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1.
Medicine (Baltimore) ; 99(1): e18445, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31895772

RESUMO

BACKGROUNDS: HER-2 positive breast cancer is a subtype of breast cancer with poor clinical outcome. The aim of this study was to identify differentially expressed genes (DEGs) for HER-2 positive breast cancer and elucidate the potential interactions among them. MATERIAL AND METHODS: Three gene expression profiles (GSE29431, GSE45827, and GSE65194) were derived from the Gene Expression Omnibus (GEO) database. GEO2R tool was applied to obtain DEGs between HER-2 positive breast cancer and normal breast tissues. Gene ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (David) online tool. Protein-protein interaction (PPI) network, hub gene identification and module analysis was conducted by Cytoscape software. Online Kaplan-Meier plotter survival analysis tool was also used to investigate the prognostic values of hub genes in HER-2 positive breast cancer patients. RESULTS: A total of 54 upregulated DEGs and 269 downregulated DEGs were identified. Among them, 10 hub genes including CCNB1, RAC1, TOP2A, KIF20A, RRM2, ASPM, NUSAP1, BIRC5, BUB1B, and CEP55 demonstrated by connectivity degree in the PPI network were screened out. In Kaplan-Meier plotter survival analysis, the overexpression of RAC1 and RRM2 were shown to be associated with an unfavorable prognosis in HER-2 positive breast cancer patients. CONCLUSIONS: This present study identified a number of potential target genes and pathways which might impact the oncogenesis and progression of HER-2 positive breast cancer. These findings could provide new insights into the detection of novel diagnostic and therapeutic biomarkers for this disease.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Ribonucleosídeo Difosfato Redutase/genética , Proteínas rac1 de Ligação ao GTP/genética , Estudos de Casos e Controles , Biologia Computacional , Regulação para Baixo , Feminino , Humanos , Receptor ErbB-2 , Transcriptoma/genética , Regulação para Cima
2.
Food Microbiol ; 85: 103301, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31500710

RESUMO

Lactobacillus paracasei is able to persist in a variety of natural and technological environments despite physico-chemical perturbations, in particular alternations between desiccation and rehydration. However, the way in which it adapts to hydric fluctuations and the genetic determinants involved are not clearly understood. To identify the genes involved in adaptation to desiccation, an annotated library of L. paracasei random transposon mutants was screened for viability after desiccation (25% relative humidity, 25 °C). We found 16 genes that have not been described as being involved in this response. Most of them are linked to either the transport of molecules or to cell wall structure and function. Our screening also identified genes encoding DNA related enzymes and an alarmone necessary for L. paracasei survival. Subsequently, the expression of the identified genes was measured at five stages of the dehydration-rehydration process to decipher the chronology of genetic mechanisms. They were classified into four different transcriptional profiles: genes upregulated during both desiccation and rehydration phases, genes upregulated during the desiccation phase only, genes downregulated during both desiccation and rehydration and genes downregulated only during the rehydration stage. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can continue or not during rehydration. The genes identified should contribute to improve the stabilization of Lactobacillus starters in dry state.


Assuntos
Dessecação , Hidratação , Lactobacillus paracasei/genética , Adaptação Fisiológica , Regulação para Baixo , Perfilação da Expressão Gênica , Lactobacillus paracasei/fisiologia , Regulação para Cima , Água
3.
Bioelectrochemistry ; 131: 107350, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31518962

RESUMO

Curcumin (Cur), the yellow pigment of well-known turmeric (Curcuma longa L.) is effective in multiple cancers including triple negative breast cancer (TNBC). In combination with electrical pulses (EP), enhanced effects of curcumin (Cur + EP) are observed in TNBC cells. To gain insights into the mechanisms of enhanced anticancer effects of Cur + EP, we studied the proteins involved in the anticancer activity of Cur + EP in MDA-MB-231, human TNBC cells using high-throughput global proteomics. A curcumin dose of 50 µM was applied with eight, 1200 V/cm, 100 µs pulses, the most commonly used electrochemotherapy (ECT) parameter in clinics. Results show that the Cur + EP treatment reduced the clonogenic ability in MDA-MB-231 cells, with the induction of apoptosis. Proteomic analysis identified a total of 1456 proteins, of which 453 proteins were differentially regulated, including kinases, heat shock proteins, transcription factors, structural proteins, and metabolic enzymes. Eight key glycolysis proteins (ALDOA, ENO2, LDHA, LDHB, PFKP, PGM1, PGAM1 and PGK1) were downregulated in Cur + EP from Cur. There was a switch in the metabolism with upregulation of 10 oxidative phosphorylation pathway proteins and 8 tricarboxylic acid (TCA) cycle proteins in the Cur + EP sample, compared to curcumin. These results provide novel systematic insights into the mechanisms of ECT with curcumin.


