RESUMO
AIMS: Glioblastoma (GBM) is the most common and aggressive intracranial tumor with poor prognosis. A large majority of clinical chemotherapeutic agents cannot achieve the desired therapeutic effect. Chelerythrine (CHE), a natural component with multitudinous pharmacological functions, has been proven to have outstanding antitumor effects in addition to antibacterial, anti-inflammatory, and hypotensive effects. However, the anti-GBM effect of CHE has not been reported to date. The purpose of this paper is to observe the anti-GBM effect of CHE and further explore the related mechanism. MATERIALS AND METHODS: GBM cell lines (U251 and T98G) and BALB/c nude mice were used in the experiments. Methyl thiazolyl tetrazolium (MTT) and clone formation assays were applied to detect the viability, proliferation and stemness of GBM cells. Flow cytometry was utilized to identify the effect of CHE on GBM apoptosis. Scratch and Transwell experiments reflected the migration and invasion of cells. In vivo, xenograft tumors were implanted subcutaneously in nude mice. The progression of tumors was assessed by ultrasound and magnetic resonance imaging. Finally, western blot, bioinformatics, and immunohistochemistry experiments were used to explore the molecular mechanisms in depth. KEY FINDINGS: In vitro tests showed that CHE inhibited the proliferation, stemness, migration, and invasion of GBM cells and induced apoptosis. In vitro, CHE was observed to restrain the progression of xenograft tumors. We eventually proved that the cytotoxicity of CHE was relevant to the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway. SIGNIFICANCE: CHE inhibited GBM progression by inhibiting the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway and is a potential chemotherapeutic drug for GBM.
Assuntos
Benzofenantridinas/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzofenantridinas/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Progressão da Doença , Relação Dose-Resposta a Droga , Glioblastoma/diagnóstico por imagem , Glioblastoma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Smad2/antagonistas & inibidores , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidoresRESUMO
Prostate cancer (PCa) is the second most prevalent cancer in men worldwide. Most cases of death from PCa are due to metastasis. Early stages of metastasis are mediated by epithelial-mesenchymal transition (EMT) process through which cancer cells acquire motility and invasive characteristics. Thus, more potent and novel therapeutic strategies must be designed based on the inhibition of EMT or metastasis. Herein, we employ a co-culture system to evaluate the anti-EMT effects of human amniotic mesenchymal stromal cells (hAMSCs) on LNCaP PCa cells. The RNA of treated (sample) and untreated cancer cells (control) and whole-cell lysates of related cells were prepared and analysed through quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Based on the results, the expression of vimentin, Snail and Zeb1 in LNCaP cells decreased and the expression of E-cadherin increased after treatment with hAMSCs. Furthermore, induction of the cellular apoptosis in LNCaP cells was detected. The anti-cancer activity of conditioned medium from hAMSCs was shown using hanging drop technique (a 3D cell culture model). Our findings support the idea that stem cells can be considered as a novel therapeutic approach to inhibit prostate cancer cells. SIGNIFICANCE OF THE STUDY: The anti-tumour activity of hAMSCs on LNCaP prostate cancer cells using 2D and 3D cell culture models via induction of apoptosis, suppression of EMT process and down-regulation of EGFR was shown. The results of the present study support this idea that hAMSCs may be a potent therapeutic tool to suppress tumour growth in LNCaP prostate cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Vimentina/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Regulação para Baixo/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Células Tumorais Cultivadas , Vimentina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Prostate cancer (PCa) is the second most diagnosed type of cancer in men worldwide. Advanced PCa is resistant to conventional therapies and high recurrence has been associated with high rates of metastasis. Cancer stem cells (CSCs) have been proposed to be responsible for this, due to their ability of selfrenewal and differentiation into other cell types. Zinc finger Eboxbinding homeobox 1 (ZEB1), a transcription factor involved in the regulation of epithelialmesenchymal transition (EMT), has been associated with the activation of several mechanisms that lead to resistance to treatment. As recent evidence has shown that CSCs may originate from nonCSCs during EMT, it was hypothesized that knocking down ZEB1 expression in PCa cell lines could revert some properties associated with CSCs. Using lentiviraltransduction, ZEB1 expression was silenced in the PCa DU145 and LNCaP cell lines. The mRNA and protein expression levels of key canonical CSC markers (Krüppellike factor 4, SOX2, CD44 and CD133) were determined using reverse transcription-quantitative PCR and western blot analysis, respectively. In addition, the colony forming ability of the ZEB1knockdown cells was evaluated, and the type of colonies formed (holoclones, paraclones and meroclones) was also characterized. Finally, the ability to form prostatospheres was evaluated in vitro. It was found that in ZEB1knockdown DU145 cells, the expression levels of CSC phenotype markers (CD44, CD133 and SOX2) were decreased compared with those in the control group. Furthermore, ZEB1knockdown cells exhibited a lower ability to form prostatospheres and to generate colonies. In conclusion, stable silencing of ZEB1 reversed CSC properties in PCa cell lines. Since ZEB1 is associated with malignancy, therapy resistance and a CSC phenotype in PCa cell lines, targeting ZEB1 may be a key factor to eradicate CSCs and improve the prognosis of patients with advanced PCa.
