RESUMO
Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.
Assuntos
Endometriose/patologia , Endométrio/patologia , Transição Epitelial-Mesenquimal/fisiologia , Estrogênios/fisiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Endometriose/cirurgia , Endométrio/efeitos dos fármacos , Endométrio/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Cistos Ovarianos/etiologia , Cistos Ovarianos/cirurgia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/biossíntese , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Microtúbulos/patologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Criança , Colesterol/metabolismo , Feminino , Furanos/farmacologia , Humanos , Ibuprofeno/farmacologia , Masculino , Camundongos Knockout , Células Sf9 , Spodoptera , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Fosfoproteínas/genética , Transativadores/genética , Animais , Neoplasias Encefálicas/genética , Glioblastoma/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Fatores de Transcrição , Proteínas de Sinalização YAP , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Laryngeal squamous cell carcinoma (LSCC) is one aggressive malignancy and accounts for 20% of all head and neck cancer. However, the role of LOC554202 in human LSCC remains unknown. The expression level of LOC554202 and miR-31 was detected in the LSCC tiussues by using qRT-PCR. Cell growth was measured by CCK-8 assay. Flow cytometry and matrigel-coated membrane was used to detect for cell cycle and invasion respectively. We indicated that lncRNA LOC554202 expression was overexpressed in LSCC tissues compared with the paired adjacent samples and higher LOC554202 expression was associated with the advanced stage. In addition, we demonstrated that the expression level of miR-31 was downregulated in LSCC tissues compared to the paired adjacent samples and lower miR-31 expression was correlated with the advanced stage. Moreover, the expression of miR-31 was negatively correlated with the expression of LOC554202 in LSCC tissues. Ectopic expression of LOC554202 promoted LSCC cell growth, cell cyle and cell invasion and overexpression of miR-31 inhibited LSCC cell growth, cell cyle and cell invasion. Elevated expression of LOC554202 suppressed miR-31 expression and promoted RhoA expression in LSCC cell, which was a direct target gene of miR-31. Furthermore, LOC554202 increased LSCC cell growth, cell cyle and cell invasion through suppressing miR-31 expression. These results suggested that LOC554202 acted as an oncogene in the development of LSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Laríngeas/metabolismo , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , MicroRNAs/genética , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Histological assessments of synovial tissues from patients with failed CoCr alloy hip prostheses demonstrate extensive infiltration and accumulation of macrophages, often loaded with large quantities of particulate debris. The resulting adverse reaction to metal debris (ARMD) frequently leads to early joint revision. Inflammatory response starts with the recruitment of immune cells and requires the egress of macrophages from the inflamed site for resolution of the reaction. Metal ions (Co2+ and Cr3+) have been shown to stimulate the migration of T lymphocytes but their effects on macrophages motility are still poorly understood. To elucidate this, we studied in vitro and in vivo macrophage migration during exposure to cobalt and chromium ions and nanoparticles. We found that cobalt but not chromium significantly reduces macrophage motility. This involves increase in cell spreading, formation of intracellular podosome-type adhesion structures and enhanced cell adhesion to the extracellular matrix (ECM). The formation of podosomes was also associated with the production and activation of matrix metalloproteinase-9 (MMP9) and enhanced ECM degradation. We showed that these were driven by the down-regulation of RhoA signalling through the generation of reactive oxygen species (ROS). These novel findings reveal the key mechanisms driving the wear/corrosion metallic byproducts-induced inflammatory response at non-toxic concentrations. STATEMENT OF SIGNIFICANCE: Adverse tissue responses to metal wear and corrosion products from CoCr alloy implants remain a great challenge to surgeons and patients. Macrophages are the key regulators of these adverse responses to the ions and debris generated. We demonstrated that cobalt, rather than chromium, causes macrophage retention by restructuring the cytoskeleton and inhibiting cell migration via ROS production that affects Rho Family GTPase. This distinctive effect of cobalt on macrophage behaviour can help us understand the pathogenesis of ARMD and the cellular response to cobalt based alloys, which provide useful information for future implant design and biocompatibility testing.