Assuntos
Antineoplásicos/uso terapêutico , Curcumina/uso terapêutico , Eletroquimioterapia/métodos , Proteínas de Neoplasias/metabolismo , Proteômica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Glicólise , Humanos , Fosforilação Oxidativa , Via de Pentose Fosfato/efeitos dos fármacos , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
4.
Chemosphere ; 240: 124905, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31563103

RESUMO

Microcystin-LR (MCLR) was commonly regarded as a potent hepatotoxin and has been reported to cause neurotoxicity. This study was aimed to investigate how maternal MCLR exposure during pregnancy alters behavioral responses in offspring mice and the possible molecular mechanism involved in this procedure. Three doses of MCLR solutions (0, 3 or 15 µg/kg body weight) were administered subcutaneously to pregnant C57bl/6 from gestation day (GD) 6-19. Our results showed that MCLR prenatal exposure led to the impairment of learning and memory function in offspring on postnatal days (PND) 35, accompanied by endoplasmic reticulum (ER) stress and neuronal apoptosis in hippocampal CA1 regions of mice. Sixteen miRNAs in hippocampus of pups on PND 35 were significantly affected by MCLR exposure with the markedly decreased transcription of miR-181a-5p. We then found that miR-181a-5p was down-regulated, accompanied by activation of ER stress after prenatal exposure to MCLR using qPCR analysis. Furthermore, glucose-regulated protein, 78kDa/binding immunoglobulin protein (Grp78/BIP), a major ER chaperone and signaling regulator, was identified as a target of miR-181a-5p. Our study showed that miR-181a could lead to a decrease in the mRNA expression and protein levels of Grp78 by directly binding to its 3'-untranslated region (3'-UTR) in primary hippocampal neurons. Our findings indicate that the up-regulation of Grp78 mediated by inhibition of miR-181a-5p is a possible mechanism resulting in ER stress and cognitive impairment in pups following prenatal MCLR exposure.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , MicroRNAs/metabolismo , Microcistinas/toxicidade , Animais , Apoptose , Regulação para Baixo , Feminino , Hipocampo/metabolismo , Masculino , Memória , Camundongos , MicroRNAs/genética , Gravidez , Regulação para Cima
5.
Gene ; 725: 144167, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31639434

RESUMO

Osteoporosis in advanced cholestatic and end-stage liver disease is related to low bone formation. Previous studies have demonstrated the deleterious consequences of lithocholic acid (LCA) and bilirubin on osteoblastic cells. These effects are partially or completely neutralized by ursodeoxycholic acid (UDCA). We have assessed the differential gene expression of osteoblastic cells under different culture conditions. The experiments were performed in human osteosarcoma cells (Saos-2) cultured with LCA (10 µM), bilirubin (50 µM) or UDCA (10 and 100 µM) at 2 and 24 h. Expression of 87 genes related to bone metabolism and other signalling pathways were assessed by TaqMan micro fluidic cards. Several genes were up-regulated by LCA, most of them pro-apoptotic (BAX, BCL10, BCL2L13, BCL2L14), but also MGP (matrix Gla protein), BGLAP (osteocalcin), SPP1 (osteopontin) and CYP24A1, and down-regulated bone morphogenic protein genes (BMP3 and BMP4) and DKK1 (Dickkopf-related protein 1). Parallel effects were observed with bilirubin, which up-regulated apoptotic genes and CSF2 (colony-stimulating factor 2) and down-regulated antiapoptotic genes (BCL2 and BCL2L1), BMP3, BMP4 and RUNX2. UDCA 100 µM had specific consequences since differential expression was observed, up-regulating BMP2, BMP4, BMP7, CALCR (calcitonin receptor), SPOCK3 (osteonectin), BGLAP (osteocalcin) and SPP1 (osteopontin), and down-regulating pro-apoptotic genes. Furthermore, most of the differential expression changes induced by both LCA and bilirubin were partially or completely neutralized by UDCA. Conclusion: Our observations reveal novel target genes, whose regulation by retained substances of cholestasis may provide additional insights into the pathogenesis of osteoporosis in cholestatic and end-stage liver diseases.


