SDF-1α Mediates Wound-Promoted Tumor Growth in a Syngeneic Orthotopic Mouse Model of Breast Cancer

Increased growth of residual tumors in the proximity of acute surgical wounds has been reported; however, the mechanisms of wound-promoted tumor growth remain unknown. Here, we used a syngeneic, orthotopic mouse model of breast cancer to study mechanisms of wound-promoted tumor growth. Our results demonstrate that exposure of metastatic mouse breast cancer cells (4T1) to SDF-1α, which is increased in wound fluid, results in increased tumor growth. Both, wounding and exposure of 4T1 cells to SDF-1α not only increased tumor growth, but also tumor cell proliferation rate and stromal collagen deposition. Conversely, systemic inhibition of SDF-1α signaling with the small molecule AMD 3100 abolished the effect of wounding, and decreased cell proliferation, collagen deposition, and neoangiogenesis to the levels observed in control animals. Furthermore, using different mouse strains we could demonstrate that the effect of wounding on tumor growth and SDF-1α levels is host dependent and varies between mouse strains. Our results show that wound-promoted tumor growth is mediated by elevated SDF-1α levels and indicate that the effect of acute wounds on tumor growth depends on the predetermined wound response of the host background and its predetermined wound response.     

Oxidative Inactivation of Brain Ecto-5′-Nucleotidase by Thiols/Fe2+ System

5-Nucleotidase, responsible for the conversion of adenosine-5-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe²⁺. The glutathione-mediated inactivation of 5-nucleotidase was potentiated remarkably by Fe²⁺, but not Cu²⁺, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe²⁺-mediated inactivation. In comparison, the glutathione/Fe²⁺ system was much more effective than the ascorbate/Fe²⁺ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H₂O₂ in the action of glutathione/Fe²⁺ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe²⁺ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5-monophosphate as substrate exhibited a modest protection against the glutathione/Fe²⁺ action, a remarkable protection was expressed by divalent metal ions such as Zn²⁺ or Mn²⁺. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe²⁺ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5-nucleotidase, were found to be more effective in potentiating the Fe²⁺- mediated inactivation. Further, kinetic analyses indicate that Fe²⁺ and thiols inhibit the 5-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe²⁺-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe²⁺ and thiols to the enzyme molecule.     

Ecto-5’-nucleotidase: Structure function relationships

Ecto-5'-nucleotidase (ecto-5'-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5'-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5'-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5'-NT is reviewed in relation to the catalytic properties and enzyme function.     

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Background

Hypoxia-mediated HIF-1α stabilization and NF-κB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth.

Methodology/Principal Findings

We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2–3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O₂) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-κB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts.

Conclusion

These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-κB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.     

HIF-1α Inhibition Reduces Nasal Inflammation in a Murine Allergic Rhinitis Model

Background

Hypoxia-inducible factor 1α (HIF-1α) is an important regulator of immune and inflammatory responses. We hypothesized that nasal allergic inflammation is attenuated by HIF-1α inhibition and strengthened by HIF-1α stabilization.

Objective

To elucidate the role of HIF-1α in a murine model of allergic rhinitis (AR).

Methods

Mice were pretreated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2) or the HIF-1α inducer cobalt chloride (CoCl₂) in an established AR murine model using ovalbumin (OVA)-sensitized BALB/c mice. HIF-1α and vascular endothelial growth factor (VEGF) expression in nasal mucosa was measured and multiple parameters of allergic responses were evaluated.

Results

HIF-1α and VEGF levels were locally up-regulated in nasal mucosa during AR. Inflammatory responses to OVA challenge, including nasal symptoms, inflammatory cell infiltration, eosinophil recruitment, up-regulation of T-helper type 2 cytokines in nasal lavage fluid, and serum OVA-specific IgE levels were present in the OVA-challenged mice. 2ME2 effectively inhibited HIF-1α and VEGF expression and attenuated the inflammatory responses. Stabilization of HIF-1α by CoCl₂ facilitated nasal allergic inflammation. HIF-1α protein levels in nasal airways correlated with the severity of AR in mice.

