RÉSUMÉ
Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.
Sujet(s)
Antimétabolites antinéoplasiques , Cytidine deaminase , Désoxycytidine , Résistance aux médicaments antinéoplasiques , Enterococcus faecium , Tumeurs de la vésicule biliaire , , Humains , Antimétabolites antinéoplasiques/métabolisme , Antimétabolites antinéoplasiques/pharmacologie , Antimétabolites antinéoplasiques/usage thérapeutique , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/composition chimique , Lignée cellulaire tumorale , Cytidine deaminase/métabolisme , Cytidine deaminase/génétique , Cytidine deaminase/composition chimique , Désoxycytidine/analogues et dérivés , Désoxycytidine/pharmacologie , Désoxycytidine/métabolisme , Désoxycytidine/composition chimique , Enterococcus faecium/enzymologie , Enterococcus faecium/génétique , Tumeurs de la vésicule biliaire/traitement médicamenteux , Tumeurs de la vésicule biliaire/génétique , Tumeurs de la vésicule biliaire/microbiologie , /métabolisme , /pharmacologie , /usage thérapeutiqueRÉSUMÉ
Gemcitabine monophosphate (dFdCMP), one of the intracellular forms of phosphorylated gemcitabine, determines its antitumor activity. A pharmaco-molecular model for determining relative gemcitabine monophosphate level based on the assessment of the activity of ENT1 and ENT2 channels as well as dCK and CDA enzymes in tumor tissue was developed. Relative gemcitabine monophosphate level is a more relevant predictive factor of gemcitabine resistance of bladder cancer as compared with the expression of individual markers related to dFdCMP formation.
Sujet(s)
, Tumeurs de la vessie urinaire , Humains , Désoxycytidine/usage thérapeutique , Désoxycytidine/métabolisme , Antimétabolites antinéoplasiques/usage thérapeutique , Tumeurs de la vessie urinaire/traitement médicamenteux , Lignée cellulaire tumoraleRÉSUMÉ
BACKGROUND: Pancreatic cancer continues to be one of the world's most lethal cancers. Chemotherapy resistance in patients with advanced pancreatic cancer often accompany with dismal prognosis, highlighting the need to investigate mechanisms of drug resistance and develop therapies to overcome chemoresistance. METHODS: This research was filed with the Chinese Clinical Trial Registry (ChiCTR2200061320). In order to isolate primary normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) samples of pancreatic ductal adenocarcinoma (PDAC) and paracancerous pancreatic tissue from individuals diagnosed with PDAC were obtained. The exosomes were obtained using ultracentrifugation, and their characteristics were determined by Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. CAF-derived miRNAs were analyzed by RT-qPCR and high-throughput sequencing. Gemcitabine (GEM) was employed to promote ferroptosis, and ferroptosis levels were determined by monitoring lipid reactive oxygen species (ROS), cell survival, and intracellular Fe2+ concentrations. To assess in vivo tumor response to GEM therapy, a xenograft tumor mouse model was utilized. RESULTS: Exosomes derived from CAFs in PDAC did not exhibit innate GEM resistance. CAFs promoted chemoresistance in PDAC cells following GEM treatment by secreting exosomes, and maintaining signaling communication with cancer cells. Mechanistically, miR-3173-5p derived from CAF exosomes sponged ACSL4 and inhibited ferroptosis after uptake by cancer cells. CONCLUSION: This work demonstrates a novel mode of acquired chemoresistance in PDAC and identifies the miR-3173-5p/ACSL4 pathway as a promising treatment target for GEM-resistant pancreatic cancer.
