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1.
Cancers (Basel) ; 15(18)2023 Sep 18.
Article in English | MEDLINE | ID: mdl-37760589

ABSTRACT

Brain tumor-initiating cells (BTICs) and tumor cell plasticity promote glioblastoma (GBM) progression. Here, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is essential for BTIC propagation and a target that mediates the suppressive effects of clemastine. Finally, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment. Collectively, these results identify pathways essential for maintaining the stemness and progenitor features of GBMs, uncover BTIC dependency on EBP, and suggest that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.

2.
Biomedicines ; 10(4)2022 Mar 23.
Article in English | MEDLINE | ID: mdl-35453502

ABSTRACT

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for MTAP-deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.

3.
Acta Neuropathol Commun ; 9(1): 178, 2021 11 03.
Article in English | MEDLINE | ID: mdl-34732238

ABSTRACT

Diffuse intrinsic pontine gliomas (DIPGs) are high-grade tumors of the brainstem that often occur in children, with a median overall survival of less than one year. Given the fact that DIPGs are resistant to chemotherapy and are not amenable to surgical resection, it is imperative to develop new therapeutic strategies for this deadly disease. The p53 pathway is dysregulated by TP53 (~ 60%) or PPM1D gain-of-function mutations (~ 30%) in DIPG cases. PPM1D gain-of-function mutations suppress p53 activity and result in DIPG tumorigenesis. While MDM2 is a major negative regulator of p53, the efficacy of MDM2 inhibitor has not been tested in DIPG preclinical models. In this study, we performed a comprehensive validation of MDM2 inhibitor RG7388 in patient-derived DIPG cell lines established from both TP53 wild-type/PPM1D-mutant and TP53 mutant/PPM1D wild-type tumors, as well in TP53 knockout isogenic DIPG cell line models. RG7388 selectively inhibited the proliferation of the TP53 wild-type/PPM1D mutant DIPG cell lines in a dose- and time-dependent manner. The anti-proliferative effects were p53-dependent. RNA-Seq data showed that differential gene expression induced by RG7388 treatment was enriched in the p53 pathways. RG7388 reactivated the p53 pathway and induced apoptosis as well as G1 arrest. In vivo, RG7388 was able to reach the brainstem and exerted therapeutic efficacy in an orthotopic DIPG xenograft model. Hence, this study demonstrates the pre-clinical efficacy potential of RG7388 in the TP53 wild-type/PPM1D mutant DIPG subgroup and may provide critical insight on the design of future clinical trials applying this drug in DIPG patients.


Subject(s)
Brain Stem Neoplasms/pathology , Diffuse Intrinsic Pontine Glioma/pathology , Protein Phosphatase 2C/genetics , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Pyrrolidines/pharmacology , para-Aminobenzoates/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Brain Stem Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Diffuse Intrinsic Pontine Glioma/genetics , Humans , Mice , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
4.
Mol Cancer Res ; 18(7): 968-980, 2020 07.
Article in English | MEDLINE | ID: mdl-32229503

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) is an invariably fatal brain tumor occurring predominantly in children. Up to 90% of pediatric DIPGs harbor a somatic heterozygous mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3.3 (H3F3A, 65%) or H3.1 (HIST1H3B, 25%). Several studies have also identified recurrent truncating mutations in the gene encoding protein phosphatase 1D, PPM1D, in 9%-23% of DIPGs. Here, we sought to investigate the therapeutic potential of targeting PPM1D, alone or in combination with inhibitors targeting specific components of DNA damage response pathways in patient-derived DIPG cell lines. We found that GSK2830371, an allosteric PPM1D inhibitor, suppressed the proliferation of PPM1D-mutant, but not PPM1D wild-type DIPG cells. We further observed that PPM1D inhibition sensitized PPM1D-mutant DIPG cells to PARP inhibitor (PARPi) treatment. Mechanistically, combined PPM1D and PARP inhibition show synergistic effects on suppressing a p53-dependent RAD51 expression and the formation of RAD51 nuclear foci, possibly leading to impaired homologous recombination (HR)-mediated DNA repair in PPM1D-mutant DIPG cells. Collectively, our findings reveal the potential role of the PPM1D-p53 signaling axis in the regulation of HR-mediated DNA repair and provide preclinical evidence demonstrating that combined inhibition of PPM1D and PARP1/2 may be a promising therapeutic combination for targeting PPM1D-mutant DIPG tumors. IMPLICATIONS: The findings support the use of PARPi in combination with PPM1D inhibition against PPM1D-mutant DIPGs.