Conclusions

HIF-1α is intimately involved in the pathogenesis of nasal allergies, and the inhibition of HIF-1α may be useful as a novel therapeutic approach for AR.     

Mithramycin A Alleviates Cognitive Deficits and Reduces Neuropathology in a Transgenic Mouse Model of Alzheimer’s Disease

Increasing evidence has shown that specificity protein 1 (Sp1) is abnormally increased in the brains of subjects with Alzheimer’s disease (AD) and transgenic AD models. However, whether the Sp1 activation plays a critical role in the AD pathogenesis and selective inhibition of Sp1 activation may have a disease-modifying effect on the AD-like phenotypes remain elusive. In this study, we reported that Sp1 mRNA and protein expression were markedly increased in the brain of APPswe/PS1dE9 transgenic mice, whereas chronic administration of mithramycin A (MTM), a selective Sp1 inhibitor, potently inhibited Sp1 activation in the APPswe/PS1dE9 mice down to the levels of wild-type mice. Specifically, we found that MTM treatment resulted in a significant improvement of learning and memory deficits, a dramatic reduction in cerebral Aβ levels and plaque burden, a profound reduction in tau hyperphosphorylation, and a marked increase in synaptic marker in the APPswe/PS1dE9 mice. In addition, MTM treatment was powerfully effective in inhibiting amyloid precursor protein (APP) processing via suppressing APP, beta-site APP cleaving enzyme 1 (BACE1), and presenilin-1 (PS1) mRNA and protein expression to preclude Aβ production in the APPswe/PS1dE9 mice. Furthermore, MTM treatment strongly inhibited phosphorylated CDK5 and GSK3β signal pathways to reduce tau hyperphosphorylation in the APPswe/PS1dE9 mice. Collectively, our findings provide evidence that Sp1 activation may contribute to the AD pathogenesis and may serve as a novel therapeutic target in the treatment of AD. The present study highlights that selective Sp1 inhibitors may be considered as disease-modifying therapeutic agents for AD.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Extracellular Adenosine Formation by Ecto-5’-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning

Background

Adenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73^-/- and wild type (WT) mice.

Methods and Results

3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73^-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73^-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73^-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73^-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73^-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73^-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.

Conclusion

The infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.     

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5′-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2 ± 1.1 and 24.5 ± 3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7 ± 0.1 and 5.7 ± 2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.     

5′-Nucleotidase in spinal meningeal compartments in the rat

Summary The distribution of 5-nucleotidase (5-Nu) is reported in spinal meninges of the rat on the basis of an immunohistochemical and enzyme histochemical investigation. Strong immunoreactivity was found in the arachnoid membrane and in the sheaths of the spinal roots as well as in septa subdividing the roots. Also the superficial layer of the ligamentum denticulatum showed enzyme staining. No immunoreactivity could be detected in the pia mater or along the spinal nerve roots outside the subarachnoid space. Within the arachnoid mater the immunoreactivity was concentrated in the basal zone of the arachnoid membrane, thus appearing as a narrow fluorescent band near the border of the dura. An accentuation of immunoreactivity could be observed in areas where small dural blood vessels approach the subarachnoid space. It is well known that adenine nucleotides released from neural and glial cells of the central nervous system finally reach the cerebrospinal fluid. We presume that 5-Nu in the arachnoid membrane and spinal root sheaths is responsible for the conversion of adenine nucleotides into adenosine and that this conversion is associated with the reabsorption process of cerebrospinal fluid which most probably also takes place in spinal meninges. Adenosine, the product of 5-nucleotidase, could play a role in the reabsorption process by its vasodilatatory effect on dural and epidural vessels.     

Stabilization of HIF-2α Induces sVEGFR-1 Production from Tumor-Associated Macrophages and Decreases Tumor Growth in a Murine Melanoma Model

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.     