Sujet(s)
Fibroblastes associés au cancer , Carcinome du canal pancréatique , Exosomes , Ferroptose , microARN , Tumeurs du pancréas , Humains , Animaux , Souris , , microARN/génétique , microARN/métabolisme , Exosomes/génétique , Exosomes/anatomopathologie , Ferroptose/génétique , Fibroblastes associés au cancer/métabolisme , Désoxycytidine/pharmacologie , Désoxycytidine/usage thérapeutique , Désoxycytidine/métabolisme , Résistance aux médicaments antinéoplasiques/génétique , Lignée cellulaire tumorale , Tumeurs du pancréas/traitement médicamenteux , Tumeurs du pancréas/génétique , Carcinome du canal pancréatique/traitement médicamenteux , Carcinome du canal pancréatique/génétique , Modèles animaux de maladie humaine , Prolifération cellulaire , Coenzyme A ligases/métabolisme , Tumeurs du pancréasRÉSUMÉ
Cytidine 5'-monophosphate (5'-CMP), a key intermediate for the production of nucleotide derivatives, has been extensively used in food, agriculture, and medicine industries. Compared to RNA degradation and chemical synthesis, the biosynthesis of 5'-CMP has attracted wide attention due to its relatively low cost and eco-friendliness. In this study, we developed a cell-free regeneration of ATP based on polyphosphate kinase 2 (PPK2) to manufacture 5'-CMP from cytidine (CR). McPPK2 from Meiothermus cerbereus exhibited high specific activity (128.5 U/mg) and was used to accomplish ATP regeneration. McPPK2 and LhUCK (a uridine-cytidine kinase from Lactobacillus helveticus) were combined to convert CR to 5'-CMP. Further, the degradation of CR was inhibited by knocking out cdd from the Escherichia coli genome to enhance 5'-CMP production. Finally, the cell-free system based on ATP regeneration maximized the titer of 5'-CMP up to 143.5 mM. The wider applicability of this cell-free system was demonstrated in the synthesis of deoxycytidine 5'-monophosphate (5'-dCMP) from deoxycytidine (dCR) by incorporating McPPK2 and BsdCK (a deoxycytidine kinase from Bacillus subtilis). This study suggests that the cell-free regeneration of ATP based on PPK2 has the advantage of great flexibility for producing 5'-(d)CMP and other (deoxy)nucleotides.
Sujet(s)
Cytidine monophosphate , Nucleoside phosphate kinase , Cytidine monophosphate/composition chimique , Cytidine monophosphate/métabolisme , Nucleoside phosphate kinase/composition chimique , Nucleoside phosphate kinase/génétique , Nucleoside phosphate kinase/métabolisme , Nucléotides , Cytidine/métabolisme , Désoxycytidine/métabolisme , Adénosine triphosphate , RégénérationRÉSUMÉ
Pancreatic ductal adenocarcinoma (PDAC) is characterised by a pro-inflammatory stroma and multi-faceted microenvironment that promotes and maintains tumorigenesis. However, the models used to test new and emerging therapies for PDAC have not increased in complexity to keep pace with our understanding of the human disease. Promising therapies that pass pre-clinical testing often fail in pancreatic cancer clinical trials. The objective of this study was to investigate whether changes in the drug-dosing regimen or the addition of cancer-associated fibroblasts (CAFs) to current existing models can impact the efficacy of chemotherapy drugs used in the clinic. Here, we reveal that gemcitabine and paclitaxel markedly reduce the viability of pancreatic cell lines, but not CAFs, when cultured in 2D. Following the use of an in vitro drug pulsing experiment, PDAC cell lines showed sensitivity to gemcitabine and paclitaxel. However, CAFs were less sensitive to pulsing with gemcitabine compared to their response to paclitaxel. We also identify that a 3D co-culture model of MIA PaCa-2 or PANC-1 with CAFs showed an increased chemoresistance to gemcitabine when compared to standard 2D mono-cultures a difference to paclitaxel which showed no measurable difference between the 2D and 3D models, suggesting a complex interaction between the drug in study and the cell type used. Changes to standard 2D mono-culture-based assays and implementation of 3D co-culture assays lend complexity to established models and could provide tools for identifying therapies that will match clinically the success observed with in vitro models, thereby aiding in the discovery of novel therapies.
