Destruction Activation Research Articles

Tumor Vascular Destruction and cGAS-STING Activation Induced by Single Drug-Loaded Nano-Micelles for Multiple Synergistic Therapies of Cancer.

Cancer and its metastasis/recurrence still threaten human health, despite various advanced treatments being employed. It is of great significance to develop simple drug formulations to enhance the efficacy and synergistic integration of various monotherapies. Herein, DMXAA, a vasodestructive agent with cGAS-STING stimulation capacity, is integrated with polyethylene glycol grafted poly (lactic-co-glycolic) acid co-polymer (PLGA-PEG), obtaining PLGA-PEG/DMXAA (PPD) nanoparticles to induce the tumor-specific vascular destruction for multiple synergistic therapies of cancer. PPD could induce the formation of blood clots in the tumor after intravenous injection, which subsequently mediate photothermal therapy and further promote the release of oxygen for enhanced radiotherapy. Meanwhile, the enhanced vascular injury can induce perfect starvation therapy of tumor. More importantly, PPD-mediated therapies could trigger potent systemic anti-tumor immunity via inducing the immunogenic death of tumor cells and activating the cGAS-STING pathway. Together with anti-PD-L1, PPD-mediated therapies could not only remove the primary tumors, but also effectively eliminate the distant tumors, metastasis, and recurrence. Therefore, the modulation of tumor composition induced by a single drug-loaded nano-micelle could be utilized to enhance the therapeutic effect of multiple treatments for synergistic and systemic antitumor response, providing a practical strategy for cancer therapy.

Receptor clustering and pathogenic complement activation in myasthenia gravis depend on synergy between antibodies with multiple subunit specificities

Myasthenia gravis is an autoimmune disorder defined by muscle weakness and fatigability associated with antibodies against proteins of the neuromuscular junction (NMJ). The most common autoantibody target is the acetylcholine receptor (AChR). Three mechanisms have been postulated by which autoantibodies might interfere with neurotransmission: direct antagonism of the receptor, complement-mediated destruction of the postsynaptic membrane, and enhanced internalization of the receptor. It is very likely that more than one of these mechanisms act in parallel. Dissecting the mechanisms of autoantibody-mediated pathology requires patient-derived, monoclonal antibodies. Using membrane antigen capture activated cell sorting (MACACS), we isolated AChR-specific B cells from patients with myasthenia gravis, and produced six recombinant antibodies. All AChR-specific antibodies were hypermutated, including isotypes IgG1, IgG3, and IgG4, and recognized different subunits of the AChR. Despite clear binding, none of the individual antibodies showed significant antagonism of the AChR measured in an in vitro neuromuscular synapse model, or AChR-dependent complement activation, and they did not induce myasthenic signs in vivo. However, combinations of antibodies induced strong complement activation in vitro, and severe weakness in a passive transfer myasthenia gravis rat model, associated with NMJ destruction and complement activation in muscle. The strongest complement activation was mediated by combinations of antibodies targeting disparate subunits of the AChR, and such combinations also induced the formation of large clusters of AChR on the surface of live cells in vitro. We propose that synergy between antibodies of different epitope specificities is a fundamental feature of this disease, and possibly a general feature of complement-mediated autoimmune diseases. The importance of synergistic interaction between antibodies targeting different subunits of the receptor can explain the well-known discrepancy between serum anti-AChR titers and clinical severity, and has implications for therapeutic strategies currently under investigation.

Structural and Dipole-Relaxation Processes in Epoxy-Multilayer Graphene Composites with Low Filler Content.

