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BACKGROUND: Reduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported. RESULTS: To examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading. CONCLUSION: mtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.
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Envejecimiento/metabolismo , ADN Mitocondrial/metabolismo , Dermis/metabolismo , Fibroblastos/metabolismo , Estrés Oxidativo , Eliminación de Secuencia , Envejecimiento de la Piel , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/patología , ADN Mitocondrial/genética , Dermis/patología , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Objective To investigate the expression of Yes-associated protein (YAP) in cutaneous squamous cell carcinoma (cSCC) and its effects on invasion and migration. Methods Immunohistochemical staining was used to detect the expression of YAP in cSCC, Bowen disease (BD), and adjacent normal tissues, and analyzed the correlation between YAP expression and clinicopathological characteristics of cSCC. A stable cell line in A431 cells with YAP gene silencing was established through lentiviral infection. Tetramethylrhodamine isothiocyanate (TRITC)-phalloidin staining was performed to analyze the distribution and number of microfilaments in A431 cells. TranswellTM chamber assay was performed to detect the invasion ability of cells, and scratch healing assay was used to determine the migration ability. Immunofluorescence cytochemistry was used to detected the expression of EMT-related markers, including epithelial-cadherin (E-cadherin), zinc-finger transcription factors Snail in A431 cells with YAP silencing. Western blot analysis was employed to detect the expression of E-cadherin, snail, ß-catenin, phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), phosphorylaedAKT (p-AKT), ribosomal proteinS6(S6), phosphorylatedS6 (p-S6), 4E-binding protein 1 (4EBP1), and phosphorylated 4EBP1 (p-4EBP1). ResultsThe expression of YAP was significantly higher in BD and cSCC tissues compared to adjacent tissues. The strong positive rate of YAP in cSCC tissues was associated with tumor size, differentiation and the level of invasion. However, there was no correlation between YAP expression and gender, age, tumor location, morphological type, or nerve and vascular invasion. After silencing the expression of YAP in A431 cells, the migration and invasion ability of tumor cells were significantly reduced, and cell microfilaments became thinner with reduced pseudopodia. The expression of E-cadherin was increased, while the expression of snail, ß-catenin, p-AKT, p-S6 and p-4EBP1were decreased. Conclusion YAP is highly expressed in cSCC tissues, and promotes the cell migration and invasion of cSCC cells by activating the PI3K/AKT signaling pathway and EMT.
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Carcinoma de Células Escamosas , Neoplasias Cutáneas , Humanos , Fosfatidilinositol 3-Quinasa , Carcinoma de Células Escamosas/genética , Proteínas Proto-Oncogénicas c-akt , beta Catenina , Fosfatidilinositol 3-Quinasas , Proteínas Señalizadoras YAP , Neoplasias Cutáneas/genética , CadherinasRESUMEN
Histone deacetylases (HDACs) are commonly overexpressed in several types of human cancers, including prostate cancer (PCa). Histone deacetylase inhibitors (HDACis) are emerging as promising tools for cancer therapy. However, there is still a need to understand their anti-tumor effects and the mechanisms underlying their action. In our study, we investigated the effects of co-treatment with CUDC-101 and docetaxel (DTX) on cell growth, clonogenicity, invasion and migration of PCa cells both in vitro, and in a xenograft mouse model. We found that the combination of CUDC-101 and DTX significantly reduced tumor growth, as evidenced by lower tumor weight and volumes. Moreover, apoptotic cell death was increased in the combination group compared to either drug alone or control. Mechanistically, we observed that the combined treatment of CUDC-101 with DTX suppressed the progression of PCa cell lines through the AKT and ERK1/2 signaling pathways. Additionally, this combination treatment reversed EMT by modulating the expression of key markers such as E-cadherin, vimentin, Snail and MMP-9. To conclude, these results demonstrated that the combination of CUDC-101 with DTX had a synergistic and significantly improved anti-carcinogenic effect. This combination may serve as a potential strategy for clinical treatment and prognosis improvement in PCa.