Assuntos
Cobalto , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Nanopartículas Metálicas , Espécies Reativas de Oxigênio/metabolismo , Proteínas rho de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese , Animais , Cobalto/efeitos adversos , Cobalto/química , Cobalto/farmacologia , Reação a Corpo Estranho/induzido quimicamente , Reação a Corpo Estranho/metabolismo , Reação a Corpo Estranho/patologia , Humanos , Macrófagos/patologia , Nanopartículas Metálicas/efeitos adversos , Nanopartículas Metálicas/química , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células U937RESUMO
MicroRNAs could mediate the targeted coding gene and the targeted non-coding RNA to form endogenous competition, which have an important regulatory role in tumorigenesis of many types of cancer, including hepatocellular carcinoma. The goal of this study was to characterize the role of miR-200b in the pathogenesis of hepatocellular carcinoma. We identified miR-200b that was predicted to regulate RhoA and circ_000839. Our data establish that miR-200b is expressed at a relatively low level in hepatocellular carcinoma ( p < 0.001). RhoA and circ_000839 are expressed at a relatively high level in hepatocellular carcinoma ( p < 0.001, respectively). Our mechanistic data indicate that RhoA is a direct target of miR-200b ( p < 0.001), binding of which affects the expression of invasion and migration in hepatocellular carcinoma cell lines ( p < 0.05). And correlation analysis showed that miR-200b was inversely correlated with RhoA and circ_000839 ( p = 0.012, p = 0.002, respectively), while RhoA was positively correlated with circ_000839 ( p < 0.001). Taken together, our data suggest that miR-200b could mediate RhoA gene and circ_000839 to form endogenous competition. And this is a direction for the association study of miR-200b and RhoA in the future.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Proteína rhoA de Ligação ao GTP/biossíntese , Adulto , Idoso , Carcinogênese , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , RNA/genética , RNA Circular , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
GTPase RhoA and its downstream Rho-associated coiled-coil-containing protein kinases (ROCKs) are frequently overexpressed in human cancers. Inhibition of the RhoA/ROCK pathway blocks angiogenesis mediated by the vascular endothelial growth factor, which led us to investigate the role of this pathway in vasculogenic mimicry (VM) - a process by which aggressive cancer cells form vessel-like structures that provide adequate blood supply for tumor growth. We showed that the expression of RhoA and its effector kinases ROCK1/2 was much higher in human osteosarcoma (OS) tissues and the human OS cell line U2OS than in nontumorous tissues and cell line hFOB 1.19 using western blot analysis and real-time PCR. Inhibition of the RhoA/ROCK signaling pathway by the pharmacological inhibitor fasudil reduced vascular-like channels of U2OS cells in Matrigel. Furthermore, we used rhodamine-phalloidin immunofluorescence, wound healing assay, and transwell migration assay to examine the effect of fasudil on tumor cell plasticity and motility, both of which play key roles in VM formation. Finally, we explored the underlying mechanisms of fasudil-induced VM destruction. In this context, we showed that the RhoA/ROCK signaling pathway is a novel regulator in VM of U2OS OS cells and suggest that fasudil in conjunction with established treatments may present a novel therapeutic strategy for OS.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Neoplasias Ósseas/irrigação sanguínea , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Plasticidade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/patologia , Imunofluorescência , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Osteossarcoma/irrigação sanguínea , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/biossíntese , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. In our study, the in vivo protective effect of danshen in prostate cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we discovered that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the migration of both androgen-dependent and androgen-independent prostate cancer cells. In addition, we noted that DT substantially inhibited the migratory ability of prostate cancer cells in both a macrophage-conditioned medium and macrophage/prostate cancer coculture medium. Mechanistically, DT both diminished the ability of prostate cancer cells to recruit macrophages and reduced the secretion of chemokine (C-C motif) ligand 2 (CCL2) from both macrophages and prostate cancer cells in a dose-dependent manner. Moreover, DT inhibited the protein expression of p-STAT3 and decreased the translocation of STAT3 into nuclear chromatin. DT also suppressed the expression of tumor epithelial-mesenchymal transition genes, including RhoA and SNAI1. In conclusion, danshen can prolong the survival rate of prostate cancer patients in Taiwan. Furthermore, DT can inhibit the migration of prostate cancer cells by interrupting the crosstalk between prostate cancer cells and macrophages via the inhibition of the CCL2/STAT3 axis. These results may provide the basis for a new therapeutic approach toward the treatment of prostate cancer progression.