Subject(s)
Aminopyridines/pharmacology , Brain Stem Neoplasms/genetics , Diffuse Intrinsic Pontine Glioma/genetics , Dipeptides/pharmacology , Mutation , Phthalazines/pharmacology , Piperazines/pharmacology , Protein Phosphatase 2C/genetics , Allosteric Regulation , Brain Stem Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Diffuse Intrinsic Pontine Glioma/drug therapy , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Protein Phosphatase 2C/antagonists & inhibitors , Rad51 Recombinase/metabolism
5.
Cell Rep ; 28(12): 3199-3211.e5, 2019 09 17.
Article in English | MEDLINE | ID: mdl-31533041

ABSTRACT

H2AX safeguards genomic stability in a dose-dependent manner; however, mechanisms governing its proteostasis are poorly understood. Here, we identify a PRMT5-RNF168-SMURF2 cascade that regulates H2AX proteostasis. We show that PRMT5 sustains the expression of RNF168, an E3 ubiquitin ligase essential for DNA damage response (DDR). Suppression of PRMT5 occurs in methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells and attenuates the expression of RNF168, leading to destabilization of H2AX by E3 ubiquitin ligase SMURF2. RNF168 and SMURF2 serve as a stabilizer and destabilizer of H2AX, respectively, via their dynamic interactions with H2AX. In supporting an important role of this signaling cascade in regulating H2AX, MTAP-deficient glioblastoma cells display higher levels of DNA damage spontaneously or in response to genotoxic agents. These findings reveal a regulatory mechanism of H2AX proteostasis and define a signaling cascade that is essential to DDR and that is disrupted by the loss of a metabolic enzyme in tumor cells.


Subject(s)
Glioblastoma/metabolism , Histones/metabolism , Neoplasm Proteins/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Proteostasis , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA Damage , Glioblastoma/genetics , Glioblastoma/pathology , Histones/genetics , Humans , Neoplasm Proteins/genetics , Protein-Arginine N-Methyltransferases/genetics , Ubiquitin-Protein Ligases/genetics
6.
Cancer Res ; 79(13): 3383-3394, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31040154

ABSTRACT

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic alterations in glioblastoma (GBM), but its pathologic consequences remain unclear. In this study, we report that loss of MTAP results in profound epigenetic reprogramming characterized by hypomethylation of PROM1/CD133-associated stem cell regulatory pathways. MTAP deficiency promotes glioma stem-like cell (GSC) formation with increased expression of PROM1/CD133 and enhanced tumorigenicity of GBM cells and is associated with poor prognosis in patients with GBM. As a combined consequence of purine production deficiency in MTAP-null GBM and the critical dependence of GSCs on purines, the enriched subset of CD133+ cells in MTAP-null GBM can be effectively depleted by inhibition of de novo purine synthesis. These findings suggest that MTAP loss promotes the pathogenesis of GBM by shaping the epigenetic landscape and stemness of GBM cells while simultaneously providing a unique opportunity for GBM therapeutics. SIGNIFICANCE: This study links the frequently mutated metabolic enzyme MTAP to dysregulated epigenetics and cancer cell stemness and establishes MTAP status as a factor for consideration in characterizing GBM and developing therapeutic strategies.


Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Neoplastic Stem Cells/pathology , Purine-Nucleoside Phosphorylase/metabolism , Purines/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Mice , Neoplastic Stem Cells/metabolism , Prognosis , Purine-Nucleoside Phosphorylase/genetics , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
Nanotechnology ; 30(27): 275101, 2019 Jul 05.
Article in English | MEDLINE | ID: mdl-30856613

ABSTRACT

Despite decades of efforts, non-invasive sensitive detection of small malignant brain tumors still remains challenging. Here we report a dual-modality 124I-labeled gold nanostar (124I-GNS) probe for sensitive brain tumor imaging with positron emission tomography (PET) and subcellular tracking with two-photon photoluminescence (TPL) and electron microscopy (EM). Experiment results showed that the developed nanoprobe has potential to reach sub-millimeter intracranial brain tumor detection using PET scan, which is superior to any currently available non-invasive imaging modality. Microscopic examination using TPL and EM further confirmed that GNS nanoparticles permeated the brain tumor leaky vasculature and accumulated inside brain tumor cells following systemic administration. Selective brain tumor targeting by enhanced permeability and retention effect and ultrasensitive imaging render 124I-GNS nanoprobe promise for future brain tumor-related preclinical and translational applications.


