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Lipids in Health and Disease Nov 2019A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols...
BACKGROUND
A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear.
METHODS
A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system.
RESULTS
The gastric tumor incidence was higher in the SULT2B1 mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction.
CONCLUSIONS
The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.
Topics: Animals; Base Sequence; CRISPR-Cas Systems; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cholesterol; Gastric Mucosa; Gene Editing; Gene Expression Regulation, Neoplastic; Humans; Hydroxycholesterols; Methylcholanthrene; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Stomach Neoplasms; Sulfotransferases; Survival Analysis
PubMed: 31757214
DOI: 10.1186/s12944-019-1149-6 -
Molecules (Basel, Switzerland) Oct 2019The prevalence of upper tract urothelial carcinoma (UTUC) in Taiwan is relatively higher than thatin Western countries. Aristolochic acid (AA), which is widely used in...
The prevalence of upper tract urothelial carcinoma (UTUC) in Taiwan is relatively higher than thatin Western countries. Aristolochic acid (AA), which is widely used in traditional Chinese herbology, is now recognized to be one of the carcinogens for UTUC. Numerous UTUC patients have chronic kidney diseases or end-stage renal diseases; however, little literature hasreported on theoncogenic pathway of AA-related UTUC. The aim of our study was to identify the potential target treatment for AA-related UTUC. Here, we established an AA pre-exposure followed bya 3-methylcholanthrene (MCA) stimulus tumorigenic cell model. We not only demonstrated that AA pre-exposure MCA stimulus tumorigenic cells have more behaviors of cell migration and invasion by enhancing the metalloproteinases (MMP) activity, which is compatible with clinical findings of AA-related UTUC, but we also validated that AA pre-exposure MCA stimulus tumorigeniccells could be activated through the mitogen-activated protein kinases (MAPK) pathway. We further dissected the route of the MAPK pathway and found that the p38 and extracellular signal regulated kinases (ERK) sub-pathways might play essential roles in AA pre-exposure urothelial cancer cell lines. This consequence was also corroborated with a tissue study in AA-exposed patients.
Topics: Aristolochic Acids; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Humans; MAP Kinase Signaling System; Matrix Metalloproteinases; Protein Kinase Inhibitors; Urologic Neoplasms; Urothelium
PubMed: 31619002
DOI: 10.3390/molecules24203707 -
Biology Open Sep 2019Recently, several promising treatments for high-grade gliomas (HGGs) failed to provide significant benefit when translated from the preclinical setting to patients....
Recently, several promising treatments for high-grade gliomas (HGGs) failed to provide significant benefit when translated from the preclinical setting to patients. Improving the animal models is fundamental to overcoming this translational gap. To address this need, we developed and comprehensively characterized a new model based on the orthotopic implantation of CT-2A cells cultured in neurospheres (NS/CT-2A). Murine CT-2A methylcholanthrene-induced HGG cells (C57BL/6 background) were cultured in monolayers (ML) or NS and orthotopically inoculated in syngeneic animals. ML/CT-2A and NS/CT-2A tumors' characterization included the analysis of tumor growth, immune microenvironment, glioma stem cells (GSCs), vascularization and metabolites. The immuno-modulating properties of NS/CT-2A and ML/CT-2A cells on splenocytes were tested Mice harboring NS/CT-2A tumors had a shorter survival than those harboring ML/CT-2A tumors (=0.0033). Compared to standard ML/CT-2A tumors, NS/CT-2A tumors showed more abundant GSCs (=0.0002 and 0.0770 for Nestin and CD133, respectively) and regulatory T cells (Tregs, =0.0074), and a strong tendency towards an increased vascularization (=0.0503). There were no significant differences in metabolites' composition between NS/ and ML/CT-2A tumors. , NS were able to drive splenocytes towards a more immunosuppressive status by reducing CD8 T cells (=0.0354) and by promoting Tregs (=0.0082), macrophages (MF, =0.0019) and their M2 subset (=0.0536). Compared to standard ML/CT-2A tumors, NS/CT-2A tumors show a more aggressive phenotype with increased immunosuppression and GSCs proliferation. Because of these specific features, the NS/CT-2A model represents a clinically relevant platform in the search for new HGG treatments aimed at reducing immunosuppression and eliminating GSCs.
