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1.
Nat Commun ; 11(1): 3904, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764536

RESUMO

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.


Assuntos
Autoantígenos/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Autoantígenos/genética , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Proteína Supressora de Tumor p53/genética , Fosfatases cdc25/metabolismo
2.
Chem Biol Interact ; 325: 109127, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437695

RESUMO

Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candidates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10 melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cytoplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle retention in G0/G1 phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126 was classified as UN GHS category 4 (LD50 > 300-2000 mg/kg), suggesting it has low acute systemic toxicity. LQFM126 can be a promising prototype for anticancer therapy.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Melanoma Experimental/patologia , Pirazóis/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Piperazinas/química , Pirazóis/síntese química , Pirazóis/química , Pirazóis/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo
3.
Int J Nanomedicine ; 15: 1997-2010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32273698

RESUMO

Background: As one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO2) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO2 toxicity under ultraviolet A (UVA) irradiation. Methods and Results: In the present study, the toxicity of nano-TiO2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 µg/mL nano-TiO2 under UVA irradiation after seven days of culture. Nano-TiO2 under UVA irradiation triggered the initiation of the TGF-ß/Smad signaling pathway, increasing the expression levels of TGF-ß1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-ß1 expression. Conclusion: Nano-TiO2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-ß/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Titânio/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Camundongos , Nanopartículas/química , Proteína Smad3/genética , Proteína Smad3/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/efeitos da radiação , Raios Ultravioleta
4.
Clin Sci (Lond) ; 134(7): 727-746, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32202295

RESUMO

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.


Assuntos
Envelhecimento/genética , Senescência Celular/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Endonucleases/deficiência , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Permeabilidade Capilar , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Superóxidos/metabolismo , Rigidez Vascular , Vasodilatação
5.
Clin Sci (Lond) ; 134(7): 889-905, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32219338

RESUMO

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.


Assuntos
Proliferação de Células , Senescência Celular , DNA Mitocondrial/metabolismo , Fibroblastos/enzimologia , Fibrose Pulmonar Idiopática/enzimologia , Pulmão/enzimologia , Nucleotidiltransferases/metabolismo , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Comunicação Parácrina , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nat Commun ; 11(1): 1335, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165639

RESUMO

Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16Ink4a/p19Arf (Cdkn2a) or p21Cip1 (Cdkn1a) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication.


Assuntos
Ciclo Celular , Senescência Celular , Interferons/metabolismo , Melanoma/imunologia , Melanoma/patologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Imunoterapia , Antígeno Ki-67/metabolismo , Linfonodos/patologia , Melanoma/terapia , Melanoma/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Análise de Sobrevida , Carga Tumoral
7.
Biochem Biophys Res Commun ; 524(3): 736-743, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32035614

RESUMO

Deferasirox (DFX) is an iron chelator approved for the treatment of iron overload diseases. However, the role of DFX in oxidative stress-induced cell apoptosis and the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this study, we found that DFX rendered resistant to H2O2-induced apoptosis in HEK293T cells, reduced the intracellular levels of the labile iron pool (LIP) and oxidative stress induced by H2O2. Furthermore, DFX inhibited the ubiquitination and degradation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) via modulation of the interaction of p21 with SCF-Skp2. DFX also showed the inhibition effect on the activation of c-Jun N-terminal kinase (JNK), pro-caspase-3 and related mitochondrial apoptosis pathway induced by H2O2. These results provide novel insights into the molecular mechanism underpinning iron-mediated oxidative stress and apoptosis, and they may represent a promising target for therapeutic interventions in related pathological conditions.


