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1.
Anticancer Res ; 41(7): 3459-3470, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230141

RESUMO

BACKGROUND/AIM: Studies have reported that the expression of c-Met and PrPC improves tumor progression. However, not much is known about their relationship. We hypothesized that c-Met and PrPC interact with each other, and enhance cancer stem cell (CSC) characteristics. MATERIALS AND METHODS: Magnetic activated cell sorting was used to examine the interaction between c-Met and PrPC The effects of the interaction on downstream signals, stem cell marker expression, and sphere formation of colorectal cancer (CRC) cells were investigated. RESULTS: We demonstrated the increased expression and binding levels of c-Met and PrPC in CRC cells compared to normal colon epithelial cells. We revealed that the c-Met and PrPC interaction induced the ERK activation and Oct4 upregulation. The inhibition of c-Met by crizotinib reduced ERK activation and Oct4 expression and suppressed CSC properties. CONCLUSION: c-Met and PrPC interact with each other, and targeting c-Met using crizotinib could be a powerful strategy for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas PrPC/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Crizotinibe/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
2.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208226

RESUMO

We investigated the role of nuclear factor of activated T cells 5 (NFAT5) under hyperosmotic conditions in human lens epithelial cells (HLECs). Hyperosmotic stress decreased the viability of human lens epithelial B-3 cells and significantly increased NFAT5 expression. Hyperosmotic stress-induced cell death occurred to a greater extent in NFAT5-knockout (KO) cells than in NFAT5 wild-type (NFAT5 WT) cells. Bcl-2 and Bcl-xl expression was down-regulated in NFAT5 WT cells and NFAT5 KO cells under hyperosmotic stress. Pre-treatment with a necroptosis inhibitor (necrostatin-1) significantly blocked hyperosmotic stress-induced death of NFAT5 KO cells, but not of NFAT5 WT cells. The phosphorylation levels of receptor-interacting protein kinase 1 (RIP1) and RIP3, which indicate the occurrence of necroptosis, were up-regulated in NFAT5 KO cells, suggesting that death of these cells is predominantly related to the necroptosis pathway. This finding is the first to report that necroptosis occurs when lens epithelial cells are exposed to hyperosmolar conditions, and that NFAT5 is involved in this process.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Cristalino/patologia , Pressão Osmótica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Soluções Hipertônicas/toxicidade , Inflamação/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pressão Osmótica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Estresse Fisiológico/efeitos dos fármacos
3.
Viruses ; 13(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202029

RESUMO

The current COVID-19 pandemic has highlighted the urgent need to develop effective therapeutic strategies. We evaluated the in vitro antiviral effect against SARS-CoV-2 of a hepatitis B virus (HBV) hexamer peptide, Poly6, which is capable of eliciting an antiviral effect against human immunodeficiency virus -1 (HIV-1), as a novel HIV-1 integrase inhibitor, and a strong anticancer immune response in an IFN-I-dependent manner, as a novel potential adjuvant in anticancer immunotherapy. Here, we report that Poly6 exerts an anti-SARS-CoV-2 effect, with an estimated 50% inhibitory concentration of 2.617 µM, in the human bronchial epithelial cell line, Calu-3 but not in Vero-E6 cells, which are deficient in type 1 interferon (IFN-I) signaling. We proved via assays based on mRNA profiles, inhibitors, or blocking antibodies that Poly6 can exert an anti-SARS-CoV-2 effect in an IFN-I-dependent manner. We also found that Poly6 inhibits IL-6 production enhanced by SARS-CoV-2 in infected Calu-3 cells at both the transcription and the translation levels, mediated via IL-10 induction in an IFN-I-dependent manner. These results indicate the feasibility of Poly6 as an IFN-I-inducing COVID-19 drug with potent antiviral and anti-inflammatory activities.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Vírus da Hepatite B/química , Interferon Tipo I/imunologia , Peptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Brônquios/citologia , Brônquios/virologia , Chlorocebus aethiops , Células Epiteliais/imunologia , Células Epiteliais/virologia , Vírus da Hepatite B/genética , Humanos , Pulmão/citologia , Pulmão/virologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Células Vero
4.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299151

RESUMO

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Túbulos Renais/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais/efeitos dos fármacos , Receptor PAR-2/genética , Edulcorantes/farmacologia
5.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209455

RESUMO

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.