Assuntos
Quimiocina CCL2/biossíntese , Medicamentos de Ervas Chinesas/farmacologia , Macrófagos/metabolismo , Fenantrenos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Medicina Tradicional Chinesa , Camundongos , Próstata/patologia , Neoplasias da Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Salvia miltiorrhiza/química , Fatores de Transcrição da Família Snail/biossíntese , Resultado do Tratamento , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
RATIONALE: Abdominal aortic aneurysms (AAAs) are characterized by pathological remodeling of the aortic wall. Although both increased Krüppel-like factor 5 (KLF5) expression and macrophage infiltration have been implicated in vascular remodeling, the role of KLF5 in macrophage infiltration and AAA formation remains unclear. OBJECTIVE: To determine the role of KLF5 in AAA formation and macrophage infiltration into AAAs. METHODS AND RESULTS: KLF5 expression was significantly increased in human AAA tissues and in 2 mouse models of experimental AAA. Moreover, in myeloid-specific Klf5 knockout mice (myeKlf5-/- mice), macrophage infiltration, medial smooth muscle cell loss, elastin degradation, and AAA formation were markedly decreased. In cell migration and time-lapse imaging analyses, the migration of murine myeKlf5-/- macrophages was impaired, and in luciferase reporter assays, KLF5 activated Myo9b (myosin IXB) transcription by direct binding to the Myo9b promoter. In subsequent coimmunostaining studies, Myo9b was colocalized with filamentous actin, cortactin, vinculin, and Tks5 in the podosomes of phorbol 12,13-dibutyrate-treated macrophages, indicating that Myo9b participates in podosome formation. Gain- and loss-of-function experiments showed that KLF5 promoted podosome formation in macrophages by upregulating Myo9b expression. Furthermore, RhoA-GTP levels increased after KLF5 knockdown in macrophages, suggesting that KLF5 lies upstream of RhoA signaling. Finally, Myo9b expression was increased in human AAA tissues, located in macrophages, and positively correlated with AAA size. CONCLUSIONS: These data are the first to indicate that KLF5-dependent regulation of Myo9b/RhoA is required for podosome formation and macrophage migration during AAA formation, warranting consideration of the KLF5-Myo9b-RhoA pathway as a therapeutic target for AAA treatment.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , Fatores de Transcrição Kruppel-Like/biossíntese , Macrófagos/metabolismo , Miosinas/biossíntese , Podossomos/metabolismo , Proteína rhoA de Ligação ao GTP/biossíntese , Animais , Linhagem Celular , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Masculino , Camundongos , Camundongos Knockout , Miosinas/deficiência , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/deficiênciaRESUMO
Global cancer burden increased to 14.1 million new cases in 2012; and breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012. Curcumin is the major bioactive ingredient extracted from the rhizome of the plant Curcuma longa (turmeric). Paclitaxel is a microtubule-stabilizing agent originally isolated from the bark of Taxus brevifolia. Curcumin and paclitaxel were evaluated with two human breast cancer cell lines as the luminal MCF-7 and the basal-like MDA-MB-231 that are either positive or negative for hormonal receptors estrogen receptor, progesterone receptor and HER2, respectively. Results indicated that curcumin combined with paclitaxel decreased c-Ha-Ras, Rho-A, p53 and Bcl-xL gene expression in comparison to control and substances alone in MCF-7 cell line. These two substances alone and combined decreased gene expression of Bcl-2 and NF-κB. However, CCND1 increased when both substances were combined in MCF-7 cells. Such substances decreased Bcl-2 and increased Bax protein expression. However, curcumin alone decreased IκBα and Stat-3 gene expression. Paclitaxel alone and combined increased IκBα and Stat-3. Curcumin alone and combined with paclitaxel increased p53, Bid, caspase-3, caspase-8 and Bax gene expression in MDA-MB-231, whereas Bcl-xL decreased such expression in MDA-MB-231 cells. When paclitaxel and curcumin were combined the expression of Bcl-2 protein was decreased. However, either substance alone and combined increased Bax protein expression corroborating the apoptotic effect of these substances. It can be concluded that curcumin may be of considerable value in synergistic therapy of breast cancer reducing the associated toxicity with use of drugs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Curcumina/farmacologia , Paclitaxel/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Feminino , Humanos , Células MCF-7 , Inibidor de NF-kappaB alfa/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteína bcl-X/biossíntese , Proteína bcl-X/genética , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.
