RESUMO
Melanoma is a type of tumor skin with high metastatic potential. Reconstructed human skin, development for pre-clinic assay, are make using primary human cells, but with same limitations. The aim this study was to characterize a cell culture model, with structure similar to human skin containing melanoma cells entirely from cell lines. Reconstructed skin with melanoma were development using human fibroblasts (MRC5), human epidermal keratinocytes (HaCat), and human melanoma (SK-MEL-28) embedded in collagen type I. The structure was characterized by hematoxylin-eosin stained, as well as points of melanoma cell invasion, which was associated with activity of MMPs (MMP-2 and MMP-9) by zymographic method. Then, the gene expression of the target molecular mechanisms involved in melanoma progression were evaluated. Here, the model development showed a region epidermis organized and separated from the dermis, with fibroblast cells confined and melanoma cells form delimited area invasion. MMP-2 and MMP-9 were identified during of cell culture and gene expression of BRAF, NRAS, and Vimentin was confirmed. The proposed model provides one more opportunity to study in vitro tumor biology of melanoma and also to allows the study of new drugs with more reliable results then whats we would find in vivo.
Assuntos
Fibroblastos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Melanoma/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Neoplasias Cutâneas/patologia , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Linhagem Celular Tumoral , Pele/metabolismo , Pele/patologia , Invasividade Neoplásica , Queratinócitos/efeitos dos fármacos , Linhagem Celular , Vimentina/metabolismo , Vimentina/genéticaRESUMO
BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.
Assuntos
Ameloblastoma , Caderinas , Canais de Cloreto , Transição Epitelial-Mesenquimal , Tumores Odontogênicos , Vimentina , Humanos , Transição Epitelial-Mesenquimal/fisiologia , Canais de Cloreto/metabolismo , Canais de Cloreto/análise , Caderinas/metabolismo , Tumores Odontogênicos/patologia , Tumores Odontogênicos/metabolismo , Ameloblastoma/patologia , Ameloblastoma/metabolismo , Vimentina/metabolismo , Adulto , Feminino , Cistos Odontogênicos/patologia , Cistos Odontogênicos/metabolismo , Masculino , Actinas/metabolismo , Adulto Jovem , Pessoa de Meia-Idade , Antígenos CD/metabolismo , AdolescenteRESUMO
Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.
Assuntos
Benzopiranos , Neoplasias da Mama , Caderinas , Transição Epitelial-Mesenquimal , Feminino , Humanos , Benzopiranos/farmacologia , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Células MCF-7 , Invasividade Neoplásica/genética , Proteínas Nucleares , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Vimentina/metabolismo , Vimentina/genéticaRESUMO
BACKGROUND: Brain metastasis (BM) is common in lung adenocarcinoma (LUAD) and has a poor prognosis, necessitating predictive biomarkers. MicroRNAs (MiRNAs) promote cancer cell growth, infiltration, and metastasis. However, the relationship between the miRNA expression profiles and BM occurrence in patients with LUAD remains unclear. METHODS: We conducted an analysis to identify miRNAs in tissue samples that exhibited different expression levels between patients with and without BM. Using a machine learning approach, we confirmed whether the miRNA profile could be a predictive tool for BM. We performed pathway analysis of miRNA target genes using a matched mRNA dataset. RESULTS: We selected 25 miRNAs that consistently exhibited differential expression between the two groups of 32 samples. The 25-miRNA profile demonstrated a strong predictive potential for BM in both Group 1 and Group 2 and the entire dataset (area under the curve [AUC] = 0.918, accuracy = 0.875 in Group 1; AUC = 0.867, accuracy = 0.781 in Group 2; and AUC = 0.908, accuracy = 0.875 in the entire group). Patients predicted to have BM, based on the 25-miRNA profile, had lower survival rates. Target gene analysis of miRNAs suggested that BM could be induced through the ErbB signaling pathway, proteoglycans in cancer, and the focal adhesion pathway. Furthermore, patients predicted to have BM based on the 25-miRNA profile exhibited higher expression of the epithelial-mesenchymal transition signature, TWIST, and vimentin than those not predicted to have BM. Specifically, there was a correlation between EGFR mRNA levels and BM. CONCLUSIONS: This 25-miRNA profile may serve as a biomarker for predicting BM in patients with LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Encefálicas , Neoplasias Pulmonares , Aprendizado de Máquina , MicroRNAs , RNA Mensageiro , Humanos , MicroRNAs/genética , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , RNA Mensageiro/genética , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Prognóstico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Transição Epitelial-Mesenquimal/genética , Conjuntos de Dados como Assunto , Vimentina/metabolismo , Vimentina/genéticaRESUMO