Assuntos
Autorrenovação Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Próstata/citologia , Próstata/patologia , Neoplasias da Próstata/patologia , Ensaio Tumoral de Célula-Tronco , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
As a potential drug/gene delivery system, the ultrasound-targeted microbubble destruction (UTMD) system can be used as a vehicle as well as increasing the permeability of biological barriers to enhance the effect of tumor treatment. However, the effect of UTMD in the tumor EMT process is unknown. In this study, we aimed to investigate the potential and mechanism of UTMD induced oxidative stress in inhibiting EMT of breast cancer. Human breast MDA231 cells were treated with microbubble (MB), ultrasound (US) and UTMD, respectively. The generation of oxidative stress, the levels of miR-200c, ZEB1 and vimentin, and the numbers of migratory cells were evaluated quantitatively and qualitatively by the measurement of intracellular reactive oxygen species (ROS), qRT-PCR, western blot assay, and transwell assay. Then, to evaluate the role of UTMD-induced oxidative stress and miR-200c in the epithelial-mesenchymal transition (EMT) inhibition, the ROS scavenger N-acetyl-L-cysteine (NAC) and miR-200c inhibitor were used before UTMD treatment. We found that UTMD induced oxidative stress, upregulated the expression of miR-200c, downregulated the expression of ZEB1 and vimentin and suppressed the MDA231 cell migration. The addition of NAC and miR-200c inhibitor had an opposite impact on the expression of miR-200c and ZEB1, thus hindered the effects of UTMD on MDA231 cells EMT. In conclusion, UTMD can inhibit the EMT characteristics of MDA231 cells. The mechanism may be related to the regulation of the miR-200c/ZEB1 axis through the generation of ROS induced by UTMD, which may provide a new strategy to prevent the tumor cells EMT under UTMD treatment.
Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Interferente Pequeno/metabolismo , Vimentina/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Acetilcisteína/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos da radiação , Feminino , Sequestradores de Radicais Livres/farmacologia , Técnicas de Transferência de Genes , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Microbolhas , Estresse Oxidativo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Ondas Ultrassônicas , Vimentina/antagonistas & inibidores , Vimentina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy. SIGNIFICANCE: Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1498/F1.large.jpg.