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Ácidos Hidroxámicos , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Docetaxel/farmacología , Línea Celular Tumoral , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Apoptosis , Proliferación CelularRESUMEN
BACKGROUND: Ezrin and YAP are abnormally expressed in various cancers, and play pivotal roles in cancer initiation and development. However, the mechanisms of Ezrin in pancreatic cancer have not been fully elucidated. In this study, we aimed to elucidate the functions and mechanisms of Ezrin in the pathogenesis of pancreatic cancer. METHODS: Effects of Ezrin deregulation on pancreatic cancer phenotype were determined in Capan-1 and BxPC-3 cells using MTT, colony formation, transwell, wound-healing, and chick chorioallantoic membrane assays. To find out the underlying mechanism of Ezrin, multiple assays were performed to detect the effect of Ezrin on Akt pathway activation and YAP expression. Then, Ezrin and YAP expression was analyzed in pancreatic cancer and normal pancreas samples. Finally, the prognostic value of Ezrin and YAP was evaluated in pancreatic cancer patients. RESULTS: Ezrin promoted proliferation, invasion, epithelial-mesenchymal transition (EMT) progression, and angiogenesis of pancreatic cancers. Mechanistically, Ezrin activated Akt/mTOR pathways and induced YAP phosphorylation and nucleus translocation. The PI3K/Akt pathway inhibitor, rapamycin, and LY294002 could partially attenuate the effect of Ezrin on cell proliferation, invasion, EMT progression, and YAP phosphorylation and translocation. Moreover, both Ezrin and YAP were significantly overexpressed in pancreatic cancer tissues compared with adjacent normal pancreas, and correlated with poor prognosis in pancreatic cancer patients. Multivariate survival analysis showed that Ezrin was an independent prognostic marker for pancreatic cancer. Furthermore, the expression status of Ezrin and YAP had positive correlations in pancreatic cancer tissues. CONCLUSION: Ezrin promoted pancreatic cancer proliferation, invasion, migration, and EMT progression, partially through activating the PI3K/Akt pathway, and also regulated YAP phosphorylation and translocation, partially through the PI3K/Akt pathway. Ezrin and YAP were significantly overexpressed in pancreatic cancers, and correlated with poor prognosis in pancreatic cancer patients.
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Ezrin acts as a dynamic linkage between plasma membrane and cytoskeleton, and thus involved in many fundamental cellular functions. Yet, its potential role in human skin is virtually unknown. Here we investigate the role of Ezrin in primary skin fibroblasts, the major cells responsible extracellular matrix (ECM) production. We report that Ezrin play an important role in the maintenance of skin fibroblast size/mechanical properties and proliferation. siRNA-mediated Ezrin knockdown decreased fibroblast size and mechanical properties, and thus impaired the nuclear translocation of YAP, a protein commonly response to cell size and mechanical force. Functionally, depletion of Ezrin significantly inhibited YAP target gene expression and fibroblast proliferation. Conversely, restoration of YAP nuclear translocation by overexpression of constitutively active YAP reversed YAP target genes expression and rescued proliferation in Ezrin knockdown cells. These data reveal a novel role for Ezrin in maintenance of fibroblast size/mechanical force and regulating YAP-mediated proliferation.
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Polyadenylate-binding protein-interacting protein 1 (Paip1) regulates translational initiation. Increasing evidence suggests that Paip1 plays important roles in cancer development and progression. This study explored the role of Paip1 in breast cancer progression and evaluated its prognostic value. The cellular location of Paip1 protein was determined using immunofluorescence. Then, Paip1 protein expression was evaluated by immunohistochemical staining in 119 breast cancers and 40 normal breast tissues. The correlation between Paip1 expression and the clinicopathologic features of breast cancer was evaluated using the χ2 test, and differences in survival curves were analyzed using log-rank tests. The role of Paip1 in breast cancer proliferation and cell cycle progression was identified by siRNA transfection. Paip1 was expressed mainly in the cytoplasm of cancer cells and tissues. Expression was observed in 60.5% of the breast cancers (72/119), which was significantly higher than in normal breast tissues (17.5%; 7/40). High expression of Paip1 protein was associated with high histologic grade, late clinical stage, and a low survival rate. Multivariate analysis indicated that Paip1 was an independent prognostic factor. Additionally, Paip1 depletion by RNAi significantly decreased cell proliferation and induced cell cycle arrest. In conclusion, our study demonstrated that Paip1 promotes the growth of breast cancers and could be a prognostic biomarker and therapeutic target.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Factores de Iniciación de Péptidos/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Neoplasias de la Mama/mortalidad , Proliferación Celular , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Factores de Iniciación de Péptidos/análisis , Pronóstico , Proteínas de Unión al ARN/análisisRESUMEN
The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-ß signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-ß signaling. CCN2 acts as a downstream mediator of TGF-ß/Smad signaling and acting synergistically with TGF-ß to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-ß/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin.