Multilayered graphene nanoplatelets (MLGs) were prepared from thermally expanded graphite flakes using an electrochemical technique. Morphological characterization of MLGs was performed using scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Raman spectroscopy (RS), and the Brunauer–Emmett–Teller (BET) method. DGEBA-epoxy-based nanocomposites filled with synthesized MLGs were studied using Static Mechanical Loading (SML), Thermal Desorption Mass Spectroscopy (TDMS), Broad-Band Dielectric Spectroscopy (BDS), and Positron Annihilation Lifetime Spectroscopy (PALS). The mass loading of the MLGs in the nanocomposites was varied between 0.0, 0.1, 0.2, 0.5, and 1% in the case of the SML study and 0.0, 1.0, 2, and 5% for the other measurements. Enhancements in the compression strength and the Young’s modulus were obtained at extremely low loadings ( 0.01%). An essential increase in thermal stability and a decrease in destruction activation energy were observed at 5%. Both the dielectric permittivity () and the dielectric loss factor () increased with increasing over the entire frequency region tested (4 Hz–8 MHz). Increased is correlated with decreased free volume when increasing . Physical mechanisms of MLG–epoxy interactions underlying the effects observed are discussed.

Matrix metalloproteinase-13 is fully activated by neutrophil elastase and inactivates its serpin inhibitor, alpha-1 antitrypsin: Implications for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage exvivo, in an MMP-dependent manner. Invitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

Pain highly correlates to collagenase-induced oa-like damage in female mice but not in males.

Pain highly correlates to collagenase-induced oa-like damage in female mice but not in males.

Scorpion alleviates bone cancer pain through inhibition of bone destruction and glia activation.

BackgroundBone cancer pain is common in patients with advanced cancers as tumor metastasizes to bone. The inefficient clinical treatment severely reduces quality of life of bone cancer pain patients. During the pain status, activated spinal astrocytes and microglia release various inflammatory cytokines, resulting in spinal inflammation and the development of neuron sensitization. Scorpion is the dry body of Buthus martensii Karsch and is often used for various pain management in clinical practice. However, its function on bone cancer pain is unclear.MethodsWe investigated the effects of intragastric administration of scorpion on bone cancer pain induced by left tibial cavity injection of Walker 256 cells. Nociceptive behavior was measured using the von Frey filaments test and the spontaneous ambulatory pain score. The bone destruction was assessed by tibial radiographs. Expression of spinal cord astrocyte marker glial fibrillary acidic protein and microglial marker Iba1 was monitored by Western blot assay and immunofluorescence. Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1β was detected by real-time polymerase chain reaction. The proliferation of Walker 256 cells was evaluated by CCK8 assay.ResultsIntragastric administration of scorpion reduced bone cancer pain behavior and relieved bone destruction, accompanied by decreased expression of spinal glial fibrillary acidic protein and Iba1 protein level and TNF-α, IL-6, and IL-1β mRNA level. Besides, scorpion inhibited proliferation of Walker 256 cells in a dose- and time-dependent manner.ConclusionOur results demonstrate that scorpion produces an analgesic effect in a rat model of bone cancer pain via inhibiting bone destruction and activation of spinal cord astrocytes and microglia.

Impact of HIV Infection and Anti-Retroviral Therapy on the Immune Profile of and Microbial Translocation in HIV-Infected Children in Vietnam.

CD4+ T-lymphocyte destruction, microbial translocation, and systemic immune activation are the main mechanisms of the pathogenesis of human immunodeficiency virus type 1 (HIV) infection. To investigate the impact of HIV infection and antiretroviral therapy (ART) on the immune profile of and microbial translocation in HIV-infected children, 60 HIV vertically infected children (31 without ART: HIV(+) and 29 with ART: ART(+)) and 20 HIV-uninfected children (HIV(−)) aged 2–12 years were recruited in Vietnam, and their blood samples were immunologically and bacteriologically analyzed. Among the HIV(+) children, the total CD4+-cell and their subset (type 1 helper T-cell (Th1)/Th2/Th17) counts were inversely correlated with age (all p < 0.05), whereas regulatory T-cell (Treg) counts and CD4/CD8 ratios had become lower, and the CD38+HLA (human leukocyte antigen)-DR+CD8+- (activated CD8+) cell percentage and plasma soluble CD14 (sCD14, a monocyte activation marker) levels had become higher than those of HIV(−) children by the age of 2 years; the CD4/CD8 ratio was inversely correlated with the plasma HIV RNA load and CD8+-cell activation status. Among the ART(+) children, the total CD4+-cell and Th2/Th17/Treg-subset counts and the CD4/CD8 ratio gradually increased, with estimated ART periods of normalization being 4.8–8.3 years, whereas Th1 counts and the CD8+-cell activation status normalized within 1 year of ART initiation. sCD14 levels remained high even after ART initiation. The detection frequency of bacterial 16S/23S ribosomal DNA/RNA in blood did not differ between HIV-infected and -uninfected children. Thus, in children, HIV infection caused a rapid decrease in Treg counts and the early activation of CD8+ cells and monocytes, and ART induced rapid Th1 recovery and early CD8+-cell activation normalization but had little effect on monocyte activation. The CD4/CD8 ratio could therefore be an additional marker for ART monitoring.