Assuntos
Apoptose/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoproteção/efeitos dos fármacos , Deferasirox/farmacologia , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Caspase 3/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio , Ferro/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Regulação para Cima/efeitos dos fármacos
8.
Cancer Genomics Proteomics ; 17(2): 169-174, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32108039

RESUMO

BACKGROUND/AIM: In this study, we aimed to investigate the prognostic role of a previously identified panel of 10 stem cell markers stratified against the catalytic subunit of telomerase (hTERT) in human breast cancer. MATERIALS AND METHODS: The mRNA copy numbers of these genes were determined using real time quantitative PCR in 124 breast cancer tissues and adjacent non-cancerous tissues. Relations between mRNA levels and survival were analysed using Kaplan-Meier plots and Cox regression analysis. RESULTS: Five genes (BMI1, NES, POU5F1, ALDH1A2 and CDKN1A) correlated with survival when stratified with hTERT and predicted overall (Wilcoxon: p=0.004; Cox: p=0.006) and disease-free (Wilcoxon: p<0.000; Cox: p=0.000) survival. CONCLUSION: This panel of genes stratified by hTERT could open new avenues for the development of new prognostic tools, as well as for the identification of new research directions regarding breast oncogenesis.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Telomerase/genética , Aldeído Desidrogenase 1/genética , Aldeído Desidrogenase 1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Humanos , Nestina/genética , Nestina/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Análise de Sobrevida , Taxa de Sobrevida , Telomerase/metabolismo
9.
J Biol Chem ; 295(10): 3055-3063, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32001619

RESUMO

In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.


Assuntos
Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
10.
FASEB J ; 34(2): 3413-3428, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31922321

RESUMO

Ror2 signaling has been shown to regulate the cell cycle progression in normal and cancer cells. However, the molecular mechanism of the cell cycle progression upon activation of Ror2 signaling still remains unknown. Here, we found that the expression levels of Ror2 in G1-arrested NIH/3T3 fibroblasts are low and are rapidly increased following the cell cycle progression induced by basic fibroblast growth factor (bFGF) stimulation. By expressing wild-type or a dominant negative mutant of E2F1, we show that E2F1 mediates bFGF-induced expression of Ror2, and that E2F1 binds to the promoter of the Ror2 gene to activate its expression. We also found that G1/S phase transition of bFGF-stimulated NIH/3T3 cells is delayed by the suppressed expression of Ror2. RNA-seq analysis revealed that the suppressed expression of Ror2 results in the decreased expression of various E2F target genes concomitantly with increased expression of Forkhead box O (FoxO) target genes, including p21Cip1 , and p27Kip1 . Moreover, the inhibitory effect of Ror2 knockdown on the cell cycle progression can be restored by suppressed expression of p21Cip1 , p27Kip1 ,or FoxO3a. Collectively, these findings indicate that E2F1-Ror2 signaling mediates the transcriptional activation and inhibition of E2F1-driven and FoxO3a-driven cell cycle-regulated genes, respectively, thereby promoting G1/S phase transition of bFGF-stimulated NIH/3T3 cells.


Assuntos
Fator de Transcrição E2F1/metabolismo , Fase G1 , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Fase S , Animais , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Transcrição E2F1/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteína Forkhead Box O3/metabolismo , Camundongos , Células NIH 3T3 , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais
11.
Int J Mol Sci ; 21(1)2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31948013

RESUMO

Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-ß-galactosidase (SA-ß-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.


Assuntos
Senescência Celular/efeitos dos fármacos , Exossomos/metabolismo , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Nanopartículas/metabolismo , Engenharia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Transmissão , Nanopartículas/uso terapêutico , Nanopartículas/ultraestrutura , Envelhecimento da Pele/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Biochem Biophys Res Commun ; 523(4): 1046-1052, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31973811