Sujet(s)
Fibroblastes associés au cancer , Carcinome du canal pancréatique , Tumeurs du pancréas , Humains , Désoxycytidine/métabolisme , Désoxycytidine/usage thérapeutique , Fibroblastes associés au cancer/métabolisme , Fibroblastes associés au cancer/anatomopathologie , Évaluation préclinique de médicament , Lignée cellulaire tumorale , Dépistage précoce du cancer , Tumeurs du pancréas/métabolisme , , Carcinome du canal pancréatique/métabolisme , Paclitaxel/métabolisme , Paclitaxel/usage thérapeutique , Microenvironnement tumoral , Tumeurs du pancréasRÉSUMÉ
Gemcitabine is widely used in the clinic as a first-line antitumor agent. However, intrinsic and acquired resistance hinders its wide clinical application. In this study, a gemcitabine prodrug nominated as WRQ-2 was designed and synthesized by conjugating gemcitabine with the indole-3-methanol analogue OSU-A9 through a carbamate linkage. WRQ-2 exhibited high cytotoxicity against six cancer cell lines (HeLa, A549, MDA-MB-231, HuH-7, MGC-803, and HCT-116) with IC50 values in low micromolar range. WRQ-2 reversed the resistance of HeLa cells to gemcitabine caused by hENT1 inhibition. Compared to gemcitabine, WRQ-2 induced a higher degree of DNA damage and apoptosis in the presence of hENT1 inhibitor. Our study suggests that compound WRQ-2 is a potential gemcitabine prodrug and worth of further antitumor activity investigation.
Sujet(s)
Tumeurs du pancréas , Promédicaments , Humains , , Promédicaments/pharmacologie , Désoxycytidine/pharmacologie , Désoxycytidine/métabolisme , Cellules HeLa , Lignée cellulaire tumorale , Tumeurs du pancréas/génétique , Tumeurs du pancréas/métabolisme , Tumeurs du pancréas/anatomopathologieRÉSUMÉ
Hypermethylation of CpG regions by human DNA methyltransferase 1 (DNMT1) silences tumor-suppression genes, and inhibition of DNMT1 can reactivate silenced genes. The 5-azacytidines are approved inhibitors of DNMT1, but their mutagenic mechanism limits their utility. A synthon approach from the analogues of S-adenosylhomocysteine, methionine, and deoxycytidine recapitulated the chemical features of the DNMT1 transition state in the synthesis of 16 chemically stable transition-state mimics. Inhibitors causing both full and partial inhibition of purified DNMT1 were characterized. The inhibitors show modest selectivity for DNMT1 versus DNMT3b. Active-site docking predicts inhibitor interactions with S-adenosyl-l-methionine and deoxycytidine regions of the catalytic site, validated by direct binding analysis. Inhibitor action with purified DNMT1 is not reflected in cultured cells. A partial inhibitor activated cellular DNA methylation, and a full inhibitor had no effect on cellular DNA methylation. These compounds provide chemical access to a new family of noncovalent DNMT chemical scaffolds for use in DNA methyltransferases.
Sujet(s)
DNA (cytosine-5-)-methyltransferase , Méthylation de l'ADN , Lignée cellulaire , ADN/métabolisme , DNA (Cytosine-5-)-methyltransferase 1/métabolisme , DNA modification methylases/métabolisme , Désoxycytidine/métabolisme , HumainsRÉSUMÉ
Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson-Crick base pairs. In addition, 5-methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C-C bond cleavage.