Development of nanotechnology of fine frozen champignon puree (agaricus bisporus)

The nanotechnology of fine-dispersed frozen champignon puree with unique characteristics using the mechanical destruction and mechanical activation processes was scientifically substantiated and developed. The new technology allows to extract the forms of BAS and biopolymers (proteins) hidden in vegetable raw materials and make fuller use of the biological potential of raw materials. Quality comparison of frozen fine additives, obtained by the innovative technology in the form of nanostructured puree with the feedstock by the content of BAS was performed in the paper. It is shown that in the freezing and low-temperature grinding of raw materials, accompanied by cryodestruction and mechanical activation processes, there is a fuller extraction of BAS from the biopolymers-bound state into the free state. Depending on the type of BAS, the increase is from 1.5...2.5 times with respect to the fresh feedstock. Comprehensive investigations have shown that the resulting fine frozen puree has fundamentally new properties. It was found that in the fine low-temperature grinding of champignons, there is a destruction of protein-chitin-mineral complexes, mechanical disruption (mechanolysis) of proteins. It was revealed that using mechanical activation in cryogenic mechanical grinding of champignons into fine puree leads to cryodestruction and mechanolysis of proteins of mushrooms to free amino acids by 70...75 %, that is, the protein is transformed (modified) into the nanostructured form, 2/3 of which consist of free amino acids and is much better dissolved and absorbed by a human body. The end result of the work is the developed and approved regulatory documentation for the product, as well as conducted testing in the industrial environment at the enterprises of the city of Kharkiv.

Multivariate Assessment of the Effect of Pump Design and Pump Gap Design Parameters on Blood Trauma.

Pump gaps are the most critical regions in a rotary blood pump when it comes to blood trauma in the form of hemolysis, protein destruction, and platelet activation. This study investigated six pump design parameters affecting the flow in a radial pump gap. A multivariate approach was employed to determine individual and quantitative parameter effects on blood trauma as well as parameter interactions. To consider the effect of shear stress and blood cell residence time, a validated numerical Lagrangian particle tracking approach was used. Based on the results, small-diameter pumps can be as blood compatible, if not more blood compatible, as large-diameter pumps as long as identical circumferential velocities and clearance gaps are maintained. Furthermore, the results indicate that an eccentric rotor position in the casing is not harmful and that a pressure difference generating washout flow and thereby reducing the cell residence time is of significant importance.