Assuntos
Bilirrubina/metabolismo , Osteoblastos/metabolismo , Osteoporose/genética , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Linhagem Celular Tumoral , Colestase/genética , Regulação para Baixo/efeitos dos fármacos , Perfil Genético , Humanos , Ácido Litocólico/farmacologia , Fígado/metabolismo , Fígado/fisiologia , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Osteoporose/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Regulação para Cima/efeitos dos fármacos , Ácido Ursodesoxicólico/farmacologia
6.
Arch Oral Biol ; 109: 104579, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634727

RESUMO

OBJECTIVES: To investigate the effect and mechanism of calcium on LS8 cell differentiation, especially on phosphatidylinositol 3 kinase (PI3K) /protein kinase B(AKT) pathway. MATERIALS AND METHODS: Ameloblast-like LS8 cell line was used and additional 0-3.5 mmol/L calcium chloride was treated for 24 h, 48 h. Cell viability and morphological changes, cell cycle and associated regulatory proteins were analyzed. RESULTS: No significant effects on morphological changes were observed. Decreased cell viability and increased S phase cells were accompanied by the significant decrease of cyclin A and cyclin B proteins, and significant increase of cyclin D protein in LS8 cells. Additionally, kallikrein-4 and amelotin expressions were significantly increased. Finally, the levels of PI3K, AKT, p-AKT and forkhead box O3 (FOXO3) significantly downregulated after calcium treatment in LS8 cells. CONCLUSIONS: Calcium inhibit proliferation and promotes differentiation in LS8 cells, this is closely related to the downregulation of PI3K/AKT signal in LS8 cells.


Assuntos
Ameloblastos/enzimologia , Cálcio/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ameloblastos/efeitos dos fármacos , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Camundongos
7.
Gut ; 69(1): 122-132, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31076405

RESUMO

OBJECTIVE: We investigated how pancreatic cancer developed resistance to focal adhesion kinase (FAK) inhibition over time. DESIGN: Pancreatic ductal adenocarcinoma (PDAC) tumours from KPC mice (p48-CRE; LSL-KRasG12D/wt; p53flox/wt) treated with FAK inhibitor were analysed for the activation of a compensatory survival pathway in resistant tumours. We identified pathways involved in the regulation of signal transducer and activator of transcription 3 (STAT3) signalling on FAK inhibition by gene set enrichment analysis and verified these outcomes by RNA interference studies. We also tested combinatorial approaches targeting FAK and STAT3 in syngeneic transplantable mouse models of PDAC and KPC mice. RESULTS: In KPC mice, the expression levels of phosphorylated STAT3 (pSTAT3) were increased in PDAC cells as they progressed on FAK inhibitor therapy. This progression corresponded to decreased collagen density, lowered numbers of SMA+ fibroblasts and downregulation of the transforming growth factor beta (TGF-ß)/SMAD signalling pathway in FAK inhibitor-treated PDAC tumours. Furthermore, TGF-ß production by fibroblasts in vitro drives repression of STAT3 signalling and enhanced responsiveness to FAK inhibitor therapy. Knockdown of SMAD3 in pancreatic cancer cells abolished the inhibitory effects of TGF-ß on pSTAT3. We further found that tumour-intrinsic STAT3 regulates the durability of the antiproliferative activity of FAK inhibitor, and combinatorial targeting of FAK and Janus kinase/STAT3 act synergistically to suppress pancreatic cancer progression in mouse models. CONCLUSION: Stromal depletion by FAK inhibitor therapy leads to eventual treatment resistance through the activation of STAT3 signalling. These data suggest that, similar to tumour-targeted therapies, resistance mechanisms to therapies targeting stromal desmoplasia may be critical to treatment durability.


Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Aminopiridinas/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/patologia , Colágeno/metabolismo , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Fibroblastos/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Camundongos Endogâmicos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 992-999, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31878995

RESUMO

Objective To investigate the effect of microRNA let-7i on the drug-resistant cells of human breast cancer and its potential molecular mechanisms. Methods Reverse transcription PCR was performed to detect the relative levels of let-7i expressed in breast cancer cell line MCF-7 as well as MCF-7 resistant to both cisplatin (CDDP) and adriamycin (ADR) (MCF-7/CDDP, MCF-7/ADR) cells. CCK-8 assay was used to measure the varied sensitivity of cell MCF-7, MCF-7/CDDP and MCF-7/ADR to ADR following chemotherapy. CCK-8 assay and colony formation assay were carried out to observe the change of sensitivity to ADR after let-7i was over-expressed or inhibited, and flow cytometry was used to determine the induced apoptosis of breast cancer cells resistant to ADR following over-expression of let-7i. ELISA was conducted to measure the activity of caspase-3/9 against breast cancer drug. Reverse transcription-PCR was performed to detect the mRNA levels of Bcl2 and K-Ras, and Western blot analysis to examine the protein levels of Bcl2, BAX and K-Ras. Results Let-7i was down-regulated in drug-resistant breast cancer cells as compared with simple MCF-7 cell line, and negatively correlated with chemotherapy resistance. Compared with negative control group, over-expression of let-7i resulted in improved cell viability and colony formation of drug-resistant breast cancer cells, and facilitated the cancer cell apoptosis induced by ADR, yet raised caspase-3/9 activity. Bcl2 level decreased, whereas BAX increased with added ADR concentration. Over-expressed let-7i significantly inhibited Bcl2 and K-Ras expression in the drug-resistant cells. Conclusion Let-7i is down-regulated in drug-resistant breast cancer cells, and negatively correlated with chemotherapy resistance. The findings suggest that let-7i may inhibit the resistance of drug-resistant breast cancer cells via inhibiting the expression of K-Ras and Bcl2.


Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Apoptose , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Regulação para Baixo , Doxorrubicina/farmacologia , Humanos , Células MCF-7
9.
Biol Res ; 52(1): 57, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767027

RESUMO

BACKGROUND: Gastric cancer is a common malignant tumor with high morbidity and mortality worldwide, which seriously affects human health. Gramicidin is a short peptide antibiotic which could be used for treating infection induced by bacteria or fungi. However, the anti-cancer effect of gramicidin on gastric cancer cells and its underlying mechanism remains largely unknown. RESULTS: Gastric cancer cells SGC-7901, BGC-823 and normal gastric mucosal cells GES-1 were treated with different concentrations of gramicidin respectively. The results of CCK-8 experiment revealed cellular toxicity of gramicidin to cancer cells while cell colony formation assay showed that gramicidin significantly inhibited the proliferation of gastric cancer cells, but had little effect on normal gastric mucosal cells. In addition, the wound healing assay showed that gramicidin inhibited the migration of SGC-7901 cell. Meanwhile, apoptosis and cell cycle analysis revealed that gramicidin induced cell apoptosis with G2/M cell cycle inhibition. Furthermore, western blot analysis demonstrated that gramicidin down-regulated the expression of cyclinD1 and Bcl-2 as well as the FoxO1 phosphorylation. CONCLUSIONS: The current study illustrated the anti-tumor activity of gramicidin on gastric cancer cells, providing a possibility for gramicidin to be applied in clinical practice for the treatment of gastric cancer.


Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gramicidina/farmacologia , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Ciclina D1/efeitos dos fármacos , Ciclina D1/metabolismo , Regulação para Baixo , Proteína Forkhead Box O1/efeitos dos fármacos , Proteína Forkhead Box O1/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
10.
Cancer Immunol Immunother ; 68(12): 2029-2039, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31709456

RESUMO

Interferon-stimulated gene 15 (ISG15) is a 15 kDa protein induced by type I interferons (IFN-α and IFN-ß) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly expressed in various malignancies, including pancreatic ductal adenocarcinoma (PDAC), suggesting a potential role of the ISG15 pathway (free ISG15 and ISG15 conjugates) in pancreatic carcinogenesis. However, very little is known about how the ISG15 pathway may contribute to pancreatic tumorigenesis. In the current study, we demonstrate that ISG15 pathway knockdown reverses the KRAS-associated phenotypes of PDAC cells such as increased proliferation and colony formation. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated ISG15 knockdown decreased tumor programmed death ligand-1 (PDL-1) expression leading to increased number of CD8+ tumor-infiltrating lymphocytes and decreased pancreatic tumor growth. In addition, the syngeneic subcutaneous mouse model revealed that knocking down the ISG15 pathway significantly decreased the rate of tumor incidence and increased the survival rate. Interestingly, the ISG15 knockdown-mediated PDL-1 downregulation in pancreatic tumors increased the efficacy of anti-programmed cell death protein-1 (PD-1) treatment. ISG15 knockdown in combination with anti-PD-1 treatment synergistically increased the number of CD8+ tumor-infiltrating lymphocytes. Additionally, ISG15 knockdown alone significantly decreased the number of tumor-infiltrating regulatory T cells (Tregs) compared to wild type tumors treated with anti-PD-1 antibody. Overall, these findings suggest that strategies to target the ISG15 pathway by itself or in combination with immunotherapy may lead to improved survival for patients diagnosed with PDAC.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Imunoterapia/métodos , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Citocinas/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Neoplasias Pancreáticas/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , RNA Interferente Pequeno/genética , Transdução de Sinais , Ubiquitinas/genética , Ubiquitinas/metabolismo
11.
Cell Physiol Biochem ; 53(5): 865-886, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31724838

RESUMO

BACKGROUND/AIMS: Heart failure is characterized by chronic low-grade vascular inflammation, which in itself can lead to endothelial dysfunction. Clinical trials showed reductions in heart failure-related hospitalizations of type 2 diabetic patients using sodium glucose co-transporter 2 inhibitors (SGLT2i's). Whether and how SGLT2i's directly affect the endothelium under inflammatory conditions is not completely understood. The aim of the study was to investigate whether the SGLT2i Empagliflozin (EMPA) and Dapagliflozin (DAPA) reduce tumor necrosis factor α (TNFα) induced endothelial inflammation in vitro. METHODS: Human coronary arterial endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were (pre-)incubated with 1 µM EMPA or DAPA and subsequently exposed to 10 ng/ml TNFα. ROS and NO were measured using live cell imaging. Target proteins were either determined by infrared western blotting or fluorescence activated cell sorting (FACS). The connection between Cav-1 and eNOS was determined by co-immunoprecipitation. RESULTS: Nitric oxide (NO) bioavailability was reduced by TNFα and both EMPA and DAPA restored NO levels in TNFα-stimulated HCAECs. Intracellular ROS was increased by TNFα, and this increase was completely abolished by EMPA and DAPA in HCAECs by means of live cell imaging. eNOS signaling was significantly disturbed after 24 h when cells were exposed to TNFα for 24h, yet the presence of both SGLT2is did not prevent this disruption. TNFα-induced enhanced permeability at t=24h was unaffected in HUVECs by EMPA. Similarly, adhesion molecule expression (VCAM-1 and ICAM-1) was elevated after 4h TNFα (1.5-5.5 fold increase of VCAM-1 and 4-12 fold increase of ICAM-1) but were unaffected by EMPA and DAPA in both cell types. Although we detected expression of SGLT2 protein levels, the fact that we could not silence this expression by means of siRNA and the mRNA levels of SGLT2 were not detectable in HCAECs, suggests aspecificity or our SGLT2 antibody and absence of SGLT2 in our cells. CONCLUSION: These data suggest that EMPA and DAPA rather restore NO bioavailability by inhibiting ROS generation than by affecting eNOS expression or signaling, barrier function and adhesion molecules expression in TNFα-induced endothelial cells. Furthermore, the observed effects cannot be ascribed to the inhibition of SGLT2 in endothelial cells.