Subject(s)
Brain Neoplasms/diagnosis , Diagnostic Imaging , Metal Nanoparticles/chemistry , Animals , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/ultrastructure , Fluorodeoxyglucose F18/chemistry , Gold/chemistry , Gold/toxicity , HEK293 Cells , Humans , Iodine Radioisotopes/chemistry , Metal Nanoparticles/toxicity , Metal Nanoparticles/ultrastructure , Mice , Optical Imaging , Organ Specificity , Positron Emission Tomography Computed Tomography
8.
Sci Rep ; 8(1): 12507, 2018 08 21.
Article in English | MEDLINE | ID: mdl-30131529

ABSTRACT

The concept that human cancer is in essence a genetic disease driven by gene mutations has been well established, yet its utilization in functional studies of cancer genes has not been fully explored. Here, we describe a simple genetics-based approach that can quickly and sensitively reveal the effect of the alteration of a gene of interest on the fate of its host cells within a heterogeneous population, essentially monitoring the genetic selection that is associated with and powers the tumorigenesis. Using this approach, we discovered that loss-of-function of TP53 can promote the development of resistance of castration in prostate cancer cells via both transiently potentiating androgen-independent cell growth and facilitating the occurrence of genome instability. The study thus reveals a novel genetic basis underlying the development of castration resistance in prostate cancer cells and provides a facile genetic approach for studying a cancer gene of interest in versatile experimental conditions.


Subject(s)
Loss of Function Mutation , Prostatic Neoplasms, Castration-Resistant/genetics , Tumor Suppressor Protein p53/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Genomic Instability , HCT116 Cells , HEK293 Cells , Humans , Male , Mice , Xenograft Model Antitumor Assays
9.
Nat Commun ; 9(1): 2087, 2018 05 25.
Article in English | MEDLINE | ID: mdl-29802247

ABSTRACT

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTpWT-IDHWT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTpWT-IDHWT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDHWT-TERTSV), and an ALT-positive subgroup (IDHWT-ALT) with mutations in ATRX or SMARCAL1.


Subject(s)
Brain Neoplasms/genetics , Genomics/methods , Glioblastoma/genetics , Isocitrate Dehydrogenase/genetics , Promoter Regions, Genetic/genetics , Telomerase/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , DNA Helicases/genetics , DNA Helicases/metabolism , Female , Glioblastoma/metabolism , Glioblastoma/pathology , HEK293 Cells , HeLa Cells , Humans , Isocitrate Dehydrogenase/metabolism , Male , Middle Aged , Mutation , Survival Analysis , Telomere Homeostasis , Young Adult
10.
Cancer Res ; 77(22): 6097-6108, 2017 11 15.
Article in English | MEDLINE | ID: mdl-28939681

ABSTRACT

Inactivating mutations in the transcriptional repression factor Capicua (CIC) occur in approximately 50% of human oligodendrogliomas, but mechanistic links to pathogenesis are unclear. To address this question, we generated Cic-deficient mice and human oligodendroglioma cell models. Genetic deficiency in mice resulted in a partially penetrant embryonic or perinatal lethal phenotype, with the production of an aberrant proliferative neural population in surviving animals. In vitro cultured neural stem cells derived from Cic conditional knockout mice bypassed an EGF requirement for proliferation and displayed a defect in their potential for oligodendrocyte differentiation. Cic is known to participate in gene suppression that can be relieved by EGFR signal, but we found that cic also activated expression of a broad range of EGFR-independent genes. In an orthotopic mouse model of glioma, we found that Cic loss potentiated the formation and reduced the latency in tumor development. Collectively, our results define an important role for Cic in regulating neural cell proliferation and lineage specification, and suggest mechanistic explanations for how CIC mutations may impact the pathogenesis and therapeutic targeting of oligodendroglioma. Cancer Res; 77(22); 6097-108. ©2017 AACR.


