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1.
Cell Death Dis ; 15(2): 131, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38346958

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Gencitabina/farmacologia , Gencitabina/uso terapêutico , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Sci Rep ; 13(1): 20530, 2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-37993628

RESUMO

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.


Assuntos
Citidina , DCMP Desaminase , Citidina/metabolismo , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Desoxicitidina , Redes e Vias Metabólicas , Citidina Desaminase/metabolismo
3.
Int J Mol Sci ; 24(17)2023 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-37685961

RESUMO

The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro. AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Proteína Semelhante a ELAV 1/genética , Gencitabina , Pâncreas , Hormônios Pancreáticos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Receptores de Hidrocarboneto Arílico/genética , RNA Mensageiro/genética , Desoxicitidina Quinase/efeitos dos fármacos , Desoxicitidina Quinase/metabolismo , Neoplasias Pancreáticas
4.
Mol Cancer Res ; 21(5): 444-457, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36757299

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most life-threatening malignancies. Although the deoxycytidine analog gemcitabine has been used as the first-line treatment for PDAC, the primary clinical challenge arises because of an eventual acquisition of resistance. Therefore, it is crucial to elucidate the mechanisms underlying gemcitabine resistance to improve treatment efficacy. To investigate potential genes whose inactivation confers gemcitabine resistance, we performed CRISPR knockout (KO) library screening. We found that deoxycytidine kinase (DCK) deficiency is the primary mechanism of gemcitabine resistance, and the inactivation of CRYBA2, DMBX1, CROT, and CD36 slightly conferred gemcitabine resistance. In particular, gene expression analysis revealed that DCK KO cells displayed a significant enrichment of genes associated with MYC targets, folate/one-carbon metabolism and glutamine metabolism pathways. Evidently, chemically targeting each of these pathways significantly reduced the survival of DCK KO cells. Moreover, the pathways enriched in DCK KO cells represented a trend similar to those in PDAC cell lines and samples of patients with PDAC with low DCK expression. We further observed that short-term treatment of parental CFPAC-1 cells with gemcitabine induces the expression of several genes, which promote synthesis and transport of glutamine in a dose-dependent manner, which suggests glutamine availability as a potential mechanism of escaping drug toxicity in an initial response for survival. Thus, our findings provide insights into novel therapeutic approaches for gemcitabine-resistant PDAC and emphasize the involvement of glutamine metabolism in drug-tolerant persister cells. IMPLICATIONS: Our study revealed the key pathways involved in gemcitabine resistance in PDAC, thus providing potential therapeutic strategies.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Desoxicitidina Quinase/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Gencitabina , Glutamina , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas
5.
Drug Resist Updat ; 67: 100932, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36706533

RESUMO

BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.


Assuntos
Antineoplásicos , Desoxicitidina Quinase , Humanos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nucleotídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína BRCA2/genética
6.
Genes (Basel) ; 13(10)2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36292719