Sujet(s)
ADN/métabolisme , Désoxycytidine/analogues et dérivés , Génome/physiologie , Animaux , Cellules CHO , Cricetulus , ADN/composition chimique , Décarboxylation , Désoxycytidine/composition chimique , Désoxycytidine/métabolisme , Deutérium/composition chimique , Souris , Isotopes de l'azote/composition chimiqueRÉSUMÉ
Human concentrative nucleoside transporters (CNTs) are responsible for cellular uptake of ribonucleosides; however, although it is important to better characterize CNT-subtype specificity to understand the systemic disposition of deoxyribonucleosides (dNs) and their analogs, the involvement of CNTs in transporting dNs is not fully understood. In this study, using COS-7 cells that transiently expressed CNT1, CNT2, or CNT3, we investigated if CNTs could transport not only ribonucleosides but also dNs, i.e., 2'-deoxyadenosine (dAdo), 2'-deoxyguanosine (dGuo), and 2'-deoxycytidine (dCyd). The cellular uptake study demonstrated that dAdo and dGuo were taken up by CNT2 but not by CNT1. Although dCyd was taken up by CNT1, no significant uptake was detected in COS-7 cells expressing CNT2. Similarly, these dNs were transported by CNT3. The apparent Km values of their uptake were as follows: CNT1, Km ï¼ 141 µM for dCyd; CNT2, Km ï¼ 62.4 µM and 54.9 µM for dAdo and dGuo, respectively; CNT3, Km ï¼ 14.7 µM and 34.4 µM for dGuo and dCyd, respectively. These results demonstrate that CNTs contribute not only to ribonucleoside transport but also to the transport of dNs. Moreover, our data indicated that CNT1 and CNT2 selectively transported pyrimidine and purine dNs, respectively, and CNT3 was shown to transport both pyrimidine and purine dNs.
Sujet(s)
Désoxyribonucléosides/métabolisme , Protéines de transport membranaire/métabolisme , Animaux , Transport biologique actif , Cellules COS , Chlorocebus aethiops , Désoxyadénosine/métabolisme , Désoxycytidine/métabolisme , Désoxyguanosine/métabolisme , Humains , Cinétique , Protéines de transport membranaire/génétique , Protéines recombinantes/génétique , Protéines recombinantes/métabolismeRÉSUMÉ
BACKGROUND: Pemetrexed (MTA) plus cisplatin combination therapy is considered the standard of care for patients with advanced non-small-cell lung cancer (NSCLC). However, in advanced NSCLC, the 5-year survival rate is below 10%, mainly due to resistance to therapy. We have previously shown that the fraction of mesenchymal-like, chemotherapy-resistant paraclone cells increased after MTA and cisplatin combination therapy in the NSCLC cell line A549. Cytidine deaminase (CDA) and thymidine phosphorylase (TYMP) are key enzymes of the pyrimidine salvage pathway. 5'-deoxy-5-fluorocytidine (5'-DFCR) is a cytidine analogue (metabolite of capecitabine), which is converted by CDA and subsequently by TYMP into 5-fluorouracil, a chemotherapeutic agent frequently used to treat solid tumors. The aim of this study was to identify and exploit chemotherapy-induced metabolic adaptations to target resistant cancer cells. METHODS: Cell viability and colony formation assays were used to quantify the efficacy of MTA and cisplatin treatment in combination with schedule-dependent addition of 5'-DFCR on growth and survival of A549 paraclone cells and NSCLC cell lines. CDA and TYMP protein expression were monitored by Western blot. Finally, flow cytometry was used to analyze the EMT phenotype, DNA damage response activation and cell cycle distribution over time after treatment. CDA expression was measured by immunohistochemistry in tumor tissues of patients before and after neoadjuvant chemotherapy. RESULTS: We performed a small-scale screen of mitochondrial metabolism inhibitors, which revealed that 5'-DFCR selectively targets chemotherapy-resistant A549 paraclone cells characterized by high CDA and TYMP expression. In the cell line A549, CDA and TYMP expression was further increased by chemotherapy in a time-dependent manner, which was also observed in the KRAS-addicted NSCLC cell lines H358 and H411. The addition of 5'-DFCR on the second day after MTA and cisplatin combination therapy was the most efficient treatment to eradicate chemotherapy-resistant NSCLC cells. Moreover, recovery from treatment-induced DNA damage was delayed and accompanied by senescence induction and acquisition of a hybrid-EMT phenotype. In a subset of patient tumors, CDA expression was also increased after treatment with neoadjuvant chemotherapy. CONCLUSIONS: Chemotherapy increases CDA and TYMP expression thereby rendering resistant lung cancer cells susceptible to subsequent 5'-DFCR treatment.
