Chemically-induced Model Research Articles

Abstract 562: Foamy Monocytes in Hypertriglyceridemia

Objective: Hypertriglyceridemia (HTG) increases risk for atherosclerotic cardiovascular disease, but the mechanisms remain poorly defined. Foamy monocytes are lipid-loaded monocytes in circulation that contribute to atherosclerosis under hypercholesterolemia. Human study has proved that HTG is associated with formation of foamy monocytes. Our study is to examine formation of foamy monocytes in HTG and their potential contribution to atherosclerosis in mouse models. Approach and results: In vivo mouse models of HTG included wild-type C57BL/6 mice on high fat diet (HFD) injected intraperitoneally with LPL inhibitor, Poloxamer 407 (P407, 0.25mg/g, every two days), as a chemically-induced model and mice with transgenic overexpression of human ApoCIII (ApoCIIItg) as a genetic model. Based on CD11c and CD36, monocytes were identified as CD36 - CD11c - , CD36 + CD11c - and CD36 + CD11c + subsets. In the first model, at 24h of the first injection, triglyceride levels increased to 367 ±84 mg/dL, higher than that of control group with saline injection (60 ±22 mg/dL, n=4, P<0.001). Meanwhile, the side scatter (SSC, representing cell granularity) values and Nile red staining for lipids of CD36 + CD11c + monocytes increased significantly, indicating formation of foamy monocytes, in mice with HTG. Furthermore, CD11c mean fluorescence intensity of CD36 + CD11c + foamy monocytes increased significantly at 2 weeks of P407 injection. In ApoCIIItg mice fed HFD (5 weeks), the percentage and SSC value of CD36 + CD11c + monocytes increased significantly (37%±5%, 247±8), also indicating elevated granularity and lipid accumulation of these monocytes, compared to wild-type mice (26%±3%, p<0.05; 226±8, p<0.05, n=4-6). In vitro treatment with human triglyceride-rich lipoprotein (hTGRL) for 24h increased the granularity and Nile red staining intensity of THP-1 monocytes, indicating foamy monocyte formation. hTGRL treatment also increased THP-1 monocyte expression of CD36, with greater uptake of cholesteryl ester-rich lipoprotein. Conclusion: High triglyceride promotes foamy monocyte formation and induces monocyte phenotypic changes in mice and tissue culture, with increased expression of CD11c and CD36, which may contribute to development of atherosclerosis under HTG.

The effect of CD26-deficiency on dipeptidyl peptidase 8 and 9 expression profiles in a mouse model of Crohn's disease.

The involvement of dipeptidyl peptidase (DP) IV/CD26 (DPP IV/CD26) family members in the pathogenesis of Crohn's disease (CD), an autoimmune inflammatory condition of the gut, is effected mainly through proteolytic cleavage of immunomodulatory substrates and DPP IV/CD26's costimulatory function. DP8 and DP9 are proteases with diverse functions including cell interactions, apoptosis, and immune response but their localization remains to be clarified. We assessed the impact of DPP IV/CD26 deficiency (CD26-/- ) on the expression profiles of DP8 and DP9 by qPCR and immunodetection as well as quantified DP8/9 enzyme activity in distinctive phases of a chemically-induced CD model in mice. CD26-/- did not affect colon DP8 mRNA expression, while the physiological concentration of DP8 protein is decreased in CD26-/- mice but rises in inflammation (P < 0.05). On the other hand, DP9 mRNA level is significantly increased in CD26-/- mice in inflammation as well as healing with the DP9 concentration being almost twofold increased (P < 0.05) in all experimental points in CD26-/- mice compared to wild-type indicating the expected up-regulation in CD26-/- conditions. Surprisingly, dominantly intracellular DP8 and DP9 were found in abundance in serum. DP8/9 activity is decreased in the inflamed colon, whereas its contribution to the overall serum DPP IV/CD26-like activity is negligible, suggesting the importance of their extra-enzymatic functions. To summarize, CD induction generated gene, protein and enzymatic changes of DP8 and DP9 so their involvement in inflammation development and/or healing process is implicated, especially in CD26-/- , and the question of their subcellular localization should be revised.