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) originates from the hepatocytes and accounts for 90% of liver cancer. The study intends to identify novel prognostic biomarkers for predicting the prognosis of HCC patients based on TCGA and GSE14520 cohorts. METHODS: Differential analysis was employed to obtain the DEGs (Differentially Expressed Genes) of the TCGA-LIHC-TPM cohort. The lasso regression analysis was applied to build the prognosis model through using the TCGA cohort as the training group and the GSE14520 cohort as the testing group. Next, based on the prognosis model, we performed the following analyses: the survival analysis, the independent prognosis analysis, the clinical feature analysis, the mutation analysis, the immune cell infiltration analysis, the tumor microenvironment analysis, and the drug sensitivity analysis. Finally, the survival time of HCC patients was predicted by constructing nomograms. RESULTS: Through the lasso regression analysis, we obtained a prognosis model of ten genes including BIRC5 (baculoviral IAP repeat containing 5), CDK4 (cyclin-dependent kinase 4), DCK (deoxycytidine kinase), HSPA4 (heat shock protein family A member 4), HSP90AA1 (heat shock protein 90 α family class A member 1), PSMD2 (Proteasome 26S Subunit Ubiquitin Receptor, Non-ATPase 2), IL1RN (interleukin 1 receptor antagonist), PGF (placental growth factor), SPP1 (secreted phosphoprotein 1), and STC2 (stanniocalcin 2). First, we found that the risk score is an independent prognosis factor and is related to the clinical features of HCC patients, covering AFP (α-fetoprotein) and stage. Second, we observed that the p53 mutation was the most obvious mutation between the high-risk and low-risk groups. Third, we also discovered that the risk score is related to some immune cells, covering B cells, T cells, dendritic, macrophages, neutrophils, etc. Fourth, the high-risk group possesses a lower TIDE score, a higher expression of immune checkpoints, and higher ESTIMATE score. Finally, nomograms include the clinical features and risk signatures, displaying the clinical utility of the signature in the survival prediction of HCC patients. CONCLUSIONS: Through the comprehensive analysis, we constructed an immune-related prognosis model to predict the survival of HCC patients. In addition to predicting the survival time of HCC patients, this model significantly correlates with the tumor microenvironment. Furthermore, we concluded that these ten immune-related genes (BIRC5, CDK4, DCK, HSPA4, HSP90AA1, PSMD2, IL1RN, PGF, SPP1, and STC2) serve as novel targets for antitumor immunity. Therefore, this study plays a significant role in exploring the clinical application of immune-related genes.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Feminino , Humanos , alfa-Fetoproteínas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Quinase 4 Dependente de Ciclina/metabolismo , Desoxicitidina Quinase/metabolismo , Proteínas de Choque Térmico/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Osteopontina/metabolismo , Fator de Crescimento Placentário/metabolismo , Complexo de Endopeptidases do Proteassoma , Receptores de Interleucina-1 , Proteína Supressora de Tumor p53 , Ubiquitinas/metabolismo
7.
Cancer Chemother Pharmacol ; 89(1): 83-91, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34825941

RESUMO

PURPOSE: Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine. METHODS: Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation. RESULTS: The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic. CONCLUSION: This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.


Assuntos
Antineoplásicos/farmacologia , Arabinonucleosídeos/farmacologia , Desoxicitidina Quinase/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Acetilação , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas , Vorinostat/farmacologia
8.
Cells ; 10(12)2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34943995

RESUMO

Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.


Assuntos
Cladribina/farmacologia , Imunomodulação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina Quinase/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Pró-Fármacos/farmacologia
9.
Neuro Oncol ; 23(6): 945-954, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33556172

RESUMO

BACKGROUND: High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown. METHODS: We examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity. RESULTS: hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients. CONCLUSIONS: The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.


Assuntos
Neoplasias Meníngeas , Meningioma , Neoplasias Pancreáticas , Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina Quinase/metabolismo , Desoxicitidina Quinase/uso terapêutico , Transportador Equilibrativo 1 de Nucleosídeo , Humanos , Neoplasias Meníngeas/tratamento farmacológico , Meningioma/tratamento farmacológico , Gencitabina
10.
Acta Haematol ; 144(5): 534-541, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33626530

RESUMO

Resistance to cytarabine is an important cause of therapy failure in persons with acute myeloid leukemia (AML). Deoxycytidine kinase, encoded by DCK, catalyzes phosphorylation of cytarabine to cytarabine monophosphate, a necessary step for eventual incorporation of cytarabine triphosphate into DNA and for clinical efficacy. Whether DCK mutations make AML cells resistant to cytarabine is controversial. We studied DCK mutations and messenger RNA (mRNA) concentrations in leukemia cells from 10 subjects with AML who received cytarabine-based therapy and relapsed and in 2 artificially induced cytarabine-resistant AML cell lines. DCK mutations were detected in 4 subjects with AML relapsing after achieving a complete remission and receiving high-dose cytarabine postremission therapy. Most mutations were in exons 4-6 and were not present before therapy. DCK was also mutated in cytarabine-resistant but not parental AML cell lines. DCK mRNA concentrations were significantly decreased in cytarabine-resistant K562 and SHI-1 cells compared with cytarabine-sensitive parental cells. Mutation frequency of DCK and mRNA concentration did not correlate with the extent of cytarabine resistance indicating other factors operate. Overexpression of wild-type DCK restored cytarabine sensitivity to previously resistant leukemia cell lines. Our data contribute to the understanding of cytarabine resistance in persons with AML.