Cervical cancer (CC) is the fourth leading cancer among women and is one of the principal gynecological malignancies. In the tumor microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role during malignant progression, exhibiting a variety of heterogeneous phenotypes. CAFs express phenotypic markers like fibroblast activation protein (FAP), vimentin, S100A4, α-smooth muscle actin (αSMA), and functional markers such as MMP9. This study aimed to evaluate the protein expression of vimentin, S100A4, αSMA, FAP, and MMP9 in mesenchymal stem cells (MSC)-CAF cells, as well as in cervical cancer samples. MSC cells were stimulated with HeLa and SiHa tumor cell supernatants, followed by protein evaluation and cytokine profile to confirm differentiation towards a CAF phenotype. In addition, automated immunohistochemistry (IHQa) was performed to evaluate the expression of these proteins in CC samples at different stages. Our findings revealed a high expression of FAP in stimulated MSC cells, accompanied by the secretion of pro/anti-inflammatory cytokines. In the other hand, CC samples were observed to have high expression of FAP, vimentin, αSMA, and MMP9. Most importantly, there was a high expression of their activation proteins αSMA and FAP during the different stages. In the early stages, a myofibroblast-like phenotype (CAFs αSMA+ FAP+), and in the late stages a protumoral phenotype (CAF αSMA- FAP+). In summary, FAP has a crucial role in the activation of CAFs during cervical cancer progression.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias do Colo do Útero , Humanos , Feminino , Fibroblastos Associados a Câncer/metabolismo , Vimentina/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias do Colo do Útero/metabolismo , Processos Neoplásicos , Fenótipo , Microambiente TumoralRESUMO
Round cell tumors are common cutaneous lesions in dogs, with increased occurrence percentages among different skin tumors. This study aimed to investigate the frequency as well as gross and pathological characteristics of round cell tumors in natural cases of tumorous dogs in relation to breed, sex, and age. Moreover, it aimed to evaluate the immunohistochemical expression of a panel of immunohistochemical stains, including vimentin, E-cadherin, and cluster of differentiation (CD45) as an adjunct technique for the differential diagnosis of cutaneous round cell neoplasm. Data were collected from 64 dogs of both sexes (36 females and 28 males), various breeds, and different ages (8 months to 7 years). The histopathological nature of neoplastic growth was reported, and neoplasm prevalence was classified using age, sex, breed, and site on the body. We observed 48 cases of transmissible venereal tumors, 12 cutaneous histiocytomas, and 4 histiocytic sarcoma. Immunohistochemical characterization revealed an intense positive immunoreactivity for vimentin in transmissible venereal tumor cells and moderate positive immunoreactivity for E-cadherin and CD45 in cutaneous histiocytoma and histiocytic sarcoma cells. In conclusion, the canine transmissible venereal tumor was the most frequent form of round cell tumor; thus, a definitive cutaneous neoplasm diagnosis should be based on histopathological morphology and immunohistochemical findings.
Assuntos
Sarcoma Histiocítico , Neoplasias Cutâneas , Tumores Venéreos Veterinários , Feminino , Masculino , Cães , Animais , Sarcoma Histiocítico/diagnóstico , Sarcoma Histiocítico/veterinária , Vimentina , Tumores Venéreos Veterinários/patologia , Imuno-Histoquímica , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/veterinária , Neoplasias Cutâneas/patologia , Caderinas/metabolismoRESUMO
Nanotechnology applications in biomedicine have increased in recent decades, primarily as therapeutic agents, drugs, and gene delivery systems. Among the nanoparticles used in medicine, we highlight cationic solid lipid nanoparticles (SLN). Given their nontoxic properties, much research has focused on the beneficial effects of SLN for drug or gene delivery system. However, little attention has been paid to the adverse impacts of SLN on the cellular environment, particularly their influence on intracellular signaling pathways. In this work, we investigate the effects triggered by cationic SLN on human prostate non-tumor cells (PNT1A) and tumor cells (PC-3). Our results demonstrate that cationic SLN enhances the migration of PC-3 prostate cancer cells but not PNT1A non-tumor prostate cells, an unexpected and unprecedented development. Furthermore, we observed that the enhanced cell migration velocity is a concentration-dependent and nanoparticle-dependent effect, and not related to any individual nanoparticle component. Moreover, cationic SLN increased vimentin expression (p < 0.05) but SLN did not affect Smad2 nuclear translocation. Meanwhile, EMT-related (epithelial-to-mesenchymal transition) proteins, such as ZEB1, underwent nuclear translocation when treated with cationic SLN, thereby affecting PC-3 cell motility through ZEB1 and vimentin modulation. From a therapeutic perspective, cationic SLN could potentially worsen a patient's condition if these results were reproduced in vivo. Understanding the in vitro molecular mechanisms triggered by nanomaterials and their implications for cell function is crucial for defining their safe and effective use.
