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1.
Anticancer Res ; 40(1): 169-176, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892565

RESUMO

BACKGROUND/AIM: Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. MATERIALS AND METHODS: Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. RESULTS: COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44+) cells showed significantly higher tumor formation capacity than CD44- cells in immunodeficient mice. CD44+ cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44+ cells. CONCLUSION: CD44+ COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.


Assuntos
Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Biomarcadores , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Fluoruracila/farmacologia , Xenoenxertos , Humanos , Receptores de Hialuronatos/genética , Camundongos
2.
Nat Commun ; 11(1): 586, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996681

RESUMO

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.


Assuntos
Biomarcadores , Endotélio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Hialuronatos/metabolismo , Transcriptoma , Animais , Aorta , Artérias , Ciclo Celular , Ciclo do Ácido Cítrico/genética , Biologia Computacional , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação para Baixo , Glicólise/genética , Gônadas , Hematopoese/fisiologia , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/genética , Ácido Hialurônico , Mesonefro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Transformador beta/metabolismo
3.
J Cancer Res Clin Oncol ; 145(11): 2649-2661, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529191

RESUMO

PURPOSE: The incidence of Urothelial carcinoma of bladder (UBC) is gradually increasing by changing lifestyle and environment. The development of a tumor has been noted to be accompanied by modifications in the extracellular matrix (ECM) consisting of CD44, hyaluronic acid (HA) and its family members. The importance of CD44 splice variants and HA family members has been studied in UBC. METHODS: The cohort of study included 50 UBC patients undergoing radical cystectomy and 50 healthy subjects. The molecular expression of CD44 and HA family members was determined. Effect of CD44 variant-specific silencing on downstream signaling in HT1376 cells was investigated. Combinatorial treatment of 4-MU (4-methylumbelliferone) with cisplatin or doxorubicin on chemosensitivity was also explored. RESULTS: Higher expression of HA, HAS2, and CD44 was observed in Indian UBC patients which also showed the trend with severity of disease. Splice variant assessment of CD44 demonstrated the distinct role of CD44v3 and CD44v6 in bladder cancer progression. shRNA-mediated downregulation of CD44v3 showed an increase effect on cell cycle, apoptosis and multiple downstream signaling cascade including pAkt, pERK and pSTAT3. Furthermore, 4-MU, an HA synthesis inhibitor, observed to complement the effect of Cisplatin or Doxorubicin by enhancing the chemosensitivity of bladder cancer cells. CONCLUSIONS: Our findings exhibit involvement of CD44 splice variants and HA family members in UBC and significance of 4-MU in enhancing chemosensitivity suggesting their novel therapeutic importance in disease therapeutics.


Assuntos
Processamento Alternativo , Receptores de Hialuronatos/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias da Bexiga Urinária/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Apoptose , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Transcrição STAT3/genética , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
4.
Kobe J Med Sci ; 65(1): E1-E9, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31341151

RESUMO

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, arising from follicular cells, and accounts for more than 80% of all thyroid malignant tumors. Although age is the strongest prognostic factor of PTC, and various cut-off ages (40-55 years) were suggested in previous studies, the molecular mechanisms causing age-related changes of PTC cell proliferation remain unclear. CD44 is a major cell surface receptor for hyaluronate and is known as a cancer stem cell marker. However, the association between CD44 and PTC is still unknown. Therefore, we determined the proliferation of primary cultured cells obtained from patients with PTC, and the CD44 mRNA expression profile to elucidate age-related association of CD44 with PTC. The results showed that cell proliferation was significantly decreased according to age. We also found that CD44v8-10 and CD44 splice variants were expressed dominantly in patients with PTC. Moreover, the CD44v8-10/CD44s mRNA expression ratio was significantly increased according to age, and there was a significant negative correlation between this expression ratio and cell proliferation. Our findings suggest that the CD44v8-10/CD44s expression ratio in PTC cells is useful for screening for aggressive PTC and may provide clinically valuable information.


Assuntos
Receptores de Hialuronatos/genética , Câncer Papilífero da Tireoide/patologia , Adolescente , Adulto , Fatores Etários , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Câncer Papilífero da Tireoide/imunologia , Adulto Jovem
5.
Vox Sang ; 114(6): 576-587, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31281973

RESUMO

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.