Assuntos
Compostos Benzidrílicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Glucosídeos/farmacologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Vasos Coronários/citologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Molécula 1 de Adesão de Célula Vascular
12.
Braz J Med Biol Res ; 52(10): e8385, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618367

RESUMO

Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.


Assuntos
Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo-Octanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Lignanas/farmacologia , Melanoma/patologia , Compostos Policíclicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , RNA Longo não Codificante , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos
13.
Life Sci ; 237: 116945, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31605710

RESUMO

AIM: Over-expression of histone deacetylase 8 (HDAC8) has been demonstrated in breast cancer. But the underlying molecular mechanism of HDAC8 on the progression of breast cancer remains unknown. MicroRNAs (miRs) are proposed as important molecules in cancer progression by targeting specific oncogenes or tumor-suppressor genes. Our overall objective was to assess the miR-216b-5p role on HDAC8; and its impacts on breast cancer (BC) progression. MAIN METHODS: We acquired cancerous and noncancerous tissues from Iran Tumor Bank (I.T.B). The MDA-MB-231, MCF-7 and MCF-10A BC cell lines were also purchased. The tissue and cell line expression levels of miR-216b-5p and HDAC8 were determined by quantitative real-time PCR (qPCR). We next measured protein levels of HDAC8 by Western blotting assay. The cell cycle, cell proliferation, and colony formation assay were determined. Finally, we investigated the role of HDAC8 using a knockout vector; and confirmed the targeting of 3' untranslated region (3'-UTR) of HDAC8 through miR-216b-5p using a luciferase reporter assay. KEY FINDINGS: Our results demonstrated a significant decrease in miR-216b-5p, and remarkable increase in HDAC8 levels within human breast cancer tissues and cell lines. The lower levels of miR-216b-5p were negatively correlated with lymph node metastasis and advanced tumor size. The overexpression of miR-216b-5p in BC cell lines inhibited cellular proliferation and progression. HDAC8 was directly down-regulated by miR-216b-5p and knockout of HDAC8 showed the similar effects as miR-216b-5p overexpression. SIGNIFICANCE: Briefly, HDAC8 is an oncogene that accelerate breast cancer proliferation and progression and miR-216b-5p modulates those functions by binding to HDAC8 3'-UTR.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , MicroRNAs/genética , Proteínas Repressoras/metabolismo , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Regulação para Baixo , Feminino , Histona Desacetilases/genética , Humanos , Prognóstico , Proteínas Repressoras/genética , Células Tumorais Cultivadas
14.
Toxicol Lett ; 317: 92-101, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31593750

RESUMO

Cigarette smoke (CS) is known to cause mitochondrial dysfunction leading to cellular senescence in lung cells. We determined the mechanism of mitochondrial dysfunction by CS in lung epithelial cells. CS extract (CSE) treatment differentially affected mitochondrial function, such as membrane potential, mitochondrial reactive oxygen species (mtROS) and mitochrondrial mass as analyzed by FACS, and were associated with altered oxidative phosphorylation (OXPHOS) protein levels (Complexes I-IV) in primary lung epithelial cells (SAEC and NHBE), and (complexes I and II) in BEAS2B cells. There were dose- and time-dependent changes in mitochondrial respiration (oxygen consumption rate parameters i.e. maximal respiration, ATP production and spare capacity, measured by the Seahorse analyzer) in control vs. CSE treated BEAS2B and NHBE/DHBE cells. Electron microscopy (EM) analysis revealed perinuclear clustering by localization and increased mitochondrial fragmentation by fragement length analysis. Immunoblot analysis revealed CS-mediated increase in Drp1 and decrease in Mfn2 levels that are involved in mitochondrial fission/fusion process. CSE treatment reduced Miro1 and Pink1 abundance that play a crucial role in the intercellular transfer mechanism and mitophagy process. Overall, these findings highlight the role of Miro1 in context of CS-induced mitochondrial dysfunction in lung epithelial cells that may contribute to the pathogenesis of chronic inflammatory lung diseases.


