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1.
Nucleic Acids Res ; 52(9): 5121-5137, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38520409

RESUMO

The S-phase checkpoint is involved in coupling DNA unwinding with nascent strand synthesis and is critical to maintain replication fork stability in conditions of replicative stress. However, its role in the specific regulation of leading and lagging strands at stalled forks is unclear. By conditionally depleting RNaseH2 and analyzing polymerase usage genome-wide, we examine the enzymology of DNA replication during a single S-phase in the presence of replicative stress and show that there is a differential regulation of lagging and leading strands. In checkpoint proficient cells, lagging strand replication is down-regulated through an Elg1-dependent mechanism. Nevertheless, when checkpoint function is impaired we observe a defect specifically at the leading strand, which was partially dependent on Exo1 activity. Further, our genome-wide mapping of DNA single-strand breaks reveals that strand discontinuities highly accumulate at the leading strand in HU-treated cells, whose dynamics are affected by checkpoint function and Exo1 activity. Our data reveal an unexpected role of Exo1 at the leading strand and support a model of fork stabilization through prevention of unrestrained Exo1-dependent resection of leading strand-associated nicks after fork stalling.


Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , Exodesoxirribonucleases , Pontos de Checagem da Fase S do Ciclo Celular , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ribonuclease H/metabolismo , Ribonuclease H/genética , Fase S/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
2.
Int J Artif Organs ; 46(7)July, 2023.
Artigo em Inglês | CONASS, Sec. Est. Saúde SP, SESSP-IDPCPROD, Sec. Est. Saúde SP | ID: biblio-1510702

RESUMO

OBJECTIVES: The reduction in size of these systems, which increases their reliability, biocompatibility and robustness, is essential to the complete implantation of the VADs, which is the main focus of the current state of art. Continuous flow VADs are actuated by brushless motors due to their reliability. The objective of the current project was to implement and simulate sensorless speed control in order to actuate VAD. METHODS: In order to increase the robustness of the system even further, a strategy that does not use Hall sensors can be implemented. The sensorless strategy to control speed that was implemented in this work aims to detect the position of the rotor by using the coil of the inactive phase in order to sense the variation in magnetic flux, which comes in the form of back-electromotive forces. RESULTS: A three phase inverter to electrically commute the motor's phases, a conditioning circuit that obtains the back-electromotive forces and a speed controller were developed. The speed control and the commutation logic were implemented by using a microcontroller. The results that were obtained in computational simulations indicated that the three-phase inverter, the commutation logic and the controller reached the project requirements. The implemented microcontroller commutation logic presented the expected behavior. Commutation signals were obtained in six stages, necessary for the correct activation of the phases of the brushless motor. The controller was validated in terms of its step response, demonstrating low overshoot and fast control action in the system. CONCLUSIONS: To further enhance the robustness of the system, an alternative strategy that eliminates the use of Hall sensors can be employed. The sensorless speed control strategy, implemented in this study, detects the position of the rotor by measuring variations in magnetic flux through the coil of the inactive phase, thus relying on back-electromotive forces for detection.


Assuntos
Simulação por Computador , Coração Auxiliar , Pontos de Checagem da Fase S do Ciclo Celular , Lógica
3.
Cancer Lett ; 544: 215804, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-35750276

RESUMO

Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.


Assuntos
Replicação do DNA , Neoplasias , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Humanos , Fator 2 Relacionado a NF-E2 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Pontos de Checagem da Fase S do Ciclo Celular , Estresse Fisiológico
4.
Curr Genet ; 68(2): 165-179, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35150303

RESUMO

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Mutação , Proteínas Serina-Treonina Quinases , Fase S/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
5.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35169076

RESUMO

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.


Assuntos
Variações do Número de Cópias de DNA/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Proteína Supressora de Tumor p53/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Genéticas , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Neoplasias/genética , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Retroelementos/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteína Supressora de Tumor p53/metabolismo
6.
PLoS One ; 17(2): e0263569, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35157703

RESUMO

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two yeast strains challenged by hydroxyurea, an inhibitor of the ribonucleotide reductase. We gained understanding of the impact on origin activation by three factors: S-phase checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Wild type W303 showed a significant reduction of fork progression accompanied by an elevated level of Rad53 phosphorylation as well as physical presence at origins compared to A364a. Moreover, a rad53K227A mutant in W303 activated more origins, accompanied by global reduction of ssDNA across all origins, compared to A364a. Sequence polymorphism in the consensus motifs of origins plays a minor role in determining strain-specific activity. Finally, we identified a new class of origins only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". Our study presents a comprehensive list of differentially used origins and provide new insights into the mechanisms of origin activation.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Fúngico/genética , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Laboratórios , Mutação , Fosforilação , Polimorfismo de Nucleotídeo Único , Pontos de Checagem da Fase S do Ciclo Celular , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Sequenciamento Completo do Genoma
7.
Int J Mol Sci ; 23(3)2022 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-35163553

RESUMO

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Ósseas/genética , Cloroquina/farmacologia , Monoterpenos/farmacologia , Osteossarcoma/genética , Tropolona/análogos & derivados , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Humanos , Osteossarcoma/tratamento farmacológico , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Tropolona/farmacologia , Proteína Supressora de Tumor p53/genética
8.
Cancer Gene Ther ; 29(7): 897-900, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34737438

RESUMO

The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the ßGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.