Characterisation of fibrosis in chemically-induced rat mammary carcinomas using multi-modal endogenous contrast MRI on a 1.5T clinical platform

ObjectivesTo determine the ability of multi-parametric, endogenous contrast MRI to detect and quantify fibrosis in a chemically-induced rat model of mammary carcinoma.MethodsFemale Sprague-Dawley rats (n=18) were administered with N-methyl-N-nitrosourea; resulting mammary carcinomas underwent nine-b-value diffusion-weighted (DWI), ultrashort-echo (UTE) and magnetisation transfer (MT) magnetic resonance imaging (MRI) on a clinical 1.5T platform, and associated quantitative MR parameters were calculated. Excised tumours were histologically assessed for degree of necrosis, collagen, hypoxia and microvessel density. Significance level adjusted for multiple comparisons was p=0.0125.ResultsSignificant correlations were found between MT parameters and degree of picrosirius red staining (r > 0.85, p < 0.0002 for ka and δ, r < -0.75, p < 0.001 for T1 and T1s, Pearson), indicating that MT is sensitive to collagen content in mammary carcinoma. Picrosirius red also correlated with the DWI parameter fD* (r=0.801, p=0.0004) and conventional gradient-echo T2* (r=-0.660, p=0.0055). Percentage necrosis correlated moderately with ultrashort/conventional-echo signal ratio (r=0.620, p=0.0105). Pimonidazole adduct (hypoxia) and CD31 (microvessel density) staining did not correlate with any MR parameter assessed.ConclusionsMagnetisation transfer MRI successfully detects collagen content in mammary carcinoma, supporting inclusion of MT imaging to identify fibrosis, a prognostic marker, in clinical breast MRI examinations.Key Points• Magnetisation transfer imaging is sensitive to collagen content in mammary carcinoma.• Magnetisation transfer imaging to detect fibrosis in mammary carcinoma fibrosis is feasible.• IVIM diffusion does not correlate with microvessel density in preclinical mammary carcinoma.

A Mouse Model of Chronic Pancreatitis Induced by an Alcohol and High Fat Diet.

Background/Aims:Study of acute pancreatitis in chemically-induced rodent models has provided useful data; models of alcoholic chronic pancreatitis have not been available in mice. The aim of the present study was to characterize a mouse model of chronic pancreatitis induced solely with an alcohol and high fat (AHF) diet.Methods:Mice were fed a liquid high fat diet containing 6% alcohol as well as a high fat supplement (57% total dietary fat) over a period of five months or as control, normal chow ad libitum. Pain related measures utilized as an index of pain included mechanical sensitivity of the hind paws determined using von Frey filaments and a smooth/rough textured plate. A modified hotplate test contributed information about higher order behavioral responses to visceral hypersensitivity. Mice underwent mechanical and thermal testing both with and without pharmacological treatment with a peripherally restricted μ-opioid receptor agonist, loperamide.Results:Mice on the AHF diet exhibited mechanical and heat hypersensitivity as well as fibrotic histology indicative of chronic pancreatitis. Low dose, peripherally restricted opiate loperamide attenuated both mechanical and heat hypersensitivity.Conclusion:Mice fed an alcohol and high fat diet develop histology consistent with chronic pancreatitis as well as opioid sensitive mechanical and heat hypersensitivity.