Assuntos
Soluções para Diálise/farmacologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Glucose/farmacologia , Inflamação/patologia , Mesoderma/metabolismo , Neovascularização Fisiológica , Diálise Peritoneal , Biomarcadores/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Viruses ; 13(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205489

RESUMO

The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC-EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.


Assuntos
Adenoviridae/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Metapneumovirus/fisiologia , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral , Células A549 , Linhagem Celular , Quimiocinas/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Células HEK293 , Humanos , Quinolinas/farmacologia
7.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204534

RESUMO

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5-400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 µg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Preparações de Plantas/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Permeabilidade da Membrana Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos , Modelos Biológicos , Transdução de Sinais , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
8.
Cells ; 10(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200372

RESUMO

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.


Assuntos
Enzima de Conversão de Angiotensina 2/fisiologia , Células Epiteliais/metabolismo , Heparina/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células A549 , Enzima de Conversão de Angiotensina 2/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Dermatan Sulfato/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Glicosaminoglicanos/farmacologia , Células HEK293 , Células HaCaT , Heparitina Sulfato/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Células Vero , Internalização do Vírus/efeitos dos fármacos
9.
Ecotoxicol Environ Saf ; 220: 112378, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34082244

RESUMO

Circular RNAs (circRNAs) have been demonstrated to play critical roles in the pathogenesis of human cancers and carcinogenesis of several environmental pollutants. Nevertheless, the function of circRNAs in cadmium carcinogenesis is unclear. circ-SHPRH is down-regulated in many cancers including non-small cell lung cancer. In our present study, during cadmium-induced transformation of human bronchial epithelial BEAS-2B cells, epithelial-mesenchymal transition (EMT) was induced. Meanwhile, at the middle and late stages of cell transformation, cadmium down-regulated the expression of circ-SHPRH, as well as QKI, a tumor suppressor protein known to prevent the proliferation and EMT during progression of human cancers, compared with passage-matched control BEAS-2B cells. Overexpression of circ-SHPRH in cadmium-transformed BEAS-2B cells promoted the expression of QKI and significantly inhibited proliferation, EMT, invasion, migration and anchorage-independent growth in soft agar of the cells. Mechanistic studies showed that circ-SHPRH functioned as a sponge of miR-224-5p to regulate QKI expression. Interestingly, QKI and circ-SHPRH could form a positive-feedback loop that perpetuated circ-SHPRH/miR-224-5p/QKI axis. Collectively, our results demonstrated that circ-SHPRH inhibited cadmium-induced transformation of BEAS-2B cells through sponging miR-224-5p to regulate QKI expression under cadmium treatment. Our study uncovered a novel molecular mechanism involved in circRNAs in the development of lung cancer due to cadmium exposure.


Assuntos
Cádmio/toxicidade , DNA Helicases/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/induzido quimicamente , MicroRNAs/metabolismo , RNA Circular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Carcinogênese/induzido quimicamente , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Regulação para Baixo , Poluentes Ambientais/toxicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
10.
Sci Rep ; 11(1): 12787, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140611

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic collapse around the world. Effective treatments to mitigate this viral infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells (HCEC) in culture challenged with IFNγ as models of the eye surface to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein to angiotensin-converting enzyme 2 (ACE2). We found that the lipid mediators, elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i) decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ-stimulated HCEC. There was also a concomitant decrease in the binding of Spike RBD with the lipid treatments. Using RNA-seq analysis, we uncovered that the lipid mediators also attenuated the expression of pro-inflammatoy cytokines participating in hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to open therapeutic avenues to counteract virus attachment and entrance to the body.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Senescência Celular/efeitos dos fármacos , Lesões da Córnea/metabolismo , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/análogos & derivados , Ácidos Docosa-Hexaenoicos/farmacologia , Descoberta de Drogas/métodos , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/metabolismo , COVID-19/virologia , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Humanos , Lipoxinas/farmacologia , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
11.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068986

RESUMO

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L-1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L-1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L-1 quinidine or the bilateral replacement of 120 Na+, 120 Cl- or 25 HCO3- reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3-. The removal of 120 Cl- had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3- via apical CFTR and basolateral Na+-HCO3- cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.