Subject(s)
Cell Differentiation/genetics , Cell Proliferation/genetics , Mutation , Neural Stem Cells/metabolism , Oligodendroglioma/genetics , Repressor Proteins/genetics , Animals , Cell Line, Tumor , Cells, Cultured , Gene Expression Profiling/methods , Humans , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Oligodendroglioma/pathology
11.
Mol Cancer Res ; 15(5): 507-520, 2017 05.
Article in English | MEDLINE | ID: mdl-28148827

ABSTRACT

IDH1 mutations occur in the majority of low-grade gliomas and lead to the production of the oncometabolite, D-2-hydroxyglutarate (D-2HG). To understand the effects of tumor-associated mutant IDH1 (IDH1-R132H) on both the neural stem cell (NSC) population and brain tumorigenesis, genetically faithful cell lines and mouse model systems were generated. Here, it is reported that mouse NSCs expressing Idh1-R132H displayed reduced proliferation due to p53-mediated cell-cycle arrest as well as a decreased ability to undergo neuronal differentiation. In vivo, Idh1-R132H expression reduced proliferation of cells within the germinal zone of the subventricular zone (SVZ). The NSCs within this area were dispersed and disorganized in mutant animals, suggesting that Idh1-R132H perturbed the NSCs and the microenvironment from which gliomas arise. In addition, tumor-bearing animals expressing mutant Idh1 displayed a prolonged survival and also overexpressed Olig2, features consistent with IDH1-mutated human gliomas. These data indicate that mutant Idh1 disrupts the NSC microenvironment and the candidate cell-of-origin for glioma; thus, altering the progression of tumorigenesis. In addition, this study provides a mutant Idh1 brain tumor model that genetically recapitulates human disease, laying the foundation for future investigations on mutant IDH1-mediated brain tumorigenesis and targeted therapy.Implications: Through the use of a conditional mutant mouse model that confers a less aggressive tumor phenotype, this study reveals that mutant Idh1 impacts the candidate cell-of-origin for gliomas. Mol Cancer Res; 15(5); 507-20. ©2017 AACR.


Subject(s)
Brain Neoplasms/pathology , Isocitrate Dehydrogenase/genetics , Lateral Ventricles/cytology , Mutation , Animals , Brain Neoplasms/genetics , Cell Differentiation , Cell Proliferation , Cells, Cultured , DNA Methylation , Gene Knock-In Techniques , Humans , Lateral Ventricles/pathology , Mice , Mice, Transgenic , Neural Stem Cells/cytology , Neural Stem Cells/pathology , Oligodendrocyte Transcription Factor 2/genetics , Promoter Regions, Genetic , Tumor Microenvironment
13.
Oncotarget ; 5(6): 1515-25, 2014 Mar 30.
Article in English | MEDLINE | ID: mdl-24722048

ABSTRACT

Frequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and the promoter of telomerase reverse transcriptase (TERT) represent two significant discoveries in glioma genomics. Understanding the degree to which these two mutations co-occur or occur exclusively of one another in glioma subtypes presents a unique opportunity to guide glioma classification and prognosis. We analyzed the relationship between overall survival (OS) and the presence of IDH1/2 and TERT promoter mutations in a panel of 473 adult gliomas. We hypothesized and show that genetic signatures capable of distinguishing among several types of gliomas could be established providing clinically relevant information that can serve as an adjunct to histopathological diagnosis. We found that mutations in the TERT promoter occurred in 74.2% of glioblastomas (GBM), but occurred in a minority of Grade II-III astrocytomas (18.2%). In contrast, IDH1/2 mutations were observed in 78.4% of Grade II-III astrocytomas, but were uncommon in primary GBM. In oligodendrogliomas, TERT promoter and IDH1/2 mutations co-occurred in 79% of cases. Patients whose Grade III-IV gliomas exhibit TERT promoter mutations alone predominately have primary GBMs associated with poor median OS (11.5 months). Patients whose Grade III-IV gliomas exhibit IDH1/2 mutations alone predominately have astrocytic morphologies and exhibit a median OS of 57 months while patients whose tumors exhibit both TERT promoter and IDH1/2 mutations predominately exhibit oligodendroglial morphologies and exhibit median OS of 125 months. Analyzing gliomas based on their genetic signatures allows for the stratification of these patients into distinct cohorts, with unique prognosis and survival.