Automated analysis of brain activity for seizure detection in zebrafish models of epilepsy

BackgroundEpilepsy is a chronic neurological condition, with over 30% of cases unresponsive to treatment. Zebrafish larvae show great potential to serve as an animal model of epilepsy in drug discovery. Thanks to their high fecundity and relatively low cost, they are amenable to high-throughput screening. However, the assessment of seizure occurrences in zebrafish larvae remains a bottleneck, as visual analysis is subjective and time-consuming. New methodFor the first time, we present an automated algorithm to detect epileptic discharges in single-channel local field potential (LFP) recordings in zebrafish. First, candidate seizure segments are selected based on their energy and length. Afterwards, discriminative features are extracted from each segment. Using a labeled dataset, a support vector machine (SVM) classifier is trained to learn an optimal feature mapping. Finally, this SVM classifier is used to detect seizure segments in new signals. ResultsWe tested the proposed algorithm both in a chemically-induced seizure model and a genetic epilepsy model. In both cases, the algorithm delivered similar results to visual analysis and found a significant difference in number of seizures between the epileptic and control group. Comparison with existing methodsDirect comparison with multichannel techniques or methods developed for different animal models is not feasible. Nevertheless, a literature review shows that our algorithm outperforms state-of-the-art techniques in terms of accuracy, precision and specificity, while maintaining a reasonable sensitivity. ConclusionOur seizure detection system is a generic, time-saving and objective method to analyze zebrafish LPF, which can replace visual analysis and facilitate true high-throughput studies.

Protective Activity of Esculetin Against 3-Nitropropionic Acid Induced Neurotoxicity Via Scavenging Reactive Oxygen Species in Male Wistar Rats

Huntington’s disease (HD) is a devastating neurodegenerative disorder with no cure till date. Many genetic or chemically induced models have been developed in rodents to study the disease. 3-Nitropropionic (3-NP) acid is a well-known neurotoxin to induce Huntington’s disease (HD) in rodents. It replicates the pathology of HD by causing oxidative stress. Esculetin is a natural compound, a coumarin, known to have neuroprotective effect in a mouse model of Parkinson’s disease. In the present study, the neuroprotective effect of esculetin on 3-NP induced oxidative stress in rat striatum was determined by behavioral and biochemical parameters. Rats were induced with 3-NP (10mg/kg) intraperitoneally for 14 days and rats induced with 3-NP were treated with esculetin (25mg/kg and 50mg/kg) for 14 days. Esculetin attenuated the behaviour of rats in morris water maze, open field, forced swim, narrow beam walk and grip strength test. Biochemical effect of esculetin was also studied on oxidative stress markers, SDH and acetylcholinesterase. Esculetin treatment alleviated the increased values of acetylcholinesterase, protein carbonyls and lipid peroxidation. On treatment with esculetin, we observed that the levels of SOD, GSH, catalase, glutathione peroxidase, SDH were increased. The present study shows that the antioxidant activity of esculetin may be responsible for its neuroprotective activity against 3- nitropropionic acid induced neurotoxicity in rats

Deficiency in Interleukin-1 receptor-associated kinase (IRAK) 4 activity attenuates manifestations of murine Lupus

Abstract IL-1R-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of MyD88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumonia, but its role in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB male mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of IRAK1, mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced TNF-α and CCL5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein (Tollip) and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background had reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, decreased numbers of splenic macrophages, total and TNF-a+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared to lupus-prone mice with functional IRAK4. Furthermore, mice expressing kinase-inactive IRAK4 had decreased proteinuria and increased survival in the chemically-induced (pristine) lupus model. Thus, IRAK4 kinase activity significantly contributes to murine lupus and could represent a new target for therapeutic interventions.

Omega-3 fatty acid supplementation decreases DNA damage in brain of rats subjected to a chemically induced chronic model of Tyrosinemia type II.

