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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 -
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 -
Cancer Drug Resistance (Alhambra,... 2020Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces...
AIM
Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces durable remissions of this tumor type. We developed three PDAC patient-derived xenograft (PDX) models with gemcitabine resistance (gemR) acquired , with which to identify mechanisms of resistance relevant to drug exposure and to evaluate novel therapies.
METHODS
Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly. Tumors initially responded, but regrew on treatment and were designated gemR. We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance [ribonucleotide reductase subunit M1 (RRM1), RRM2, human concentrative nucleoside transporter 1 (hCNT1), human equilibrative nucleoside transporter 1 (hENT1), cytidine deaminase (CDA), and deoxycytidine kinase (dCK)] in gemR and respective gemcitabine-naive parental tumors.
RESULTS
Parental and gemR tumors did not differ in tumor cell morphology, amount of tumor-associated stroma, or expression of stem cell markers. No consistent pattern of expression of the six gemR marker proteins was observed among the models. Increases in RRM1 and CDA were consistent with -derived gemR models. However, rather than the expected decreases of hCNT1, hENT1, and dCK, gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.
CONCLUSION
These models are the first PDAC PDX models with gemcitabine resistance acquired . The data indicate that mechanisms identified in models with resistance acquired are unlikely to be the predominant mechanisms when resistance is acquired . Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models.
PubMed: 33073205
DOI: 10.20517/cdr.2020.35 -
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 -
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 -
Journal of Molecular Endocrinology Feb 2020This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of...
This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of pancreatic cancer cells to gemcitabine in vitro and in vivo. The GLP-1R and protein kinase A (PKA) levels were compared between the human pancreatic cancer cell line PANC-1 and the gemcitabine-resistant cell line PANC-GR. The in vitro effects of liraglutide on the cell proliferation and apoptosis as well as the nuclear factor-kappa B NF-κB expression levels of PANC-GR cells were evaluated. In addition, a mouse xenograft model of human pancreatic cancer was established by s.c. injection of PANC-1 cells, and the effects of liraglutide on the chemosensitivity were evaluated in vitro and in vivo. In contrast to PANC-1 cells, PANC-GR cells exhibited lower expression levels of GLP-1R and PKA. Incubation with liraglutide dose dependently inhibited the growth, promoted the apoptosis, and increased the expression of GLP-1R and PKA of PANC-GR cells. Similar effects of liraglutide were observed in another human pancreatic cancer cell line MiaPaCa-2/MiaPaCa-2-GR. Either the GLP-1R antagonist Ex-9, the PKA inhibitor H89, or the NF-κB activator lipopolysaccharide (LPS) could abolish the antiproliferative and proapoptotic activities of liraglutide. Additionally, each of these agents could reverse the expression of NF-κB and ABCG2, which was decreased by liraglutide treatment. Furthermore, liraglutide treatment increased the chemosensitivity of pancreatic cancer cells to gemcitabine, as evidenced by in vitro and in vivo experiments. Thus, GLP-1R agonists are safe and beneficial for patients complicated with pancreatic cancer and diabetes, especially for gemcitabine-resistant pancreatic cancer.
Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Deoxycytidine; Flow Cytometry; Glucagon-Like Peptide-1 Receptor; Humans; Immunohistochemistry; Liraglutide; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Gemcitabine
PubMed: 31855560
DOI: 10.1530/JME-19-0186 -
Journal of Nuclear Medicine : Official... May 2021We developed an Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living...
We developed an Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. Anti-PD-L1 underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine followed by Zr radiolabeling. CT26 colon cancer cells and PD-L1-overexpressing CT26/PD-L1 cells underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics, biodistribution, and PET imaging were evaluated in mice. Zr-anti-PD-L1 synthesis was straightforward and efficient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that reduction produced half-antibody fragments, and matrix-assisted laser desorption ionization time-of-flight analysis estimated 2.18 conjugations per antibody, indicating specific conjugation at the hinge-region disulfide bonds. CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater Zr-anti-PD-L1 binding than that of weakly expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction in binding by excess cold antibody. Intravenous Zr-anti-PD-L1 followed biexponential blood clearance. PET/CT image analysis demonstrated decreases in major organ activity over 7 d, whereas high CT26/PD-L1 tumor activity was maintained. Again, this was suppressed by excess cold antibody. Treatment of CT26 cells with gemcitabine for 24 h augmented PD-L1 protein to 592.4% ± 114.2% of the control level and increased Zr-anti-PD-L1 binding, accompanied by increased AKT (protein kinase B) activation and reduced phosphatase and tensin homolog (PTEN). In CT26 tumor-bearing mice, gemcitabine treatment substantially increased tumor uptake from 1.56% ± 0.48% to 6.24% ± 0.37% injected dose per gram (tumor-to-blood ratio, 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease in PTEN. Zr-anti-PD-L1 showed specific targeting with favorable imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased Zr-anti-PD-L1 uptake. Zr-anti-PD-L1 PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.