Assuntos
Caderinas/genética , Cateninas/genética , Diferenciação Celular/genética , Endoderma/crescimento & desenvolvimento , Células-Tronco Embrionárias Murinas , Animais , Blastocisto/metabolismo , Caderinas/biossíntese , Cateninas/biossíntese , Adesão Celular/genética , Linhagem da Célula/genética , Polaridade Celular/genética , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/genética , Endoderma/metabolismo , Humanos , Camundongos , Imagem Óptica , Células-Tronco Pluripotentes/metabolismo , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genética , delta CateninaRESUMO
Rho GTPase-activating proteins (RhoGAPs) are implicated in the development and progression of ovarian cancer. ARHGAP10 is a member of RhoGAP proteins and inactivates Cdc42 by converting GTP-bound form to GDP-bound form. Here, we aimed to evaluate ARHGAP10 expression profile and functions in ovarian cancer. The decreased expression of ARHGAP10 was found in 77.3% (58/75) of ovarian cancer tissues, compared with their non-tumorous counterparts. Furthermore, overall survival in ovarian cancer patients with higher expression of ARHGAP10 was longer than those with lower expression. Ectopic expression of ARHGAP10 in two ovarian cancer cell lines with lower expression of ARHGAP10 (A2780 and HO-8910) dramatically suppressed cell proliferation in vitro. In nude mice, its stable overexpression significantly inhibited the tumorigenicity of A2780 cells. We further demonstrated that overexpression of ARHGAP10 significantly inhibited cell adhesion, migration and invasion, resulted in cell arrest in G1 phase of cell cycle and a significant increase of apoptosis. Moreover, ARHGAP10 interacted with Cdc42 and overexpression of ARHGAP10 inhibited the activity of Cdc42 in A2780 cells. Gene set enrichment analysis on The Cancer Genome Atlas dataset showed that KEGG cell cycle, replication and base excision repair (BER) pathways were correlatively with the ARHGAP10 expression, which was further confirmed in ovarian cancer cells by western blotting. Hence, ARHGAP10 may serve as a tumor suppressor through inactivating Cdc42, as well as inhibiting cell cycle, replication and BER pathways. Our data suggest an important role of ARHGAP10 in the molecular etiology of cancer and implicate the potential application of ARHGAP10 in cancer therapy.