Tyrosinemia type II is an inborn error of metabolism caused by a mutation in a gene encoding the enzyme tyrosine aminotransferase leading to an accumulation of tyrosine in the body, and is associated with neurologic and development difficulties in numerous patients. Because the accumulation of tyrosine promotes oxidative stress and DNA damage, the main aim of this study was to investigate the possible antioxidant and neuroprotective effects of omega-3 treatment in a chemically-induced model of Tyrosinemia type II in hippocampus, striatum and cerebral cortex of rats. Our results showed chronic administration of L-tyrosine increased the frequency and the index of DNA damage, as well as the 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the hippocampus, striatum and cerebral cortex. Moreover, omega-3 fatty acid treatment totally prevented increased DNA damage in the striatum and hippocampus, and partially prevented in the cerebral cortex, whereas the increase in 8-OHdG levels was totally prevented by omega-3 fatty acid treatment in hippocampus, striatum and cerebral cortex. In conclusion, the present study demonstrated that the main accumulating metabolite in Tyrosinemia type II induce DNA damage in hippocampus, striatum and cerebral cortex, possibly mediated by free radical production, and the supplementation with omega-3 fatty acids was able to prevent this damage, suggesting that could be involved in the prevention of oxidative damage to DNA in this disease. Thus, omega-3 fatty acids supplementation to Tyrosinemia type II patients may represent a new therapeutic approach and a possible adjuvant to the curren t treatment of this disease.

ANTI-ULCER STUDY OF STANDARDIZED ETHANOL ROOT EXTRACT OF AGANOSMA DICHOTOMA AND ISOLATED URSOLIC ACID

Objective: Aganosma dichotoma K. Schum (Apocynaceae) has been traditionally used as an Ayurvedic ulcer treatment and the study scientifically validates the antiulcer effect of A. dichotoma ethanol root extract (EAD).Methods: The studies included the isolation, quantification of ursolic acid through HPTLC. Acute and sub-acute toxicity study of EAD for 28 d and antiulcer effect of ursolic acid (50 mg/kg, p. o.), EAD (100, 200 and 400 mg/kg, p. o.) were also evaluated on both the physical (pyloric ligation, PL; cold restrain stress, CRS;) and chemical (absolute ethanol, aspirin, ASP;) induced ulcer in Charles Foster albino rats for 7 d. The various gastric-ulcer parameters, viz. gastric pH, volume, acid-pepsin output, DNA content, H+K+-ATPase activity, mucus content, microvascular permeability, antioxidant enzyme, and gastric histopathological study were performed.Results: The isolated ursolic acid was characterized by NMR and mass spectrometer and quantified through HPTLC in EAD (4.26% w/w). Acute oral toxicity study indicated that LD50 of extract was ≥ 5 g/kg. EAD at the dose of 200 and 400 mg/kg, p. o. reduced the ulcer score in both physical and chemical-induced ulcer models. In PL model EAD (400 mg/kg, p. o.) and ursolic acid (50 mg/kg, p. o.) showed antisecretory property by inhibiting aggressive factors [increase in gastric pH (35.02%, 26.73%), whereas decreased gastric volume (43.55%, 34.35%) and acid-pepsin output (75.23%, 68.81%), respectively]. EAD at 400 mg/kg p. o. showed significant effect on proton pump inhibition while ursolic acid didn’t showed any effect.Conclusion: The effects of EAD were accredited mainly to the offensive mechanism and justify its traditional usage in the treatment of gastric ulcers.

Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice.

New treatment options and drug targets for colorectal carcinoma are a pressing medical need. Inflammation and pro-inflammatory cytokines produced by Th1 and Th17 cells like IL-6, TNF, IL-17 and IL-23 promote the development and growth of colorectal cancer (CRC). The immunoproteasome is a proteasome subtype highly expressed in immune cells but also in the intestine. Since the immunoproteasome promotes Th1 and Th17 differentiation and pro-inflammatory cytokine production, we investigated here whether deficiency or inhibition of the immunoproteasome subunit LMP7 would interfere with CRC development and exacerbation in preventive and therapeutic mouse models. Treatment with the LMP7 inhibitor ONX 0914 blocked tumor initiation and progression in either chemically-induced (AOM/DSS) or transgenic mouse models (ApcMin/+) of colon carcinogenesis. ONX 0914 treatment strongly reduced tumor numbers and CRC-associated loss of body weight while the survival rates were significantly enhanced. Moreover, genetic LMP7 deficiency markedly reduced the tumor burden in AOM/DSS induced wild type and ApcMin/+ mice. In conclusion, we show that the immunoproteasome is involved in CRC development and progression and we identify LMP7 as a new potential drug target for the treatment of CRC.

Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii Shows a Protective Effect on DNBS and DSS-Induced Colitis Model in Mice through Inhibition of NF-κB Pathway.

Faecalibacterium prausnitzii and its supernatant showed protective effects in different chemically-induced colitis models in mice. Recently, we described 7 peptides found in the F. prausnitzii supernatant, all belonging to a protein called Microbial Anti-inflammatory Molecule (MAM). These peptides were able to inhibit NF-κB pathway in vitro and showed anti-inflammatory properties in vivo in a DiNitroBenzene Sulfate (DNBS)-induced colitis model. In this current proof we tested MAM effect on NF-κB pathway in vivo, using a transgenic model of mice producing luciferase under the control of NF-κB promoter. Moreover, we tested this protein on Dextran Sodium Sulfate (DSS)-induced colitis in mice. To study the effect of MAM we orally administered to the mice a Lactococcus lactis strain carrying a plasmid containing the cDNA of MAM under the control of a eukaryotic promoter. L. lactis delivered plasmids in epithelial cells of the intestinal membrane allowing thus the production of MAM directly by host. We showed that MAM administration inhibits NF-κB pathway in vivo. We confirmed the anti-inflammatory properties of MAM in DNBS-induced colitis but also in DSS model. In DSS model MAM was able to inhibit Th1 and Th17 immune response while in DNBS model MAM reduced Th1, Th2, and Th17 immune response and increased TGFβ production.

Antinociceptive activity of methanol extract of leaves of Solanum sisymbriifolium in heat and chemical-induced pain

Solanum sisymbriifolium Lamk. (Solanaceae) is a perennial prickly herb traditionally used in folk medicine to treat different ailments. This study evaluated the methanol extract of S. sisymbriifolium leaf (MSS) (100, 200, and 400 mg/kg; p.o.) for analgesic activity using heat- and chemical-induced pain models such as hot plate, tail immersion, formalin-induced licking and acetic acid-induced writhing. Interaction with the opioid receptor system was verified using naloxone to antagonize the effect of MSS, if any. MSS demonstrated significant and dose-dependent antinociceptive activity, though moderate, in both hot plate and tail immersion test (p < 0.05). Naloxone reversed the antinociceptive effect in both tests. MSS produced profound antinociceptive effect in formalin test and acetic acid-induced writhing at 400 mg/kg dose. These results imply that the antinociceptive effect of MSS is mainly due to its central effect and peripheral effects also contribute to some extent. These results provide preliminary evidence of antinociceptive activity of S. sisymbriifolium leaves and the potential of the plant in treating different painful conditions.

Effects of raloxifene against letrozole-induced bone loss in chemically-induced model of menopause in mice