PubMed: 31511246
DOI: 10.1242/bio.044552 -
Autophagy Jun 2020Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator...
UNLABELLED
Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of mice. VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of siRNA in , but not in VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, gene deficiency mimicked the phenotype of depletion by presenting reduced autophagy and increased senescence and . Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence.
ABBREVIATIONS
3-MA: 3 methyladenine; ACTA2/SM-actin: actin, alpha 2, smooth muscle, aorta; ACTB/β-actin: actin beta; AGT II: angiotensin II; ATG5: autophagy related 5; BECN1: beclin 1; CAT: catalase; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); Chl: chloroquine; CTSD: cathepsin D; CYCS: cytochrome C, somatic; DHE: dihydroethidium; DPBS: Dulbecco's phosphate-buffered saline; EL: elastic lamina; EM: extracellular matrix; FDG: fluorescein-di-β-D-galactopyranoside; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; γH2AFX: phosphorylated H2A histone family, member X, HDCFDA: 2',7'-dichlorodihydrofluorescein diacetate; LAMP2: lysosomal-associated membrane protein 2; MASMs: mouse vascular smooth muscle cells; MEF: mouse embryonic fibroblast; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; MTOR: mechanistic target of rapamycin kinase; NFE2L2: nuclear factor, erythroid derived 2, like 2; NOX1: NADPH oxidase 1; OPTN: optineurin; PFA: paraformaldehyde; PFU: plaque-forming units; PPARGC1A/PGC-1α: peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; Ptdln3K: phosphatidylinositol 3-kinase; RASMs: rat vascular smooth muscle cells; ROS: reactive oxygen species; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SIRT1: sirtuin 1; Spautin 1: specific and potent autophagy inhibitor 1; SQSTM1/p62: sequestosome 1; SOD: superoxide dismutase; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFRC: transferrin receptor; TRP53/p53: transformation related protein 53; TUBG1: tubulin gamma 1; VSMCs: vascular smooth muscle cells; WT: wild type.
Topics: Animals; Aorta; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Benzylamines; Brain; Cathepsin D; Cellular Senescence; Lysosomal-Associated Membrane Protein 2; Lysosomes; Male; Methylcholanthrene; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Myocytes, Smooth Muscle; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quinazolines; RNA, Small Interfering; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Transferrin; Sequestosome-1 Protein; Sirolimus; Up-Regulation
PubMed: 31441382
DOI: 10.1080/15548627.2019.1659612 -
Journal of Experimental & Clinical... Aug 2019The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1,...
BACKGROUND
The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.
METHODS
Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.
RESULTS
We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.
CONCLUSIONS
In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.
Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1B1; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Methylcholanthrene; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Protein Transport; Receptors, Aryl Hydrocarbon; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Structure-Activity Relationship
PubMed: 31370872
DOI: 10.1186/s13046-019-1337-2 -
Molecular and Cellular Biology Oct 2019Transgenic mice expressing a constitutively active form of the aryl hydrocarbon receptor in keratinocytes (AhR-CA mice) develop severe dermatitis that substantially...
Transgenic mice expressing a constitutively active form of the aryl hydrocarbon receptor in keratinocytes (AhR-CA mice) develop severe dermatitis that substantially recapitulates the pathology of human atopic dermatitis. The neurotrophic factor artemin (Artn) is highly expressed in the epidermis of AhR-CA mice and causes hypersensitivity to itch (alloknesis) by elongating nerves into the epidermis. However, whether the gene is regulated directly by AhR or indirectly through complex regulation associated with AhR remains unclear. To this end, we previously conducted chromatin immunoprecipitation-sequencing analyses of the locus and found a xenobiotic response element (XRE) motif located far upstream (52 kb) of the gene. Therefore, in this study, we addressed whether the XRE actually regulates the gene expression by deleting the region containing the motif. We generated two lines of Artn mice. In the mouse epidermis, inducible expression of the gene by the AhR agonist 3-methylcholanthrene was substantially suppressed compared to that in wild-type mice. Importantly, in AhR-CA::Artn mice, expression was significantly suppressed, and alloknesis was improved. These results demonstrate that the gene is indeed regulated by the distal XRE-containing enhancer, and alloknesis in AhR-CA mice is provoked by the AhR-mediated direct induction of the gene.