Assuntos
Regulação para Baixo , Proteínas Ativadoras de GTPase/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese , Animais , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Supressoras de Tumor/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1) was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1). Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA) mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05). Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4), ITGA5, and integrin beta 1 (ITGB1); otherwise, RhoA expression was significantly decreased (p < 0.05). Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Integrinas/biossíntese , Securina/metabolismo , Trofoblastos/metabolismo , Proteína rhoA de Ligação ao GTP/biossíntese , Linhagem Celular , Humanos , Integrinas/genética , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Securina/genética , Trofoblastos/citologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: It is difficult to achieve rhythm control in patients with long-standing persistent atrial fibrillation (AF). The radiofrequency maze procedure is an effective means in curing AF with a variable recurrence rate depending on patient characteristics and AF duration. In these patients, the characteristics of the atrial substrate have not been well investigated. Because the inflammatory process has been shown to be important in the pathogenesis of AF, we sought to characterize the proinflammatory gene expression in left atria obtained from patients with AF undergoing mitral valve surgery combined with the maze procedure to distinguish the changes associated with AF and its recurrence after the surgical ablation. METHODS: Left atrial appendages from 35 patients receiving mitral valve surgery were used for study. Ten patients had sinus rhythm (SR) and 25 patients had persistent AF for more than 1 year and underwent the maze procedure. Among the AF patients, 13 patients remained in SR (AF-SR) and 12 patients had recurrent AF during the 1-year clinical follow-up (AF-AF). The nCounter Human Inflammation Array (NanoString Technologies, Seattle, Wash) was used for evaluating proinflammatory gene expression. Quantitative polymerase chain reaction, Western blot, and immunohistochemistry were applied for studying messenger RNA and protein expression. RESULTS: Of 144 expressed proinflammatory genes, the inflammation array analysis revealed that 32 genes were differentially expressed between AF (including AF-SR and AF-AF) and SR. Thirteen genes were differentially expressed between AF-SR and AF-AF. The array and quantitative polymerase chain reaction produced parallel results in analyzing the expression of particular genes. Concordant with the gene expression difference between AF and SR patients, rapid pacing increased the expressions of SHC1, RHOA, PDGFA, and TRAF2 in HL-1 myocytes, implicating a causative effect of tachyarrhythmia on these genes. Compared with AF-SR, AF-AF expressed more intense oxidative stress, upregulations of collagen, transforming growth factor beta 1, and intranuclear nuclear factor of activated T-cells. Regression analysis showed that increased left atrial diameter was associated with the expression of RHOA and STAT1. CONCLUSIONS: Differential expression profiles of proflammatory genes were presented between SR and AF and between maintained SR and recurrent AF after the maze procedure. The identified inflammatory molecules associated with AF and failed surgical ablation may provide clues for developing new potential therapeutic targets to improve AF rhythm control.
Assuntos
Fibrilação Atrial/cirurgia , Procedimentos Cirúrgicos Cardíacos/métodos , Ablação por Cateter/métodos , Regulação da Expressão Gênica , Insuficiência da Valva Mitral/cirurgia , Fator de Transcrição STAT1/genética , Proteína rhoA de Ligação ao GTP/genética , Fibrilação Atrial/complicações , Fibrilação Atrial/genética , Western Blotting , Células Cultivadas , Átrios do Coração/patologia , Humanos , Imuno-Histoquímica , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Insuficiência da Valva Mitral/complicações , Insuficiência da Valva Mitral/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Reação em Cadeia da Polimerase , RNA/genética , Fator de Transcrição STAT1/biossíntese , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
The importance of tumor-stromal cell interactions in breast tumor progression and invasion is well established. Here, an evaluation of differential genomic profiles of carcinoma-associated fibroblasts (CAFs) compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs) revealed altered focal adhesion pathways. These data were validated through confocal assays. To verify the possible role of fibroblasts in lymph node invasion, we constructed a tissue microarray consisting of primary breast cancer samples and corresponding lymph node metastasis and compared the expression of adhesion markers RhoA and Rac1 in fibroblasts located at these different locations. Two distinct tissue microarrays were constructed from the stromal component of 43 primary tumors and matched lymph node samples, respectively. Fibroblasts were characterized for their expression of α-smooth muscle actin (α-SMA) and vimentin. Moreover, we verified the level of these proteins in the stromal compartment from normal adjacent tissue and in non-compromised lymph nodes. Our immunohistochemistry revealed that 59 % of fibroblasts associated with primary tumors and 41 % of the respective metastatic lymph nodes (p = 0.271) displayed positive staining for RhoA. In line with this, 57.1 % of fibroblasts associated with primary tumors presented Rac1-positive staining, and the frequency of co-positivity within the lymph nodes was 42.9 % (p = 0.16). Expression of RhoA and Rac1 was absent in fibroblasts of adjacent normal tissue and in compromised lymph nodes. Based on our findings that no significant changes were observed between primary and metastatic lymph nodes, we suggest that fibroblasts are active participants in the invasion of cancer cells to lymph nodes and support the hypothesis that metastatic tumor cells continue to depend on their microenvironment.