Assuntos
Acroleína/análogos & derivados , Antineoplásicos Fitogênicos/farmacologia , Bicarbonatos/metabolismo , Células Epiteliais/metabolismo , Trato Gastrointestinal/metabolismo , Canal de Cátion TRPA1/agonistas , Acroleína/farmacologia , Animais , Células Epiteliais/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Suínos
12.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070855

RESUMO

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA/genética , Regulação da Expressão Gênica , Reparo de DNA por Recombinação , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Antineoplásicos Fitogênicos/farmacologia , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Etoposídeo/farmacologia , Técnicas de Inativação de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Fatores de Transcrição/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
13.
Nat Commun ; 12(1): 3356, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099663

RESUMO

Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.


Assuntos
Ciclina D/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Células MCF-7 , Camundongos , Microscopia de Fluorescência , Piperazinas/farmacologia , Ligação Proteica , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo
14.
FASEB J ; 35(7): e21519, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34137477

RESUMO

Globally, COPD remains a major cause of disability and death. In the United States alone, it is estimated that approximately 14 million people suffer from the disease. Given the high disease burden and requirement for chronic, long-term medical care associated with COPD, it is essential that new disease modifying agents are developed to complement the symptomatic therapeutics currently available. In the present report, we have identified a potentially novel therapeutic agent through the use of a high throughput screen based on the knowledge that cigarette smoke induces the proteolytic enzyme MMP1 leading to destruction of the lung in COPD. A construct utilizing the cigarette responsive promoter element of MMP-1 was conjugated to a luciferase reporter and utilized in an in vitro assay to screen the NIH Molecular Libraries Small Molecule Repository to identify putative targets that suppressed luciferase expression in response to cigarette smoke extract (CSE). Selective serotonin reuptake inhibitors potently inhibited luciferase expression and were further validated. SSRI treatment suppressed MMP-1 production in small airway epithelial cells exposed to (CSE) in vitro as well as in smoke exposed rabbits. In addition, SSRI treatment inhibited inflammatory cytokine production while rescuing cigarette smoke induced downregulation in vivo of the anti-inflammatory lipid transporter ABCA1, previously shown by our laboratory to be lung protective. Importantly, SSRI treatment prevented lung destruction in smoke exposed rabbits as measured by morphometry. These studies support further investigation into SSRIs as a novel therapeutic for COPD may be warranted.


Assuntos
Fumar Cigarros/efeitos adversos , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Metaloproteinase 1 da Matriz/química , Pneumonia/tratamento farmacológico , Enfisema Pulmonar/tratamento farmacológico , Inibidores de Captação de Serotonina/farmacologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Pulmão/metabolismo , Pulmão/patologia , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/patologia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/enzimologia , Enfisema Pulmonar/patologia , Coelhos , Serotonina/metabolismo
15.
FASEB J ; 35(7): e21699, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34151459

