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1.
J Cell Sci ; 135(8)2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35343565

RESUMO

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.


Assuntos
Ciclina D1 , Proteína Supressora de Tumor p53 , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Fosforilação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
Int J Mol Sci ; 25(17)2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39273527

RESUMO

NME6 belongs to the family of nucleoside diphosphate kinase enzymes, whose major role is to transfer the terminal phosphate from NTPs, mostly ATP, to other (d)NDPs via a high-energy intermediate. Beside this basic enzymatic activity, the family, comprising 10 genes/proteins in humans, executes a number of diverse biochemical/biological functions in the cell. A few previous studies have reported that NME6 resides in the mitochondria and influences oxidative phosphorylation while interacting with RCC1L, a GTPase involved in mitochondrial ribosome assembly and translation. Considering the multifunctional role of NME family members, the goal of the present study was to assess the influence of the overexpression or silencing of NME6 on fundamental cellular events of MDA-MB-231T metastatic breast cancer cells. Using flow cytometry, Western blotting, and a wound-healing assay, we demonstrated that the overexpression of NME6 reduces cell migration and alters the expression of EMT (epithelial-mesenchymal transition) markers. In addition, NME6 overexpression influences cell cycle distribution exclusively upon DNA damage and impacts the MAPK/ERK signaling pathway, while it has no effect on apoptosis. To conclude, our results demonstrate that NME6 is involved in different cellular processes, providing a solid basis for future, more precise investigations of its role.


Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Mitocôndrias , Humanos , Linhagem Celular Tumoral , Mitocôndrias/metabolismo , Mitocôndrias/genética , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Apoptose/genética , Nucleosídeo Difosfato Quinase D/metabolismo , Nucleosídeo Difosfato Quinase D/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Ciclo Celular/genética
3.
Int J Mol Sci ; 24(1)2022 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-36613518

RESUMO

Metastatic melanoma is one of the most aggressive tumors, with frequent mutations affecting components of the MAPK pathway, mainly protein kinase BRAF. Despite promising initial response to BRAF inhibitors, melanoma progresses due to development of resistance. In addition to frequent reactivation of MAPK or activation of PI3K/AKT signaling pathways, recently, the p53 pathway has been shown to contribute to acquired resistance to targeted MAPK inhibitor therapy. Canonical tumor suppressor p53 is inactivated in melanoma by diverse mechanisms. The TP53 gene and two other family members, TP63 and TP73, encode numerous protein isoforms that exhibit diverse functions during tumorigenesis. The p53 family isoforms can be produced by usage of alternative promoters and/or splicing on the C- and N-terminus. Various p53 family isoforms are expressed in melanoma cell lines and tumor samples, and several of them have already shown to have specific functions in melanoma, affecting proliferation, survival, metastatic potential, invasion, migration, and response to therapy. Of special interest are p53 family isoforms with increased expression and direct involvement in acquired resistance to MAPK inhibitors in melanoma cells, implying that modulating their expression or targeting their functional pathways could be a potential therapeutic strategy to overcome resistance to MAPK inhibitors in melanoma.


Assuntos
Melanoma , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Genes p53 , Proteínas Proto-Oncogênicas B-raf/genética , Fosfatidilinositol 3-Quinases/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral
4.
Int J Mol Sci ; 23(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36077308

RESUMO

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico
5.
Carcinogenesis ; 37(12): 1161-1169, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27655834

RESUMO

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.


Assuntos
Acetofenonas/administração & dosagem , Adenocarcinoma/metabolismo , Esôfago de Barrett/metabolismo , Neoplasias Esofágicas/metabolismo , Refluxo Gastroesofágico/metabolismo , Ácidos/efeitos adversos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Antioxidantes/administração & dosagem , Proteína BRCA1/biossíntese , Esôfago de Barrett/tratamento farmacológico , Esôfago de Barrett/etiologia , Esôfago de Barrett/patologia , Ácidos e Sais Biliares/efeitos adversos , Ácidos e Sais Biliares/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/etiologia , Neoplasias Esofágicas/patologia , Ácido Gástrico/metabolismo , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/patologia , Humanos , Espécies Reativas de Oxigênio/metabolismo
6.
Carcinogenesis ; 34(3): 522-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23188674