Sujet(s)
Carcinome pulmonaire non à petites cellules/génétique , Cytidine deaminase/métabolisme , Désoxycytidine/analogues et dérivés , Tumeurs du poumon/génétique , Carcinome pulmonaire non à petites cellules/traitement médicamenteux , Désoxycytidine/métabolisme , Humains , Tumeurs du poumon/traitement médicamenteuxSujet(s)
Antimétabolites antinéoplasiques/administration et posologie , Désoxycytidine/analogues et dérivés , Systèmes de délivrance de médicaments/méthodes , Microbulles , Tumeurs du pancréas/traitement médicamenteux , Ultrasonothérapie/méthodes , Antimétabolites antinéoplasiques/métabolisme , Désoxycytidine/administration et posologie , Désoxycytidine/métabolisme , Humains , Tumeurs du pancréas/diagnostic , Tumeurs du pancréas/métabolisme , Pharmacologie clinique/méthodes ,RÉSUMÉ
18F-FAC (2'-deoxy-2'-18F-fluoro-ß-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor 18F-FAC PET images with 14C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by 18F-FAC. Methods:18F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of 18F-FAC and 14C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both 18F-FAC and 14C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-KrasG12D LSL-p53R172H) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and 14C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Results: Tumor-to-muscle ratios of 14C-gemcitabine and 18F-FAC correlated well across all PDX models and treatments (R2 = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised 14C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, P = 0.013). PET/MR 18F-FAC images showed a 12% increase in tumor 18F-FAC uptake after PEGPH20 treatment (P = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. Conclusion:18F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.
Sujet(s)
Désoxycytidine/analogues et dérivés , Tumeurs du pancréas/imagerie diagnostique , Tumeurs du pancréas/métabolisme , Tomographie par émission de positons , Animaux , Désoxycytidine/composition chimique , Désoxycytidine/métabolisme , Désoxycytidine/usage thérapeutique , Modèles animaux de maladie humaine , Vecteurs de médicaments/composition chimique , Vecteurs de médicaments/métabolisme , Hyaluronoglucosaminidase/métabolisme , Souris , Tumeurs du pancréas/traitement médicamenteux , Polyéthylène glycols/composition chimique , Polyéthylène glycols/métabolisme ,RÉSUMÉ
PURPOSE: KRAS is the most frequently mutated gene in human cancers, and ~ 90% of pancreatic cancers exhibit KRAS mutations. Despite the well-known role of KRAS in malignancies, directly inhibiting KRAS is challenging. METHODS: In this study, we successfully synthesized apolipoprotein E3-based liposomes for the co-delivery of gemcitabine (GEM) and a small interfering RNA targeting KRAS (KRAS-siRNA) to improve the efficacy of pancreatic cancer treatment. RESULTS: Apolipoprotein E3 self-assembly on the liposome surface led to a substantial increase in its internalization in PANC1 human pancreatic cancer cells. KRAS-siRNA led to downregulated KRAS protein expression and KRAS-dependent carcinogenic pathways, resulting in the inhibition of cell proliferation, cell cycle arrest, increased apoptosis, and suppression of tumor progression. The combination of KRAS-siRNA and GEM induced a synergistic improvement in cell apoptosis and significantly lower cell viability compared with single-agent therapy. The low IC50 value of A3-SGLP might be attributed to potentiation of the anticancer effect of GEM by siRNA-mediated silencing of KRAS mutations, thereby inducing synergistic effects on cancer cells. CONCLUSION: A3-SGLP led to a marked decrease in the overall tumor burden and did not show any signs of toxicity. Therefore, the combination of KRAS-siRNA and GEM holds great potential for the treatment of pancreatic cancer.