RESUMO

FUT2, a protein that uses l-fucose to mediate fucosylation of intestinal epithelial cells, is one of the detected gene variants in IBD patients. We aimed to investigate whether exogenous l-fucose could be an enteral nutritional supplement to protect intestinal barrier function. The effect of l-fucose on the restoration of epithelial barrier function in both the DSS-induced colitis mouse model and LPS-stimulated Caco-2 cells was investigated, and the impact on fucosylation of epithelial cells was examined. The severity of DSS-induced colitis was significantly reduced by l-fucose. Restoration of epithelial barrier function by l-fucose was detected. Direct l-fucose-mediated protection of tight junctions was observed in Caco-2 cells. Moreover, exogenous l-fucose promoted the exogenous metabolic pathway of l-fucose, and fucosylation of epithelial cells both in vivo and in vitro. Moreover, knockout of the FUT2 gene restrained fucosylation and the protective effect of l-fucose on barrier function. The severity of colitis was not improved by l-fucose in Fut2 knockout mice. Therefore we conclude that exogenous l-fucose protects intestinal barrier function and relieves intestinal inflammation via upregulation of FUT2-mediated fucosylation of intestinal epithelial cells.


Assuntos
Colite/prevenção & controle , Células Epiteliais/efeitos dos fármacos , Fucose/farmacologia , Fucosiltransferases/fisiologia , Inflamação/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
16.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071419

RESUMO

Interleukin (IL)-33 is a member of the interleukin (IL)-1 family of cytokines linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has a direct effect on human gastric epithelial cells (GES-1), the human gastric adenocarcinoma cell line (AGS), and the gastric carcinoma cell line (NCI-N87) by assessing its role in the regulation of cell proliferation, migration, cell cycle, and apoptosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assays, migration by wound healing assay, and apoptosis by caspase 3/7 activity assay and annexin V assay. Cell cycle was analyzed by means of propidium iodine assay, and gene expression regulation was assessed by RT-PCR profiling. We found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell lines, and it can stimulate proliferation and reduce apoptosis in normal epithelial cell lines. These effects were also confirmed by the analysis of cell cycle gene expression, which showed a reduced expression of pro-proliferative genes in cancer cells, particularly in genes involved in G0/G1 and G2/M checkpoints. These results were confirmed by gene expression analysis on bioptic and surgical specimens. The aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell-type-dependent manner.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Interleucina-33/farmacologia , Proteínas Recombinantes/farmacologia , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-33/genética , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
17.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071686

RESUMO

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.


Assuntos
Comunicação Celular/efeitos dos fármacos , Dinoprostona/farmacologia , Células Epiteliais/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Células Madin Darby de Rim Canino , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
18.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068694

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease characterized by the proliferation of myofibroblasts and deposition of extracellular matrix that results in irreversible distortion of the lung structure and the formation of focal fibrosis. The molecular mechanism of IPF is not fully understood, and there is no satisfactory treatment. However, most studies suggest that abnormal activation of transforming growth factor-ß1 (TGF-ß1) can promote fibroblast activation and epithelial to mesenchymal transition (EMT) to induce pulmonary fibrosis. Deglycosylated azithromycin (Deg-AZM) is a compound we previously obtained by removing glycosyls from azithromycin; it was demonstrated to exert little or no antibacterial effects. Here, we discovered a new function of Deg-AZM in pulmonary fibrosis. In vivo experiments showed that Deg-AZM could significantly reduce bleomycin-induced pulmonary fibrosis and restore respiratory function. Further study revealed the anti-inflammatory and antioxidant effects of Deg-AZM in vivo. In vitro experiments showed that Deg-AZM inhibited TGF-ß1 signaling, weakened the activation and differentiation of lung fibroblasts, and inhibited TGF-ß1-induced EMT in alveolar epithelial cells. In conclusion, our findings show that Deg-AZM exerts antifibrotic effects by inhibiting TGF-ß1-induced myofibroblast activation and EMT.


Assuntos
Azitromicina/uso terapêutico , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Transdução de Sinais , Animais , Azitromicina/química , Azitromicina/farmacologia , Bleomicina , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Inflamação/patologia , Pulmão/patologia , Camundongos , Modelos Biológicos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Células NIH 3T3 , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
20.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064412

RESUMO

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO's properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Boranos/farmacologia , Regulação Neoplásica da Expressão Gênica , Nanopartículas/química , Oligonucleotídeos Antissenso/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Boranos/síntese química , Boranos/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HeLa , Humanos , Cinética , Células MCF-7 , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de Sinais
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