RESUMO

The p53 activities are due, at least in part, to its ability to form oligomers that bind to specific DNA sequences and activate transcription. Since some mutant p53 proteins and ΔNp73 isoforms form heterocomplexes with TAp73, we asked whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73. Moreover, it has already been found that some isoforms form complex with wtp53 and some of them inhibit p53 tumor-suppressor functions. Therefore, we studied the complex formation and co-immunoprecipitation assays show that all six p53 isoforms examined can form complexes with TAp73ß, whereas only Δ133p53α/ß/γ isoforms form complex with TAp73α. All p53 isoforms counteract TAp73ß transactivation function but with different efficiency and in a promoter-dependent manner. Furthermore, apoptotic activity of TAp73ß was augmented by coexpression of p53ß, whereas Δ133p53α and ß inhibit its apoptotic activity most efficiently. We have determined the half-life of different p53 isoforms: p53γ isoform has the shortest half-life, whereas Δ133p53γ has the longest half-life. Inhibitory interactions of two proteins in complex often lead to their stabilization. However, only three isoforms (Δ133p53α, Δ133p53ß and Δ40p53α) stabilize TAp73ß. We are convinced that defining the interactions between p53/p73 would give a new insight into how the p53 isoforms modulate the p73 functions in tumorigenesis.


Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Meia-Vida , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Transcrição Gênica , Proteína Tumoral p73
7.
Life (Basel) ; 13(12)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38137892

RESUMO

Diabetes mellitus is a complex metabolic disease associated with reduced synaptic plasticity, atrophy of the hippocampus, and cognitive decline. Cognitive impairment results from several pathological mechanisms, including increased levels of advanced glycation end products (AGEs) and their receptors, prolonged oxidative stress and impaired activity of endogenous mechanisms of antioxidant defense, neuroinflammation driven by the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), decreased expression of brain-derived neurotrophic factor (BDNF), and disturbance of signaling pathways involved in neuronal survival and cognitive functioning. There is increasing evidence that dietary interventions can reduce the risk of various diabetic complications. In this context, flavonols, a highly abundant class of flavonoids in the human diet, are appreciated as a potential pharmacological intervention against cognitive decline in diabetes. In preclinical studies, flavonols have shown neuroprotective, antioxidative, anti-inflammatory, and memory-enhancing properties based on their ability to regulate glucose levels, attenuate oxidative stress and inflammation, promote the expression of neurotrophic factors, and regulate signaling pathways. The present review gives an overview of the molecular mechanisms involved in diabetes-induced cognitive dysfunctions and the results of preclinical studies showing that flavonols have the ability to alleviate cognitive impairment. Although the results from animal studies are promising, clinical and epidemiological studies are still needed to advance our knowledge on the potential of flavonols to improve cognitive decline in diabetic patients.

8.
Cancers (Basel) ; 13(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207603

RESUMO

The p53 tumor suppressor protein is crucial for cell growth control and the maintenance of genomic stability. Later discovered, p63 and p73 share structural and functional similarity with p53. To understand the p53 pathways more profoundly, all family members should be considered. Each family member possesses two promoters and alternative translation initiation sites, and they undergo alternative splicing, generating multiple isoforms. The resulting isoforms have important roles in carcinogenesis, while their expression is dysregulated in several human tumors including colorectal carcinoma, which makes them potential targets in cancer treatment. Their activities arise, at least in part, from the ability to form tetramers that bind to specific DNA sequences and activate the transcription of target genes. In this review, we summarize the current understanding of the biological activities and regulation of the p53/p73 isoforms, highlighting their role in colorectal tumorigenesis. The analysis of the expression patterns of the p53/p73 isoforms in human cancers provides an important step in the improvement of cancer therapy. Furthermore, the interactions among the p53 family members which could modulate normal functions of the canonical p53 in tumor tissue are described. Lastly, we emphasize the importance of clinical studies to assess the significance of combining the deregulation of different members of the p53 family to define the outcome of the disease.

