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ELife Mar 2024Host-directed therapy (HDT) is an emerging approach to overcome antimicrobial resistance in pathogenic microorganisms. Specifically, HDT targets host-encoded factors...
Host-directed therapy (HDT) is an emerging approach to overcome antimicrobial resistance in pathogenic microorganisms. Specifically, HDT targets host-encoded factors required for pathogen replication and survival without interfering with microbial growth or metabolism, thereby eliminating the risk of resistance development. By applying HDT and a drug repurposing approach, we demonstrate that ()-DI-87, a clinical-stage anticancer drug and potent inhibitor of mammalian deoxycytidine kinase (dCK), mitigates abscess formation in organ tissues upon invasive bloodstream infection. Mechanistically, ()-DI-87 shields phagocytes from staphylococcal death-effector deoxyribonucleosides that target dCK and the mammalian purine salvage pathway-apoptosis axis. In this manner, ()-DI-87-mediated protection of immune cells amplifies macrophage infiltration into deep-seated abscesses, a phenomenon coupled with enhanced pathogen control, ameliorated immunopathology, and reduced disease severity. Thus, pharmaceutical blockade of dCK represents an advanced anti-infective intervention strategy against which staphylococci cannot develop resistance and may help to fight fatal infectious diseases in hospitalized patients.
Topics: Animals; Humans; Staphylococcus aureus; Deoxycytidine Kinase; Abscess; Staphylococcal Infections; Anti-Infective Agents; Mammals
PubMed: 38512723
DOI: 10.7554/eLife.91157 -
Asian Pacific Journal of Cancer... Jul 2021Epigenetic alterations play an important role in tumorigenesis. Hypermethylation of CpG islands within the promoter regions of tumor suppressor genes (TSGs) and histone... (Comparative Study)
Comparative Study
Effect of Decitabine (5-aza-2'-deoxycytidine, 5-aza-CdR) in Comparison with Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) on DNMT1, DNMT3a and DNMT3b, HDAC 1-3, SOCS 1, SOCS 3, JAK2, and STAT3 Gene Expression in Hepatocellular Carcinoma HLE and LCL-PI 11 Cell Lines.
BACKGROUND
Epigenetic alterations play an important role in tumorigenesis. Hypermethylation of CpG islands within the promoter regions of tumor suppressor genes (TSGs) and histone deacetylation lead to the silencing of the genes resulting in cancer induction. The suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of this family has been reported in several cancers, the protein of the SOCS family inhibit the cytokine-activated Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway to modulate cellular responses. Previously, we evaluated the effects of DNA demethylating agents and histone deacetylase inhibitors on hepatocellular carcinoma (HCC). The current study aimed to investigate the effect of decitabine (5-aza-2'-deoxycytidine, 5-aza-CdR) in comparison to vorinostat (suberoylanilide hydroxamic acid, SAHA) on DNMT1, DNMT3a and DNMT3b, HDAC 1-3, SOCS 1, SOCS 3, JAK2, and STAT3 gene expression, cell growth inhibition, and apoptosis induction of HCC HLE and LCL-PI 11 cell lines.
MATERIAL AND METHODS
The HLE and LCL-PI 11 cells were treated with 5-aza-CdR and SAHA and then the MTT assay, flow cytometry assay, and quantitative real-time RT-PCR were achieved to determine cell viability, cell apoptosis, and relative gene expression respectively.
RESULTS
The result indicated that both compounds inhibited cell growth, induced apoptosis, and down-regulated DNMT1, DNMT3a DNMT3b, HDAC 1-3, JAK2, and STAT3 and up-regulated HDAC 1-3, SOCS 1, and SOCS 3 genes expression significantly. The apoptotic effect of SAHA was stronger than that of 5-Aza-CdR.
CONCLUSION
5-Aza-CdR and SAHA can induce cell growth inhibition and apoptosis induction through the JAK/STAT pathway.
Topics: Antigens, Neoplasm; Apoptosis; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cell Survival; Decitabine; Gene Expression; Histone Deacetylases; Humans; Janus Kinases; Liver Neoplasms; Repressor Proteins; STAT Transcription Factors; Signal Transduction; Vorinostat
PubMed: 34319031
DOI: 10.31557/APJCP.2021.22.7.2089 -
Frontiers in Oncology 2022The use of immune adjuvants such as toll-like receptor (TLR) agonists reflects a novel strategy in prostate cancer (PCa) therapy. However, interleukin-1 receptor...