Sujet(s)
Antimétabolites antinéoplasiques/pharmacologie , Apolipoprotéine E3/métabolisme , Désoxycytidine/analogues et dérivés , Techniques de transfert de gènes , Lipides/composition chimique , Nanoparticules , Tumeurs du pancréas/thérapie , Protéines proto-oncogènes p21(ras)/génétique , Petit ARN interférent/métabolisme , Thérapie par l'interférence par ARN , Animaux , Antimétabolites antinéoplasiques/composition chimique , Antimétabolites antinéoplasiques/métabolisme , Apolipoprotéine E3/composition chimique , Apoptose/effets des médicaments et des substances chimiques , Points de contrôle du cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Association thérapeutique , Désoxycytidine/composition chimique , Désoxycytidine/métabolisme , Désoxycytidine/pharmacologie , Préparation de médicament , Humains , Liposomes , Mâle , Souris de lignée BALB C , Souris nude , Mutation , Tumeurs du pancréas/génétique , Tumeurs du pancréas/métabolisme , Tumeurs du pancréas/anatomopathologie , Protéines proto-oncogènes p21(ras)/métabolisme , Petit ARN interférent/génétique , Récepteurs aux lipoprotéines LDL/métabolisme , Tests d'activité antitumorale sur modèle de xénogreffe ,RÉSUMÉ
The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.
Sujet(s)
ADN catalytique/métabolisme , Biocatalyse , ADN catalytique/composition chimique , Désoxycytidine/analogues et dérivés , Désoxycytidine/composition chimique , Désoxycytidine/métabolisme , Structure moléculaire , Uridine/analogues et dérivés , Uridine/composition chimique , Uridine/métabolismeRÉSUMÉ
Gastric cancer is a frequently occurring cancer with high mortality each year worldwide. Finding new and effective therapeutic strategy against human gastric cancer is still urgently required. Hence, we have established a new method to achieve treatment-actuated modifications in a tumor microenvironment by utilizing synergistic activity between two potential anticancer drugs. Dual drug delivery of gemcitabine (GEM) and Camptothecin-11 (CPT-11) exhibits a great anti-cancer potential, as GEM enhances the effect of CPT-11 treatment of human gastric cells by providing microenvironment stability. However, encapsulation of GEM and CPT-11 obsessed by poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) is incompetent owing to unsuitability between the binary free GEM and CPT-11 moieties and the polymeric system. Now, we display that CPT-11 can be prepared by hydrophobic covering of the drug centers with dioleoylphosphatidic acid (DOPA). The DOPA-covered CPT-11 can be co-encapsulated in PLGA NPs alongside GEM to stimulate excellent anticancer property. The occurrence of the CPT-11 suggestively enhanced the encapsulations of GEM into PLGA NPs (GEM-CPT-11 NPs). Formation of the nanocomposite (GEM-CPT-11 NPs) was confirmed by FTIR and X-ray spectroscopic techniques. Further, the morphology of GEM NPs, CPT-11 NPs, and GEM-CPT-11 NPs and NP size was examined by transmission electron microscopy (TEM), respectively. Furthermore, GEM-CPT-11 NPs induced significant apoptosis in human gastric NCI-N87 and SGC-791 cancer cells in vitro. The morphological observation and apoptosis were confirmed by the various biochemical assays (AO-EB, nuclear staining, and annexin V-FITC). In addition, evaluation of the hemolysis assay with erythrocytes of human shows excellent biocompatibility of free GEM, free CPT-11, GEM NPs, CPT-11 NPs, and GEM-CPT-11 NPs. The results suggest that GEM-CPT-11 NPs are one of the promising nursing cares for human gastric cancer therapeutic candidates worthy of further investigations.