Subject(s)
Glioma/classification , Glioma/genetics , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Promoter Regions, Genetic/genetics , Telomerase/genetics , Adult , Biomarkers, Tumor/genetics , Female , Glioma/mortality , Glioma/pathology , Humans , Male , Middle Aged , Neoplasm Grading , Prognosis , Survival Rate
14.
PLoS One ; 9(1)2014.
Article in English | MEDLINE | ID: mdl-29220837

ABSTRACT

[This corrects the article DOI: 10.1371/journal.pone.0045667.].

15.
Oncotarget ; 4(11): 2144-53, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24240169

ABSTRACT

KMT2D (lysine (K)-specific methyltransferase 2D), formerly named MLL2 (myeloid/lymphoid or mixed-lineage leukemia 2, also known as ALR/MLL4), is a histone methyltransferase that plays an important role in regulating gene transcription. In particular, it targets histone H3 lysine 4 (H3K4), whose methylations serve as a gene activation mark. Recently, KMT2D has emerged as one of the most frequently mutated genes in a variety of cancers and in other human diseases, including lymphoma, medulloblastoma, gastric cancer, and Kabuki syndrome. Mutations in KMT2D identified thus far point to its loss-of-function in pathogenesis and suggest its role as a tumor suppressor in various tissues. To determine the effect of a KMT2D deficiency on neoplastic cells, we used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic colorectal and medulloblastoma cancer cell lines that differ with respect to their endogenous KMT2D status. We found that a KMT2D deficiency resulted in attenuated cancer cell proliferation and defective cell migration. Analysis of histone H3 modifications revealed that KMT2D was essential for maintaining the level of global H3K4 monomethylation and that its enzymatic SET domain was directly responsible for this function. Furthermore, we found that a majority of KMT2D binding sites are located in regions of potential enhancer elements. Together, these findings revealed the role of KMT2D in regulating enhancer elements in human cells and shed light on the tumorigenic role of its deficiency. Our study supports that KMT2D has distinct roles in neoplastic cells, as opposed to normal cells, and that inhibiting KMT2D may be a viable strategy for cancer therapeutics.


Subject(s)
DNA-Binding Proteins/metabolism , Histone Acetyltransferases/metabolism , Histone Demethylases/metabolism , Histones/metabolism , Neoplasm Proteins/metabolism , Cell Growth Processes/physiology , Cell Movement/physiology , DNA-Binding Proteins/genetics , Histone Acetyltransferases/genetics , Histone Demethylases/genetics , Histones/genetics , Humans , Methylation , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/metabolism , Transfection
16.
PLoS One ; 7(9): e45667, 2012.
Article in English | MEDLINE | ID: mdl-23029172

ABSTRACT

The incidence and severity of allergic asthma have increased over the last century, particularly in the United States and other developed countries. This time frame was characterized by marked environmental changes, including enhanced hygiene, decreased pathogen exposure, increased exposure to inhaled pollutants, and changes in diet. Although iron is well-known to participate in critical biologic processes such as oxygen transport, energy generation, and host defense, iron deficiency remains common in the United States and world-wide. The purpose of these studies was to determine how dietary iron supplementation affected the severity of allergic inflammation in the lungs, using a classic model of IgE-mediated allergy in mice. Results showed that mice fed an iron-supplemented diet had markedly decreased allergen-induced airway hyperreactivity, eosinophil infiltration, and production of pro-inflammatory cytokines, compared with control mice on an unsupplemented diet that generated mild iron deficiency but not anemia. In vitro, iron supplementation decreased mast cell granule content, IgE-triggered degranulation, and production of pro-inflammatory cytokines post-degranulation. Taken together, these studies show that iron supplementation can decrease the severity of allergic inflammation in the lung, potentially via multiple mechanisms that affect mast cell activity. Further studies are indicated to determine the potential of iron supplementation to modulate the clinical severity of allergic diseases in humans.


