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1.
J Lipid Res ; : 100609, 2024 Jul 29.
Article in English | MEDLINE | ID: mdl-39084491

ABSTRACT

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids, typically reported as rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate whether GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, GD2), epithelial-mesenchymal transition (EMT) markers (EpCAM, TROP2, CD9), and lineage markers (CD45, CD31, CD90) at the single-cell level. As a next step, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

2.
Cancer Immunol Immunother ; 72(5): 1209-1224, 2023 May.
Article in English | MEDLINE | ID: mdl-36376516

ABSTRACT

Recent studies have underscored the importance of gamma-delta (γδ) T cells in mediating potent MHC-unrestricted cytotoxicity in numerous malignancies. Here, we analyzed Vδ1 and Vδ2 γδ T cell subsets in newly diagnosed chronic myeloid leukemia (CML) patients (n = 40) who had initiated tyrosine kinase inhibitor (TKI) therapy including imatinib (n = 22), nilotinib (n = 14) and dasatinib (n = 4). Patient peripheral blood samples were analyzed at diagnosis and monitored prospectively at 3, 6, 12 and 18 months post-TKI. γδ T cells isolated from healthy donors and CML patients were used against K562, LAMA-84 and KYO-1 cell lines and against primary CML cells in cytotoxicity assays. We found large expansions of Vδ1 and Vδ2 T cells in patients at diagnosis compared to age-matched healthy donors (n = 40) (p < 0.0001). The γδ T cell reconstitution in patients on imatinib and also on nilotinib showed significant reductions of Vδ1 T cell and Vδ2 T cell absolute counts at 3 months compared to diagnosis. Importantly, Vδ1 and Vδ2 T absolute cell counts remained at normal levels from 3 months throughout the follow-up. Next, we observed susceptibility to specific lysis of primary CML tumor cells by Vδ1 T cells from healthy donors. Furthermore, we determined inherent cytotoxic reactivity by autologous patients' Vδ1 T lymphocytes against primary CML tumor cells. Finally, the TCR clonality profiles showed in CML patients mostly polyclonal repertoires regardless of the TKI. Our results provide further evidence into γδ T cell antileukemia immunity in CML that might be beneficial for long-term disease control and treatment outcome.


Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Leukemia, Myeloid , Humans , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets , Cell Line , Leukemia, Myeloid/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
3.
Am J Pathol ; 192(9): 1321-1335, 2022 09.
Article in English | MEDLINE | ID: mdl-35750257

ABSTRACT

Toll-like receptor 3 (TLR3) is an endosomal receptor expressed in several immune and epithelial cells. Recent studies have highlighted its expression also in solid tumors, including prostate cancer (PCa), and have described its role primarily in the proinflammatory response and induction of apoptosis. It is up-regulated in some castration-resistant prostate cancers. However, the role of TLR3 in prostate cancer progression remains largely unknown. The current study experimentally demonstrated that exogenous TLR3 activation in PCa cell lines leads to a significant induction of secretion of the cytokines IL-6, IL-8, and interferon-ß, depending on the model and chemoresistance status. Transcriptomic analysis of TLR3-overexpressing cells revealed a functional program that is enriched for genes involved in the regulation of cell motility, migration, and tumor invasiveness. Increased motility, migration, and invasion in TLR3-overexpressing cell line were confirmed by several in vitro assays and using an orthotopic prostate xenograft model in vivo. Furthermore, TLR3-ligand induced apoptosis via cleavage of caspase-3/7 and poly (ADP-ribose) polymerase, predominantly in TLR3-overexpressing cells. These results indicate that TLR3 may be involved in prostate cancer progression and metastasis; however, it might also represent an Achilles heel of PCa, which can be exploited for targeted therapy.