Assuntos
Quinases Ciclina-Dependentes , Pontos de Checagem da Fase S do Ciclo Celular , Ciclo Celular , Ciclina A/genética , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citocinas , Humanos , Fase S
9.
J Ethnopharmacol ; 284: 114832, 2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-34775036

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC is a traditional medicinal plant. It is widely used in clinical treatment and disease prevention in China, India and other regions. Modern studies have reported the phytotoxicity, cytotoxicity and the animal toxicity of Zanthoxylum armatum DC, and the damage of genetic material has been observed in plants, but the detailed mechanism has not been explored. Besides, the toxicity of normal mammalian cells has not been evaluated. AIM OF THE STUDY: To evaluate the effects and underlying mechanism of genetic material damage in BRL 3A cells induced by Zanthoxylum armatum DC. MATERIALS AND METHODS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry was used for identification of compounds in methanol extract of Zanthoxylum armatum DC. BRL 3A cells were incubated with different concentrations of methanol extract of Zanthoxylum armatum DC (24 h). The cytotoxicity of extract was assessed with cell viability, LDH release rate, and ROS production. The damage of genetic material was assessed with OTM value of comet cells, cell cycle and the expression levels of p-ATM, p- Chk2, Cdc25A, and CDK2. RESULTS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry investigation revealed the presence of compounds belonging to flavonoid, fatty acid and alkaloid groups. The viability of BRL 3A cells was reduced in a time-dose dependent manner treated by methanol extract of Zanthoxylum armatum DC. It increased LDH release rate and ROS production, activated the DNA double strand damage marker of γH2AX and produced comet cells. In addition, methanol extract of Zanthoxylum armatum DC caused ATM-mediated DNA damage, further phosphorylated Chk2, inhibited cell cycle related proteins, and arrested the G1/S cycle. CONCLUSIONS: Methanol extract of Zanthoxylum armatum DC induces DNA damage and further leads G1/S cell cycle arrest by triggering oxidative stress in the BRL 3A cells. This study provides some useful evidences for its development as an antitumor drug via activation of ATM/Chk2.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/efeitos dos fármacos , Extratos Vegetais/farmacologia , Zanthoxylum/química , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Sobrevivência Celular , Quinase do Ponto de Checagem 2/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/química , Ratos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
10.
ACS Appl Mater Interfaces ; 14(1): 20-31, 2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-34914354

RESUMO

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Pulmonares/metabolismo , Células A549 , Animais , Técnicas de Cultura de Células/instrumentação , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ácidos Graxos/metabolismo , Feminino , Humanos , Fenômenos Mecânicos , Metabolômica , Camundongos Endogâmicos BALB C , Camundongos Nus , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia
11.
Mol Med Rep ; 25(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34935054

RESUMO

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT­29 was examined. First, five solvent fractions [n­hexane, dichloromethane (DCM), ethyl acetate, n­butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose­ and time­dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT­29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose­dependent manner, which was associated with the protein expression levels of apoptosis­related proteins, including increased Fas, Bad and Bax, and decreased pro­caspase­8, Bcl­2, Bcl­xL, pro­caspase­9, pro­caspase­7 and pro­caspase­3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose­dependent manner in HT­29 cells treated with DCM. In addition, cell cycle analysis confirmed S­phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle­related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT­29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S­phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT­29 colorectal cells. Further research on the composition of the DCM fraction is warranted.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Calystegia/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Cloreto de Metileno/química
12.
Biochem Biophys Res Commun ; 584: 7-14, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34753066

RESUMO

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.