IntroductionThe deleterious effects of letrozole, an aromatase inhibitor, used in the adjuvant treatment of breast cancer in postmenopausal women, on bone are well-documented and represent a major drawback to its clinical use. Raloxifene, a selective estrogen receptor modulator and a clinically approved anti-osteoporotic drug, has been recently demonstrated to be efficacious in women with breast cancer. The present study evaluated the effects of preventive and curative treatment with raloxifene on letrozole-induced alterations of bone microarchitecture and turnover markers in a chemically-induced menopause model in mice. MethodSwiss strain albino female mice were made menopausal by inducing ovotoxicity using vinyl cyclohexene di epoxide (VCD, 160 mg/kg for 15 days followed by 30 days drug-free period) confirmed by ovarian histology and serum estradiol levels. Effects on femoral and lumbar bones were evaluated by micro CT determination of bone volume, trabecular number, separation, thickness, connective density and trabecular pattern factor and bone turnover markers including ALP, TRAP5b, hydroxyproline and RANKL. In addition to these, markers of Wnt signaling (sclerostin and dickkopf-1) were also evaluated. To rule out the involvement of pharmacokinetic interaction, plasma levels of letrozole and raloxifene were measured following drugs alone and in combination. ResultsThough bone loss was observed in VCD treated mice (as indicated by micro CT measurements), it was further enhanced with letrozole administration (1 mg/kg) for one month particularly in epiphysis of femoral bones. Raloxifene (15 mg/kg), whether administered concurrently or post-letrozole was able to revert the structural alterations and changes in turnover markers caused by letrozole to varying degrees (p < 0.01 or p < 0.001). Further, estrogen deficiency following letrozole treatment in ovotoxic mice was associated with significant increase in sclerostin and dickkopf-1 in both lumbar and femur bones (p < 0.001) which was attenuated with preventive and curative treatment with raloxifene (p < 0.05). The plasma levels of letrozole remained unaffected by raloxifene administration and vice versa. ConclusionsOur study indicates the potential of raloxifene in preventing and attenuating letrozole-induced bone loss. Further, these effects were found to be independent of a pharmacokinetic interaction between the two drugs.

Vagotomy and Gastric Tumorigenesis.

Vagotomy reduces gastric acid secretion and was therefore introduced as a surgical treatment for peptic ulcers in the 1970s. Later, it was replaced by acid reducing medication, such as histamine type 2 (H2) receptor antagonists and proton pump inhibitors (PPIs). A large body of evidence has indicated that drug-induced hypochlorhydria per se does not increase the risk of gastric cancer. Early studies on the effects of vagotomy in chemically-induced rodent models of gastric cancer reported an increased risk of developing gastric cancer. This was most likely due to a delayed gastric emptying, which later has been accounted for by including an additional drainage procedure, e.g. pyloroplasty. In a recent study using three different mouse models of gastric cancer (including genetically engineered, chemically-induced and Helicobacter pylori-infected mice), either unilateral vagotomy or bilateral truncal vagotomy with pyloroplasty was found to significantly attenuate tumorigenesis in the denervated side of the stomach at early preneoplastic stages as well as at later stages of tumorigenesis. Consistently, pharmacological denervation using botulinum toxin A or muscarinic acetylcholine receptor 3 (M3R) blockade inhibited tumorigenesis. Moreover, it was found that recurrence of gastric cancer was reduced in patients following vagotomy. Thus, these new findings suggest the potential treatment strategies to target the nerve, neurotransmitters, corresponding receptors and their downstream signaling pathways for the malignancy.

Analgesic Activity of Novel GABA Esters after Transdermal Delivery

Analgesic activity of novel GABA esters with l-menthol (1), thymol (2), carvacrol (3) and guaiacol (4) was investigated by pharmacological models of thermal and chemical stimuli in mice after topical application in an ointment (2% w/w). The initial terpenes - l-menthol (5), thymol (6), carvacrol (7) and guaiacol (8) have also been studied under the same experimental conditions. All studied compounds were found to produce an antinociceptive effect in both thermal- and chemical-induced models of acute pain after their topical application. In the hot plate test, compound 1 demonstrated maximum analgesic activity and attenuated acute pain more than the reference drug BZC. GABA esters with l-menthol (1) and carvacrol (3), as well as l-menthol (5) itself exhibited analgesic activity which is the same or better in comparison with benzocaine in chemical-stimulated models of pain caused by either formalin or capsaicin.

Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical.

Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chemically-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clinical investigations. The primary route of isothiocyanate metabolism is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymatic activity.

The receptor for advanced glycation end products is required for β-catenin stabilization in a chemical-induced asthma model.