Assuntos
Transfusão de Sangue , Eritrócitos/fisiologia , Vesículas Extracelulares/fisiologia , Glicoproteínas de Membrana/fisiologia , Monócitos/fisiologia , Fagocitose , Antígeno CD47/análise , Antígeno CD47/genética , Antígenos CD55/análise , Antígenos CD55/genética , Antígenos CD59/análise , Antígenos CD59/genética , Eritrócitos/citologia , Eritrócitos/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/genética , Fosfatidilserinas/análise
6.
Int J Mol Sci ; 20(15)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357721

RESUMO

Combinatorial therapeutic strategies using siRNA and small molecules to eradicate tumors are emerging. Targeting multiple signaling pathways decreases the chances of cancer cells switching and adapting new signaling processes that may occur when using a single therapeutic modality. Aberrant functioning of Notch-1, Wnt/ß-catenin, and STAT3 proteins and their crosstalk signaling pathways have been found to be involved in tumor survival, drug resistance, and relapse. In the current study, we describe a therapeutic potential of single and combinations of siRNA designed for silencing Notch-1, Wnt/ß-catenin, and STAT3 in MCF7_DoxS (wild type) and MCF7_DoxR (doxorubicin resistant) breast cancer cells. The MCF7_DoxR cells were developed through treatment with a gradual increase in doxorubicin concentration, the expression of targeted genes was investigated, and the expression profiling of CD44/CD24 of the MCF7_DoxS and MCF7_DoxR cells were detected by flow cytometry. Both MCF7_DoxS and MCF7_DoxR breast cancer cells were treated with single and combinations of siRNA to investigate synergism and were analyzed for their effect on cell proliferation with and without doxorubicin treatment. The finding of this study showed the overexpression of targeted genes and the enrichment of the CD44-/CD24+ phenotype in MCF7_DoxR cells when compared to MCF7_DoxS cells. In both cell lines, the gene silencing efficacy showed a synergistic effect when combining STAT3/Notch-1 and STAT3/Notch-1/ß-catenin siRNA. Interestingly, the chemosensitivity of MCF7_DoxS and MCF7_DoxR cells to doxorubicin was increased when combined with siRNA treatment. Our study shows the possibility of using single and combinations of siRNA to enhance the chemosensitivity of cancer cells to conventional antitumor chemotherapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor Notch1/genética , Fator de Transcrição STAT3/genética , beta Catenina/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antígeno CD24/genética , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Células MCF-7 , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , beta Catenina/antagonistas & inibidores
7.
Mol Med Rep ; 20(2): 951-958, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173237

RESUMO

Prostate cancer (PCa) is the most common malignancy among males worldwide, and is one of the leading causes of cancer­related mortality. MicroRNAs (miRs) are a type of endogenous, noncoding RNA that serve a key role in pathological processes, and have been demonstrated to be involved in the formation and progression of PCa. Previous studies have reported that miR­106b acts as an oncogene; however, the specific effects of miR­106b on PCa have not been fully elucidated. The present study aimed to investigate the role and underlying molecular mechanisms of miR­106b in the initiation and progression of PCa. In this study, miR­106b was reported to be overexpressed and la­related protein 4B (LARP4B) was downregulated in PCa tissues compared with paracancerous tissues. In addition, LARP4B was identified as a target gene of miR­106b by bioinformatics prediction analysis and a dual luciferase reporter gene assay. Furthermore, MTT, wound healing and Transwell assays were performed to evaluate PCa cell viability, and migration and invasive abilities. The data revealed that inhibition of miR­106b significantly suppressed the viability, migration and invasion of PCa cells. In addition, inhibition of miR­106b significantly suppressed the mRNA and protein expression of cancer­related genes, including matrix metalloproteinase­2, cluster of differentiation 44 and Ki­67, and increased that of the tumor suppressor, mothers against decapentaplegic homolog 2. Collectively, the findings of the present study indicated that miR­106b may target LAR4B to inhibit cancer cell viability, migration and invasion, and may be considered as a novel therapeutic target in PCa.