Assuntos
Lipossomos , Nanopartículas , Neoplasias da Próstata , Masculino , Humanos , Lipídeos/toxicidade , Vimentina , Próstata , Linhagem Celular Tumoral , Plasmídeos , Nanopartículas/toxicidade , DNARESUMO
The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.
Assuntos
Membrana Epirretiniana , Quinases Associadas a rho , Humanos , Actinas/metabolismo , Células Ependimogliais/metabolismo , Vimentina/metabolismo , Sobrevivência Celular , Membrana Epirretiniana/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismoRESUMO
Vimentin is a cytoskeletal protein important for many cellular processes, including proliferation, migration, invasion, stress resistance, signaling, and many more. The vimentin-deficient mouse has revealed many of these functions as it has numerous severe phenotypes, many of which are found only following a suitable challenge or stress. While these functions are usually related to vimentin as a major intracellular protein, vimentin is also emerging as an extracellular protein, exposed at the cell surface in an oligomeric form or secreted to the extracellular environment in soluble and vesicle-bound forms. Thus, this review explores the roles of the extracellular pool of vimentin (eVIM), identified in both normal and pathological states. It focuses specifically on the recent advances regarding the role of eVIM in wound healing and cancer. Finally, it discusses new technologies and future perspectives for the clinical application of eVIM.
Assuntos
Neoplasias , Animais , Camundongos , Vimentina/genética , Vimentina/metabolismo , Transdução de Sinais , Cicatrização/genética , Movimento CelularRESUMO
BACKGROUND: Skin tissue engineering is a rapidly evolving field of research that effectively combines stem cells and biological scaffolds to replace damaged tissues. Human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) are essential to generate tissue constructs, due to their potent immunomodulatory effects and release of paracrine factors for tissue repair. Here, we investigated whether hWJ-MSC grown on human acellular dermal matrix (hADM) scaffolds and exposed to a proinflammatory environment maintain their ability to produce in vitro growth factors involved in skin injury repair and promote in vivo wound healing. METHODS: We developed a novel method involving physicochemical and enzymatic treatment of cadaveric human skin to obtain hADM scaffold. Subsequently, skin bioengineered constructs were generated by seeding hWJ-MSCs on the hADM scaffold (construct 1) and coating it with human platelet lysate clot (hPL) (construct 2). Either construct 1 or 2 were then incubated with proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) for 12, 24, 48, 72 and 96 h. Supernatants from treated and untreated constructs and hWJ-MSCs on tissue culture plate (TCP) were collected, and concentration of the following growth factors, bFGF, EGF, HGF, PDGF, VEGF and Angiopoietin-I, was determined by immunoassay. We also asked whether hWJ-MSCs in the construct 1 have potential toward epithelial differentiation after being cultured in an epithelial induction stimulus using an air-liquid system. Immunostaining was used to analyze the synthesis of epithelial markers such as filaggrin, involucrin, plakoglobin and the mesenchymal marker vimentin. Finally, we evaluated the in vivo potential of hADM and construct 1 in a porcine full-thickness excisional wound model. RESULTS: We obtained and characterized the hADM and confirmed the viability of hWJ-MSCs on the scaffold. In both constructs without proinflammatory treatment, we reported high bFGF production. In contrast, the levels of other growth factors were similar to the control (hWJ-MSC/TCP) with or without proinflammatory treatment. Except for PDGF in the stimulated group. These results indicated that the hADM scaffold maintained or enhanced the production of these bioactive molecules by hWJ-MSCs. On the other hand, increased expression of filaggrin, involucrin, and plakoglobin and decreased expression of vimentin were observed in constructs cultured in an air-liquid system. In vivo experiments demonstrated the potential of both hADM and hADM/hWJ-MSCs constructs to repair skin wounds with the formation of stratified epithelium, basement membrane and dermal papillae, improving the appearance of the repaired tissue. CONCLUSIONS: hADM is viable to fabricate a tissue construct with hWJ-MSCs able to promote the in vitro synthesis of growth factors and differentiation of these cells toward epithelial lineage, as well as, promote in a full-thickness skin injury the new tissue formation. These results indicate that hADM 3D architecture and its natural composition improved or maintained the cell function supporting the potential therapeutic use of this matrix or the construct for wound repair and providing an effective tissue engineering strategy for skin repair.