Topics: Animals; B7-H1 Antigen; Cell Line, Tumor; Colonic Neoplasms; Deoxycytidine; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Immunoconjugates; Isotope Labeling; Mice; Positron Emission Tomography Computed Tomography; Radioisotopes; Tissue Distribution; Zirconium; Gemcitabine
PubMed: 32917780
DOI: 10.2967/jnumed.120.250720 -
Journal of Ethnopharmacology Dec 2022Artemisia argyi H. Lév. & Vaniot (Asteraceae), also called "Chinese mugwort", is frequently used as a herbal medicine in China, Japan, Korea, and eastern parts of...
ETHNOPHARMACOLOGICAL RELEVANCE
Artemisia argyi H. Lév. & Vaniot (Asteraceae), also called "Chinese mugwort", is frequently used as a herbal medicine in China, Japan, Korea, and eastern parts of Russia. It is known as "ai ye" in China and "Gaiyou" in Japan. In ancient China, the buds and leaves of A. argyi were commonly consumed before and after Tomb-sweeping Day. It is used to treat malaria, hepatitis, cancer, inflammatory diseases, asthma, irregular menstrual cycle, sinusitis, and pathologic conditions of the kidney and liver. Although A. argyi extract (AAE) has shown anti-tumor activity against various cancers, the therapeutic effect and molecular mechanism of AAE remains to be further studied in lung cancer.
AIM OF THE STUDY
This study aimed to demonstrate the anti-tumor effect of AAE and its associated biological mechanisms in CL1-0 parent and gemcitabine-resistant (CL1-0-GR) lung cancer cells.
EXPERIMENTAL PROCEDURE
Human lung cancer cells CL1-0 and CL1-0-GR cells were treated with AAE. Cell viability was assessed using the MTT, colony, and spheroid formation assays. Migration, invasion, and immunofluorescence staining were used to determine the extent of epithelial- mesenchymal transition (EMT). JC-1 and MitoSOX fluorescent assays were performed to investigate the effect of AAE on mitochondria. Apoptosis was detected using the TUNEL assay and flow cytometry with Annexin V staining.
RESULT
We found that A. argyi significantly decreased cell viability and induced apoptosis, accompanied by mitochondrial membrane depolarization and increased ROS levels in both parent cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0-GR). AAE-induced apoptosis is regulated via the PI3K/AKT and MAPK signaling pathways. It also prevents CL1-0 and CL1-0-GR cancer cell invasion, migration, EMT, colony formation, and spheroid formation. In addition, AAE acts cooperative with commercial chemotherapy drugs to enhance tumor spheroid shrinkage.
CONCLUSION
Our study provides the first evidence that A. argyi treatment suppresses both parent and gemcitabine-resistant lung cancer cells by inducing ROS, mitochondrial membrane depolarization, and apoptosis, and reducing EMT. Our finding provides insights into the anti-cancer activity of A. argyi and suggests that A. argyi may serve as a chemotherapy adjuvant that potentiates the efficacy of chemotherapeutic agents.
Topics: Annexin A5; Apoptosis; Artemisia; Cell Line, Tumor; Deoxycytidine; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Gemcitabine
PubMed: 36075273
DOI: 10.1016/j.jep.2022.115658 -
Genes Sep 2019Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer...
Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the genomic library of knockout and knockdown (YKO/KD) strains to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.
Topics: Antimetabolites, Antineoplastic; Cytarabine; Deoxycytidine; Deoxycytidine Kinase; Epistasis, Genetic; Gene Ontology; Gene Regulatory Networks; High-Throughput Screening Assays; Humans; Nucleosides; Pharmacogenetics; Phenomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Gemcitabine
PubMed: 31575041
DOI: 10.3390/genes10100770 -
Frontiers in Pharmacology 2022Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors....
Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC). Although transfection resulted in a significant expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, and expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.
PubMed: 36278182
DOI: 10.3389/fphar.2022.991751