Subject(s)
Prostatic Neoplasms , Toll-Like Receptor 3 , Animals , Apoptosis , Cell Line, Tumor , Humans , Male , Poly I-C/pharmacology , Prostate/pathology , Prostatic Neoplasms/pathology , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism
4.
Int J Mol Sci ; 22(17)2021 Aug 25.
Article in English | MEDLINE | ID: mdl-34502101

ABSTRACT

Sphingolipids (SLs), glycosphingolipids (GSLs), and eicosanoids are bioactive lipids, which play important roles in the etiology of various diseases, including cancer. However, their content and roles in cancer cells, and in particular in the exosomes derived from tumor cells, remain insufficiently characterized. In this study, we evaluated alterations of SL and GSL levels in transformed cells and their exosomes, using comparative HPLC-MS/MS analysis of parental human bronchial epithelial cells HBEC-12KT and their derivative, benzo[a]pyrene-transformed HBEC-12KT-B1 cells with the acquired mesenchymal phenotype. We examined in parallel SL/GSL contents in the exosomes released from both cell lines. We found significant alterations of the SL/GSL profile in the transformed cell line, which corresponded well with alterations of the SL/GSL profile in exosomes derived from these cells. This suggested that a majority of SLs and GSLs were transported by exosomes in the same relative pattern as in the cells of origin. The only exceptions included decreased contents of sphingosin, sphingosin-1-phosphate, and lactosylceramide in exosomes derived from the transformed cells, as compared with the exosomes derived from the parental cell line. Importantly, we found increased levels of ceramide phosphate, globoside Gb3, and ganglioside GD3 in the exosomes derived from the transformed cells. These positive modulators of epithelial-mesenchymal transition and other pro-carcinogenic processes might thus also contribute to cancer progression in recipient cells. In addition, the transformed HBEC-12KT-B1 cells also produced increased amounts of eicosanoids, in particular prostaglandin E2. Taken together, the exosomes derived from the transformed cells with specifically upregulated SL and GSL species, and increased levels of eicosanoids, might contribute to changes within the cancer microenvironment and in recipient cells, which could in turn participate in cancer development. Future studies should address specific roles of individual SL and GSL species identified in the present study.


Subject(s)
Cell Transformation, Neoplastic , Exosomes/metabolism , Respiratory Mucosa/metabolism , Sphingolipids/metabolism , Benzo(a)pyrene/toxicity , Bronchi/cytology , Carcinogens/toxicity , Cell Line , Humans , Respiratory Mucosa/drug effects
5.
Int J Mol Sci ; 22(13)2021 Jun 22.
Article in English | MEDLINE | ID: mdl-34206240

ABSTRACT

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.


Subject(s)
Adenocarcinoma/metabolism , Colonic Neoplasms/metabolism , Fatty Acids/metabolism , Gene Expression Regulation, Neoplastic , Lipid Metabolism , Phospholipids/metabolism , Adenocarcinoma/enzymology , Adenocarcinoma/genetics , Aged , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Fatty Acid Desaturases/genetics , Fatty Acid Desaturases/metabolism , Fatty Acid Elongases/genetics , Fatty Acid Elongases/metabolism , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Female , Humans , Lipidomics , Lipogenesis , Male , Stearoyl-CoA Desaturase/genetics , Stearoyl-CoA Desaturase/metabolism
6.
Arch Biochem Biophys ; 671: 18-26, 2019 08 15.
Article in English | MEDLINE | ID: mdl-31176685

ABSTRACT

P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.


Subject(s)
Mitogen-Activated Protein Kinase 14/metabolism , Mouse Embryonic Stem Cells/metabolism , NADPH Oxidase 2/metabolism , Superoxides/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Gene Knockdown Techniques , Gene Knockout Techniques , Membrane Potential, Mitochondrial/physiology , Mice , Mitochondria/metabolism , Mitogen-Activated Protein Kinase 14/genetics , NADPH Oxidase 2/genetics
7.
Int J Mol Sci ; 20(24)2019 Dec 13.
Article in English | MEDLINE | ID: mdl-31847237