Assuntos
Autoantígenos/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Ribonucleoproteínas/genética , Idoso , Antagomirs/genética , Antagomirs/metabolismo , Autoantígenos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo
8.
Curr Med Sci ; 39(3): 403-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209810

RESUMO

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Ácido Láctico/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Simportadores/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Via de Sinalização Wnt/genética
9.
Cancer Sci ; 110(8): 2643-2651, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31222839

RESUMO

Scirrhous-type gastric cancer (SGC) is one of the most intractable cancer subtypes in humans, and its therapeutic targets have been rarely identified to date. Exploration of somatic mutations in the SGC genome with the next-generation sequencers has been hampered by markedly increased fibrous tissues. Thus, SGC cell lines may be useful resources for searching for novel oncogenes. Here we have conducted whole exome sequencing and RNA sequencing on 2 SGC cell lines, OCUM-8 and OCUM-9. Interestingly, most of the mutations thus identified have not been reported. In OCUM-8 cells, a novel CD44-IGF1R fusion gene is discovered, the protein product of which ligates the amino-terminus of CD44 to the transmembrane and tyrosine-kinase domains of IGF1R. Furthermore, both CD44 and IGF1R are markedly amplified in the OCUM-8 genome and abundantly expressed. CD44-IGF1R has a transforming ability, and the suppression of its kinase activity leads to rapid cell death of OCUM-8. To the best of our knowledge, this is the first report describing the transforming activity of IGF1R fusion genes. However, OCUM-9 seems to possess multiple oncogenic events in its genome. In particular, a novel BORCS5-ETV6 fusion gene is identified in the OCUM-9 genome. BORCS5-ETV6 possesses oncogenic activity, and suppression of its message partially inhibits cell growth. Prevalence of these novel fusion genes among SGC awaits further investigation, but we validate the significance of cell lines as appropriate reagents for detailed genomic analyses of SGC.


Assuntos
Adenocarcinoma Esquirroso/genética , Oncogenes/genética , Neoplasias Gástricas/genética , Células 3T3 , Adenocarcinoma Esquirroso/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Proteínas de Membrana/genética , Camundongos , Mutação/genética , Receptor IGF Tipo 1/genética , Estômago/patologia , Neoplasias Gástricas/patologia
10.
BMC Immunol ; 20(1): 20, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226944

RESUMO

BACKGROUND: CD44 is a multifunctional molecule that plays major roles in both leukocyte recruitment and tissue proliferation. Since mucosal hyperplasia and leukocyte infiltration of the middle ear cavity are major features of otitis media, we evaluated the role of CD44 in the pathophysiology and course of this disease in a mouse model of middle ear infection. Expression of genes related to CD44 function were evaluated using gene arrays in wild-type mice. The middle ears of mice deficient in CD44 were inoculated with non-typeable Haemophilus influenzae. Histopathology and bacterial clearance were compared to that seen in wild-type controls. RESULTS: We observed strong up-regulation of CD44 and of genes related to its role in leukocyte extravasation into the middle ear, during the course of acute otitis media. Mice deficient in CD44 exhibited reduced early mucosal hyperplasia and leukocyte recruitment, followed by delayed resolution of infection and persistent inflammation. CONCLUSIONS: CD44 plays an important role in OM pathogenesis by altering the mucosal growth and neutrophil enlistment. Targeted therapies based on CD44 could be useful adjuncts to the treatment of middle ear infections.


Assuntos
Orelha Média/imunologia , Infecções por Haemophilus/imunologia , Haemophilus influenzae/fisiologia , Receptores de Hialuronatos/metabolismo , Membrana Mucosa/imunologia , Neutrófilos/imunologia , Otite Média/imunologia , Animais , Movimento Celular , Modelos Animais de Doenças , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos
11.
Mol Cell Biochem ; 459(1-2): 35-47, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073886