Assuntos
Citarabina/farmacologia , Desoxicitidina Quinase , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda , Mutação , Proteínas de Neoplasias , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
11.
J Clin Lab Anal ; 34(11): e23457, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32671914

RESUMO

BACKGROUND: Several biomarkers of gemcitabine effectiveness have been studied in cancers, but less so in hepatocellular carcinoma (HCC), which is identified as the fifth most common cancer worldwide. Investigation of human equilibrative nucleoside transporter-1 (HENT-1) and deoxycytidine kinase (DCK), genes involved in gemcitabine uptake and metabolism, can be beneficial in the selection of potential cancer patients who could be responding to the treatment. AIM: To study HENT-1 and DCK gene expression in HCC patients with different protocols of treatment. METHODS: Using real-time PCR, we analyzed expression levels of HENT-1 and DCK genes from peripheral blood samples of 109 patients (20 controls & 89 HCC patients) between March 2015 and March 2017. All the 89 HCC patients received the antioxidants selenium (Se) and vitamin E (Vit.E) either alone (45 patients) or in combination with gemcitabine (24 patients) or radiofrequency ablation (RFA) (20 patients). RESULTS: There was a significant increase in HENT-1 expression levels in HCC patients treated with Se and Vit.E alone as compared to controls (P Ë‚ .0001), while there was no significant difference between HCC patients treated with gemcitabine or RFA as compared to controls. In contrast, expression of DCK was significantly increased in all groups of HCC patients as compared to controls (P Ë‚ .0001). CONCLUSIONS: HENT-1 and DCK mRNA expressions are important markers of HCC and for GEM effect and GEM sensitivity in patients with HCC. This could be beneficial in the selection of HCC patients sensitive to gemcitabine to avoid subjecting resistant patients to unnecessary chemotherapy.


Assuntos
Carcinoma Hepatocelular , Desoxicitidina Quinase , Desoxicitidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo , Neoplasias Hepáticas , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Estudos Transversais , Desoxicitidina/uso terapêutico , Desoxicitidina Quinase/sangue , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Egito , Transportador Equilibrativo 1 de Nucleosídeo/sangue , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Resultado do Tratamento , Gencitabina
12.
Artigo em Inglês | MEDLINE | ID: mdl-32727269

RESUMO

Dexamethasone (DEX) is often used in the initial treatment of leukemia. Earlier we demonstrated that DEX decreased the activity of deoxycytidine kinase (dCK) which is essential for the activation of cytarabine (ara-C). Therefore we investigated the effect of DEX on the in vivo sensitivity of acute myeloid leukemia (AML) to ara-C and another deoxycytidine analog, gemcitabine, in the Brown Norway Myeloid Leukemia (BNML) rat model for AML, and its ara-C resistant variant B-araC, in relation to the effects on dCK activity.The antileukemic effect was evaluated as survival of the rats, while dCK activity was measured in leukemic spleen (completely consisting of BNML cells) with liver as representative normal tissue, 24 hr after treatment with ara-C or DEX with radioactive deoxycytidine (CdR) as a substrate.Treatment with ara-C increased life-span of BNML by 200%, which was not affected by DEX. Gemcitabine was ineffective. In the liver of BNML bearing rats DEX decreased dCK activity 33%, while ara-C increased dCK activity slightly (to 129%), but in the combination of ara-C/DEX dCK activity was also decreased. In the livers of Bara-C bearing rats dCK was 2.7-fold higher compared to BNML rats, which was increased 179% in the gemcitabine-DEX treated rats. In BNML leukemic spleens DEX decreased dCK activity 41% and gem/dex 46%, but ara-C increased dCK activity to 123%, but in the combination this effect was neutralized. In Bara-C spleens only ara-C/dex decreased dCK activity (32%).In conclusion; in an AML rat model DEX did not affect the antileukemic effect of ara-C, nor the dCK activity.