ABSTRACT

Effects of airborne particles on the expression status of markers of cellular toxic stress and on the release of eicosanoids, linked with inflammation and oxidative damage, remain poorly characterized. Therefore, we proposed a set of various methodological approaches in order to address complexity of PM0.5-induced toxicity. For this purpose, we used a well-characterized model of A549 pulmonary epithelial cells exposed to a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Electron microscopy confirmed accumulation of PM0.5 within A549 cells, yet, autophagy was not induced. Expression profiles of various cellular stress response genes that have been previously shown to be involved in early stress responses, namely unfolded protein response, DNA damage response, and in aryl hydrocarbon receptor (AhR) and p53 signaling, were analyzed. This analysis revealed induction of GREM1, EGR1, CYP1A1, CDK1A, PUMA, NOXA and GDF15 and suppression of SOX9 in response to PM0.5 exposure. Analysis of eicosanoids showed no oxidative damage and only a weak anti-inflammatory response. In conclusion, this study helps to identify novel gene markers, GREM1, EGR1, GDF15 and SOX9, that may represent a valuable tool for routine testing of PM0.5-induced in vitro toxicity in lung epithelial cells.


Subject(s)
Air Pollutants/toxicity , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Lung/metabolism , Particulate Matter/toxicity , Signal Transduction/drug effects , A549 Cells , Aerosols , Epithelial Cells/pathology , Humans , Lung/pathology
8.
Int J Mol Sci ; 20(23)2019 Nov 30.
Article in English | MEDLINE | ID: mdl-31801289

ABSTRACT

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.


Subject(s)
Colonic Neoplasms/enzymology , Gene Expression Regulation, Neoplastic , Lactosylceramides/metabolism , Lipid Metabolism/genetics , Sphingolipids/metabolism , Acid Ceramidase/genetics , Acid Ceramidase/metabolism , Alkaline Ceramidase/genetics , Alkaline Ceramidase/metabolism , Animals , Ceramides/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Disease Models, Animal , Humans , Lysophospholipids/metabolism , Neutral Ceramidase/genetics , Neutral Ceramidase/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine N-Acyltransferase/genetics , Sphingosine N-Acyltransferase/metabolism , Tumor Cells, Cultured
10.
Stem Cells ; 31(4): 641-51, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23355370

ABSTRACT

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.


Subject(s)
Melanoma/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rhodamine 123/pharmacology , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Chromones/pharmacology , Cyclin D1/genetics , Cyclin D1/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunohistochemistry , Morpholines/pharmacology , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Tumor Cells, Cultured
11.
Cell Mol Neurobiol ; 34(1): 1-15, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24132391

ABSTRACT

Notch and gp130 signaling are involved in the regulation of multiple cellular processes across various tissues during animal ontogenesis. In the developing nervous system, both signaling pathways intervene at many stages to determine cell fate-from the first neural lineage commitment and generation of neuronal precursors, to the terminal specification of cells as neurons and glia. In most cases, the effects of Notch and gp130 signaling in these processes are similar. The aim of the current review was to summarize the knowledge regarding the roles of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells during animal ontogenesis, from early embryo to adult. Recent data show a direct crosstalk between these signaling pathways that seems to be specific for a particular type of neural progenitors.


Subject(s)
Cytokine Receptor gp130/metabolism , Neural Stem Cells/metabolism , Receptors, Notch/metabolism , Signal Transduction , Animals , Humans , Neurogenesis , Receptor Cross-Talk
12.
Transcription ; : 1-20, 2024 Mar 28.
Article in English | MEDLINE | ID: mdl-38547312

ABSTRACT

Aryl hydrocarbon receptor (AhR) is a transcription factor that is primarily known as an intracellular sensor of environmental pollution. After five decades, the list of synthetic and toxic chemicals that activate AhR signaling has been extended to include a number of endogenous compounds produced by various types of cells via their metabolic activity. AhR signaling is active from the very beginning of embryonal development throughout the life cycle and participates in numerous biological processes such as control of cell proliferation and differentiation, metabolism of aromatic compounds of endogenous and exogenous origin, tissue regeneration and stratification, immune system development and polarization, control of stemness potential, and homeostasis maintenance. AhR signaling can be affected by various pharmaceuticals that may help modulate abnormal AhR signaling and drive pathological states. Given their role in immune system development and regulation, AhR antagonistic ligands are attractive candidates for immunotherapy of disease states such as advanced prostate cancer, where an aberrant immune microenvironment contributes to cancer progression and needs to be reeducated. Advanced stages of prostate cancer are therapeutically challenging and characterized by decreased overall survival (OS) due to the metastatic burden. Therefore, this review addresses the role of AhR signaling in the development and progression of prostate cancer and discusses the potential of AhR as a drug target for the treatment of advanced prostate cancer upon entering the phase of drug resistance and failure of first-line androgen deprivation therapy.Abbreviation: ADC: antibody-drug conjugate; ADT: androgen deprivation therapy; AhR: aryl hydrocarbon receptor; AR: androgen receptor; ARE: androgen response element; ARPI: androgen receptor pathway inhibitor; mCRPC: metastatic castration-resistant prostate cancer; DHT: 5a-dihydrotestosterone; FICZ: 6-formylindolo[3,2-b]carbazole; 3-MC: 3-methylcholanthrene; 6-MCDF: 6-methyl-1,3,8-trichlorodibenzofuran; MDSCs: myeloid-derived suppressor cells; PAHs: polycyclic aromatic hydrocarbons; PCa: prostate cancer; TAMs: tumor-associated macrophages; TF: transcription factor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TME: tumor microenvironment; TRAMP: transgenic adenocarcinoma of the mouse prostate; TROP2: tumor associated calcium signal transducer 2.