Assuntos
Derme Acelular , Células-Tronco Mesenquimais , Geleia de Wharton , Humanos , Animais , Suínos , Proteínas Filagrinas , Vimentina/metabolismo , Derme Acelular/metabolismo , gama Catenina/metabolismo , gama Catenina/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismoRESUMO
As lesões odontogênicas epiteliais benignas constituem um grupo heterogêneo de lesões. A proteína CLIC4 atua na regulação dos processos de parada de crescimento e apoptose, participando também do processo de transdiferenciação dos fibroblastos em miofibroblastos que passam a expressar α-SMA. Além disso, a expressão de CLIC4 pode interferir no processo de transição epitélio-mesenquima (TEM) em neoplasias. Este trabalho avaliou a imunoexpressão de CLIC4, α-SMA, E-caderina e Vimentina em ameloblastomas (AM) (n = 16), ceratocistos odontogênicos (n = 20) e tumores odontogênicos adenomatóides (TOA) (n = 8). A análise da expressão imunoistoquímica das proteínas CLIC4, E-caderina e vimentina no componente epitelial das lesões e de CLIC4 e α-SMA no tecido conjuntivo foi realizada de forma semi-quantitativa por um avaliador previamente calibrado. A expressão no componente epitelial de CLIC4 foi analisada separadamente no núcleo e no citoplasma, bem como a marcação de E-caderina que foi avaliada na membrana e no citoplasma. As comparações dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de CLIC4, α-SMA, E-caderina e Vimentina foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foram observados diferentes padrões de marcação entre os grupos analisados, observando-se que a imunoexpressão exclusivamente citoplasmática da CLIC4 no componente epitelial dos AM (p < 0,001) e TOA (p < 0,001) foi significativamente superior a dos CO, não demonstrarando significância estatística entre os AM e TOA. A imunoexpressão (nuclear e citoplasmática) da CLIC4 no revestimento epitelial CO foi significativamente superior à encontrada no componente epitelial dos AM (p < 0,001) e dos TOA (p < 0,001). A imunoexpressão estromal de CLIC4 foi significativamente superior nos AM (p = 0,009) e CO (p = 0,004) quando comparados aos TOA. A imunoexpressao de α-SMA significativamente maior em AM (p = 0,016) e CO (p = 0,034) quando comparados aos TOA. Para a imunoexpressão membranar da E-caderina em CO foi significativamente superior em comparação à encontrada nos AM (p = 0,009) e nos TOA (p = 0,024). Foi observada maior imunoexpressão de E-caderina (membranar e citoplasmática) nos COs, quando comparados aos AM (p < 0,001) e aos TOAs (p < 0,001). A expressão de Ecaderina citoplasmática foi significativamente maior nos AM e TOA (p < 0,001) quando comparados aos CO. Observou-se diferença estatisticamente significativa na imunoexpressão de vimentina entre os casos de AM e os casos de TOA (p = 0,038) e CO (p < 0,001), bem como entre o TOA e CO (p < 0,001). As correlações testadas entre os escores das proteínas estudadas evidenciou que no grupo dos AM foi possível evidenciar moderada correlação positiva e estatisticamente significativa (r = 0,527; p = 0,036) entre a expressão citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina. Também foi verificada fraca correlação negativa e estatisticamente significativa (r = -0,499; p = 0,049) entre a expressão núcleo-citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina nos AM. Além disso, uma moderada correlação positiva e estatisticamente significativa entre a expressão estromal da CLIC4 e a expressão da α-SMA nos AM (r = 0,648; p = 0,007) e nos CO (r = 0,541; p = 0,014). Foi observada forte correlação negativa e estatisticamente significativa (r = -0,813; p < 0,001) entre a expressão da E-caderina e a expressão da vimentina nos AM. Os resultados deste estudo sugerem um potencial envolvimento de CLIC4 no processo de transdiferenciação de miofibroblastos, e que a presença destas células é mais frequentemente associada a lesões de comportamento biológico mais agressivo como os AM e CO, além de uma possível atuação desta proteína na regulação do ciclo celular e na TEM nas lesões estudadas (AU).