Assuntos
Inibidores da Angiogênese/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adulto , Idoso , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína 1 Semelhante à Quitinase-3/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lignanas/farmacologia , Lignanas/uso terapêutico , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Microambiente Tumoral/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidoresRESUMO
Purpose/Aim: More and more evidences suggest that airway remodeling of fibrotic lung diseases may be associated with epithelial-mesenchymal transition (EMT) of human A549 cells induced by transforming growth factor (TGF)-ß1. Schisandrin B (Sch B) is the highest content of dibenzocyclooctadiene lignans in Schisandra chinensis. In this study, we assessed the inhibitory influences of Sch B on TGF-ß1-stimulated EMT in human A549 cells. Materials and Methods: The influences of Sch B on cell viability, invasion and metastasis in TGF-ß1-induced human A549 cells were detected by MTT, wound healing and transwell invasion assays. The expression levels of α-SMA, E-cadherin, ZEB1 and Twist1 were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. The enrichment of H3K4me3 and H3K9me3 at the ZEB1 promoter was determined by ChIP analysis. Results: Experimental results showed that Sch B increased the expression of the epithelial phenotype marker E-cadherin and inhibited the expression of the mesenchymal phenotype marker α-SMA during EMT induced by TGF-ß1. The enhancement in invasion and migration of TGF-ß1-induced A549 cells was inhibited by Sch B. Sch B also repressed the expression of ZEB1 transcription factor in EMT, by increasing the enrichment of H3K9me3 at the ZEB1 promoter to repress its transcription while the expression of the Twist1 transcription factor was unaffected. Conclusions: Our data suggest that Sch B can prevent TGF-ß1-stimulated EMT in A549 cells through epigenetic silencing of ZEB1, which may be clinically related to the efficient treatment of EMT-associated fibrotic diseases.
Assuntos
Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Lignanas/farmacologia , Compostos Policíclicos/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Células A549 , Antineoplásicos/uso terapêutico , Ciclo-Octanos/farmacologia , Ciclo-Octanos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Epigênese Genética/efeitos dos fármacos , Humanos , Lignanas/uso terapêutico , Fitoterapia , Compostos Policíclicos/uso terapêutico , Schisandra , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE: To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms. PATIENTS AND METHODS: Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity. RESULTS: The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together. CONCLUSIONS: Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.
Assuntos
Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Antagomirs/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
OBJECTIVE: In order to investigate the role of the zinc finger E-box-binding homeobox 1 (ZEB1) expression in the incidence of non-small cell lung cancer (NSCLC), the effects of the ZEB1 expression on the pathogenesis and prognosis of NSCLC were investigated. PATIENTS AND METHODS: Correlations of the expression of ZEB1 in 88 clinical patients with NSCLC with clinicopathological features were determined. The patients were followed up for 60 months to study the correlation between the ZEB1 expression and the prognosis of NSCLC patients. To further explore the role of the ZEB1 expression in the incidence of NSCLC, the expressions of ZEB1 and E-cadherin in three NSCLC cell lines (A549, NCI-H1299 and NCI-H1975) and human normal lung epithelial BESA-2B cell line were measured, and the cell invasion ability was detected. The role of ZEB1 in NSCLC cell invasion was verified through the knockdown of ZEB1 by the short hairpin ribonucleic acids (shRNAs). In addition, its effects on the proliferation and apoptosis of NSCLC cells were confirmed by the overexpression of ZEB1. RESULTS: The expression of ZEB1 in NSCLC tissues was remarkably higher than that in normal tissues, and the expression level of ZEB1 was significantly related to the cancer stage and tumor size. The lower the expression of ZEB1 was, the higher the overall survival rate and the longer the survival time would be. The expression of ZEB1 was negatively correlated with that of E-cadherin in cell lines, and ZEB1-shRNAs markedly reduced the invasion ability of NSCLC cells. The overexpression of ZEB1 resulted in an increase in the proliferation activity and a significant decrease in the apoptosis of A549 cells. CONCLUSIONS: The expression of ZEB1 is closely related to the incidence and prognosis of NSCLC. Increased expression of ZEB1 is helpful for promoting the invasion of NSCLC and enhancing the proliferation activity of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Apoptose , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Incidência , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