13.
Sci Rep ; 14(1): 7827, 2024 04 03.
Article in English | MEDLINE | ID: mdl-38570556

ABSTRACT

Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.


Subject(s)
Melanoma , Neoplastic Cells, Circulating , Skin Neoplasms , Animals , Humans , Melanoma/pathology , Neoplastic Cells, Circulating/pathology , Skin Neoplasms/pathology , Lymphatic Metastasis , Flow Cytometry
14.
Environ Toxicol Pharmacol ; 107: 104424, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38522766

ABSTRACT

The role of benzo[a]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFß1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression.


Subject(s)
Benzo(a)pyrene , Epithelial Cells , Humans , Benzo(a)pyrene/toxicity , Ligands , Epithelial Cells/metabolism , DNA Damage , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism
16.
Arch Toxicol ; 87(4): 681-98, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23196670

ABSTRACT

Although the tumor-promoting effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar polychlorinated biphenyls (PCBs), and related compounds in liver tissue are primarily attributed to the activation of the aryl hydrocarbon receptor (AhR), the underlying molecular mechanisms are still unclear. Liver progenitor (oval) cells have been suggested to constitute a potential target for hepatocarcinogenic chemicals. To better understand AhR-driven pathways, we analyzed the transcriptional program in response to coplanar PCB 126 in contact-inhibited rat liver progenitor WB-F344 cells using high-density microarrays. After 6-h treatment, we identified 145 significantly deregulated genes considered to be direct AhR-dependent target genes. The number of differentially regulated genes increased to 658 and 968 genes after 24 and 72 h, respectively. Gene ontology analysis revealed that these genes were primarily involved in drug and lipid metabolism, cell cycle and growth control, cancer developmental processes, cell-cell communication, and adhesion. Interestingly, the Wnt and TGF-ß signaling pathways, both being involved in developmental and tumorigenic processes, belonged to the most affected pathways. AhR- and ARNT-dependent regulation of selected target genes of interest was then confirmed using TCDD as a model AhR agonist, together with pharmacological inhibition of the AhR and by RNA-interference techniques. We demonstrated AhR-dependent regulation of emerging and novel AhR target genes, such as Fst, Areg, Hbegf, Ctgf, Btg2, and Foxq1. Among them, the transcription factor Foxq1, recently suggested to contribute to tumor promotion and/or progression, was found to be regulated at both mRNA and protein levels by AhR/ARNT activation.


Subject(s)
Epithelial Cells/metabolism , Gene Expression Regulation/genetics , Liver/metabolism , Receptors, Aryl Hydrocarbon/genetics , Stem Cells/metabolism , Animals , Cell Line , Epithelial Cells/drug effects , Epithelial Cells/pathology , Estrogen Antagonists/toxicity , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Liver/drug effects , Liver/pathology , Oligonucleotide Array Sequence Analysis , Polychlorinated Biphenyls/toxicity , Rats , Receptors, Aryl Hydrocarbon/metabolism , Stem Cells/drug effects , Stem Cells/pathology
17.
Arch Toxicol ; 87(3): 491-503, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23085979