Assuntos
Fumar Cigarros/efeitos adversos , Células Epiteliais/metabolismo , Pulmão/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Proteínas rho de Ligação ao GTP/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Metabolismo Energético , Células Epiteliais/ultraestrutura , Humanos , Pulmão/ultraestrutura , Mitocôndrias/ultraestrutura , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Transdução de Sinais
15.
Anticancer Res ; 39(10): 5297-5310, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570424

RESUMO

BACKGROUND/AIM: Low-molecular weight heparins (LMWHs) may possess putative antitumoral properties; however, the underlying mechanism(s) remains elusive. We evaluated the antiproliferative and antimigratory effects of enoxaparin (a LMWH) in lung adenocarcinoma A549 cells, and assessed the possible mechanism involved, and the effect on doxorubicin's efficacy. MATERIALS AND METHODS: Proliferation and migration were evaluated using BrdU and transwell assays, respectively. Immunoblotting was used to measure PAR-1, PAR-2, MMP-2, ERK1/2 and Akt proteins. Apoptosis and cell cycle studies examined the combined effect of enoxaparin and doxorubicin. RESULTS: Enoxaparin inhibited A549 cell proliferation and migration. Following PAR-1 gene knock down, enoxaparin's effect on A549 cell proliferation was diminished compared to scrambled siRNA. Our experiments verified that enoxaparin-mediated down-regulation of MAPK and PI3K, reduced MMP-2 expression and inhibited A549 cell migration. Additionally, enoxaparin increased doxorubicin's efficacy by enhancing apoptosis, while no effect on cell-cycle progression was observed. CONCLUSION: Results suggest that the anticancer activity of enoxaparin in A549 cells was mediated by the interference of two major PAR-1 downstream signaling pathways, MAPK/ERK and PI3K/Akt, which in turn inhibit proliferation and migration. Therefore, enoxaparin may be promising as an adjunct to traditional chemotherapy for lung cancer and warrants further investigation.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Enoxaparina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo
16.
Anticancer Res ; 39(10): 5311-5327, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570425

RESUMO

BACKGROUND/AIM: MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3. MATERIALS AND METHODS: CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed. RESULTS: The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays. CONCLUSION: MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Deleção de Sequência/genética , Regiões 3' não Traduzidas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Oncogenes/genética , Células PC-3 , Neoplasias da Próstata/genética , Regulação para Cima/genética , Proteínas rho de Ligação ao GTP/genética
17.
Anticancer Res ; 39(10): 5361-5367, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570430

RESUMO

BACKGROUND/AIM: The mechanism responsible for B-cell translocation gene 1 (BTG1) down-regulation in breast carcinoma remains unknown. We examined the BTG1 expression status in breast carcinoma cells and investigated the mechanism underlying the observed alterations. MATERIALS AND METHODS: Four breast carcinoma cell lines (SK-BR-3, MDA-MB-231, T-47D, and MCF-7), and one normal mammary epithelial cell line (MCF-10A) were analyzed. BTG1 expression was examined using quantitative reverse transcription polymerase chain reaction (PCR) and western blot. Methylation status of the BTG1 promoter was analyzed using methylation-specific PCR (MSP). To investigate the effect of methylation on BTG1, the cells were treated with a demethylating agent. RESULTS: The carcinoma cells expressed significantly lower levels of BTG1 mRNA and protein than normal cells. The BTG1 promoter was highly methylated in the carcinoma cells. 5-aza-2-deoxycytidine significantly restored BTG1 expression. CONCLUSION: Down-regulation of BTG1 expression through epigenetic repression is involved in mammary carcinogenesis. BTG1 is a potential diagnostic marker and therapeutic target for breast carcinoma.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA/genética , Regulação para Baixo/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , RNA Mensageiro/genética
18.
Chem Biol Interact ; 314: 108847, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610155