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Molecule-targeted therapy has achieved great progress in cancer therapy. Effective drug combinations are one way to enhance the therapeutic efficacy and combat resistance. Here, we determined the effect of the PI3K/mTOR dual inhibitor BEZ235 and the histone deacetylase inhibitor Trichostatin A (TSA) on human breast cancer. We demonstrated that the combination of BEZ235 and TSA results in significant synergistic growth inhibition of multiple breast cancer cell lines. Mechanistic studies revealed that the combined therapy induced apoptosis in a caspase-dependent manner, which might be related to the further depression of the PI3K/Akt/mTOR signalling pathway. Additionally, co-treatment with BEZ235 and TSA enhanced autophagic cell death by up-regulating the expression of LC3B-II and Beclin-1. The vivo tumour modelling studies revealed that BEZ235 combined with TSA blocked tumour growth without noticeable side effects. These data suggest that the combination of BEZ235 and TSA may be a new selective strategy, which may have significant clinical application in the treatment of breast cancer patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Imidazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/uso terapéutico , Imidazoles/administración & dosificación , Células MCF-7 , Ratones , Ratones Desnudos , Quinolinas/administración & dosificación , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate the clinicopathological significance of ezrin and SIX1 overexpression in alpha fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). METHODS: The EnVision immunohistochemical method was used to detect the protein expression of ezrin and SIX1 in the tumor tissues and corresponding adjacent normal liver tissues from 50 AFP-negative HCC patients. The correlations between the overexpression of ezrin and SIX1 and the prognosis of the patients with HCC were analyzed by Chi-square test and Spearman analysis. RESULTS: The positive rates of ezrin and SIX1 protein expression in AFP-negative HCC tissues were 68.0% (34/50) and 60.0% (30/50), respectively, and they were significantly higher than the expression rates [38.0% (19/50) and 26.0% (13/50)] in adjacent normal liver tissues. Chi-square test showed that the expression of both ezrin and SIX1 proteins were related to TNM stage and lymphovascular infiltration, and Spearman analysis revealed a positive correlation between ezrin and SIX1 expression in AFP-negative HCC. CONCLUSION: Both Ezrin and SIX1 proteins are highly expressed in AFP-negative HCC, and significantly related with the TNM stage and lymphovascular infiltration of patients with AFP-negative HCC. In addition, there is a positive correlation between ezrin and SIX1 expression in AFP-negative HCC.
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Biomarcadores de Tumor/biosíntesis , Carcinoma Hepatocelular/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Proteínas de Homeodominio/biosíntesis , Neoplasias Hepáticas/metabolismo , alfa-Fetoproteínas/metabolismo , Adulto , Anciano , Carcinoma Hepatocelular/patología , Distribución de Chi-Cuadrado , Femenino , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. NSCLC development and progression have recently been correlated with the heightened activation of histone deacetylases (HDACs) and PI3K/Akt signaling pathways. Targeted inhibition of these proteins is promising approach for the development of novel therapeutic strategies to treat patients with advanced NSCLC. For this reason, we combined a dual PI3K and mTOR inhibitor, BEZ235 with the HDAC inhibitor Trichostatin A (TSA), to determine their combined effects on human NSCLC. In this study, we initially discovered that co-treatment with BEZ235 and TSA showed a synergistic effect on inhibition of NSCLC cell proliferation and induction of apoptosis. The combination treatment also synergistically suppressed NSCLC migration, invasion and the NSCLC epithelial-mesenchymal transition (EMT) in vitro. The synergistic effect was also evidenced by declines in xenograft growth and metastasis rates and in ki-67 protein expression in vivo. Together, these results indicated that BEZ235 and TSA combination treatment significantly increased anti-tumor activities compared with BEZ235 and TSA alone, supporting a further evaluation of combination treatment for NSCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Células A549 , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/farmacología , Imidazoles/administración & dosificación , Imidazoles/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinolinas/administración & dosificación , Quinolinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Resultado del TratamientoRESUMEN
Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.