Topics: Air Pollutants; Animals; Antibodies, Neutralizing; Basic Helix-Loop-Helix Transcription Factors; Dermatitis, Atopic; Enhancer Elements, Genetic; Gene Expression Regulation; Humans; Methylcholanthrene; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Receptors, Aryl Hydrocarbon; Response Elements; Skin; Xenobiotics
PubMed: 31358547
DOI: 10.1128/MCB.00190-19 -
Journal For Immunotherapy of Cancer Jun 2019Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T...
BACKGROUND
Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown.
METHODS
Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed.
RESULTS
IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs.
CONCLUSION
IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance.
Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Female; Granzymes; Humans; Interferon Type I; Mice, Inbred C57BL; Mice, Knockout; Receptor, Interferon alpha-beta; STAT3 Transcription Factor; Signal Transduction; T-Lymphocytes, Cytotoxic
PubMed: 31228946
DOI: 10.1186/s40425-019-0635-8 -
JCI Insight Jul 2019Cancer development is influenced by hereditary mutations, somatic mutations due to random errors in DNA replication, or external factors. It remains unclear how distinct...
Cancer development is influenced by hereditary mutations, somatic mutations due to random errors in DNA replication, or external factors. It remains unclear how distinct cell-intrinsic and -extrinsic factors impact oncogenesis within the same tissue type. We investigated murine soft tissue sarcomas generated by oncogenic alterations (KrasG12D activation and p53 deletion), carcinogens (3-methylcholanthrene [MCA] or ionizing radiation), and in a novel model combining both factors (MCA plus p53 deletion). Whole-exome sequencing demonstrated distinct mutational signatures in individual sarcoma cohorts. MCA-induced sarcomas exhibited high mutational burden and predominantly G-to-T transversions, while radiation-induced sarcomas exhibited low mutational burden and a distinct genetic signature characterized by C-to-T transitions. The indel to substitution ratio and amount of gene copy number variations were high for radiation-induced sarcomas. MCA-induced tumors generated on a p53-deficient background showed the highest genomic instability. MCA-induced sarcomas harbored mutations in putative cancer-driver genes that regulate MAPK signaling (Kras and Nf1) and the Hippo pathway (Fat1 and Fat4). In contrast, radiation-induced sarcomas and KrasG12Dp53-/- sarcomas did not harbor recurrent oncogenic mutations, rather they exhibited amplifications of specific oncogenes: Kras and Myc in KrasG12Dp53-/- sarcomas, and Met and Yap1 for radiation-induced sarcomas. These results reveal that different initiating events drive oncogenesis through distinct mechanisms.
Topics: Animals; Carcinogenesis; Carcinogens; DNA Mutational Analysis; Genomic Instability; Humans; Methylcholanthrene; Mice; Neoplasms, Experimental; Neoplasms, Radiation-Induced; Oncogenes; Proto-Oncogene Proteins p21(ras); Sarcoma; Tumor Suppressor Protein p53; Exome Sequencing
PubMed: 31112524
DOI: 10.1172/jci.insight.128698 -
International Journal of Molecular... May 2019TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR...
TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC- mice. Our study reveals that mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.
Topics: Adipose Tissue; Animals; Azo Compounds; Chylous Ascites; Cytokines; Fatty Liver; Gene Expression Regulation, Enzymologic; Inflammation; Inflammation Mediators; Liver; Male; Methylcholanthrene; Mice, Knockout; Poly(ADP-ribose) Polymerases; Polychlorinated Dibenzodioxins; Pyrazoles; Receptors, Aryl Hydrocarbon; Signal Transduction; Survival Analysis
PubMed: 31083300
DOI: 10.3390/ijms20092312 -
Molecular Biology of the Cell Jun 2019Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored....
Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.
Topics: Actins; Animals; Carcinogenesis; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Methylcholanthrene; Mice; Myosin-Light-Chain Kinase; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB
PubMed: 30995168
DOI: 10.1091/mbc.E18-12-0790