Sujet(s)
Protocoles de polychimiothérapie antinéoplasique/administration et posologie , Désoxycytidine/analogues et dérivés , Systèmes de délivrance de médicaments/méthodes , Irinotécan/administration et posologie , Tumeurs de l'estomac/traitement médicamenteux , Antimétabolites antinéoplasiques/administration et posologie , Antimétabolites antinéoplasiques/métabolisme , Antinéoplasiques/administration et posologie , Antinéoplasiques/métabolisme , Protocoles de polychimiothérapie antinéoplasique/métabolisme , Lignée cellulaire tumorale , Survie cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/physiologie , Désoxycytidine/administration et posologie , Désoxycytidine/métabolisme , Relation dose-effet des médicaments , Humains , Irinotécan/métabolisme , Tumeurs de l'estomac/métabolisme , Résultat thérapeutique ,RÉSUMÉ
Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.
Sujet(s)
Tumeurs du sein/génétique , Réplication de l'ADN/génétique , DNA-(apurinic or apyrimidinic site) lyase/génétique , Désoxycytidine/analogues et dérivés , Instabilité du génome/génétique , Tumeurs de l'ovaire/génétique , 5-Méthyl-cytosine/métabolisme , Animaux , Tumeurs du sein/traitement médicamenteux , Tumeurs du sein/métabolisme , Lignée cellulaire , Lignée cellulaire tumorale , DNA-(apurinic or apyrimidinic site) lyase/métabolisme , Désoxycytidine/métabolisme , Résistance aux médicaments antinéoplasiques/génétique , Femelle , Humains , Souris , Souris nude , Tumeurs expérimentales/traitement médicamenteux , Tumeurs expérimentales/génétique , Tumeurs expérimentales/métabolisme , Tumeurs de l'ovaire/traitement médicamenteux , Tumeurs de l'ovaire/métabolisme , Phtalazines/pharmacologie , Pipérazines/pharmacologie , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologieRÉSUMÉ
Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.
Sujet(s)
5-Méthyl-cytosine/métabolisme , Réparation de l'ADN , Désoxycytidine/analogues et dérivés , Épigenèse génétique , Génome , Cellules souches pluripotentes/métabolisme , Animaux , Isotopes du carbone , Lignée cellulaire , ADN/génétique , ADN/métabolisme , Méthylation de l'ADN , Désoxycytidine/métabolisme , Marquage isotopique , Souris , Souris transgéniques , Oxydoréduction , Cellules souches pluripotentes/cytologieRÉSUMÉ
Pancreatic ductal adenocarcinoma remains one of the challenging malignancies to treat, and chemotherapy is the primary treatment strategy available to most patients. Gemcitabine, one of the oldest chemotherapeutic drugs approved for pancreatic cancer, has limited efficacy, due to low drug distribution to the tumor and chemoresistance following therapy. In this study, we delivered gemcitabine monophosphate using lipid calcium phosphate nanoparticles, to desmoplastic pancreatic tumors. Monophosphorylation is a critical, rate-limiting step following cellular uptake of gemcitabine and precursor of the pharmacologically active gemcitabine triphosphate. Our drug delivery strategy enabled us to achieve robust tumor regression with a low parenteral dose in a clinically relevant, KRAS mutant, syngeneic orthotopic allograft, lentivirus-transfected KPC cell line-derived model of pancreatic cancer. Treatment with gemcitabine monophosphate significantly increased apoptosis of cancer cells, enabled reduction in the proportion of immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells, and did not increase expression of cancer stem cell markers. Overall, we could trigger a strong antitumor response in a treatment refractory PDAC model, while bypassing critical hallmarks of gemcitabine chemoresistance.