Elevated Red Cell Microparticles (RMP) in Hematologic and Thrombotic Disorders

Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders.Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP".Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p<0.01). Of 138 TP patients with elevated RMP, 31 were TBS(+) and 107 were TBS(-). Levels of RMP for TBS(+) and TBS(-) were 4,698 ±3,208 and 3,012 ±1,503, counts/mL, respectively (p <0.001). (III) RMP in HCS: Of 175 HCS with elevated RMP, 116 were TBS(+) and 59 were TBS(-). The levels of RMP for TBS(+) and TBS(-) were 4,062 ±3,285 and 2,987 ±1,454, counts/mL, respectively (p <0.02). Among these 116 TBS(+) patients, 22 did not receive antithrombotic treatment at the time of assay (HCS-1), 84 received treatment at time of assay (HCS-2), and 10 developed recurrent TBS despite therapy (HCS-3). Levels of RMP for HCS-1, HCS-2, and HCS-3 were 5,091 ±3,804, 3,973 ±3,044, and 6,191 ±3,763, respectively. Of these, HCS-2 differed significantly from HCS-3, p < 0.05. (IV) TBS with vs. without risk factors: Of 251 TBS with elevated RMP, 226 had known hematological or coagulation disorders, or risk factors for TBS (such as HA, TP, MPD, thrombocytosis, cancers, atrial fibrillation, APS, lupus, or markers for HCS). The remaining 25 without risk factors nevertheless had TBS and elevated RMP, suggesting RMP may be a useful biomarker for thrombotic risk.Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. DisclosuresNo relevant conflicts of interest to declare.

An in vitro investigation of the influence of stenosis severity on the flow in the ascending aorta

Cardiovascular diseases can lead to abnormal blood flows, some of which are linked to hemolysis and thrombus formation. Abnormal turbulent flows of blood in the vessels with stenosis create strong shear stresses on blood elements and may cause blood cell destruction or platelet activation. We implemented a Lagrangian (following the fluid elements) measurement technique of three dimensional particle tracking velocimetry that provides insight on the evolution of viscous and turbulent stresses along blood element trajectories. We apply this method to study a pulsatile flow in a compliant phantom of an aorta and compare the results in three cases: the reference case (called “healthy” case), and two cases of abnormal flows due to mild and severe stenosis, respectively. The chosen conditions can mimic a clinical application of an abnormal flow due to a calcific valve.We estimate the effect of aortic stenosis on the kinetic energy of the mean flow and the turbulent kinetic energy, which increases about two orders of magnitude as compared with the healthy flow case. Measuring the total flow stress acting on a moving fluid element that incorporates viscous stresses and the apparent turbulent-induced stresses (the so-called Reynolds stresses) we find out similar increase of the stresses with the increased severity of the stenosis. Furthermore, these unique Lagrangian measurements provide full acceleration and, consequently, the forces acting on the blood elements that are estimated to reach the level that can considerably deform red blood cells. These forces are strong and abrupt due to the contribution of the turbulent fluctuations which is much stronger than the typically measured phase-averaged values.

Thermal stability of ammonium chlorocobaltates(II)

The thermal destruction of ammonium penta-, tetra-, and trichlorocobaltates(II) was studied. Thermogravimetric analysis (TGA) data was used to calculate the thermal destruction activation energy, which was 8.6 kJ/mol for (NH4)3CoCl5, 14.8 kJ/mol for (NH4)2CoCl4, and 117.4 kJ/mol for NH4CoCl3. The decomposition enthalpy of 35.9 kJ/mol for (NH4)3CoCl5, 12.1 kJ/mol for (NH4)2CoCl4, and 166.3 kJ/mol for NH4CoCl3 was determined from differential scanning calorimetry (DSC) data.

Effect of the spatial network structure and cross-link density of diene rubbers on their thermal stability and fire hazard

The paper presents the results of investigating the effect of the density and spatial network structure of diene rubbers (NBR and SBR) on their thermal properties and fire hazard. The rubbers were either conventionally cross-linked by means of sulfur and organic peroxide or nonconventionally with the use of iodoform (CH3I). Based on thermo-kinetic analysis, the destruction activation energy of the elastomers investigated and their vulcanizates was determined under air and inert gas. The analysis of particular stages of their thermal decomposition was also presented. During the combustion of the elastomeric materials obtained, it has been found that their fire hazard depends not only on the elastomer chemical structure but also on the method of its cross-linking.