RESUMO

Recent study implicates that gastric cancer stem cells (CSCs) are capable of generating multiple types of cells to promote tumor growth and heterogeneity important for the development of gastric cancer. However, knowledge is limited regarding the expression and characteristics of marker-positive gastric CSCs. Therefore, gastric CSCs from a series of human gastric cancer cell lines (SNU-5, SNU-16, BGC-823, PAMC-82, MKN-45, and NCI-N87) using four putative CSC surface markers (CD44, CD90, CD133, and epithelial-cell adhesion molecule) were investigated the underlying mechanisms regulating such subpopulations. Only SNU-5 and SNU-16 exhibited independent co-expression of CD44+ and CD90+, which exhibited spheroid-colony formation in vitro and tumor formation in immunodeficient mice. Functional studies revealed that CD44+ cells were more invasive compared with CD90+ cells, whereas CD90+ cells exhibited higher levels of proliferation than CD44+ cells. Furthermore, serial xenotransplantation in mice of CD44+/CD90+ cells derived from SNU-5 and SNU-16 revealed rapid growth of CD90+ cells in subcutaneous lesions and a high metastatic capacity of CD44+ cells in the lung. Mechanistic analyses revealed that CD44+ cells underwent epithelial-to-mesenchymal transition (EMT) following acquisition of mesenchymal features, whereas CD90+ cells enhanced the activation of retinoblastoma phosphorylation at Ser780 and oncogenic cell cycle regulators. The expression of CD44 and CD90 in gastric cancer tissues was associated with distant metastasis and the differentiation state of tumors. These results demonstrated that CD44 and CD90 are specific biomarkers capable of identifying and isolating metastatic and tumorigenic CSCs through their ability to regulate EMT and the cell cycle in gastric cancer cell lines.


Assuntos
Biomarcadores Tumorais/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/biossíntese , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/metabolismo , Antígenos Thy-1/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Receptores de Hialuronatos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Antígenos Thy-1/genética
12.
Oncol Rep ; 42(1): 91-102, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115569

RESUMO

Ovarian cancer (OC) has the highest mortality rate among female malignant tumors, and OC commonly relapses and metastasizes. The mechanisms underlying the occurrence and development of ovarian cancer are numerous and complicated. The aim of the present study was to explore an important molecular mechanism that may provide a theoretical basis for the clinical treatment of ovarian cancer. In the present study, the expression level of miR­21 was analyzed in clinical specimens, normal ovarian epithelial cells and three different ovarian cancer epithelial cell lines. Then, in vitro experiments were performed following the transient transfection of miR­21 mimics and inhibitors into SKOV3 cells. RT­PCR, western blot analysis, colony formation assay, and Transwell migration and invasion assays were used to explore the role of miR­21 in ovarian cancer. In addition, Wnt signaling pathway inhibitors and activators were used to validate the hypothesis that the miR­21/Wnt/CD44v6 pathway plays an important role in OC. In ovarian cancer tissues and cells, miR­21 was highly expressed, and the high expression of miR­21 could activate the Wnt signaling pathway to regulate the expression of CD44v6 and affect the proliferation, invasion and migration of OC cells. miR­21 regulated the expression of CD44v6 by activating the Wnt signaling pathway, which plays an important role in the development of ovarian cancer. These findings provide a potential new therapeutic target for the clinical diagnosis and treatment of ovarian cancer.


Assuntos
Receptores de Hialuronatos/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Regulação para Cima , Adulto , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Via de Sinalização Wnt
13.
Fertil Steril ; 112(2): 323-335.e2, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31056312

RESUMO

OBJECTIVE: To elucidate the potential regulatory function of miR-23a/b-3p on spermatogenesis-specific genes. DESIGN: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, Northern blot, dual luciferase assay, and Western blot confirmation. SETTING: University research and clinical institutes. PATIENT(S): A total of 115 men presenting at an infertility clinic. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Significant higher abundance levels of miR-23a/b-3p and lower abundance levels of PFKFB4, HMMR, SPATA6, and TEX15 in oligoasthenozoospermic men compared with those in normozoospermic men. RESULT(S): In oligoasthenozoospermic men, the abundance levels of miR-23a/b-3p were significantly higher when compared with controls as determined by RT-qPCR. After in silico prediction of potential targets of miR-23a/b-3p, PFKFB4, HMMR, SPATA6, and TEX15 have been identified as direct targets by dual luciferase assays. Mutations in the miR-23a/b-3p binding site within the 3'UTRs resulted in abrogated responsiveness to miR-23a/b-3p. PFKFB4, HMMR, SPATA6, and TEX15 mRNA and HMMR and SPATA6 protein levels were significantly lower in oligoasthenozoospermic men compared with in normozoospermic men. Correlation analysis showed that the sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with PFKFB4, HMMR, SPATA6, and TEX15 abundance levels (lower ΔCt, the higher abundance levels). CONCLUSION(S): This study establishes a link between up-regulation of miR-23a/b-3p and the coincident down-regulation of four expressed genes in the sperm of men with oligoasthenozoospermia, compared with men with normozoospermia. This study provides a novel insight into some of the mechanisms leading to male subfertility, offering a possible therapeutic target for treatment, or even for male contraception.