ABSTRACT

The aryl hydrocarbon receptor (AhR) contributes to the control of cell-to-cell communication, cell adhesion, migration or proliferation. In the present study, we investigated the regulation of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) during the AhR-dependent disruption of contact inhibition in non-tumorigenic liver epithelial cells. The contact inhibition of cell proliferation is a process restricting the cell division of confluent non-transformed cells, which is frequently abolished in cancer cells; however, the mechanisms contributing to its disruption are still only partially understood. Disruption of contact inhibition, which was induced by toxic AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or polycyclic aromatic hydrocarbons in epithelial WB-F344 cells, reduced Cx43 protein levels, possibly via enhanced proteasomal degradation, significantly decreased the amount of gap junction plaques and downregulated GJIC, in an AhR-dependent manner. Although both intracellular and membrane Cx43 pools were markedly reduced in cells released from contact inhibition by TCDD, siRNA-mediated Cx43 knock-down was not sufficient to stimulate proliferation in contact-inhibited cells. Our data suggest that downregulation of Cx43/GJIC in non-transformed epithelial cells is an inherent part of disruption of contact inhibition, which occurs at the post-transcriptional level. This process runs in parallel with alterations of other forms of cell-to-cell communication, thus suggesting that toxic AhR agonists may simultaneously abrogate contact inhibition and reduce GJIC, two essential mechanisms linked to deregulation of cell-to-cell communication during tumor promotion and progression.


Subject(s)
Carcinogens/toxicity , Cell Communication/drug effects , Connexin 43/metabolism , Contact Inhibition/drug effects , Epithelial Cells/drug effects , Gap Junctions/drug effects , Liver/drug effects , Receptors, Aryl Hydrocarbon/agonists , Signal Transduction/drug effects , Animals , Benz(a)Anthracenes/toxicity , Cell Line , Cell Proliferation , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Connexin 43/genetics , Dose-Response Relationship, Drug , Down-Regulation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fluorenes/toxicity , Gap Junctions/metabolism , Gap Junctions/pathology , Gene Knockdown Techniques , Indoles/pharmacology , Ligands , Liver/metabolism , Liver/pathology , Liver Neoplasms/chemically induced , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Phloroglucinol/analogs & derivatives , Phloroglucinol/pharmacology , Phosphorylation , Polychlorinated Dibenzodioxins/toxicity , Proteasome Endopeptidase Complex/metabolism , RNA Interference , Rats , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Time Factors , Transfection
18.
J Cell Biochem ; 113(2): 563-70, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21948563

ABSTRACT

Retinoic acid (RA) is able to induce the differentiation of embryonic stem cells into neuronal lineages. The mechanism of this effect is unknown but it has been evidenced to be dependent on the formation of floating spheroids called embryoid bodies. Results presented here show that the inhibition of phosphoinositide 3-kinase signaling pre-determines mouse embryonic stem cells to RA induced neurogenesis in monolayer culture with no need of embryoid bodies formation.


Subject(s)
Chromones/pharmacology , Embryonic Stem Cells/physiology , Morpholines/pharmacology , Neurogenesis/drug effects , Phosphoinositide-3 Kinase Inhibitors , Tretinoin/pharmacology , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Culture Techniques , Cell Shape/drug effects , Cells, Cultured , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental/drug effects , Genes, Reporter , Keratin-8/genetics , Keratin-8/metabolism , Luciferases/biosynthesis , Luciferases/genetics , Mice , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Signal Transduction/drug effects , Transcription, Genetic , Tubulin/genetics , Tubulin/metabolism
19.
Biocell ; 36(3): 121-6, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23682427

ABSTRACT

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.


Subject(s)
Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/physiology , Electron Transport Complex I/metabolism , Cell Line , Cell Line, Tumor , Fluorescent Dyes/pharmacology , Gene Silencing , HeLa Cells , Humans , Image Processing, Computer-Assisted , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Phenanthridines/pharmacology , Superoxides/metabolism , Telomerase/metabolism , Telomere/ultrastructure
20.
Cancers (Basel) ; 14(17)2022 Aug 31.
Article in English | MEDLINE | ID: mdl-36077780

ABSTRACT

The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

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