Thermal properties and flammability of ethylene-vinyl acetate rubbers (EVM) and their cross-linked blends with nitrile rubber (NBR)

This paper presents the investigation results concerning the thermal stability, fire hazard and flammability of non-cross-linked and cross-linked EVM rubbers as well as their cross-linked blends with NBR. Thermal analysis was carried out under both air and inert gas within the temperature range of T=−100 to 700°C. The destruction activation energy of the materials investigated was determined with the use of two non-isothermal methods: Flynn-Wall-Ozawa's and Kissinger's methods. The fire hazard of the vulcanizates investigated was determined by means of a micro-calorimeter and the coupled TG-FTIR method. Polycyclic aromatic hydrocarbons in the gaseous thermal decomposition products of the material tested were determined by the GC–MS technique.

Innovative technologies of vitamin fruitberry iсе-cream production using frozen fine-dispersed additives made of plant raw materials

The objective of the paper is the scientific substantiation of technologies for producing new kinds of fruit-berry ice-cream and fine-dispersed additives using a cryogenic “shock” freezing and low-temperature grinding for producing healthy products with high content of BaS. In the paper the quality of frozen fine-dispersed additives made by the innovative technology in the form of nanostructured puree was compared with feedstock in regard to BaS content. It is shown, that under conditions of feedstock freezing and low-temperature grinding, accompanied by cryogenic destruction and mechanical activation processes, BaS is more fully extracted from a biopolymer-bound into a free state. The increase rate of BaS extraction depends on its kind and varies from 1.6 to 2.9 times with respect to fresh feedstock. Technology, technological scheme and three formulations of new fruit-berry ice-cream (“Vitaminchik”, “limonchik”, “Tropik”) were developed, with their quality analysis in respect to BaS content. It is shown that new healthy fruit-berry ice-cream is better than the native analogues by its chemical composition and competes with the best foreign analogues, as it contains a great amount of BaS: vitamin C, phenol compounds, tanning substances, organic acids, pectin and mineral substances. Thus, 100 g of the fruit-berry ice-cream “Vitaminchik” contains the daily norm of vitamin C, “limonchik” and “Tropik” have half of the daily norm of this vitamin. Moreover, new kinds of ice-cream have a significant content of phenol compounds, tanning, pectin and mineral substances, etc. The quality of Ir spectrums of new kinds of ice-cream was compared to their analogue (“Snihovyk” fruit-berry ice-cream, “Khladoprom” PrJSC). The final result of the work is the development of the Scientific research Project on nanostructured puree and new kinds of fruit-berry ice-cream. Furthermore, new kinds of ice-cream have passed degustation and approbation in production environment at Kharkov enterprises (“Khladoprom” PrJSC, “Polus LTD” SUIS LLC).

Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host–parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian.

Neurotropic viral infections leading to epilepsy: focus on Theiler's murine encephalomyelitis virus.

Neurotropic viruses cause viral encephalitis and are associated with the development of seizures/epilepsy. The first infection-driven animal model for epilepsy, the Theiler's murine encephalomyelitis virus-induced seizure model is described herein. Intracerebral infection of C57BL/6 mice with Theiler's murine encephalomyelitis virus induces acute seizures from which the animals recover. However, once the virus is cleared, a significant portion of the animals that experienced acute seizures later develop epilepsy. Components of the innate immune response to viral infection, including IL-6 and complement component 3, have been implicated in the development of acute seizures. Multiple mechanisms, including neuronal cell destruction and cytokine activation, play a role in the development of acute seizures. Future studies targeting the innate immune response will lead to new therapies for seizures/epilepsy.

Perspectives on the Role of Photodynamic Therapy in the Treatment of Pancreatic Cancer

Photodynamic therapy (PDT) is a noninvasive procedure involving a photosensitizing agent that is activated by light to produce reactive oxygen species (ROS) that selectively destroy tumor cells. In recent years, PDT has been used in the treatment of pancreatic cancer (PC). The antitumor effects of PDT include three main mechanisms: direct tumor cell death (necrosis, apoptosis, and autophagy), vascular destruction, and immune system activation. The present paper systematically summarizes the effects of PDT in the treatment of PC from the experimental studies to the clinical studies and discusses the mechanisms of PDT-induced PC destruction.