Assuntos
Neoplasias da Mama/genética , Invasividade Neoplásica/genética , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The association of TMEM45A with various cancers has been recently reported. However, the biological function of TMEM45A in ovarian cancer remains unclear. The present study aimed to elucidate the role of TMEM45A in regulating the biological behavior of ovarian cancer cells. We compared the expression of TMEM45A between ovarian cancer tissues and normal tissues based on RNA-sequencing data of the ovarian cancer cohort from The Cancer Genome Atlas (TCGA) project and our real-time PCR data from 25 pairs of ovarian cancer and their matched non-cancerous tissue samples. The expression of TMEM45A was then suppressed in two ovarian cancer cell lines, HO-8910 and A2780, by RNA interference. Cell proliferation, cell cycle distribution, adhesion and invasive ability were then detected using the Cell Counting Kit-8 assay (CCK-8), propidium iodide (PI) staining, and cell adhesion and Transwell assays, respectively. In addition, the mRNA and protein levels of transforming growth factor-ß (TGF-ß1 and TGF-ß2), Ras homolog family member A (RhoA) and Rho-associated kinase 2 (ROCK2) were detected with real-time PCR and western blotting, respectively. TCGA data and our real-time PCR results demonstrated the overexpression of TMEM45A in ovarian cancer. Silencing of TMEM45A significantly inhibited cell proliferation and significantly increased the cell population in the G1 phase. Moreover, knockdown of TMEM45A also inhibited cell adhesion as well as cell invasion. More importantly, suppression of TMEM45A notably downregulated the expression of TGF-ß1, TGF-ß2, RhoA and ROCK2. In conclusion, TMEM45A may function as an oncogene for ovarian cancer, and inhibition of TMEM45A may be a therapeutic strategy for ovarian cancer.
Assuntos
Proteínas de Membrana/genética , Neoplasias Ovarianas/genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta2/biossíntese , Quinases Associadas a rho/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/antagonistas & inibidores , Invasividade Neoplásica/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Interferência de RNA , RNA Mensageiro/biossínteseRESUMO
Recent studies have suggested that contactin-1 has a key role in cancer cell proliferation and migration, however the detailed mechanism of this process is still unclear. Here, human gastric cancer cell line MKN45 was employed. It was found that under hypoxia conditions contactin-1 mRNA and protein levels were both up-regulated by HIF-1alpha expression. Furthermore, although hypoxia increased the migration rate of MKN45 cells, contactin-1 (CNTN1) shRNA reversed this process. Meanwhile, RhoA V14 and RhoA V14N19 mutation constructs were employed, and it was found that constitutively active form of RhoA reversed the cell migration suppression induced by contactin-1 knockdown, while dominant-negative form of RhoA blocked hypoxia induced hypermigration. Apart from this, contactin-1 displayed the ability to phosphorylate the RhoA activator p115 RhoGEF. Thus, under hypoxia conditions, elevated HIF-1alpha seems to up-regulate contactin-1 expression and by this activate RhoA and facilitate migration of cancer cells.
Assuntos
Movimento Celular/genética , Contactina 1/biossíntese , Neoplasias Gástricas/genética , Proteína rhoA de Ligação ao GTP/biossíntese , Hipóxia Celular/genética , Linhagem Celular Tumoral , Contactina 1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , RNA Mensageiro/biossíntese , Fatores de Troca de Nucleotídeo Guanina Rho/biossíntese , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Neoplasias Gástricas/patologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway.