Subject(s)
Hypersensitivity/prevention & control , Iron/administration & dosage , Pneumonia/prevention & control , Animals , Female , Male , Mast Cells/immunology , Mice , Mice, Inbred C57BL
17.
PLoS One ; 7(7): e41797, 2012.
Article in English | MEDLINE | ID: mdl-22848611

ABSTRACT

Mutations that increase susceptibility to inflammatory bowel disease (IBD) have been identified in a number of genes in both humans and mice, but the factors that govern how these mutations contribute to IBD pathogenesis and result in phenotypic presentation as ulcerative colitis (UC) or Crohn disease (CD) are not well understood. In this study, mice deficient in both TNF and IL-10 (T/I mice) were found to spontaneously develop severe colitis soon after weaning, without the need for exogenous triggers. Colitis in T/I mice had clinical and histologic features similar to human UC, including a markedly increased risk of developing inflammation-associated colon cancer. Importantly, development of spontaneous colitis in these mice was prevented by antibiotic treatment. Consistent with the known role of Th17-driven inflammation in response to bacteria, T/I mice had elevated serumTh17-type cytokines when they developed spontaneous colitis and after systemic bacterial challenge via NSAID-induced degradation of the mucosal barrier. Although TNF production has been widely considered to be be pathogenic in IBD, these data indicate that the ability to produce normal levels of TNF actually protects against the spontaneous development of colitis in response to intestinal colonization by bacteria. The T/I mouse model will be useful for developing new rationally-based therapies to prevent and/or treat IBD and inflammation-associated colon cancer and may further provide important insights into the pathogenesis of UC in humans.


Subject(s)
Colitis, Ulcerative/metabolism , Colonic Neoplasms/metabolism , Disease Models, Animal , Animals , Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Breeding , Colitis, Ulcerative/microbiology , Colitis, Ulcerative/pathology , Colitis, Ulcerative/prevention & control , Colonic Neoplasms/complications , Colonic Neoplasms/pathology , Colonic Neoplasms/prevention & control , Disease Susceptibility , Enterobacteriaceae/drug effects , Enterobacteriaceae/physiology , Female , Humans , Inflammation/complications , Interleukin-10/deficiency , Male , Mice , Mice, Inbred C57BL , Tumor Necrosis Factor-alpha/deficiency
18.
Inflamm Bowel Dis ; 16(12): 2012-21, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20848493

ABSTRACT

BACKGROUND: Bromelain, a mixture of proteolytic enzymes typically derived from pineapple stem, decreases production of proinflammatory cytokines and leukocyte homing to sites of inflammation. We previously showed that short-term oral treatment with bromelain purified from pineapple stem decreased the severity of colonic inflammation in C57BL/6 Il10(-/-) mice with chronic colitis. Since fresh pineapple fruit contains similar bromelain enzymes but at different proportions, this study aimed to determine whether long-term dietary supplementation with pineapple (supplied as juice) could decrease colon inflammation and neoplasia in Il10(-/-) mice with chronic colitis as compared with bromelain derived from stem. METHODS: Colitis was triggered in Il10(-/-) mice by exposure to the non-steroidal anti-inflammatory drug piroxicam. Mice with colitis were supplemented with fresh vs. boiled pineapple juice or bromelain purified from stem for up to 6 months. RESULTS: Experimental mice readily consumed fresh pineapple juice at a level that generated mean stool proteolytic activities equivalent to 14 mg bromelain purified from stem, while control mice received boiled juice with inactive enzymes. Survival was increased in the group supplemented with fresh rather than boiled juice (P = 0.01). Mice that received fresh juice also had decreased histologic colon inflammation scores and a lower incidence of inflammation-associated colonic neoplasia (35% versus 66%; P < 0.02), with fewer neoplastic lesions/colon (P = 0.05). Flow cytometric analysis of murine splenocytes exposed to fresh pineapple juice in vitro demonstrated proteolytic removal of cell surface molecules that can affect leukocyte trafficking and activation. CONCLUSIONS: These results demonstrate that long-term dietary supplementation with fresh or unpasteurized frozen pineapple juice with proteolytically active bromelain enzymes is safe and decreases inflammation severity and the incidence and multiplicity of inflammation-associated colonic neoplasia in this commonly used murine model of inflammatory bowel disease.