Understanding the Villain: DMBA-Induced Preantral Ovotoxicity Involves Selective Follicular Destruction and Primordial Follicle Activation through PI3K/Akt and mTOR Signaling

7,12-Dimethylbenz-[a]anthracene (DMBA) is an environmental carcinogen which has a potent ovotoxic affect on rat and mouse ovaries, causing complete follicular depletion resulting in premature ovarian failure. Although the overall effects of DMBA on ovarian folliculogenesis are well known, little is known about the exact molecular mechanisms behind its ovotoxicity. In this study, we characterized the mechanisms behind DMBA-induced ovotoxicity in immature follicles. Microarray analysis of neonatal mouse ovaries exposed to DMBA in vitro revealed a multilayered mechanism of DMBA-induced neonatal ovotoxicity involving a distinct cohort of genes and ovarian signaling pathways primarily associated with follicular atresia, tumorigenesis, and follicular growth. Histomorphological and immunohistological analysis supported the microarray data, showing evidence of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Further immunohistological analysis identified increased Akt1 phosphorylation, mTOR activation, and decreased FOXO3a expression in DMBA-treated primordial oocytes. Our results reveal a novel mechanism of DMBA-induced preantral ovotoxicity involving selective immature follicle destruction and primordial follicle activation involving downstream members of the PI3K/Akt and mTOR signaling pathways.

Abstract 1988: A bi-directional feedback loop between Wnt and ERK is important in maintaining the proliferative phenotype of normal human urothelial cells

Abstract Introduction: The urothelial lining of the bladder is ordinarily mitotically quiescent, but can rapidly regenerate in response to injury. This regenerative capacity is thought to be important in restoring urinary barrier function and offers a primary target for deregulation in cancer. Previous work in our laboratory has implicated autocrine EGFR activation in this process, but other regulatory pathways may be involved. Wnt signalling has been shown to be important in the renewal of a number of adult epithelial tissues, but to date, no conclusive evidence for a role for Wnt in urothelial regeneration has been demonstrated. In the absence of Wnt ligand, β-catenin is sequestered via the destruction complex. Activation of the canonical Wnt pathway by ligand, inactivates the destruction complex, allowing β-catenin to translocate to the nucleus and drive proliferation. The aim of this study was to assess whether Wnt signaling was functional in normal human urothelial (NHU) cells, and to analyze the effects of Wnt and downstream EGFR signaling on proliferation. Materials and Methods: NHU cells were grown as finite lifespan cell lines in proliferating cultures. Destruction complex antagonists were used to activate the Wnt pathway. Proliferation assays, immunofluorescence labeling and TOPFLASH luciferase reporter assays were used to assess NHU cell responses. Pathway crosstalk was analyzed using shRNA targeted to β-catenin, as well as antagonists of the EGFR signaling pathways. Results: Pharmacological inhibition of the destruction complex or activation of the Wnt pathway with exogenous Wnt ligand led to nuclear accumulation of β-catenin and an increase in TCF/LEF transcription factor activity. These effects were only apparent when autocrine EGFR signaling was blocked. Knockdown of β-catenin led to significant changes in p-ERK and p-AKT downstream signaling. Conclusion: Wnt signaling can be activated in NHU cells, verifying the presence of a functional pathway. Activation of the pathway is however masked by EGFR signaling, indicating pathway crosstalk. The effect of β-catenin knockdown on p-ERK and p-AKT indicates a bi-directional positive feedback loop between the Wnt and EGFR signaling cascades in NHU cells. This study identifies the Wnt signaling pathway as an important signaling cascade in regulating the regenerative phenotype of NHU cells and indicates at least two pathways that may be arrogated by urothelial cells during neoplastic transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2011-1988