Assuntos
Infertilidade Masculina/genética , MicroRNAs/genética , Adulto , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , MicroRNAs/fisiologia , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Adulto Jovem
14.
Pathobiology ; 86(4): 182-189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31132784

RESUMO

CD47 activates signal regulatory protein alpha expressed on macrophages and suppresses its phagocytic ability; therefore, CD47 is drawing attention as an immune checkpoint in the innate immune system. Expression of CD47 in cancer is thought to allow cancer cells to escape antitumor immunity of the innate immune system. In this study, expression of CD47 was examined by immunostaining in colorectal cancer (CRC) and compared with the expression of CD44, which is a marker for cancer stem cells. In 95 cases of stage II-IV CRC, CD47 and CD44 showed overexpression in 82 and 80 cases, respectively. Both expression levels correlated with distant metastasis. Moreover, the expression of CD47 and CD44 in each case showed a significant correlation. In stage III cases, disease-free survival of cases showing high expression of CD47 and CD44 was worse than that of the cases with low expression. Furthermore, 3 of the stage IV cases were administered nivolumab, a checkpoint inhibitor of the acquired immune system, and 2 patients showed recurrence thereafter. All recurrent tumors highly expressed CD47 and CD44 and showed the epithelial-mesenchymal transition (EMT) phenotype. Our results suggest that CD47 promotes the malignancy of CRC in association with EMT and enhances the stemness of cancer cells. Moreover, our study suggests that CD47 and CD44 are involved in imparting resistance to programmed cell death (PD)-1/PD-ligand 1 inhibitors.


Assuntos
Antígeno CD47/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores de Hialuronatos/genética , Adulto , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas/citologia , Nivolumabe/uso terapêutico
15.
Oncol Rep ; 41(6): 3393-3403, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002352

RESUMO

Scientific evidence linking vitamin D with various cancer types is growing, but the effects of vitamin D on ovarian cancer stem cell­like cells (CSCs) are largely unknown. The present study aimed to examine whether vitamin D was able to restrain the stemness of ovarian cancer. A side population (SP) from malignant ovarian surface epithelial cells was identified as CSCs, in vitro and in vivo. Furthermore, 1α,25­dihydroxyvitamin D3 [1α,25(OH)2D3] treatment inhibited the self­renewal capacity of SP cells by decreasing the sphere formation rate and by suppressing the mRNA expression levels of cluster of differentiation CD44, NANOG, OCT4, SOX2, Krüppel­like factor 4 and adenosine triphosphate binding cassette subfamily G member 2. Additionally, 1α,25(OH)2D3 treatment decreased the expression of Cyclin D1, whereas it increased the expression of ß­catenin and vitamin D receptor (VDR). Notably, immunofluorescence staining verified that 1α,25(OH)2D3 promoted the expression of ß­catenin in the cytoplasm. Furthermore, vitamin D3 delayed the onset of tumor formation derived from injection of ovarian CSCs to nude mice, by reducing CD44 and enhancing ß­catenin expressions in vivo. In conclusion, 1α,25(OH)2D3 restrains the stem cell­like properties of ovarian cancer cells by enhancing the expression of VDR, by promoting the expression of ß­catenin in the cytoplasm, and by suppressing the expression of CD44. These findings provide a novel insight into the functions of vitamin D in diminishing the stemness of cancer CSCs.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados , Vitamina D/genética , Autorrenovação Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Fatores de Transcrição Kruppel-Like/genética , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição SOXB1/genética , Vitamina D/metabolismo , Vitamina D/farmacologia , beta Catenina/genética
16.
RNA ; 25(7): 813-824, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988101