RESUMO

Although tissue aging is accompanied with cellular senescence, it is much complicated than senescence given both types and number of cells change with age. Alternative polyadenylation (APA) had shown tissue specificity and APA-mediated 3' untranslated region (3' UTR) lengthening could regulate senescence-associated phenotypes. However, whether tissue aging shows similar trends remains unknown. Here, we performed a comprehensive analysis on RNA-seq datasets derived from multiple cells and rat tissues of young and old age. Although APA-mediated 3' UTR lengthening in various senescent cells reinforced the previous discovery, tissue aging showed much more complexity in APA. Interestingly, testis was the only tissue displaying dramatic 3' UTR lengthening and decreased expression trend of corresponding genes in aged rat. Genes with longer 3' UTR in aged testis were enriched in senescence-associated pathways, among which, Mdm2, encoding an E3 ligase of p53, favored distal poly(A) site resulting in lengthened 3' UTR and decreased expression. Longer 3' UTR of Mdm2 generated less protein, and decreased Mdm2 expression led to senescence-associated phenotypes along with increased p53 and p21 protein abundance, which could all be reversed by Mdm2 overexpression. Our work revealed complicated APA changes during tissue aging and discovered APA-mediated 3' UTR lengthening of Mdm2 is a hidden layer in regulating the well-known senescence-related p53-p21 signal axis during testis aging, and also has potential implications regarding declined male fertility along aging.


Assuntos
Regiões 3' não Traduzidas/genética , Envelhecimento/metabolismo , Senescência Celular , Poliadenilação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Testículo/citologia , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Humanos , Masculino , Fenótipo , Ratos , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo
13.
Cancer Chemother Pharmacol ; 85(2): 273-284, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907647

RESUMO

OBJECTIVE: Senescence mechanisms are vital to resistance to long-term olaparib maintenance treatment. Recently, peroxisome proliferator-activated receptor-γ agonists (e.g., rosiglitazone) have been reported to ameliorate the senescence-like phenotype by modulating inflammatory mediator production. This study examined synergistic effects on the anti-tumor activity of rosiglitazone combined with olaparib in ovarian cancer treatment. METHODS: A2780 and SKOV3 mouse subcutaneous xenograft models were established for observing anti-tumor effects in living organisms and were randomly split into combination (both olaparib and rosiglitazone), rosiglitazone (10 mg/kg), olaparib (10 mg/kg), control (solvent) groups that received treatment once every 2 or 3 days (n = 6 per group). Cell counting kit-8 (CCK-8) assays were used to test the influences of rosiglitazone and olaparib on cell proliferation. PI and Annexin-V-FITC staining was used with flow cytometry to assess the cell cycle distribution and cell apoptosis. Senescence-associated ß-galactosidase (SA-ß-Gal) staining was used to observe cellular senescence. We performed quantitative real-time polymerase chain reaction assays to study the senescence-related secretory phenotype (SASP). RESULTS: Olaparib and rosiglitazone were observed to synergistically retard subcutaneous ovarian cancer growth in vivo, and synergistically suppress ovarian cancer cell proliferation in vitro. Compared with olaparib alone, the percentage of positive cells expressed SA-ß-gal and SASP were significantly decreased in the treatment of combination of olaparib and rosiglitazone. Furthermore, olaparib plus rosiglitazone increased the percentage of apoptosis in ovarian cancer cell compared with olaparib alone. In A2780 cells, it showed lower expression of P53, phospho-p53 (Ser15), P21, and P18 protein in combination treatment compared with olaparib alone. While, in SKOV3 cells, it showed lower expression of phosphor-retinoblastoma protein (Rb) (Ser807/811), and higher expression of cyclin D1, P21, and P16 protein in combination treatment compared with olaparib alone. CONCLUSIONS: Rosiglitazone combined with olaparib can help manage ovarian cancer by ameliorating olaparib-induced senescence and improving anti-tumor effects.


Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Neoplasias Ovarianas/induzido quimicamente , Neoplasias Ovarianas/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rosiglitazona/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
14.
Med Sci Monit ; 26: e920266, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31919338