BACKGROUND: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. METHODS: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. RESULTS: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect. CONCLUSIONS: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/radioterapia , Proteína Quinase C-épsilon/antagonistas & inibidores , Tolerância a Radiação/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Reparo do DNA/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Mimetismo Molecular , Células PC-3 , Proteína Quinase C-épsilon/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Osteosarcoma (OS) patients often exhibit pulmonary metastasis, which results in high patient mortality. Our present study established the doxorubicin (Dox) resistant human OS MG-63 and HOS cells and named them MG-63/Dox and HOS/Dox, respectively. The Dox resistant OS cells had greater invasion ability than that of parental cells. The expression of ZEB1, while not FOXM1, Snail, HIF-1α, or Sp1, was significantly increased in Dox resistant OS cells. Silencing of ZEB1 can attenuate the metastasis and increase Dox sensitivity of MG-63/Dox and HOS/Dox cells. The upregulation of ZEB1 can increase of the expression of interlukin-6 (IL-6). Anti-IL-6 inhibited the invasion and increase the Dox sensitivity of MG-63/Dox and HOS/Dox cells. There was no significant difference of ZEB1 mRNA between Dox resistant and control cells. The upregulation of ZEB1 in Dox resistant OS cells can be attributed to the increase of protein half-life. This was confirmed by results that the inhibitor of proteasomal degradation can increase ZEB1 in Dox resistant OS cells. Over expression of SIAH1 can inhibit the expression of ZEB1 and increase the Dox sensitivity of MG-63/Dox and HOS/Dox cells. Collectively, we confirmed that SIAH1 induced ZEB1 is involved in the Dox resistance of OS cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interleucina-6/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Osteossarcoma/tratamento farmacológico , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Antibióticos Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Interleucina-6/metabolismo , Proteínas Nucleares/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
AIM: To investigate whether long non-coding RNA (lncRNA) ZEB1 antisense 1 (ZEB1-AS1) affects cell migration and invasion of cervical cancer by regulating epithelial-mesenchymal transition (EMT) via the p38MAPK pathway. METHODS: Human cervical cancer cell line Hela was classified into Control, NC siRNA, ZEB1-AS1 siRNA, SB203580 (p38MAPK pathway inhibitor) and ZEB1-AS1 siRNA + Anisomycin (p38MAPK pathway activator) groups. Quantitative real-time polymerase chain reaction was performed for ZEB1-AS1 expression, Western blotting to measure p38MAPK signaling pathway-/EMT-related proteins, and Wound-healing and Transwell assays to evaluate cell migration and invasion respectively. RESULTS: ZEB1-AS1 was upregulated in cancer tissues and related to major clinicopathological features of cervical cancer. Besides, patients with lower-ZEB1-AS1-expression had a higher 5-year survival rate than those patients with higher-ZEB1-AS1-expression. High ZEB1-AS1 expression and advanced Federation of Gynecology and Obstetrics stage were independent risk factors for patients' prognosis. Both ZEB1-AS1 siRNA and SB203580 effectively reduced p-p38 expression and the migration and invasion of Hela cells, with elevation of E-cadherin and reduction of Vimentin and N-cadherin. However, inhibitory effects of ZEB1-AS1 siRNA on EMT as well as cell migration and invasion of the Hela cell were reversed by Anisomycin. CONCLUSION: Inhibition of ZEB1-AS1 can block the p38MAPK signaling pathway, ultimately restricting the EMT and suppressing cell migration and invasion of cervical cancer cells.
Assuntos
Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Sistema de Sinalização das MAP Quinases/fisiologia , RNA Longo não Codificante/fisiologia , Neoplasias do Colo do Útero/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Antígenos CD/análise , Caderinas/análise , Feminino , Expressão Gênica , Células HeLa , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Prognóstico , RNA Antissenso/antagonistas & inibidores , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Interferente Pequeno/fisiologia , Transfecção , Vimentina/análise , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidoresRESUMO
Ovarian cancer is one of the deadliest gynecological malignancies in women. Chemoresistance has been a major obstacle for ovarian cancer treatment. Zinc finger E-box-binding homeobox 1 (ZEB1) is an important regulator of tumor development in various types of cancer. Abnormal expression of SLC3A2 (CD98hc), a type 2 transmembrane cell surface molecule, has been described in several cancers. This study was designed to investigate the role of ZEB1 and SLC3A2 in the chemoresistance to cisplatin in ovarian cancer cells. We found that ZEB1 was increased in cisplatin-resistant SKOV3/DPP cells. Downregulation of ZEB1 significantly decreased cell viability in response to cisplatin, increased cis-platin-induced apoptosis, and decreased migration and invasion in the presence of cisplatin. In addition, downregulation of ZEB1 decreased the