Assuntos
Antineoplásicos/farmacologia , Citarabina/farmacologia , Desoxicitidina Quinase/metabolismo , Dexametasona/farmacologia , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/enzimologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citarabina/uso terapêutico , Interações Medicamentosas , Ratos
13.
Technol Cancer Res Treat ; 19: 1533033820934133, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32588770

RESUMO

BACKGROUND: The biological function of deoxycytidine kinase in tumor is not yet clear, and there are a few studies relating to the correlation of deoxycytidine kinase gene with the occurrence and development of liver cancer. METHODS: The messenger RNA expression of deoxycytidine kinase was analyzed with the use of the UALCAN and GEPIA database. Moreover, we assessed the function of deoxycytidine kinase on clinical prognosis with Kaplan-Meier plotter database. The relationship between deoxycytidine kinase and cancer immune infiltrates was investigated via Tumor Immune Estimation Resource site. Furthermore, Tumor Immune Estimation Resource was also used to evaluate the correlations between the expression of deoxycytidine kinase and gene marker sets of immune infiltrates. RESULTS: The deoxycytidine kinase messenger RNA level significantly upregulated in patients with liver cancer compared to normal liver samples. Moreover, the increased expression of deoxycytidine kinase messenger RNA was closely associated with reduced overall survival and disease-free survival in all liver cancers. In addition, deoxycytidine kinase expression displayed a strong correlation with infiltrating levels of macrophages, neutrophils, and dendritic cells in liver cancer, and deoxycytidine kinase expression was positively correlated with diverse immune marker sets in liver cancer. CONCLUSIONS: All the above findings suggested that increased expression of deoxycytidine kinase was significantly related to unfavorable prognosis in patients with liver cancer. And deoxycytidine kinase is correlated with immune infiltrating levels, including those of B cells, macrophages, neutrophils, and dendritic cells in patients with liver cancer. These findings suggest that deoxycytidine kinase can be used as a prognostic biomarker for determining prognosis and immune infiltration in liver cancer. And deoxycytidine kinase is a potential target for liver cancer therapy, and these preliminary findings require further study to determine whether deoxycytidine kinase-targeting reagents might be developed for clinical application in liver cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Desoxicitidina Quinase/metabolismo , Neoplasias Hepáticas/patologia , Linfócitos do Interstício Tumoral/imunologia , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/imunologia , Masculino , Prognóstico , Taxa de Sobrevida
14.
Mol Cancer Ther ; 19(8): 1623-1635, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32430484

RESUMO

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.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Caseína Quinase Idelta/antagonistas & inibidores , Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Desoxicitidina/farmacologia , Desoxicitidina Quinase/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina , Neoplasias Pancreáticas
15.
Nucleosides Nucleotides Nucleic Acids ; 39(10-12): 1347-1358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189555

RESUMO

Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are required for the activation of thymidine and deoxycytidine analogs used in antiviral and anticancer therapies. Many anticancer drugs cause oxidative stress, and the rise of GSSG and other reactive oxygen species may lead to alteration in gene expression, protein, nucleic acids and lipid modifications. Here, we investigated the effects of oxidative stress and nucleoside analog on the expression and activity of TK1 and dCK. Treatment with GSSG resulted in glutathionylation of dCK and dGK but not TK1 and Dm-dNK, and glutathionylation led to increased dCK activity but decreased dGK activity. Treatment with hydrogen peroxide resulted in induction of TK1, however, the TK1 activity did not correlate with TK1 protein levels, indicating that TK1 protein was inactive. The cellular expression of dCK, however, was reduced but dCK activity was not affected at concentration ≤ 4 mM. Treatment with TFT or 5FdU resulted in downregulation of both TK1 and dCK. However, araC and dFdC treatment led to increased dCK protein but decreased dCK activity. In contrast, both TK1 protein and activity were upregulated after araC and dFdC treatment. Doxorubicin treatment led to upregulation of the TK1 but downregulation of dCK. In conclusion TK1 and dCK expression and activity are apparently affected by oxidative stress and treatment by nucleoside analogs. These results demonstrate the pharmacokinetic importance of characterizing the expression and activity of TK1 and dCK during chemotherapy with thymidine and deoxycytidine analogs in order to optimize their efficacy.


Assuntos
Desoxicitidina Quinase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Nucleosídeos/química , Nucleosídeos/farmacologia , Timidina Quinase/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster/enzimologia , Glutationa/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos
16.
Genes (Basel) ; 10(10)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31575041

RESUMO

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 Saccharomycescerevisiae 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.