RESUMO

BACKGROUND Prostate cancer, non-cutaneous malignant tumor, is the second common cause of cancer related mortalities in American men and is responsible for 13% of deaths related to cancer. The present study investigated the anti-cancer effects of 3,6-diazabicyclo[3.3.1]heptane on LNCaP and PC3 prostate cancer cells in vitro and on tumor growth in vivo in BALB/C nude mice. MATERIAL AND METHODS Reduction of cell viability by 3,6-diazabicyclo[3.3.1]heptane was evaluated by sulphorhodamine-B staining and apoptosis onset using annexin V and propidium iodide (PI) staining. The 2',7'-dichlorofluorescein-diacetate stain was used for assessment of reactive oxygen species (ROS) formation while as western blotting for analysis of protein expression. RESULTS The viability of LNCaP and PC3 cells was reduced significantly (P<0.05) by 3,6-diazabicyclo[3.3.1]heptane in dose-based manner. At 30 µM of 3,6-diazabicyclo[3.3.1]heptane the viability of LNCaP and PC3 cells was reduced to 32 and 28%, respectively. The 3,6-diazabicyclo[3.3.1]heptane treatment increased apoptosis in LNCaP cells to 43.31% at 30 µM. The cell cycle in LNCaP cells was arrested in G1 phase on treatment with 3,6-diazabicyclo[3.3.1]heptane. The expression of cyclin D1 and p21 proteins was significantly increased by 3,6-diazabicyclo[3.3.1]heptane in LNCaP and PC3 cells. The growth of prostate tumor was also suppressed in vivo in mice by 3,6-diazabicyclo[3.3.1]heptane treatment. CONCLUSIONS In summary, the study demonstrated that LNCaP and PC3 prostate cancer cell viability is suppressed by 3,6-diazabicyclo[3.3.1]heptane treatment. The suppression of prostate cancer cell viability by 3,6-diazabicyclo[3.3.1]heptane involves apoptosis induction, cell cycle arrest and upregulation of p21 expression. Therefore, 3,6-diazabicyclo[3.3.1]heptane can be a potential chemotherapeutic agent for prostate cancer.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Heptanos/farmacologia , Neoplasias da Próstata/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células PC-3/efeitos dos fármacos , Próstata/metabolismo
15.
Dis Markers ; 2020: 6520259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31998417

RESUMO

Background: Previous studies have demonstrated that P21 (WAF1/CIP1) is a valuable prognostic factor in several malignant tumors. However, it is not known whether P21 can predict the prognosis in patients with esophageal cancer (EC). The aim of this research was to investigate the contribution of P21 expression to the clinicopathological characteristics and of EC. Methods: A systematic review and meta-analysis of study focusing on P21 expression, clinicopathological characteristics, and clinical outcomes in patients with EC was performed using seven databases (PubMed, Embase, Web of Science, and four Chinese databases). Pooled hazard ratios and odds ratios were used to explore the association between P21 expression, clinicopathological characteristics, and outcomes in patients with EC. The heterogeneity of the studies was classified by the I 2 statistic. The sensitivity analysis was then utilized to assess the robustness of the results. Finally, the funnel plot and Begg's test were used to evaluate the publication bias. Results: Forty-five studies with 3098 patients were eligible for inclusion in the meta-analysis. Thirty of these studies reported on clinicopathological characteristics and 15 on clinical outcomes. The pooled hazard ratio of 1.456 (95% confidence intervals 1.033-2.053, P = 0.032) for overall survival indicated that a low P21 expression level was an unfavorable prognostic factor for a clinical outcome in patients with EC. Furthermore, the pooled odds ratio confirmed an association between decreased P21 expression and poor clinicopathological characteristics, including differentiation, lymph node metastasis, invasion, and higher grade and clinical stage. Notably, high P21 expression was a significant predictor of a favorable response to chemotherapy. There was no evidence of publication bias. Conclusion: Reduced P21 expression is associated with a poor outcome in patients with EC.