Cytoplasmic retention of β-catenin will lead to its nuclear translocation and subsequent interaction with the transcription factor TCF/LEF that regulates target gene expression. We have previously demonstrated aberrant expression of β-catenin in a model of asthma induced by toluene diisocyanate (TDI). The aim of this study was to examine whether the receptor for advanced glycation end products (RAGE) can regulate β-catenin expression in TDI-induced asthma. Male BALB/c mice were sensitized and challenged with TDI to generate a chemically-induced asthma model. Inhibitors of RAGE, FPS-ZM1 and the RAGE antagonist peptide (RAP), were injected i.p. after each challenge. Airway resistance was measured in vivo and bronchoalveolar lavage fluid was analysed. Lungs were examined by histology and immunohistochemistry. Western blotting and quantitative PCR were also used. Expression of RAGE and of its ligands HMGB1, S100A12, S100B, HSP70 was increased in TDI-exposed lungs. These increases were inhibited by FPS-ZM1 or RAP. Either antagonist blunted airway reactivity, airway inflammation and goblet cell metaplasia, and decreased release of Th2 cytokines. TDI exposure decreased level of membrane β-catenin, phosphorylated Akt (Ser(473) ), inactivated GSK3β (Ser(9) ), dephosphorylated β-catenin at Ser(33) /(37) /Thr(41) , which controls its cytoplasmic degradation, increased phosphorylated β-catenin at Ser(552) , raised cytoplasmic and nuclear levels of β-catenin and up-regulated its targeted gene expression (MMP2, MMP7, MMP9, VEGF, cyclin D1, fibronectin), all of which were reversed by RAGE inhibition. RAGE was required for stabilization of β-catenin in TDI-induced asthma, identifying protective effects of RAGE blockade in this model.

Abstract 3327: The tumor associated sialyltransferase ST6Gal-I promotes a cancer stem cell phenotype and upregulates stem-related transcription factors

Abstract Altered glycosylation is a key hallmark of tumor cells; still, the role of individual glycosyltransferases remains unclear. ST6Gal-I is a tumor-associated sialyltransferase which catalyzes the addition of a sialic acid sugar to substrate glycoproteins. Addition of the negatively-charged sialic acid by ST6Gal-I has been shown to alter receptor conformation, clustering, and surface retention, leading to changes in downstream signaling. In this study we assayed ST6Gal-I by immunohistochemistry and report the great majority of patient ovarian and pancreatic tumors express this enzyme. In contrast, the normal epithelium expresses minimal ST6Gal-I. Enzyme expression in ovarian cancers is enriched during metastasis and correlates with worse progression-free and overall survival. Recent evidence points to ST6Gal-I activity in stem/progenitor cells. In light of this, we investigated whether ST6Gal-I functionally promotes a cancer stem cell (CSC) phenotype, i.e. resistance to chemotherapy, survival as tumorspheroids, and ability to initiate tumors. We previously reported that ST6Gal-I activity confers resistance to cisplatin; we now show its activity additionally confers resistance to gemcitabine in pancreatic tumor cells. ST6Gal-I expressing cells are enriched in patient derived xenografts (PDX) treated with gemcitabine suggesting that these cells preferentially survive chemotherapy in vivo. In addition to chemoresistance, ST6Gal-I promotes the growth of pancreatic and ovarian cell lines in tumorspheroid culture. Moreover, ST6Gal-I expressing primary tumor cells isolated from ovarian cancer ascites or PDX tumors survive in tumorspheroid culture, whereas ST6Gal-I negative cells do not. Conversely, forced expression of ST6Gal-I protects tumor cells exposed to the ascites fluid milieu in vitro, while non-ST6Gal-I expressing cells succumb to this inflammatory environment. In a limiting dilution tumor initiating assay, ST6Gal-I expressing cells have a higher tumor incidence and form larger tumors compared to cells with ST6Gal-I knockdown. We next created a conditional mouse model with forced ST6Gal-I expression in the intestinal tract and used AOM-DSS chemically-induced carcinogenesis model to evaluate tumor formation. Compared with wildtype mice, ST6Gal-I knock-in mice have a greater tumor burden, evidenced by increased tumor number and area. As a novel mechanistic link beteween ST6Gal-I and the CSC phenotype, direct modulation of ST6Gal-I levels in tumor cells regulates the expression of stem-related transcription factors, Sox9 and Slug, implicated in tumor progression. The finding that a distinct glycosyltransferase governs the expression of key transcription factors highlights the tumor glycome as a driving factor in CSC behavior. Citation Format: Matthew J. Schultz, Andrew T. Holdbrooks, Asmi Chakraborty, William E. Grizzle, Charles N. Landen, Donald J. Buchsbaum, Michael G. Conner, Rebecca C. Arend, Karina J. Yoon, Chris A. Klug, Daniel C. Bullard, Robert A. Kesterson, Patsy G. Oliver, Amber K. O’Connor, Bradley K. Yoder, Susan L. Bellis. The tumor associated sialyltransferase ST6Gal-I promotes a cancer stem cell phenotype and upregulates stem-related transcription factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3327.