Assuntos
Tumor de Células de Leydig/genética , Proteínas Proto-Oncogênicas/genética , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética , Amidas/administração & dosagem , Animais , Anexina A5/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Tumor de Células de Leydig/tratamento farmacológico , Tumor de Células de Leydig/patologia , Masculino , Proteínas Proto-Oncogênicas/biossíntese , Piridinas/administração & dosagem , RNA Interferente Pequeno , Ratos , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/biossíntese , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
PURPOSE: Persistent pulmonary hypertension remains a major cause of mortality and morbidity in cases of congenital diaphragmatic hernia (CDH). Recently, RhoA/Rho-kinase-mediated vasoconstriction has been reported to be important in the pathogenesis of pulmonary hypertension (PH). Several recent reports have described that fasudil, a potent Rho-kinase inhibitor and vasodilator, could represent a potential therapeutic option for PH. We designed this study to investigate the hypothesis that the expression level of RhoA is increased in the nitrofen-induced CDH rat model. The expression level of Wnt11, an activator of RhoA, was also evaluated. METHODS: Pregnant rats were treated with or without nitrofen on gestational day 9 (D9). Fetuses were sacrificed on D17, D19 and D21 and were divided into control and CDH groups. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression levels of both Wnt11 and RhoA. An immunofluorescence study was also performed to evaluate the expression and localization of RhoA. RESULTS: The relative mRNA expression levels of pulmonary Wnt11 and RhoA on D21 were significantly increased in the CDH group compared with the control group (p=0.016 and p=0.008, respectively). The immunofluorescence study confirmed the overexpression of RhoA in the pulmonary vessels of CDH rats on D21. CONCLUSIONS: Our results provide evidence that the RhoA/Rho-kinase-mediated pathway is involved in the pathogenesis of PH in the nitrofen-induced CDH rat model. Our data also suggest that the fasudil, a Rho-kinase inhibitor, could represent a therapeutic option for the treatment of PH in CDH.
Assuntos
Hérnias Diafragmáticas Congênitas/genética , Pulmão/metabolismo , Prenhez , RNA Mensageiro/genética , Proteína rhoA de Ligação ao GTP/genética , Animais , Modelos Animais de Doenças , Feminino , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/metabolismo , Éteres Fenílicos/toxicidade , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Proteína rhoA de Ligação ao GTP/biossínteseRESUMO
Increasingly, in castration-resistant prostate cancer, patients are often treated with docetaxel and the bisphosphonate zoledronic acid concurrently, yet there is still a paucity in the literature regarding the molecular basis of how this drug combination works. The study was performed on the hormone-resistant cell line PC-3. Cells were treated with clinically relevant concentrations of docetaxel and zoledronic acid either as single agents or in sequence and combination. Cell viability and apoptosis were assessed along with the prenylation status of the GTPases Ras and RhoA. Following 1-mM zoledronic acid treatment, inhibition of the prenylation of H-Ras and Rho A was observed along with an increase in the unprenylated form in the cytoplasm. Docetaxel 1 nM and zoledronic acid 1 mM also showed an increase in the unprenylated form of both small GTP-binding proteins in the cytoplasm and a reduction of protein in the membrane fraction. Overall, zoledronic acid followed by docetaxel was the best regimen producing the greatest reduction in cell viability and increase in apoptosis. At the highest concentrations of zoledronic acid and docetaxel, zoledronic acid followed by docetaxel was also the most effective at reducing the prenylation of both H-Ras and RhoA at the membrane. We have demonstrated that clinically achievable concentrations of zoledronic acid and docetaxel cause a reduction in the prenylation of both H-Ras and Rho A and a reduction of protein movement into the membrane. The most effective regimen overall was high-dose zoledronic acid followed by docetaxel, suggesting that this regimen may be of benefit in clinical practice.