Assuntos
Biomarcadores Tumorais/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Esofágicas/metabolismo , Biomarcadores Tumorais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Metástase Neoplásica
16.
Life Sci ; 244: 117342, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978450

RESUMO

AIMS: Microvascular endothelial cell dysfunction is a leading cause of radiation-induced heart disease (RIHD). BRCA1 plays an important role in DNA damage repair. The study aims to explore the effect of BRCA1 in endothelial cells involved in RIHD. MATERIALS AND METHODS: BRCA1 and p21 expression were detected in human umbilical vein endothelial cells (HUVECs) and in mouse heart tissue after irradiation exposure. The effects of BRCA1 on cell proliferation, cell cycle and radiosensitivity were determined in HUVECs with overexpression and knockdown of BRCA1. A mouse model of RIHD was established. Heart damage was detected in C57BL/6J mice and endothelial cell specific knockout BRCA1 mice (EC-BRCA1-/-). KEY FINDINGS: BRCA1 and p21 expression was significantly increased both in vitro and vivo response to irradiation. BRCA1 overexpression in endothelial cells enhanced cell growth and G1/S phase arrest, and the opposite results were observed in BRCA1 knockdown endothelial cells. BRCA1 downregulated endothelial cell cycle-related genes cyclin A, cyclin D1, cyclin E and p-Rb through increasing p21 expression, and HUVECs with BRCA1 gene knockdown were more sensitive to radiation. In vivo, a decrease in cardiac microvascular density, as well as cardiomyocyte hypoxia and apoptosis were observed in a time-dependent manner. EC-BRCA1-/- mice were more prone to severe RIHD than EC-BRCA1+/- mice after 16Gy radiation exposure due to endothelial dysfunction caused by loss of BRCA1, and p21 was declined in EC-BRCA1-/- mice heart. SIGNIFICANCE: These findings indicate that BRCA1 plays a protective role in RIHD by regulating endothelial cell cycle arrest mediated by p21 signal.


Assuntos
Proteína BRCA1/metabolismo , Pontos de Checagem do Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Neovascularização Patológica/prevenção & controle , Substâncias Protetoras/administração & dosagem , Tolerância a Radiação , Animais , Proteína BRCA1/genética , Proteína BRCA1/fisiologia , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/etiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Radiação Ionizante
17.
PLoS Genet ; 16(1): e1008580, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31940341

RESUMO

RNA-binding proteins (RBPs) associate with the primary, precursor, and mature microRNAs, which in turn control post-transcriptional gene regulation. Here, by small RNAseq, we show that RBP FXR1 controls the expression of a subset of mature miRNAs, including highly expressed miR301a-3p in oral cancer cells. We also confirm that FXR1 controls the stability of miR301a-3p. Exoribonuclease PNPT1 degrades miR301a-3p in the absence of FXR1 in oral cancer cells, and the degradation is rescued in the FXR1 and PNPT1 co-knockdown cells. In vitro, we show that PNPT1 is unable to bind and degrade the miRNA once the FXR1-miRNA complex forms. Both miR301a-3p and FXR1 cooperatively target the 3'-UTR of p21 mRNA to promote its degradation. Thus, our work illustrates the unique role of FXR1 that is critical for the stability of a subset of mature miRNAs or at least miR301a-3p to target p21 in oral cancer.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , MicroRNAs/genética , Neoplasias Bucais/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/genética
18.
Life Sci ; 241: 117134, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31811854

RESUMO

AIMS: Non-small cell lung cancer (NSCLC), characterized by extensive metastasis and poor prognosis, is the most common type of lung cancer. Dysregulation of certain lncRNAs is known to be linked to the tumorigenesis of NSCLC. However, the specific roles in NSCLC for many other lncRNAs, such as linc01088, remain largely unknown. MATERIALS AND METHODS: The expression patterns of linc01088, p21 and EZH2 were examined both in NSCLC tissues and cell lines using RT-qPCR assay. CCK-8, colony formation, immunofluorescence staining, and flow cytometry assays were employed to evaluate the effects of linc01088 on NSCLC cell proliferation properties. RNA immunoprecipitation (RIP) assay was performed to determine the direct binding relationship between linc01088 and zeste homolog 2 (EZH2). Western blot and RT-qPCR analysis were performed to assess p21 level within knockdown of either linc01088 or EZH2. Nude mouse subcutaneous NSCLC models were constructed for further validating the effects and mechanisms of linc01088 in vivo. KEY FINDINGS: linc01088 and EZH2 were highly expressed both in NSCLC tissues and cell lines. Knockdown of linc01088 suppressed the proliferation of NSCLC cells, and prolonged the G1 phase while shortened S and G2-M phases. RIP assay revealed the direct binding relationship between linc01088 and EZH2. Knockdown of either linc01088 or EZH2 induced up-regulation of p21 expression, which subsequently inhibited the tumor growth. SIGNIFICANCE: We demonstrated that linc01088 could promote cell proliferation via binding with EZH2 to repress p21, which aggravates the tumorigenesis of NSCLC. Therefore, linc01088 might be a potential oncogene and target for novel anti-tumor therapies.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biomed Pharmacother ; 121: 109640, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31810114