The use of immune adjuvants such as toll-like receptor (TLR) agonists reflects a novel strategy in prostate cancer (PCa) therapy. However, interleukin-1 receptor associated kinase 1 (IRAK1), a central effector of TLR signaling, has been shown to be responsible for resistance to radiation-induced tumor cell death. In order to better understand the function and epigenetic regulation of IRAK1 in PCa, we performed cell culture experiments together with integrative bioinformatic studies using the latest single-cell RNA-sequencing data of human PCa and normal prostate (NOR), and data from The Cancer Genome Atlas. We focused on key effectors of TLR signaling, the Myddosome-complex components IRAK1, IRAK4 and MYD88 (myeloid differentiation primary response 88), and TRAF6 (tumor-necrosis-factor receptor associated factor 6). In PCa, -mRNA was specifically enriched in luminal epithelial cells, representing 57% of all cells, whereas and were predominantly expressed in leukocytes, and , in endothelial cells. Compared to NOR, only was significantly overexpressed in PCa (Benjamini-Hochberg adjusted p<2x10), whereas the expression of , , and was unchanged in PCa, and -expression was inversely correlated with a specific differentially methylated region (-DMR) within a predicted promoter region enriched for H3K27ac (Spearman correlation r<-0.36; Fisher's test, p<10). Transcription factors with high binding affinities in -DMR were significantly enriched for canonical pathways associated with viral infection and carcinogenic transformation in the Kyoto Encyclopedia of Gene and Genomes analysis. DU145 cells, exhibiting hypermethylated -DMR and low -expression, reacted with 4-fold increased -expression upon combined treatment with 5-aza-2-deoxycytidine and trichostatin A, and were unresponsive to infection with the uropathogenic strain UTI89. In contrast, PC3 and LNCaP cells, exhibiting hypomethylated -DMR and high endogenous -mRNA levels, responded with strong activation of -expression to UTI89 infection. In summary, exclusive overexpression of was observed in luminal epithelial cells in PCa, suggesting it has a role in addition to Myddosome-dependent TLR signaling. Our data show that the endogenous epigenetic status of PCa cells within -DMR is decisive for expression and should be considered as a predictive marker when selective IRAK1-targeting therapies are considered.
PubMed: 36226067
DOI: 10.3389/fonc.2022.991368 -
EBioMedicine Aug 2019TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency...
BACKGROUND
TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2 mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days.
METHODS
To enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP.
FINDINGS
Parenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients.
INTERPRETATIONS
These data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FUND: National Institutes of Health P01HD32062.
Topics: Animals; Biological Availability; Blood-Brain Barrier; DNA, Mitochondrial; Deoxyribonucleosides; Disease Models, Animal; Enzyme Activation; Humans; Mice; Mice, Knockout; Mice, Transgenic; Mitochondria; Muscle, Skeletal; Organ Specificity; Oxidative Phosphorylation; Phenotype; Thymidine Kinase
PubMed: 31383553
DOI: 10.1016/j.ebiom.2019.07.037 -
Molecular Therapy. Methods & Clinical... Jun 2023Pancreatic cancer remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We previously demonstrated that the...
Pancreatic cancer remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We previously demonstrated that the intra-tumoral gene transfer of somatostatin receptor 2, to combat tumor aggressiveness, or of deoxycytidine kinase and uridylate monophosphate kinase, to sensitize to gemcitabine chemotherapy, has anti-tumoral potential in experimental models of cancer. Here, we describe the development of the CYL-02 non-viral gene therapy product that comprises a DNA-plasmid encoding for the three aforementioned genes, which expression is targeted to tumor cells, and complexed with polyethyleneimine non-viral vector. We performed pre-clinical toxicology, bio-distribution, and therapeutic activity studies of CYL-02 in two rodent models of pancreatic cancer. We found that CYL-02 is safe, does not increase gemcitabine toxicity, is rapidly cleared from blood following intravenous administration, and sequestered in tumors following intra-tumoral injection. CYL-02 drives the expression of therapeutic genes in cancer cells and strongly sensitizes tumor cells to gemcitabine, both and , with significant inhibition of tumor cells dissemination. This study was instrumental for the later use of CYL-02 in patients with advanced pancreatic cancer, demonstrating that rigorous and thorough preclinical investigations are informative for the clinical transfer of gene therapies against this disease.
PubMed: 37063483
DOI: 10.1016/j.omtm.2023.03.005 -
Acta Biochimica Polonica Jul 2019DNA methylation and histone modifications are major components of cellular epigenetic pattern determining gene expression. Cancer cells have their own epigenetic array,...
Epigenetic modifiers 5-aza-2'-deoxycytidine and valproic acid differentially change viability, DNA damage and gene expression in metastatic and non-metastatic colon cancer cell lines.
DNA methylation and histone modifications are major components of cellular epigenetic pattern determining gene expression. Cancer cells have their own epigenetic array, which can be different in cells of primary and metastatic tumors. In the present work we investigated effects of 1 mM valproic acid (VPA), a histone deacetylase inhibitor and 0.2 μM 5-aza-2'-deoxycytidine (5-aza-dC), a DNA demethylation agent, singly or in combination in two colorectal cancer cell lines Caco-2 (non-metastatic) and LoVo (metastatic). Cell viability, DNA damage and the mRNA expression of the CDC25C (cell division cycle 25C), CDKN1A (cyclin dependent kinase inhibitor 1A) and CHEK1 (checkpoint kinase 1), SQSTM1 (sequestosome 1) and ULK1 (unc-51 like autophagy activating kinase 1), RELA (RELA proto-oncogene, NF-κB subunit) and TP53BP1 (tumor protein p53 binding protein 1) genes important in cell cycle regulation, autophagy and cancer progression were investigated. Both drugs induced a moderate decrease in cell viability and significant DNA damage in both cell lines. LoVo cells were more sensitive to VPA and combined treatment than Caco-2. LoVo cells also showed higher expression of genes that are often associated with more aggressive tumors than Caco-2 cells and treatment with the modifiers increased this difference. In conclusion, 5-aza-dC and VPA can induce different effects in metastatic and non-metastatic cancer cell lines and this may be important in determination of epigenetic profile responsible for metastatic properties of cancer cells.