A Validated Mathematical Model of Tumor Growth Including Tumor–Host Interaction,Cell-Mediated Immune Response and Chemotherapy

We consider a dynamical model of cancer growth including three interacting cell populations of tumor cells, healthy host cells and immune effector cells. The tumor–immune and the tumor–host interactions are characterized to reproduce experimental results. A thorough dynamical analysis of the model is carried out, showing its capability to explain theoretical and empirical knowledge about tumor development. A chemotherapy treatment reproducing different experiments is also introduced. We believe that this simple model can serve as a foundation for the development of more complicated and specific cancer models.     

Adenosine 5′-Triphosphate-Sensitive Potassium Channel Activator Induces the Up-Regulation of Caveolin-1 Expression in a Rat Brain Tumor Model

This study was performed to determine whether minoxidil sulfate (MS), a selective Adenosine 5′-triphosphate-sensitive potassium channel (K _ATP channel) activator, has an effect on the expression of caveolin-1 in the rat’s brain tumor tissue. Using a rat brain glioma (C6) model, we found that the expression of caveolin-1 protein at tumor sites was greatly increased after intracarotid infusion of MS at a dose of 30 μg/kg/min for 15, 30, and 60 min via Western blot analysis. And the peak value of the caveolin-1 expression was observed in rats with glioma after 15 min of MS perfusion, which was significantly attenuated by reactive oxygen species (ROS) scavenger (N-2-mercaptopropionyl glycine, MPG). In addition, MPG also significantly inhibited the increase of blood–brain tumor barrier (BTB) permeability which was induced by MS. This led to the conclusion that the MS-induced BTB permeability increase may be related to the accelerated formation of caveolin-1 protein, and could be mediated by ROS.     

A Novel Small-molecule Tumor Necrosis Factor α Inhibitor Attenuates Inflammation in a Hepatitis Mouse Model

Overexpression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions. To explore chemical inhibitors against TNFα activity, we applied computer-aided drug design combined with in vitro and cell-based assays and identified a lead chemical compound, (E)-4-(2-(4-chloro-3-nitrophenyl) (named as C87 thereafter), which directly binds to TNFα, potently inhibits TNFα-induced cytotoxicity (IC₅₀ = 8.73 μm) and effectively blocks TNFα-triggered signaling activities. Furthermore, by using a murine acute hepatitis model, we showed that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival. Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat TNFα-mediated inflammatory diseases.     

Impaired Sense of Smell in a Drosophila Parkinson’s Model

Parkinson’s disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3–5 day-old flies, from 15–20 and from 27–30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.     

Heterozygosity for a Hypomorphic Polβ Mutation Reduces the Expansion Frequency in a Mouse Model of the Fragile X-Related Disorders

The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5’ UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.     

Knockdown of PPAR δ Gene Promotes the Growth of Colon Cancer and Reduces the Sensitivity to Bevacizumab in Nude Mice Model

The role of peroxisome proliferator – activated receptor- δ (PPAR δ) gene in colon carcinogenesis remains highly controversial. Here, we established nude mice xenograft model using a human colon cancer cell line KM12C either with PPAR δ silenced or normal. The xenografts in PPAR δ-silenced group grew significantly larger and heavier with less differentiation, promoted cell proliferation, increased expression of vascular endothelial growth factor (VEGF) and similar apoptosis index compared with those of PPAR δ-normal group. After treated with the specific VEGF inhibitor bevacizumab, the capacities of growth and proliferation of xenografts were decreased in both groups while still significantly higher in PPAR δ-silenced group than in PPAR δ-normal group. Administration of PPAR δ agonist significantly decreased VEGF expression in PPAR δ-normal KM12C cells but not in PPAR δ-silenced cells. These findings demonstrate that, knockdown of PPAR δ promotes the growth of colon cancer by inducing less differentiation, accelerating the proliferation and VEGF expression of tumor cells in vivo, and reduces tumor sensitivity to bevacizumab. This study indicates that PPAR δ attenuates colon carcinogenesis.