Sujet(s)
Antimétabolites antinéoplasiques/administration et posologie , Désoxycytidine/analogues et dérivés , Nanoparticules/administration et posologie , Tumeurs du pancréas/traitement médicamenteux , Tumeurs du pancréas/génétique , Protéines proto-oncogènes p21(ras)/génétique , Animaux , Antimétabolites antinéoplasiques/métabolisme , Lignée cellulaire tumorale , Désoxycytidine/administration et posologie , Désoxycytidine/métabolisme , Systèmes de délivrance de médicaments/méthodes , Humains , Souris , Souris de lignée C57BL , Mutation/génétique , Nanoparticules/métabolisme , Tumeurs du pancréas/métabolisme , Charge tumorale/effets des médicaments et des substances chimiques , Charge tumorale/physiologie , , Tumeurs du pancréasRÉSUMÉ
Gemcitabine (Gem) is a key drug for pancreatic cancer, yet limited by high systemic toxicity, low bioavailability and poor pharmacokinetic profiles. To overcome these limitations, Gem prodrug amphiphiles were synthesised with oleyl, linoleyl and phytanyl chains. Self-assembly and lyotropic mesophase behaviour of these amphiphiles were examined using polarised optical microscopy and Synchrotron SAXS (SSAXS). Gem-phytanyl was found to form liquid crystalline inverse cubic mesophase. This prodrug was combined with phospholipids and cholesterol to create biomimetic Gem-lipid prodrug nanoparticles (Gem-LPNP), verified by SSAXS and cryo-TEM to form liposomes. In vitro testing of the Gem-LPNP in several pancreatic cancer cell lines showed lower toxicity than Gem. However, in a cell line-derived pancreatic cancer mouse model Gem-LPNP displayed greater tumour growth inhibition than Gem using a fraction (<6 %) of the clinical dose and without any systemic toxicity. The easy production, improved efficacy and low toxicity of Gem-LPNP represents a promising new nanomedicine for pancreatic cancer.
Sujet(s)
Matériaux biomimétiques/usage thérapeutique , Désoxycytidine/analogues et dérivés , Nanoparticules/usage thérapeutique , Tumeurs du pancréas/traitement médicamenteux , Promédicaments/usage thérapeutique , Animaux , Matériaux biomimétiques/composition chimique , Carboxylesterase/métabolisme , Lignée cellulaire tumorale , Désoxycytidine/métabolisme , Désoxycytidine/usage thérapeutique , Dimyristoylphosphatidylcholine/composition chimique , Liposomes/composition chimique , Souris de lignée NOD , Souris SCID , Nanoparticules/composition chimique , Pancréas/anatomopathologie , Tumeurs du pancréas/anatomopathologie , Promédicaments/composition chimique , Promédicaments/métabolisme , Suidae ,RÉSUMÉ
Gemcitabine (GEM) is used to treat various cancers such as breast, pancreatic, non-small lung, ovarian, bladder, and cervical cancers. GEM, however, has the problem of non-selectivity. Water-soluble, fluorescent, and mono-dispersed carbon dots (CDs) were fabricated by ultrasonication of sucrose. The CDs were further conjugated with GEM through amide linkage. The physical and morphological properties of these carbon dot-gemcitabine (CD-GEM) conjugates were determined using different analytical techniques. In vitro cytotoxicity and apoptosis studies of CD-GEM conjugates were evaluated by various bioactivity assays on human cell lines, MCF-7 (human breast adenocarcinoma), and HeLa (cervical cancer) cell lines. The results of kinetic studies have shown a maximum drug loading efficacy of 17.0 mg of GEM per 50.0 mg of CDs. The CDs were found biocompatible, and the CD-GEM conjugates exhibited excellent bioactivity and exerted potent cytotoxicity against tumor cells with an IC50 value of 19.50 µg ml-1 in HeLa cells, which is lower than the IC50 value of pure GEM (â¼20.10 µg ml-1). In vitro studies on CD-GEM conjugates demonstrated the potential to replace the conventional administration of GEM. CD-GEM conjugates are more stable, have a higher aqueous solubility, and are more cytotoxic as compared to GEM alone. The CD-GEM conjugates show reduced side effects in the normal cells along with excellent cellular uptake. Hence, CD-GEM conjugates are more selective toward cancerous cell lines as compared to non-cancerous cells. Also, the CD-GEM conjugates successfully induced early and late apoptosis in cancer cell lines and might be effective and safe to use for in vivo applications.