Benign epithelial odontogenic lesions constitute a heterogeneous group of lesions. the CLIC4 protein acts in the regulation of growth arrest and apoptosis processes, also participating in the process of transdifferentiation of fibroblasts Into myofibroblasts that begin to express α-SMA. Furthermore, CLIC4 expression can interfere with the epithelialmesenchymal transition (EMT) process in neoplasms. This work evaluated the immunoexpression of CLIC4, α-SMA, e-cadherin and vimentin in ameloblastomas (AM) (n = 16), odontogenic keratocysts (OK) (n = 20) and adenomatoid odontogenic tumors (AOT) (n = 8). The analysis of the immunohistochemical expression of the proteins CLIC4, ecadherin and vimentin in the epithelial component of the lesions and of CLIC4 and α-SMA in the connective tissue was carried out in a semi-quantitative way by a previously calibrated evaluator. Expression in the epithelial component of CLIC4 was analyzed separately in the nucleus and cytoplasm, as well as e-cadherin labeling, which was evaluated in the membrane and cytoplasm. Comparisons of the percentages of immunoreactivity in relation to the studied groups were carried out using the nonparametric kruskal-wallis and mann-whitney tests. Possible correlations between the expression of CLIC4, α-SMA, e-cadherin and vimentin were evaluated using the spearman correlation test. The significance level was set at 5% (p < 0.05). Different staining patterns were observed between the groups analyzed, observing that the exclusively cytoplasmic immunoexpression of CLIC4 in the epithelial component of AM (p < 0.001) and AOT (p < 0.001) was significantly higher than that of OK, not demonstrating statistical significance between the AM and AOT. The immunoexpression (nuclear and cytoplasmic) of CLIC4 in the co epithelial lining was significantly higher than that found in the epithelial component of AM (p < 0.001) and AOT (p < 0.001). Stromal CLIC4 immunoexpression was significantly higher in AM (p = 0.009) and OK (p = 0.004) when compared to AOT. The immunoexpression of α-SMA is significantly higher in AM (p = 0.016) and OK (p = 0.034) when compared to AOT. For e-cadherin membrane immunoexpression in co was significantly higher compared to that found in AM (p = 0.009) and AOT (p = 0.024). Greater immunoexpression of e-cadherin (membrane and cytoplasmic) was observed in OK, when compared to AM (p < 0.001) and AOT (p < 0.001). Cytoplasmic ecadherin expression was significantly higher in AM and AOT (p < 0.001) when compared to OK. A statistically significant difference in vimentin immunoexpression was observed between cases of AM and cases of AOT (p = 0.038) and OK (p < 0.001), as well as between AOT and OK (p < 0.001). The correlations tested between the scores of the proteins studied showed that in the am group it was possible to demonstrate a moderate positive and statistically significant correlation (r = 0.527; p = 0.036) between the cytoplasmic expression of clic4 and the cytoplasmic expression of e-cadherin. A weak and statistically significant negative correlation (r = -0.499; p = 0.049) was also found between the nucleus-cytoplasmic expression of clic4 and the cytoplasmic expression of e- cadherin in AM. Furthermore, a moderate positive and statistically significant correlation between the stromal expression of CLIC4 and the expression of α-SMA in AM (r = 0.648; p = 0.007) and OK (r = 0.541; p = 0.014). Additionally, a strong negative and statistically significant correlation (r = -0.813; p < 0.001) was observed between the expression of ecadherin and the expression of vimentin in AM. The results of this study suggest a potential involvement of CLIC4 in the myofibroblast transdifferentiation process, and that the presence of these cells is more frequently associated with lesions with more aggressive biological behavior such as AM and OK, in addition to a possible role of this protein in the regulation of cell cycle and EMT in the lesions studied (AU).
Assuntos
Ameloblastoma/patologia , Cistos Odontogênicos/patologia , Caderinas/metabolismo , Epitélio/lesões , Vimentina/metabolismo , Estudos Transversais/métodos , Estudos Retrospectivos , Estatísticas não Paramétricas , Miofibroblastos/patologia , Transição Epitelial-MesenquimalRESUMO
Cervical cancer (CC) is one of the most common and deadly types of female cancer worldwide. Late diagnosis in CC increases the risk of tumor cells spreading to distant organs (metastasis). The epithelial-mesenchymal transition (EMT) is a fundamental process of cancer metastasis. Inflammation can lead to tumor progression, EMT induction, and metastasis. The inflammatory microenvironment is a potent inducer of EMT; inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and Transforming growth factor-beta (TGF-ß1) activate transcriptional factors such as STAT3, Snail, Smad, and the Nuclear Factor kappa light-chain-enhancer of activated beta cells (NF-κΒ), which drive EMT. Anti-inflammatory compounds may be an option in the disruption of EMT. PenToXifylline (PTX) possesses potent anti-inflammatory effects by inhibiting NF-κB activity. In addition, PTX exerts an anti-fibrotic effect by decreasing Smad2/3/4. We hypothesize that PTX could exert anti-EMT effects. CaSki human cervical tumor cells were exposed to TNF-α 10 ng/mL and TGF-ß1 alone or in combination for 5 days. Our results revealed that TNF-α and TGF-ß1 induced N-cadherin and Vimentin, confirming the induction of EMT. Furthermore, the combination of cytokines synergized the expression of mesenchymal proteins, enhanced IκBα and p65 phosphorylation, and upregulated Serpin family E member 1 (SERPINE1) mRNA. PTX pretreatment prior to the addition of TNF-α and TGF-ß1 significantly reduced N-cadherin and Vimentin levels. To our knowledge, this is the first time that this effect of PTX has been reported. Additionally, PTX reduced the phosphorylation of IκB-α and p65 and significantly decreased SERPINE1 expression, cell proliferation, migration, and invasion. In conclusion, PTX may counteract EMT in cervical cancer cells by decreasing the NF-κB and SERPINE1.