volume and weight of implanted tumors. SLC3A2 was decreased in cisplatin-resistant SKOV3/DPP cells. Upregulation of SLC3A2 significantly decreased cell viability in response to cisplatin, increased cisplatin-induced apoptosis, and decreased migration and invasion in the presence of cisplatin. Moreover, upregulation of SLC3A2 decreased the volume and weight of implanted tumors. Downregulation of ZEB1 resulted in a significant increase of SLC3A2 expression. Moreover, downregulation of SLC3A2 significantly inhibited ZEB1 knockdown-mediated inhibition of cisplatin-resistance. ZEB1-mediated regulation of SLC3A2 was involved in the chemoresistance to cisplatin in ovarian cancer cells. Overall, we provide new insights into the mechanism of chemoresistance to cisplatin in ovarian cancer cells. ZEB1/SLC3A2 may be promising therapeutic targets for enhancement of the sensitivity of ovarian cancer cells to cisplatin-mediated chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Transplante Heterólogo , Regulação para Cima/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
BACKGROUND/AIMS: Long noncoding RNAs (lncRNAs) have been a research hotspot, as they play important roles in tumor development. However, their expression pattern and biological function in osteosarcoma have not yet been clarified. METHODS: Differentially expressed lncRNAs in osteosarcoma and paracarcinoma tissues were identified by screening an lncRNA microarray, and candidate lncRNAs were verified by quantitative real-time PCR (qRT-PCR). A series of bioinformatics and molecular biological methods were adopted to investigate the interaction among lncRNA, microRNA (miRNA), and miRNA target genes during the development and occurrence of osteosarcoma. Cell viability was measured using a Cell Counting Kit-8 assay. RESULTS: Chip microarray screening combined with the validation of differentially expressed candidate lncRNAs showed that the lncRNA small nucleolar RNA host gene 16 (SNHG16) had the largest fold change. SNHG16 was highly expressed in osteosarcoma tissues and cell lines, and its downregulation led to the suppressed proliferation of osteosarcoma cells. Further investigations revealed that SNHG16 could upregulate zinc finger E-box-binding homeobox 1 (ZEB1) expression by acting as an endogenous sponge of miR-205. Moreover, rescue assays proved that the effects of SNHG16 on the proliferation of osteosarcoma cells were dependent on miR-205. CONCLUSION: SNHG16 can significantly enhance the proliferation of osteosarcoma cells. In addition, SNHG16, miR-205, and ZEB1 interact in a common pathway during the development and occurrence of osteosarcoma, providing novel targets for intervention in the treatment of osteosarcoma.
Assuntos
Proliferação de Células , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Regiões 3' não Traduzidas , Antagomirs/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Bases de Dados Genéticas , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Mammalian palate separates the oral and nasal cavities for normal feeding, breathing and speech. The palatal shelves are a pair of maxillary prominences that consist of the neural crest-derived mesenchyme and surrounding epithelium. Palatogenesis is completed by the fusion of the midline epithelial seam (MES) after the medial edge epithelium (MEE) cells make contact between the palatal shelves. Various cellular and molecular events, such as apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT), are involved in palatogenesis. The Zeb family of transcription factors is an essential player during normal embryonic development. The distinct role of the Zeb family has not been thoroughly elucidated to date. In mouse palate, the Zeb family factors are expressed in the palatal mesenchyme until MEE contact. Interestingly, the expression of the Zeb family has also been observed in MES, which is already fused with the mesenchymal region. The regulatory roles of the Zeb family in palatogenesis have not been elucidated to date. The purpose of this study is to determine the Zeb family effects on the cellular events. To investigate the functions of the Zeb family, siRNA targeting Zeb family was used to treat in vitro organ culture for temporary inhibition of the Zeb family during palatogenesis. In the cultured palate containing siRNA, MES was clearly observed, and E-cadherin, an epithelial marker, was still expressed. Inhibition of the Zeb family results in the suppression of apoptosis, increased cell proliferation, and defective cell migration in the developing palate. Our data suggest that the Zeb family plays multiple roles in the stimulation and inhibition of apoptosis and cell proliferation and efficient mesenchymal cell migration during palatogenesis.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Palato/embriologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/fisiologia , Animais , Movimento Celular , Proliferação de Células , Células Epiteliais , Proteínas de Homeodomínio/fisiologia , Camundongos , Técnicas de Cultura de Órgãos , Palato/crescimento & desenvolvimento , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidoresRESUMO
Non-small cell lung cancer (NSCLC) is known as highly metastatic disease because it is difficult to diagnose at early stage. More than 60% of NSCLC patients' overexpress receptor tyrosine kinase (RTK) such as EGFR that has been proved to display resistance to receptor tyrosine kinase inhibitor (TKI) through PI3K signaling, while single PI3K inhibitors increase RTK expression as feedback. So, to select the proper targeted agent or target an assortment of molecular subsets, such as EGFR mutations for different subgroups of patients with NSCLC is urgent. Compound BENC-511, a potent PI3K inhibitor, had effects on inhibiting cancer cell survival and delaying tumor growth, but the effects and mechanisms on cancer metastasis are not clear. Methods of Scratch assay, Transwell system, experimental metastasis mice models, plasmid transfection, quantitative real-time PCR and Western blot were used. Results showed that BENC-511 could significantly inhibit lung cancer cells invasion and metastasis both in vitro and in vivo. And it not only inhibited PI3K/Akt signal pathway, but also directly suppressed phosphorylation of EGFR and nuclear translocation of ß-catenin. Moreover, our study firstly reported BENC-511 seemed more sensitive to NSCLC cells that highly expressed Zinc-finger E-box binding protein 1 (ZEB1), one of the epithelial-mesenchymal transition (EMT) inducer, and knockdown of ZEB1 could improve the effects of this compound. These findings suggested that BENC-511 should be a promising lead molecule for anti-metastasis therapy by targeting ß-catenin/ZEB1 regulatory loop and serve as a therapeutic agent to inhibit metastasis of NSCLC.
Assuntos
Benzopiranos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , beta Catenina/metabolismo , Animais , Benzopiranos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transplante Homólogo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
BACKGROUND/AIMS: MicroRNA-142-3p (miR-142-3p) is dysregulated in many malignancies and may function as a tumor suppressor or oncogene in tumorigenesis and tumor development. However, few studies have investigated the clinical significance and biological function of miR-142-3p in hepatocellular carcinoma (HCC). METHODS: The expression levels of taurine upregulated gene 1 (TUG1), miR-142-3p, and zinc finger E-box-binding homeobox 1 (ZEB1) were evaluated in HCC tissues and cell lines by quantitative real-time PCR. MTT and colony formation assays were used to detect cell proliferation ability, transwell assays were used to assess cell migration and invasion, and luciferase reporter assays were used to examine the interaction between the long noncoding RNA TUG1 and miR-142-3p. Tumor formation was evaluated through in vivo experiments. RESULTS: miR-142-3p was significantly downregulated in HCC tissues, but TUG1 was upregulated in HCC tissues. Knockdown of TUG1 and upregulation of miR-142-3p inhibited cell proliferation, cell migration, cell invasion, and the epithelial-mesenchymal transition (EMT). miR-142-3p was found to be a prognostic factor of HCC, and the mechanism by which TUG1 upregulated ZEB1 was via direct binding to miR-142-3p. In vivo assays showed that TUG1 knockdown suppressed cell proliferation and the EMT in nude mice. CONCLUSION: The results of this study suggest that the TUG1/miR-142-3p/ ZEB1 axis contributes to the formation of malignant behaviors in HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Antagomirs/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion-migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 ± 2.35 µg/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cyclamen/química , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/metabolismo , Extratos Vegetais/farmacologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Células A549 , Antígenos CD , Caderinas , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Long non-coding RNA OIP5-AS1 has been studied in human diseases, including several kinds of cancers. It was not studied or reported in hepatoblastoma, so we chose it to do our research in hepatoblastoma. We thought OIP5-AS1 was oncogenic in hepatoblastoma for the high expression of it in hepatoblastoma tissues and cells. OIP5-AS1 knockdown was carried out in cancer cells. Unsurprisingly, this action was verified to be able to inhibit cell proliferation, metastasis and EMT progress in hepatoblastoma. We then measured the low expression level of miR-186a-5p and the high expression level of ZEB1 in hepatoblastoma tissues and cells. The relevance among them was analyzed by using correlation analysis. In order to prove the ceRNA pattern in this study, nuclear separation experiment, RIP assay and dual luciferase assays were all put into use. We discovered OIP5-AS1 is located in nucleus of cancerous cells. It could target to miR-186a-5p and up-regulate the target gene of miR-186a-5p (ZEB1). Finally, rescue assay was utilized and proved the effect of OIP5-AS1-miR-186a-5p-ZEB1 axis on hepatoblastoma cell activities. Based on all above findings, we came into a conclusion that OIP5-AS1 is a ceRNA in Hepatoblastoma cells through modulating miR-186a-5p/ZEB1.