RESUMO

Lead (Pb) is one of the toxic heavy metals that have several toxicological implications including cytotoxicities and oxidative stress. The release of reactive oxygen species (ROS) usually initiates lipid peroxidation and resulting in inflammation and tissue injury. However, the detailed identification of the Pb-produced lipid hydroperoxides has received little attention. Furthermore, the mechanisms behind such effects are less informed. Therefore, this study firstly investigated Pb-produced lipid hydroperoxides in human HepG2 cells using LC/MS. The effects of Pb on the antioxidant enzymes were additionally examined using qPCR and their dependent activities. As a protection trial, the ameliorative effects of rosmarinic (RMA) and ascorbic (ASA) acids on Pb-induced cytotoxicity and oxidative stress and their regulatory effects on Nrf2/Keap1 pathway were investigated. The achieved results confirmed cytotoxicity and oxidative damage of Pb on HepG2 cells. In addition, 20 lipid hydroperoxides (LOOH) were identified including 11 phosphatidylcholine hydroperoxides (PCOOH), 5 triacylglycerol hydroperoxides (TGOOH) and 4 cholesteryl ester hydroperoxides (CEOOH). The most dominant LOOH species were PCOOH 34:2, PCOOH 34:3, PCOOH 38:7, TGOOH 60:14, TGOOH 60:15, CEOOH 18:3 and CEOOH 20:4. Pb significantly downregulated Nrf2-regulated antioxidant enzymes at both the pretranscriptional and functional levels. Co-exposure of HepG2 cells to RMA and ASA significantly reduced Pb-produced adverse outcomes. This protection occurred via activation Nrf2-Keap1 antioxidant pathway.


Assuntos
Antioxidantes/metabolismo , Ácido Ascórbico/química , Cinamatos/química , Depsídeos/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Chumbo/química , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Ascórbico/farmacologia , Cromatografia Líquida de Alta Pressão , Cinamatos/farmacologia , Depsídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Humanos , Chumbo/toxicidade , Peróxidos Lipídicos/análise , Peróxidos Lipídicos/metabolismo , Espectrometria de Massas , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
19.
Chem Biol Interact ; 314: 108848, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610156

RESUMO

Cardiomyocyte injury induced by acute myocardial infarction contributes to myocardial dysfunction. Accumulating evidence has demonstrated that pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a cytoprotective protein that protects against various adverse injuries. However, whether PHLPP2 participates in regulating myocardial-infarction-induced cardiomyocyte injury remains unknown. In the present study, we aimed to investigate the biological role and molecular mechanism of PHLPP2 in regulating hypoxia-induced cardiomyocyte injury. Cardiomyocytes were cultured in an anaerobic chamber for 24 h to induce hypoxic injury in vitro. The expression of PHLPP2 was determined by real-time quantitative PCR and Western blot analysis. Cell viability was measured by MTT assay. Cell apoptosis was assessed by TUNEL and caspase-3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA probe. PHLPP2 expression was highly upregulated in hypoxia-injured cardiomyocytes. Inhibition of PHLPP2 by small interfering RNA (siRNA)-mediated gene silencing significantly improved the viability of hypoxia-injured cardiomyocytes and attenuated hypoxia-induced apoptosis and ROS production. In contrast, PHLPP2 overexpression exacerbated hypoxia-induced apoptosis and ROS production in cardiomyocytes. Mechanism research revealed that PHLPP2 silencing increased the phosphorylation of glycogen synthase kinase (GSK)-3ß and promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, PHLPP2 inhibition promoted Nrf2/antioxidant response element (ARE) transcriptional activity. However, Nrf2 silencing markedly reversed PHLPP2-inhibition-mediated cardioprotection, while GSK-3ß inhibition partially blocked the PHLPP2-overexpression-induced adverse effect. Taken together, these findings demonstrate that PHLPP2 inhibition alleviates hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE antioxidant signaling via inactivating GSK-3ß, a pathway that highlights the importance of the PHLPP2/GSK-3ß/Nrf2/ARE signaling axis in regulation of cardiomyocyte injury. Our study suggests a potential relevance for PHLPP2 in acute myocardial infarction, and this protein may serve as a promising target for cardioprotection.


Assuntos
Elementos de Resposta Antioxidante/genética , Hipóxia Celular , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Cell Physiol Biochem ; 53(4): 731-745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613064

RESUMO

BACKGROUND/AIMS: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas. METHODS: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis. RESULTS: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival. CONCLUSION: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.


Assuntos
Adenosina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Adenosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Dano ao DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Mapas de Interação de Proteínas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , S-Adenosil-Homocisteína/metabolismo
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