Topics: Caco-2 Cells; Cell Cycle Checkpoints; Cell Survival; Checkpoint Kinase 1; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA Methylation; Decitabine; Drug Therapy, Combination; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Mas; Transcription Factor RelA; Valproic Acid
PubMed: 31284710
DOI: 10.18388/abp.2019_2814 -
Clinical Science (London, England :... Apr 2021The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a...
RATIONALE
The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation.
METHODS
Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation.
RESULTS
Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity.
CONCLUSION
These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.
Topics: Acute Lung Injury; Animals; Cells, Cultured; Decitabine; Disease Models, Animal; Endothelial Cells; Epigenesis, Genetic; Humans; Lipopolysaccharides; Lung Injury; Male; Mice, Inbred C57BL; Myosin-Light-Chain Kinase; Pneumonia; Polymorphism, Single Nucleotide; Respiratory Distress Syndrome; Stress, Mechanical; Tumor Necrosis Factor-alpha; Mice
PubMed: 33792658
DOI: 10.1042/CS20201448 -
Molecular Cancer Therapeutics Aug 2020Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the...
Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Casein Kinase Idelta; Cell Proliferation; Deoxycytidine; Deoxycytidine Kinase; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays; Gemcitabine
PubMed: 32430484
DOI: 10.1158/1535-7163.MCT-19-0997 -
Journal of the American Chemical Society Aug 2020Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies...
Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2'-azidocytidine (2'-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2'-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2'-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2'-AzCyd labeling. Further, 2'-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.
Topics: Cytidine; DNA-Directed RNA Polymerases; HeLa Cells; Humans; RNA
PubMed: 32786764
DOI: 10.1021/jacs.0c04566 -
Oncology Reports Mar 2021Overcoming chemo‑ and radio‑resistance is a major challenge in pancreatic cancer treatment. Therefore, there is an urgent need to discover novel therapeutic...
Overcoming chemo‑ and radio‑resistance is a major challenge in pancreatic cancer treatment. Therefore, there is an urgent need to discover novel therapeutic approaches to avoid chemo‑ and radio‑resistance in pancreatic cancer. Catechol is a phytochemical found in some fruits and vegetables. A few studies have reported on the potential anticancer effects of pure catechol. The present study aimed to explore the chemo‑ and radio‑sensitizing effects of catechol in Panc‑1 human pancreatic cancer cells. The effects of catechol on Panc‑1 cell proliferation, clonogenic survival, invasion, and migration were assessed using MTT, cell migration, and Transwell invasion assays. The chemo‑ and radio‑sensitizing effects of catechol on Panc‑1 cells were evaluated via MTT assay and flow cytometry. Western blotting was conducted to analyze the expression of proteins involved in several mechanisms induced by catechol in Panc‑1 cells, including growth inhibition, apoptosis, suppression of epithelial‑mesenchymal transition (EMT), and chemo‑ and radio‑sensitizing activities. The results indicated that catechol inhibited proliferation, promoted apoptosis, and suppressed cell migration, invasion, and EMT in Panc‑1 cells in a dose‑dependent manner. Catechol treatment also induced the phosphorylation of AMP‑activated protein kinase (AMPK) with a concomitant reduction in the expression of Hippo signaling pathway components, including Yes‑associated protein, cysteine‑rich angiogenic inducer 61, and connective tissue growth factor. In addition, catechol enhanced the chemosensitivity of Panc‑1 cells to gemcitabine, a commonly used chemotherapy in pancreatic cancer treatment. A combination of catechol and radiation enhanced apoptosis and increased the expression of two radiation‑induced DNA damage markers, p‑ATM and p‑Chk2. Collectively, the present results demonstrated that catechol, a naturally occurring compound, could suppress the proliferation of pancreatic cancer cells, reduce the expression of EMT‑related proteins, and enhance the chemo‑ and radio‑sensitivity of Panc‑1 cells by targeting AMPK/Hippo signaling.
Topics: AMP-Activated Protein Kinases; Adaptor Proteins, Signal Transducing; Apoptosis; Catechols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxycytidine; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Pancreatic Neoplasms; Phosphorylation; Radiation Tolerance; Signal Transduction; Transcription Factors; YAP-Signaling Proteins; Gemcitabine
PubMed: 33650657
DOI: 10.3892/or.2021.7924