RESUMO

Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Fatores de Processamento de RNA/química , Acetilação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cromatina/genética , Proteína p300 Associada a E1A/genética , Éxons , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Regiões Promotoras Genéticas , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Transcrição Genética , Células Tumorais Cultivadas
17.
J Ethnopharmacol ; 238: 111877, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-30995545

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Luffa cylindrica (L.) M.Roem is a climbing plant its parts have been used as traditional medicine for the treatment of different types of diseases including diarrhea, inflammation, cancer and viral infections. The parts used include fruit, seeds and leaves. AIM OF THE STUDY: Our study aims to investigate the effect of the aqueous-ethanol extract of Luffa cylindrica leaves on breast cancer stem cells CD44+/24- and other cell sub-populations using clinical samples with different molecular sub-types of breast cancer in vitro. MATERIALS AND METHODS: Breast tissues were obtained from patients undergoing surgery for the removal of breast tumors after complete clinical and pathological investigations. Tissue samples were processed to cell suspensions and treated with the extract in the tissue culture laboratory. Percentages of cell sub-populations within tumors and viability were measured by flowcytometry using clusters of differentiation as cell markers. RESULTS: Our results revealed that there were decreases in the total cell viability, CD44+/24- and total CD24+ cell sub-populations percentages after treatment with the extract, this may be an important indication of using Luffa leaves extract in the treatment of breast cancer or in combination with the traditional treatments. CONCLUSION: Luffa cylindrica has proven to have anticancer activity on three different subtypes of breast cancer including luminal A, luminal B and Her2/neu enriched more over it has cytotoxic effect on both bulk tumor cells as well as cancer stem cells sub population CD44+/24- which possess high tumorigenic potency, these results were confirmed by measuring their viable number after treatment and sphere formation assay results.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Luffa/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antígeno CD24/genética , Antígeno CD24/metabolismo , Etanol , Feminino , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/classificação , Extratos Vegetais/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Técnicas de Cultura de Tecidos
18.
Lupus ; 28(5): 621-628, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30907297

RESUMO

BACKGROUND: Adhesion molecule CD44 contributes to T cell migration into target organs. A higher expression of CD44v3 and v6 isoforms has been identified on T cells from systemic lupus erythematosus (SLE) patients. The aim of this study was to investigate the expression of CD44v3/v6 on T cells of SLE patients in order to evaluate their correlation with clinical features. METHODS: Sixteen healthy subjects (HSs) and 33 SLE female patients were enrolled. Fifteen patients were in remission (Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K) = 0) and 18 patients had an active disease (SLEDAI-2K ≥ 4). Experiments were conducted by flow cytometry. RESULTS: Expression of CD44v3 on CD4+ and CD8+ T cells was higher in active patients compared to HSs ( p = 0.0097 and p = 0.0096). CD44v3 on CD8+ T cells was also higher in active patients compared to patients in remission ( p = 0.038). CD44v6 was higher on CD4+ and CD8+ T cells from active patients compared to HSs ( p = 0.003 and p = 0.0036) and to patients in remission ( p = 0.01 and p = 0.02). In active patients the ratio CD44v3/v6 was unbalanced towards isoform v6 on both T cell populations. In a receiver operating characteristic curve analysis, CD44v6 on CD4+ T cells was the most sensitive and specific one (specificity of 81.8%, sensitivity of 75%). Expression of CD44v6 on CD4+ and CD8+ T cells correlated with the SLEDAI-2K ( p = 0.03, r = 0.38 and p = 0.02, r = 0.39). CD44v6 and CD44v3 on CD8+ T cells associated with nephritis and arthritis ( p = 0.047 and p = 0.023). CONCLUSIONS: CD44v3/v6 can be used as biomarkers of disease activity and phenotypes; isoform v6 on CD4+ T cells can be useful as a diagnostic biomarker.


