of tumours from NK cell-mediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-xL -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell-secreted IFN-γ. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.

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 host support niche as a control point for tumor dormancy: implications for tumor development and beyond.

An increasingly appreciated focus of carcinogenesis research is on mechanisms governing tumor growth after the fact of cancer cell creation. Of particular interest are dynamical interactions between tumor and host cell populations that can themselves strongly impact the fate of established cancer lesions. Regardless of tumor type, all cancers face the common problem of having to breach the barrier of angiogenic competency in order to advance from a microscopic lesion to symptomatic disease. If pre-angiogenic tumor cells are held in dormancy due to cell cycle arrest, this will postpone the need to traverse this higher-level barrier. On the other hand, the barrier itself may prove limiting to a tumor at its diffusion-limited size, creating a population-level dormancy characterized by balanced proliferation and cell death. In both cases of dormancy, the "angiogenic switch" has not yet occurred. We here describe and mathematically quantify an underappreciated third dormancy state defined by an angiogenic balance following the angiogenic switch. In this state we term "post-vascular dormancy," a tumor has attained angiogenic competency, but again demonstrates balanced proliferation and cell death because ambient pro- and anti-angiogenic influences are offsetting. Interestingly, autopsies have shown virtually all of us carry latent tumors in pre- or post-vascular states, many of which lie under the threshold of routine clinical detection. We show how, in the post-vascular case, tumor latency can arise from an elaborate mechanism of self-controlled growth, mediated through the tumor-vascular interaction. Underlying this observation is the finding that a tumor produces both angiogenesis stimulators and inhibitors, with the latter having greater influence, both locally and systemically, as the tumor grows-a mechanism we hypothesize is an aberrant co-option of normal organogenic regulation. That a tumor can limit its own growth raises the prospect that chronic therapies aimed at suppressing this tumor-host dynamic may compare favorably to current strategies which often yield favorable short-term responses but fail to deliver long-term tumor suppression.