Assuntos
Pentoxifilina , Neoplasias do Colo do Útero , Feminino , Humanos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Transição Epitelial-Mesenquimal , Vimentina/metabolismo , Pentoxifilina/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Caderinas/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral , Inibidor 1 de Ativador de Plasminogênio/genéticaRESUMO
The interactions between mitochondria and the cytoskeleton have been found to alter mitochondrial function; however, the mechanisms underlying this phenomenon are largely unknown. Here, we explored how the integrity of the cytoskeleton affects the cellular organization, morphology and mobility of mitochondria in Xenopus laevis melanocytes. Cells were imaged in control condition and after different treatments that selectively affect specific cytoskeletal networks (microtubules, F-actin and vimentin filaments). We observed that mitochondria cellular distribution and local orientation rely mostly on microtubules, positioning these filaments as the main scaffolding of mitochondrial organization. We also found that cytoskeletal networks mold mitochondria shapes in distinct ways: while microtubules favor more elongated organelles, vimentin and actin filaments increase mitochondrial bending, suggesting the presence of mechanical interactions between these filaments and mitochondria. Finally, we identified that microtubule and F-actin networks play opposite roles in mitochondria shape fluctuations and mobility, with microtubules transmitting their jittering to the organelles and F-actin restricting the organelles motion. All our results support that cytoskeleton filaments interact mechanically with mitochondria and transmit forces to these organelles molding their movements and shapes.
Assuntos
Actinas , Citoesqueleto , Citoesqueleto de Actina , Filamentos Intermediários , Microtúbulos , Vimentina , AnimaisRESUMO
There is an urgent need to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions involved in virus spread and pathogenesis, which might contribute to the identification of new therapeutic targets. In this study, we investigated the presence of SARS-CoV-2 in postmortem lung, kidney, and liver samples of patients who died with coronavirus disease (COVID-19) and its relationship with host factors involved in virus spread and pathogenesis, using microscopy-based methods. The cases analyzed showed advanced stages of diffuse acute alveolar damage and fibrosis. We identified the SARS-CoV-2 nucleocapsid (NC) in a variety of cells, colocalizing with mitochondrial proteins, lipid droplets (LDs), and key host proteins that have been implicated in inflammation, tissue repair, and the SARS-CoV-2 life cycle (vimentin, NLRP3, fibronectin, LC3B, DDX3X, and PPARγ), pointing to vimentin and LDs as platforms involved not only in the viral life cycle but also in inflammation and pathogenesis. SARS-CoV-2 isolated from a patient´s nasal swab was grown in cell culture and used to infect hamsters. Target cells identified in human tissue samples included lung epithelial and endothelial cells; lipogenic fibroblast-like cells (FLCs) showing features of lipofibroblasts such as activated PPARγ signaling and LDs; lung FLCs expressing fibronectin and vimentin and macrophages, both with evidence of NLRP3- and IL1ß-induced responses; regulatory cells expressing immune-checkpoint proteins involved in lung repair responses and contributing to inflammatory responses in the lung; CD34+ liver endothelial cells and hepatocytes expressing vimentin; renal interstitial cells; and the juxtaglomerular apparatus. This suggests that SARS-CoV-2 may directly interfere with critical lung, renal, and liver functions involved in COVID-19-pathogenesis.
Assuntos
COVID-19 , Humanos , COVID-19/patologia , Fibronectinas , Vimentina , SARS-CoV-2 , Células Endoteliais , Proteína 3 que Contém Domínio de Pirina da Família NLR , PPAR gama , Pulmão , Inflamação/patologia , Rim , FígadoRESUMO
PURPOSE: To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. METHODS: Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. RESULT: HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial-mesenchymal transition such as E-cadherin, were increased, while vimentin and α-SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α-SMA was increased. In addition, the level of ß-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. CONCLUSION: HIF-1α contributed to EMT and metastasis through activation of canonical ß-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma.