Assuntos
Aminoidrolases/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Administração Oral , Aminoidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Feminino , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Enzimas Multifuncionais/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
BMC Cancer ; 21(1): 1234, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34789190

RESUMO

BACKGROUND: ß-lapachone (ß-lap), the NQO1 bioactivatable drug, is thought to be a promising anticancer agent. However, the toxic side effects of ß-lap limit the drug use, highlighting the need for a thorough understanding of ß-lap's mechanism of action. ß-lap undergoes NQO1-dependent futile redox cycling, generating massive ROS and oxidative DNA lesions, leading to cell death. Thus, base excision repair (BER) pathway is an important resistance factor. XRCC1, a scaffolding component, plays a critical role in BER. METHODS: We knocked down XRCC1 expression by using pLVX-shXRCC1 in the MiaPaCa2 cells and BxPC3 cells and evaluated ß-lap-induced DNA lesions by γH2AX foci formation and alkaline comet assay. The cell death induced by XRCC1 knockdown + ß-lap treatment was analysed by relative survival, flow cytometry and Western blotting analysis. RESULTS: We found that knockdown of XRCC1 significantly increased ß-lap-induced DNA double-strand breaks, comet tail lengths and cell death in PDA cells. Furthermore, we observed combining XRCC1 knockdown with ß-lap treatment switched programmed necrosis with ß-lap monotherapy to caspase-dependent apoptosis. CONCLUSIONS: These results indicate that XRCC1 is involved in the repair of ß-lap-induced DNA damage, and XRCC1 loss amplifies sensitivity to ß-lap, suggesting targeting key components in BER pathways may have the potential to expand use and efficacy of ß-lap for gene-based therapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Quebras de DNA de Cadeia Dupla , Naftoquinonas/farmacologia , Neoplasias Pancreáticas/terapia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/deficiência , Antineoplásicos/efeitos adversos , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaio Cometa , Reparo do DNA , DNA de Neoplasias/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular , Histonas/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/efeitos adversos , Naftoquinonas/metabolismo , Necroptose/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribose) Polimerase-1/biossíntese , Pontos de Checagem da Fase S do Ciclo Celular
14.
Int J Mol Sci ; 22(20)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34681640

RESUMO

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.


Assuntos
Adenoma/patologia , Aldosterona/metabolismo , Proliferação de Células , ATPase Trocadora de Sódio-Potássio/genética , Adenoma/metabolismo , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Mutação , Ouabaína/farmacologia , Fosforilação/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcriptoma , Quinases da Família src/metabolismo
15.
J Inorg Biochem ; 225: 111622, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34624670

RESUMO

The studies of iridium (III) complexes as potent anticancer reagents have attracted great attention. Here, a new iridium (III) complex [Ir(bzq)2(PYIP)](PF6) (Ir1, bzq = benzo[h]quinoline, PYIP = 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) was synthesized and its liposomes (Ir1Lipo) was prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cytotoxic activity of Ir1 and Ir1Lipo on HepG2, SGC-7901, BEL-7402, HeLa, B16, A549 and normal NIH3T3 cells. The complex Ir1 displays no obvious inhibitory effect on the growth of BEL-7402 cells, while the Ir1Lipo shows significant cytotoxic activity on BEL-7402 cells (IC50 = 2.6 ± 0.03 µM). In further studies, Ir1Lipo induced apoptosis by the mitochondrial pathways, such as increasing intracellular reactive oxygen species (ROS) content and intracellular Ca2+ level, decreasing the mitochondrial membrane potential (MMP). In addition, after incubation with Ir1Lipo, the colony formation of BEL-7402 cells was significantly inhibited. Moreover, flow cytometry was used to detect the impact of Ir1Lipo on cell cycle distribution, and western blot was used to detect the expression of caspases and Bcl-2 (B-cell lymphoma-2) family proteins. Furthermore, Ir1Lipo exhibited significant antitumor activity in vivo with an inhibitory rate of 65.8%. These results indicated that Ir1Lipo induces apoptosis in BEL-7402 cells through intrinsic mitochondrial pathway.


Assuntos
Antineoplásicos/uso terapêutico , Complexos de Coordenação/uso terapêutico , Portadores de Fármacos/química , Lipossomos/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/farmacologia , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Irídio/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Células NIH 3T3 , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
BMC Cancer ; 21(1): 1088, 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625047

RESUMO

BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.


Assuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Isoquinolinas/farmacologia , NF-kappa B/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Antineoplásicos/uso terapêutico , Fator de Transcrição E2F1/antagonistas & inibidores , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Humanos , Isoquinolinas/uso terapêutico , Masculino , Pessoa de Meia-Idade , Células PC-3 , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Pontos de Checagem da Fase S do Ciclo Celular , Fatores de Tempo , Análise Serial de Tecidos
17.
J Inorg Biochem ; 225: 111616, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34555601