Subject(s)
Ananas , Colitis/drug therapy , Colonic Neoplasms/prevention & control , Interleukin-10/deficiency , Phytotherapy , Administration, Oral , Animals , Bromelains/therapeutic use , Colitis/etiology , Colitis/metabolism , Colonic Neoplasms/etiology , Colonic Neoplasms/metabolism , Cytokines/metabolism , Dietary Supplements , Disease Models, Animal , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
19.
Exp Dermatol ; 19(6): 518-26, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20100190

ABSTRACT

Hair loss (alopecia) can result from a variety of metabolic, endocrine, immunologic, and environmental causes. This investigation was undertaken to determine the mechanisms underlying the sporadic development of alopecia in litters from C57BL/6 interleukin-10-deficient (Il10(-/-)) mice. All pups in affected litters demonstrated alopecia by postnatal days 17-19, with hair loss from their trunks but not from their head, base of tail, or feet. Histopathology revealed distorted hair follicles containing broken hair shafts and prominent dermal infiltrates containing increased numbers of activated mast cells. Hair re-growth began soon after weaning, suggesting that the alopecia was triggered by factors transmitted during lactation. Milk from Il10(-/-) dams induced macrophage secretion of pro-inflammatory cytokines in vitro regardless of whether or not their pups developed alopecia. Feeding dams a diet containing 3-6 ppm iron increased the percentage of litters with alopecia to 100% for pups with mast cells, with 0% alopecia in mast cell-deficient pups. When dams were fed a diet containing 131 ppm iron, significantly lower haemoglobin and hematocrit values were observed in pups from litters with alopecia (71%; 5 of 7 litters) compared to litters without alopecia. Genetic or pharmacologic inhibition of c-kit that resulted in depletion of mast cells in pups prevented hair loss in at-risk litters. These studies demonstrate that maternal iron-restricted diets enhance the incidence of alopecia in IL-10-deficient mouse pups and suggest mast cells as potential effector cells. Further studies are indicated to further explore the mechanisms involved and to determine how mast cells may contribute to alopecia in humans.


Subject(s)
Alopecia/etiology , Interleukin-10/deficiency , Iron Deficiencies , Proto-Oncogene Proteins c-kit/genetics , Alopecia/genetics , Alopecia/pathology , Anemia/complications , Anemia/pathology , Animals , Animals, Suckling , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cell Degranulation , Cell Line , Chemokines/metabolism , Cytokines/metabolism , Diet , Female , Hair Follicle/pathology , Interleukin-10/genetics , Macrophage Activation/immunology , Macrophages/immunology , Macrophages/metabolism , Male , Mast Cells/drug effects , Mast Cells/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Mutant Strains , Milk/immunology , Pregnancy , Proto-Oncogene Proteins c-kit/immunology , Skin/pathology
20.
Clin Immunol ; 126(3): 345-52, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18160345

ABSTRACT

Oral bromelain has been anecdotally reported to decrease inflammation in ulcerative colitis (UC). Proteolytically active bromelain is known to decrease expression of mRNAs encoding pro-inflammatory cytokines by human leukocytes in vitro. To assess the effect of bromelain on mucosal secretion of cytokines in inflammatory bowel disease (IBD), endoscopic colon biopsies from patients with UC, Crohn's disease (CD), and non-IBD controls were treated in vitro with bromelain or media, then cultured. Secretion of pro-inflammatory cytokines and chemokines was measured. Significant increases in granulocyte colony-stimulating factor (G-CSF), interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF) were detected in the media from actively inflamed areas in UC and CD as compared with non-inflamed IBD tissue and non-IBD controls. In vitro bromelain treatment decreased secretion of G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-gamma, CCL4/macrophage inhibitory protein (MIP)-1beta, and TNF by inflamed tissue in IBD. Bromelain may be a novel therapy for IBD.


Subject(s)
Biopsy , Bromelains/pharmacology , Colon/drug effects , Colon/metabolism , Cytokines/metabolism , Adolescent , Adult , Colon/pathology , Female , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Male , Tissue Culture Techniques
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