Assuntos
Desoxicitidina Quinase/genética , Nucleosídeos/toxicidade , Farmacogenética/métodos , Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina Quinase/metabolismo , Epistasia Genética , Ontologia Genética , Redes Reguladoras de Genes , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fenômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Gencitabina
17.
Cell Death Dis ; 10(6): 390, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101804

RESUMO

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines' sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hidroxiureia/farmacologia , Leucemia Mieloide Aguda/patologia , Zidovudina/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citarabina/farmacologia , Dano ao DNA/efeitos dos fármacos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Sinergismo Farmacológico , Histonas/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Ubiquitinação , Zidovudina/uso terapêutico
18.
Sci Rep ; 9(1): 7643, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113993

RESUMO

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.


Assuntos
Antineoplásicos/toxicidade , Biomarcadores Tumorais/genética , Monofosfato de Citidina/análogos & derivados , Desoxicitidina Quinase/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/genética , Neoplasias Pancreáticas/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Ensaios Clínicos Fase I como Assunto , Monofosfato de Citidina/uso terapêutico , Monofosfato de Citidina/toxicidade , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Desoxicitidina/toxicidade , Desoxicitidina Quinase/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Gencitabina
19.
Molecules ; 24(10)2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31130654

RESUMO

This study aimed to investigate whether the anti-tumor effect of gemcitabine (GEM) in non-small-cell lung cancer (NSCLC) treatment was affected by Danggui Buxue decoction (DBD), and explore the potential mechanisms. The combined use of GEM and DBD showed an enhanced tumor growth inhibition effect in a murine Lewis lung carcinoma (LLC) model. LC-MS/MS results showed that the pharmacokinetic behaviors of a GEM active metabolite, gemcitabine triphosphate (dFdCTP), were found to be altered remarkably in the peripheral blood mononuclear cells (PBMC) of DBD co-administration rats. In addition, after co-administration of DBD with GEM, Western Blot and qPCR results confirmed that the expression of deoxycytidine kinase (dCK) in tumor tissues of LLC-bearing mice were markedly increased. DBD co-administration also reversed the upregulation of P-glycoprotein (P-gp) in tumor tissues induced by GEM. Moreover, DBD could notably up-regulate the IL-12p70 and GM-CSF expression in mice serum, suggesting potential immunomodulatory activities in tumor-bearing mice. Meanwhile, DBD inhibited the P-gp efflux activity in A549 cells. Therefore, the regulation of dCK and P-gp played important roles in the alternation of GEM pharmacokinetics and the enhancement of the anti-tumor effect of GEM. DBD being a potential dCK promoter could work as an adjuvant agent to boost the anticancer effect of GEM.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Medicamentos de Ervas Chinesas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Animais , Desoxicitidina/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Gencitabina
20.
Cancer Res ; 79(10): 2775-2783, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30940660

RESUMO

Deoxycytidine kinase (DCK) is a key enzyme for the activation of a broad spectrum of nucleoside-based chemotherapy drugs (e.g., gemcitabine); low DCK activity is one of the most important causes of cancer drug-resistance. Noninvasive imaging methods that can quantify DCK activity are invaluable for assessing tumor resistance and predicting treatment efficacy. Here we developed a "natural" MRI approach to detect DCK activity using its natural substrate deoxycytidine (dC) as the imaging probe, which can be detected directly by chemical exchange saturation transfer (CEST) MRI without any synthetic labeling. CEST MRI contrast of dC and its phosphorylated form, dCTP, successfully discriminated DCK activity in two mouse leukemia cell lines with different DCK expression. This dC-enhanced CEST MRI in xenograft leukemic cancer mouse models demonstrated that DCK(+) tumors have a distinctive dynamic CEST contrast enhancement and a significantly higher CEST contrast than DCK(-) tumors (AUC0-60 min = 0.47 ± 0.25 and 0.20 ± 0.13, respectively; P = 0.026, paired Student t test, n = 4) at 1 hour after the injection of dC. dC-enhanced CEST contrast also correlated well with tumor responses to gemcitabine treatment. This study demonstrates a novel MR molecular imaging approach for predicting cancer resistance using natural, nonradioactive, nonmetallic, and clinically available agents. This method has great potential for pursuing personalized chemotherapy by stratifying patients with different DCK activity. SIGNIFICANCE: A new molecular MRI method that detects deoxycytidine kinase activity using its natural substrate deoxycytidine has great translational potential for clinical assessment of tumor resistance and prediction of treatment efficacy.


Assuntos
Desoxicitidina Quinase/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/metabolismo , Feminino , Xenoenxertos , Leucemia/enzimologia , Leucemia/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade por Substrato
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