RESUMO

Ruthenium-containing complexes have emerged as good alternative to the currently used platinum-containing drugs for malignant tumor therapy. In this work, cytotoxic effects of recently synthesized ruthenium polypyridyl complexes [Ru(bpy)2(CFPIP)](ClO4)2 (bpy = 2,2'-bipyridine, CFPIP = (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ru(II)-1), [Ru(phen)2(CFPIP)](ClO4)2 (phen = 1,10-phenanthroline, Ru(II)-2) and [Ru(dmb)2(CFPIP)](ClO4)2 (dmb = 4,4'-dimethyl-2,2'-bipyridine, Ru(II)-3) toward different tumor cells were investigated in vitro and compared with cisplatin, the most widely used chemotherapeutic drug against hepatocellular carcinoma (HepG-2). The results demonstrate that target complexes show excellent cytotoxicity against HepG-2 cells with low IC50 value of 21.4 ± 1.5, 18.0 ± 2.1 and 22.3 ± 1.7 µM, respectively. It was important noting that target Ru(II) complexes exhibited better antitumor activity than cisplatin (IC50 = 28.5 ± 2.4 µM) against HepG-2 cells, and has no obvious toxicity to normal cell LO2. DNA binding results suggest that Ru(II)-1, Ru(II)-2 and Ru(II)-3 interact with CT DNA (calf thymus DNA) through intercalative mode. Complexes exerted its antitumor activity through increasing anti-migration and inducing cell cycle arrest at the S phase. In addition, the apoptosis was tested by AO (acridine orange)/EB (ethidium bromide) staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and colocalization tests were also evaluated by ImageXpress Micro XLS system. Overall, the results show that the ruthenium polypyridyl complexes induce apoptosis in HepG-2 cells through ROS-mediated mitochondria dysfunction pathway.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Piridinas/síntese química , Piridinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
18.
Blood Cells Mol Dis ; 91: 102594, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34520986

RESUMO

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.


Assuntos
Quimiocinas CXC/metabolismo , Células Eritroides/citologia , Granulócitos/citologia , Macrófagos/citologia , Células-Tronco/citologia , Animais , Proliferação de Células , Células Cultivadas , Células Eritroides/metabolismo , Granulócitos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Pontos de Checagem da Fase S do Ciclo Celular , Células-Tronco/metabolismo
19.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360807

RESUMO

This study investigated the roles of low-molecular-weight fucoidan (LMWF) in enhancing the anti-cancer effects of fluoropyrimidine-based chemotherapy. HCT116 and Caco-2 cells were treated with LMWF and 5-FU. Cell viability, cell cycle, apoptosis, and migration were analyzed in both cell types. Potential mechanisms underlying how LMWF enhances the anti-cancer effects of fluoropyrimidine-based chemotherapy were also explored. The cell viability of HCT116 and Caco-2 cells was significantly reduced after treatment with a LMWF--5FU combination. In HCT116 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through the (1) induction of cell cycle arrest in the S phase and (2) late apoptosis mediated by the Jun-N-terminal kinase (JNK) signaling pathway. In Caco-2 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through both the c-mesenchymal-epithelial transition (MET)/Kirsten rat sarcoma virus (KRAS)/extracellular signal-regulated kinase (ERK) and the c-MET/phosphatidyl-inositol 3-kinases (PI3K)/protein kinase B (AKT) signaling pathways. Moreover, LMWF enhanced the suppressive effects of 5-FU on tumor cell migration through the c-MET/matrix metalloproteinase (MMP)-2 signaling pathway in both HCT116 and Caco-2 cells. Our results demonstrated that LMWF is a potential complementary therapy for enhancing the efficacies of fluoropyrimidine-based chemotherapy in colorectal cancers (CRCs) with the wild-type or mutated KRAS gene through different mechanisms. However, in vivo studies and in clinical trials are required in order to validate the results of the present study.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células CACO-2 , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Células HCT116 , Humanos , Polissacarídeos/farmacologia
20.
Inorg Chem ; 60(17): 13051-13061, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34369147

RESUMO

The facile modification of the ligands in organometallic Ru(II)-arene complexes offers more opportunities to optimize their pharmacological profiles. Herein, three Ru(II)-arene complexes containing a glutathione S-transferase (GST) inhibitor (NBDHEX) in chelate ligand have been designed and synthesized in this study. In vitro results indicated that the ligation with NBDHEX significantly increased the activities and selectivities of the organometallic Ru(II)-arene complexes against tumor cells, especially complex 3, which was the most active compound among the tested compounds. DFT calculations and hydrolysis results demonstrated that complex 3 with more alkyl groups in the arene ligand has increased electron density at the Ru(II) center as compared with complexes 1 and 2, thus resulting in the improved hydrolysis rate, which may be responsible for its higher anticancer activity. Further studies showed that complexes 1-3 can cause the loss of the mitochondrial membrane potential and upregulate the expression of Bcl-2 and Bax in A549 cells, suggesting that complexes 1-3-induced cell death may be mediated via the mitochondrial apoptotic pathway. Thus, these findings suggested that simultaneous modification of the chelate ligands and arene rings in the organometallic Ru(II)-arene complexes is an effective way to improve their pharmacological properties.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
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