RESUMO

Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling, which ultimately leads to pulmonary arterial hypertension (PAH). Osthole has been previously shown to inhibit tumor cell growth. Our previous experiments demonstrated that osthole could prevent monocrotaline-induced PAH and pulmonary artery remodeling in rats and that its effects might be associated with inhibiting PASMC proliferation. However, the exact mechanism remains unclear. In this study, we observed the inhibitory effect of osthole on platelet-derived growth factor (PDGF)-BB-induced rat PASMC growth, cell cycle progression and proliferating cell nuclear antigen (PCNA) expression, as measured by CCK-8 assay, flow cytometric analysis and western blotting, respectively. We also detected the expression and activities of the cell cycle regulators cyclin D1/CDK4, cyclin E1/CDK2, p53, p27 and p21 and the TGF-ß1/Smad/p38 signaling pathways in rat PASMCs by western blotting. Our results show that osthole effectively suppressed PDGF-BB-stimulated proliferation, PCNA protein expression, and cell cycle progression in rat PASMCs in vitro. We further demonstrated that treatment with osthole significantly induced cell cycle arrest at the G0/G1 phase in PASMCs, which was supported by the finding that osthole significantly decreased cyclin D1/CDK4 and cyclin E1/CDK2 protein levels and increased p53, p27 and p21 protein levels. These effects may partly be attributed to the downregulation of TGF-ß1/Smad/p38 signaling pathway activation. Our findings suggest that osthole is a potential therapeutic candidate that warrants further investigation regarding its potential use for the treatment of PAH.


Assuntos
Cumarínicos/farmacologia , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
20.
J Exp Clin Cancer Res ; 38(1): 490, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831018

RESUMO

BACKGROUND: N-myc downstream-regulated gene 1 (NDRG1) has been shown to play a key role in tumor metastasis. Recent studies demonstrate that NDRG1 can suppress tumor growth and is related to tumor proliferation; however, the mechanisms underlying these effects remain obscure. METHODS: Immunohistochemistry (IHC) was used to detect NDRG1 and p21 protein expression in colorectal cancer tissue, and clinical significance of NDRG1 was also analyzed. CCK-8 assay, colony formation assay, flow cytometry, and xenograft model were used to assess the effect of NDRG1 on tumor proliferation in vivo and in vitro. The mechanisms underlying the effect of NDRG1 were investigated using western blotting, immunofluorescence, immunoprecipitation, and ubiquitylation assay. RESULTS: NDRG1 was down-regulated in CRC tissues and correlated with tumor size and patient survival. NDRG1 inhibited tumor proliferation through increasing p21 expression via suppressing p21 ubiquitylation. NDRG1 and p21 had a positive correlation both in vivo and in vitro. Mechanistically, E3 ligase NEDD4 could directly interact with and target p21 for degradation. Moreover, NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21, increasing p21 expression and inhibit tumor proliferation. CONCLUSION: Our study could fulfill potential mechanisms of the NDRG1 during tumorigenesis and metastasis, which may serve as a tumor suppressor and potential target for new therapies in human colorectal cancer.


Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Prognóstico , Proteólise , Análise de Sobrevida , Carga Tumoral , Ubiquitinação
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