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Coriocarcinoma , beta Catenina , Gravidez , Feminino , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Vimentina/metabolismo , Caderinas/genética , Caderinas/metabolismo , Coriocarcinoma/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transição Epitelial-Mesenquimal , Movimento Celular , Regulação Neoplásica da Expressão GênicaRESUMO
The central function of telomerase is maintaining the telomere length. However, several extra-telomeric roles have been identified for this protein complex. In this study, we evaluated the effect of the silencing of the catalytic subunit of telomerase (TERT) on the expression of candidate microRNAs, cell activation markers and glial-related genes in a glioblastoma cell line (T98G). The silencing was performed by a siRNA and the qPCR method was used to analyze the expression of TERT and downstream genes. Flow cytometry was used to quantify the TERT protein, and bioinformatics analysis was carried out to analyze the functions of microRNA target genes. Here, it was observed that after a 50% reduction of the TERT gene, the expression of ARG1 (Arginase 1) was upregulated, whereas NES (Nestin), GLUL (Glutamate-Ammonia Ligase), VIM (Vimentin) and the hsa-miR-29b-3p microRNA were downregulated (P-value <0.05). A bioinformatic analysis showed that target genes of hsa-miR-29b are associated with focal adhesion, the PI3K-Akt signaling pathway, among others. These results are important because they contribute to the knowledge of extratelomeric functions by providing relevant evidence about novel genes modulated by TERT.
Assuntos
MicroRNAs , Telomerase , Vimentina/metabolismo , Arginase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Telomerase/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem CelularRESUMO
In cancer, the Epithelial to Mesenchymal Transition (EMT) is the process in which epithelial cells acquire mesenchymal features that allow metastasis, and chemotherapy resistance. Growth hormone (GH) has been associated with melanoma, breast, and endometrial cancer progression through an autocrine regulation of EMT. Since exogenous and autocrine expression of GH is known to have different molecular effects, we investigated whether exogenous GH is capable of regulating the EMT of cancer cells. Furthermore, we investigated whether exogenous GH could promote EMT in non-cancerous cells. To study the effect of GH (100 ng/ml) on cancer and non-cancer cells, we used HeLa and HEK293 cell lines, respectively. We evaluated the loss of cell-cell contacts, by cell scattering assay and migration by wound-healing assay. Additionally, we evaluated the morphological changes by phalloidin-staining. Finally, we evaluated the molecular markers E-cadherin and vimentin by flow cytometry. GH enhances cell scattering and the migratory rate and promotes morphological changes such as cell area increase and actin cytoskeleton filaments formation on HeLa cell line. Moreover, we found that GH favors the expression of the mesenchymal protein vimentin, followed by an increase in E-cadherin's epithelial protein expression, characteristics of an epithelial-mesenchymal hybrid phenotype that is associated with metastasis. On HEK293cells, GH promotes morphological changes, including cell area increment and filopodia formation, but not affects scattering, migration, nor EMT markers expression. Our results suggest that exogenous GH might participate in cervical cancer progression favoring a hybrid EMT phenotype but not on non-cancerous HEK293 cells.
Assuntos
Transição Epitelial-Mesenquimal , Hormônio do Crescimento , Humanos , Células HeLa , Células HEK293 , Hormônio do Crescimento/farmacologia , Vimentina , Linhagem Celular Tumoral , Caderinas/metabolismo , Fatores de Transcrição , Movimento CelularRESUMO
The most significant interest in veterinary oncology is occupied by tumors of a mixed nature, which are represented by the direct interaction of epithelial and mesenchymal components. According to some data, the formation of cell lineage of mesenchymal cells occurs due to the transformation of epidermal basket cells surrounding the glandular epithelium. In this case, the formation of a cartilage anlage with its further transformation into differentiated bone tissue occurs. The article contains information about the structure and features of the location and interaction of mixed tumors' glandular and stromal components in female dogs (n=29). It was revealed that in addition to simple mixed neoplasms, tumors with proplasia of the mesenchymal component into highly differentiated cartilage or bone tissue were also often registered. To confirm subcellular changes in the expression of certain types of proteins, such as a-SMA and vimentin, an IHC study was used. The expression of the studied biomarkers was established in mesenchymal fibroblastic differon cells and myoepithelial cells. The intensity of immunoreactive material expression ranged from moderate (2+) to strong (3+), indicating changes in myoepithelial cells' genotype during the formation of mixed mammary neoplasms.(AU)