Assuntos
Biomarcadores Tumorais/biossíntese , Transição Epitelial-Mesenquimal/fisiologia , Hepatoblastoma/metabolismo , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , Homeobox 1 de Ligação a E-box em Dedo de Zinco/biossíntese , Idoso , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Proliferação de Células/fisiologia , Regulação para Baixo/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Células Hep G2 , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Regulação para Cima/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidoresRESUMO
Epithelial-Mesenchymal Transition (EMT) is a biological program that plays key roles in various developmental and pathological processes. Although much work has been done on signaling pathways and transcription factors regulating EMT, the epigenetic regulation of EMT remains not well understood. Histone variants have been recognized as a key group of epigenetic regulators. Among them, macroH2A1 is involved in stem cell reprogramming and cancer progression. We postulated that macroH2A1 may play a role in EMT, a process involving reprogramming of cellular states. In this study, we demonstrate that expression of macroH2A1 is dramatically reduced during EMT induction in immortalized human mammary epithelial cells (HMLE). Moreover, ectopic expression of the macroH2A1.1 isoform, but not macroH2A1.2, can suppress EMT induction and reduce the stem-like cell population in HMLE. Interestingly, macroH2A1.1 overexpression cannot revert stable mesenchymal cells back to the epithelial state, suggesting a stage-specific role of macroH2A1.1 in EMT. We further pinpointed that the function of macroH2A1.1 in EMT suppression is dependent on its ability to bind the NAD+ metabolite PAR, in agreement with the inability to suppress EMT by macroH2A1.2, which lacks the PAR binding domain. Thus, our work discovered a previously unrecognized isoform-specific function of macroH2A1 in regulating EMT induction.
Assuntos
Transição Epitelial-Mesenquimal , Histonas/metabolismo , Antígeno CD24/metabolismo , Caderinas/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Histonas/deficiência , Histonas/genética , Humanos , Receptores de Hialuronatos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Vimentina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
The ataxia-telangiectasia-mutated (ATM) and rad3-related (ATR) checkpoint pathway plays an essential role in modulating cellular responses to replication stress and DNA damage to maintain genomic stability. In various tumors, cancer cells have increased dependence on ATR signaling for survival, making ATR a promising target for cancer therapy. ATR inhibitors sensitize multiple tumor cell types to radiation and DNA-damaging agents, but application of an ATR inhibitor alone shows limited efficacy. In the present study, we investigated the role of epithelial-to-mesenchymal transition (EMT) and the EMT transcription factor ZEB1 in regulating cell sensitivity to the ATR inhibitor VE-821. We found that VE-821 induced EMT with concomitant ZEB1 upregulation and promoted migration in cells in which the anti-proliferative effect of VE-821 was limited. Knocking down ZEB1 using siRNA partially reversed VE-821-induced EMT, and sensitized cells to VE-821 via effective attenuation of migration and AKT/ERK signaling. Moreover, ZEB1 inhibition promoted Chk1 phosphorylation and induced S-phase arrest by enhancing TopBP1 expression, which suggests a distinctive modulatory effect of ZEB1 on Chk1. Finally, combining VE-821 with ZEB1 inhibition enhanced DNA damage accumulation. These results demonstrate that EMT represents a novel mechanism for limiting the effectiveness of an ATR inhibitor, and thus suggest that ZEB1 inhibition might represent a new approach to increasing the efficiency of, or reversing resistance to, ATR inhibitors.