Assuntos
Marcadores Genéticos , Receptores de Hialuronatos/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T/imunologia , Adulto , Estudos de Casos e Controles , Progressão da Doença , Feminino , Citometria de Fluxo , Expressão Gênica , Variação Genética , Humanos , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Índice de Gravidade de Doença
19.
J Exp Clin Cancer Res ; 38(1): 132, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890157

RESUMO

BACKGROUND: Cancer-initiating cell (CIC) exosomes (CIC-TEX) are suggested reprogramming Non-CIC. Mode of message transfer and engagement of CIC-markers being disputed, we elaborated the impact of CD44v6 and Tspan8 on the response of Non-CIC. METHODS: Non-metastasizing CD44v6- and Tspan8-knockdown (kd) pancreatic cancer cells served as Non-CIC. CIC-TEX coculture-induced changes were evaluated by deep-sequencing and functional assays. Tumor progression was surveyed during in vivo CIC-TEX treatment. RESULTS: Deep-sequencing of CIC-TEX-cocultured CD44v6kd-Non-CIC revealed pronounced mRNA changes in signaling, transport, transcription and translation; altered miRNA affected metabolism, signaling and transcription. CIC-TEX coculture-induced changes in Tspan8kd-Non-CIC mostly relied on CIC-TEX-Tspan8 being required for targeting. CIC-TEX transfer supported apoptosis resistance and significantly promoted epithelial mesenchymal transition, migration, invasion and (lymph)angiogenesis of the kd Non-CIC in vitro and in vivo, deep-sequencing allowing individual mRNA and miRNA assignment to altered functions. Importantly, CIC-TEX act as a hub, initiated by CD44v6-dependent RTK, GPCR and integrin activation and involving CD44v6-assisted transcription and RNA processing. Accordingly, a kinase inhibitor hampered CIC-TEX-fostered tumor progression, which was backed by an anti-Tspan8 blockade of CIC-TEX binding. CONCLUSIONS: This in depth report on the in vitro and in vivo impact of CIC-TEX on CD44v6kd and Tspan8kd Non-CIC unravels hub CIC-TEX activity, highlighting a prominent contribution of the CIC-markers CD44v6 to signaling cascade activation, transcription, translation and miRNA processing in Non-CIC and of Tspan8 to CIC-TEX targeting. Blocking CIC-TEX binding/uptake and uptake-initiated target cell activation significantly mitigated the deleterious CIC-TEX impact on CD44v6kd and Tspan8kd Non-CIC.


Assuntos
Reprogramação Celular/genética , Exossomos/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Linhagem Celular Tumoral , Exossomos/genética , Xenoenxertos , Humanos , Receptores de Hialuronatos/genética , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
20.
Dev Comp Immunol ; 96: 135-143, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30885554

RESUMO

CD44 gene is a cell surface receptor which undergoes complex alternative splicing and extensive post-translational modifications. Although many studies have showed that CD44 is involved in the process of host defense, the function of piscine CD44 in antibacterial or antiviral defense response remains unclear. In the present study, we report the functional characterization of zebrafish CD44c, which is more similar to CD44b antigen isoforms rather than CD44a based on amino acid composition and phylogenetic analysis. The expression of zebrafish CD44c was inducible in response to bacterial and viral infections. During SVCV infection, the in vivo studies revealed that CD44c overexpression led to the increased virus loads and decreased survival rate. The attenuated response by zebrafish CD44c in response to SVCV infection were characterized by the impaired production of inflammatory cytokines and the impaired expressions of IFNs, IFN-stimulated genes, MHC class I and II genes. During Edwardsiella piscicida infection, the overexpression of zebrafish CD44c facilitated bacterial growth and dissemination, but did not impact on larvae survival. The detrimental role of CD44c in host defense against E. piscicida infection was supported by a decreased production of several antibacterial molecules including defbl2, defbl3, NK-lysin and RNase3. All together, these results firstly demonstrate the negative regulation of piscine CD44c in viral and bacterial infection.


Assuntos
Infecções por Enterobacteriaceae/imunologia , Doenças dos Peixes/imunologia , Receptores de Hialuronatos/imunologia , Infecções por Rhabdoviridae/imunologia , Peixe-Zebra/imunologia , Processamento Alternativo/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Citocinas/imunologia , Citocinas/metabolismo , Edwardsiella/imunologia , Edwardsiella/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/microbiologia , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Imunidade Inata , Larva/imunologia , Larva/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Rhabdoviridae/imunologia , Rhabdoviridae/patogenicidade , Infecções por Rhabdoviridae/virologia , Relação Estrutura-Atividade , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/virologia
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