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Animais , Feminino , Neoplasias Mamárias Animais/diagnóstico , Mioepitelioma/veterinária , Vimentina/análise , Biomarcadores Tumorais/análise , CãesRESUMO
BACKGROUND/AIMS: The renal inflammatory response and kidney regeneration in ischemia-reperfusion injury (IRI) are associated with Toll-like receptor 4 (TLR4). Here we study the role of TLR4 during IRI in the renal cortex and medulla separately, using wild-type (TLR4-WT) and Knockout (TLR4-KO) TLR4 mice. METHODS: We used 30 minutes of bilateral renal ischemia, followed by 48 hours of reperfusion in C57BL/6 mice. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and proteostasis in the renal cortex and medulla by qRT-PCR and Western blot. In addition, we studied kidney morphology by H/E and PAS. RESULTS: Renal ischemia (30min) and reperfusion (48hrs) induced the mRNA and protein of TLR4 in the renal cortex. In addition, Serum Creatinine (SCr), blood urea nitrogen (BUN), Neutrophil gelatinase-associated lipocalin (NGAL), and acute tubular necrosis (ATN) were increased in TLR4-WT by IRI. Interestingly, the SCr and BUN had normal levels in TLR-KO during IRI. However, ATN and high levels of NGAL were present in the kidneys of TLR4-KO mice. The pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (Foxp3 and IL-10) markers increased by IRI only in the cortex of TLR4-WT but not in TLR4-KO mice. Furthermore, the M1 (CD38 and Frp2) and M2 (Arg-I, Erg-2, and c-Myc) macrophage markers increased by IRI only in the cortex of TLR4-WT. The TLR4-KO blunted the IRI-upregulation of M1 but not the M2 macrophage polarization. Vimentin increased in the renal cortex and medulla of TLR4-WT animals but not in the cortex of TLR4-KO mice. In addition, iNOS and clusterin were increased by IRI only in the cortex of TLR4-WT, and the absence of TLR4 inhibited only clusterin upregulation. Finally, Hsp27 and Hsp70 protein levels increased by IRI in the cortex and medulla of TLR4-WT and TRL4-KO lost the IRI-upregulation of Hsp70. In summary, TLR4 participates in renal ischemia and reperfusion through pro-inflammatory and anti-inflammatory responses inducing impaired kidney function (SCr and BUN). However, the IRI-upregulation of M2 macrophage markers (cortex), iNOS (cortex), IL-6 (medulla), vimentin (medulla), and Hsp27 (cortex and medulla) were independent of TLR4. CONCLUSION: The TLR4 inactivation during IRI prevented the loss of renal function due to the inactivation of inflammation response, avoiding M1 and preserving the M2 macrophage polarization in the renal cortex.
Assuntos
Nefropatias , Traumatismo por Reperfusão , Animais , Camundongos , Clusterina/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Inflamação/complicações , Interleucina-6/genética , Interleucina-6/metabolismo , Isquemia , Rim/metabolismo , Córtex Renal/metabolismo , Nefropatias/complicações , Lipocalina-2/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração , Traumatismo por Reperfusão/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Vimentina/metabolismoRESUMO
BACKGROUND/AIMS: Acute kidney injury (AKI) carries high morbidity and mortality, and the inducible nitric oxide synthase (iNOS) is a potential molecular target to prevent kidney dysfunction. In previous work, we reported that the pharmacological inhibitions of iNOS before ischemia/reperfusion (I/R) attenuate the I/R-induced AKI in mice. Here, we study the iNOS inhibitor 1400W [N-(3-(Aminomethyl)benzyl] acetamide, which has been described to be much more specific to iNOS inhibition than other compounds. METHODS: We used 30 minutes of bilateral renal ischemia, followed by 24 hours of reperfusion in Balb/c mice. 1400w (10 mg/kg i.p) was applied before I/R injury. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and nephrogenic genes by qRT-PCR in the renal cortex and medulla. The Periodic Acid-Schiff (PAS) was used to study the kidney morphology. RESULTS: Remarkably, we found that 1400W affects the renal cortex and medulla in different ways. Thus, in the renal cortex, 1400W prevented the I/R-upregulation of 1. NGAL, Clusterin, and signs of morphological damage; 2. IL-6 and TNF-α; 3. TGF-ß; 4. M2(Arg1, Erg2, cMyc) and M1(CD38, Fpr2) macrophage polarization makers; and 5. Vimentin and FGF2 levels but not in the renal medulla. CONCLUSION: 1400W conferred protection in the kidney cortex compared to the kidney medulla. The present investigation provides relevant information to understand the opportunity to use 1400W as a therapeutic approach in AKI treatment.