9.
Mater Sci Eng C Mater Biol Appl ; 91: 486-495, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30033280

RESUMO

Gold nanoparticles (AuNPs) were synthesized in the presence of citrate (Au-CIT), glutathione (Au-GSH) and aminodextran (Au-DEX) in order to modify AuNPs surfaces and to increase their cellular uptake in the breast cancer cells MDA-MB-231. AuNPs were characterized with respect to their particle size, shape and colloidal stability in an aqueous solution and cell media. The mass accumulation of each AuNP type inside cancer cells was determined quantitatively, using Inductive Coupled Plasma - mass spectroscopy. The sub-cellular accumulation was studied using Transmission Electron Microscopy (TEM). It was found that gold nanoparticles applied to cancer cells were localized in cytoplasmic vesicles and that the highest uptake was shown in the presence of Au-GSH nanoparticles. The effect of AuNPs on the cell cycle was investigated using flow cytometry and Western blot analysis. The gold nanoparticles alone did not affect the cell cycle, as shown by flow cytometry. Furthermore, the cancer cells were irradiated using conventional clinically relevant high-energy X-ray radiation of 6 MV in the dose of 4 Gy. The results on cells only irradiated showed an S phase arrest six and 8 h after irradiation, and a G2/M arrest 24 and 48 h after irradiation. The irradiation of breast cancer cells treated with AuNPs has shown no significant variation in cell cycle distribution as opposed to X-ray radiation alone.


Assuntos
Neoplasias da Mama/patologia , Ciclo Celular , Ouro/química , Nanopartículas Metálicas/química , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Endocitose/efeitos dos fármacos , Feminino , Humanos , Hidrodinâmica , Nanopartículas Metálicas/ultraestrutura , Espectrofotometria Ultravioleta , Frações Subcelulares/metabolismo , Raios X
10.
Oncogene ; 37(37): 5054-5065, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29849123

RESUMO

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.


Assuntos
Autofagia/fisiologia , Células Epiteliais/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Proteína Supressora de Tumor p14ARF/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Células HCT116 , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Humanos , Transdução de Sinais/fisiologia , Estômago/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima/fisiologia , Fatores de Virulência/metabolismo
11.
Sci Rep ; 7(1): 9956, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855537

RESUMO

Gastroesophageal reflux disease (GERD) is the strongest known risk factor for esophageal adenocarcinoma. In the center of tumorigenic events caused by GERD is repeated damage of esophageal tissues by the refluxate. In this study, we focused on a genotoxic aspect of exposure of esophageal cells to acidic bile reflux (BA/A). Analyzing cells generated from patients with Barrett's esophagus and human esophageal specimens, we found that BA/A cause significant DNA damage that is mediated by reactive-oxygen species. ROS originate from mitochondria and NADPH oxidases. We specifically identified NOX1 and NOX2 enzymes to be responsible for ROS generation. Inhibition of NOX2 and NOX1 with siRNA or chemical inhibitors significantly suppresses ROS production and DNA damage induced by BA/A. Mechanistically, our data showed that exposure of esophageal cells to acidic bile salts induces phosphorylation of the p47phox subunit of NOX2 and its translocation to the cellular membrane. This process is mediated by protein kinase C, which is activated by BA/A. Taken together, our studies suggest that inhibition of ROS induced by reflux can be a useful strategy for preventing DNA damage and decreasing the risk of tumorigenic transformation caused by GERD.


Assuntos
Esôfago de Barrett/patologia , Dano ao DNA , Células Epiteliais/patologia , NADPH Oxidase 1/metabolismo , NADPH Oxidase 2/metabolismo , Ácidos e Sais Biliares/toxicidade , Células Cultivadas , Humanos , Espécies Reativas de Oxigênio/toxicidade
13.
Curr Pharm Des ; 17(6): 591-602, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21391909

RESUMO

The p53 family consists of three members: p53, p63 and p73. All three of them have a role in cell cycle arrest and induction of apoptosis. However, despite structural and partly functional similarity, there are striking differences in their functions and each of them has its own and unique identity. All three genes encode multiple variants with opposing functions in cancer development - full length transactivation forms with proapoptotic and antiproliferative functions, and dominant-negative transactivation-deficient forms with anti-apoptotic (oncogenic) functions. The functional interactions between family members are crucial to gain insight and understand their role in cancer biology. The discovery of p53/p73 network could have a major clinical impact in prognostic use and targeted drug design. In the current review we present the recent achievements in p73 research including very complex and sophisticated p73 regulation and response to DNA damage, and functional interactions among family members. We discuss how p73 has affected drug discovery. According to the p73 tumor suppressor function, we outline current aspects of anticancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Humanos , Terapia de Alvo Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores
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