Abstract 3820: Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma

Abstract Background and aims Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies. Methods Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC. Results Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p&amp;lt;0.0001). In a GEMM model, IGF2-overexpression accelerated HCC progression and reduced survival (p = 0.02). Conversely, IGF2-blockage using an IGF1/2-monoclonal antibody (mAb) or specific shRNAs against IGF2 reduced viability and proliferation in vitro (p&amp;lt;0.05), and inhibited IGF-Insulin pathway activation without disturbing insulin metabolism. IGF1/2-mAb delayed tumor growth and increased survival in vivo compared to placebo or sorafenib (p&amp;lt;0.0001), through antiproliferative and antiangiogenic mechanisms. Conclusions IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients. Citation Format: Iris Martinez-Quetglas, Roser Pinyol, Daniel Dauch, Sara Torrecilla, Victoria Tovar, Agrin Moeini, Clara Alsinet, Anna Portela, Augusto Villanueva, Manel Esteller, Lars Zender, Josep M Llovet. Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3820.

Abstract 536: Excessive HCK kinase activity in the tumor stroma promotes colorectal cancer progression

Abstract Excessive activation of the myeloid-specific Src-family kinase Hematopoietic Cell Kinase (HCK) acts as a tumor intrinsic oncogene by promoting proliferation and survival of immune cells, and confers a poor prognosis for leukemia. However, the role of HCK in the tumor stroma of solid malignancies remains unexplored. We analyzed the expression level of HCK in matched biopsies from sporadic colorectal cancer patients and observed elevated HCK phosphorylation in tumors compared to unaffected colons. Analysis of corresponding RNAseq data revealed a striking correlation between tumors with high HCK gene expression and a gene signature indicative of a tumor-promoting alternatively-activated macrophage (AAM) endotype. To functionally assess this observation, we exploited HckCA mice [Ernst et al., J Exp Med 2002] that express a constitutively active form of Hck as a knockin mutation, and subjected these mice to a chemically-induced model of sporadic colorectal cancer. HckCA mice developed more and larger tumors compared to wild-type (WT) animals, and this was associated with a significant bias towards CD206+ AAMs in tumors of HckCA mice without affecting the total number of tumor-associated leukocyte and lymphocytes. Likewise, adoptive bone-marrow transfer experiments revealed enhanced tumor formation and AAM differentiation in WT mice reconstituted with HckCA bone marrow, with a reciprocal decrease of these parameters in HckCA mice reconstituted with WT marrow. Finally, pharmacologic targeting of the catalytic activity of Hck with a small molecule inhibitor significantly reduced tumor progression and impaired AAM polarisation. Collectively, our findings suggest that excessive Hck activity in the tumor stroma promotes the progression of solid cancers by modulating the endotype of tumor-associated myeloid cells. Therefore, Hck represents a rational therapeutic target for macrophage re-education in solid cancers by limiting polarization of tumor-promoting AAM. Citation Format: Matthias Ernst, Ashleigh Poh, Frederic Masson, Tracy Putoczki, Robert O’Donoghue. Excessive HCK kinase activity in the tumor stroma promotes colorectal cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 536.