RESUMO
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
Caveolin-1 (CAV1) is a membrane-bound protein that suppresses tumor development yet also promotes metastasis. E-cadherin is important in CAV1-dependent tumor suppression and prevents CAV1-enhanced lung metastasis. Here, we used murine B16F10 and human A375 melanoma cells with low levels of endogenous CAV1 and E-cadherin to unravel how co-expression of E-cadherin modulates CAV1 function in vitro and in vivo in WT C57BL/6 or Rag-/- immunodeficient mice and how a pro-inflammatory environment generated by treating cells with prostaglandin E2 (PGE2) alters CAV1 function in the presence of E-cadherin. CAV1 expression augmented migration, invasion, and metastasis of melanoma cells, and these effects were abolished via transient co-expression of E-cadherin. Importantly, exposure of cells to PGE2 reverted the effects of E-cadherin expression and increased CAV1 phosphorylation on tyrosine-14 and metastasis. Moreover, PGE2 administration blocked the ability of the CAV1/E-cadherin complex to prevent tumor formation. Therefore, our results support the notion that PGE2 can override the tumor suppressor potential of the E-cadherin/CAV1 complex and that CAV1 released from the complex is phosphorylated on tyrosine-14 and promotes migration/invasion/metastasis. These observations provide direct evidence showing how a pro-inflammatory environment caused here via PGE2 administration can convert a potent tumor suppressor complex into a promoter of malignant cell behavior.
Assuntos
Dinoprostona , Melanoma Experimental , Animais , Humanos , Camundongos , Caderinas/metabolismo , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Dinoprostona/farmacologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Tirosina/farmacologiaRESUMO
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
A 7-year-old captive female jaguar (Panthera onca) was presented with a 7-day history of dyspnoea and weight loss. Clinical examination revealed hepatomegaly and elevated serum alanine aminotransferase activity. Pulmonary ultrasonography revealed comet-tail images and an alveolar pattern was detected on thoracic radiography. Due to the poor prognosis, the jaguar was euthanized after 10 days. At necropsy, the main gross findings were hepatomegaly, splenomegaly and multifocal to coalescent, slightly elevated grey areas in the lungs. Histological examination revealed neoplastic proliferation of pleomorphic histiocytes arranged in cohesive sheets in the lungs, liver, spleen, kidneys and lymph nodes. Neoplastic cells had intense immunolabelling for vimentin and ionized calcium-binding adaptor molecule 1, and were immunonegative for pancytokeratin, E-cadherin, CD20, CD3 and CD79α. These findings were compatible with a systemic histiocytic disorder, distinct from any well-defined histiocytic proliferative disease in domestic animals.
Assuntos
Panthera , Animais , Feminino , Hepatomegalia/veterinária , Histiócitos , Autopsia/veterinária , Animais de ZoológicoRESUMO
Abstract Objective MicroRNA-29a-3p has been reported in a variety of cancers, but its role in hypopharyngeal cancer remains unclear. This study was to determine the role of microRNA-29a-3p in the occurrence and development of hypopharyngeal cancer. Methods 40 patients with hypopharyngeal cancer who underwent surgery in the Affiliated Hospital of Jining Medical University from April 2013 to November 2017 were selected for this study. The cancer tissue samples of the patients were collected, and the patients were followed up for three years. The expression of microRNA-29a-3p in tissue samples was detected by in situ hybridization with fluorescent probe, and the relationships among microRNA-29a-3p and clinicopathological factors, postoperative recurrent-metastasis, survival time were studied. Immunohistochemical was used to detect the expression of Ki67 and E-cadherin in tissue samples. Results Combined with HE staining results showed that microRNA-29a-3p expression was relatively high in non-cancer tissue cells (red blood cells and fibroblasts in tumor interstitial vessels), but was relatively low in cancer tissue and cells. According to the follow-up data of 40 patients with hypopharyngeal cancer, tumor size, T-stage, tumor differentiation, postoperative recurrent-metastasis of hypopharyngeal cancer patients were significantly negatively correlated with microRNA-29a-3p (p< 0.05). Immunohistochemica results further confirmed that microRNA-29a-3p was negatively correlated with the expression of Ki67 and E-cadherin. The survival time of patients positively related with microRNA-29a-3p expression (p< 0.05). Moreover, ROC curve analysis showed that the area under the curve of the combined detection of miRNA-29a-3p+Ki67+E-cadherin was larger than that of the single detection of the three indexes. Conclusions The expression of microRNA-29a-3p is closely related to the occurrence, development and prognosis of hypopharyngeal cancer, and it affects the proliferation and invasion. This indicates that microRNA-29a-3p serves as a therapeutic target for the occurrence and development of hypopharyngeal cancer. The evidence of study designs of this study is IV using "Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence".
RESUMO
OBJECTIVE: MicroRNA-29a-3p has been reported in a variety of cancers, but its role in hypopharyngeal cancer remains unclear. This study was to determine the role of microRNA-29a-3p in the occurrence and development of hypopharyngeal cancer. METHODS: 40 patients with hypopharyngeal cancer who underwent surgery in the Affiliated Hospital of Jining Medical University from April 2013 to November 2017 were selected for this study. The cancer tissue samples of the patients were collected, and the patients were followed up for three years. The expression of microRNA-29a-3p in tissue samples was detected by in situ hybridization with fluorescent probe, and the relationships among microRNA-29a-3p and clinicopathological factors, postoperative recurrent-metastasis, survival time were studied. Immunohistochemical was used to detect the expression of Ki67 and E-cadherin in tissue samples. RESULTS: Combined with HE staining results showed that microRNA-29a-3p expression was relatively high in non-cancer tissue cells (red blood cells and fibroblasts in tumor interstitial vessels), but was relatively low in cancer tissue and cells. According to the follow-up data of 40 patients with hypopharyngeal cancer, tumor size, T-stage, tumor differentiation, postoperative recurrent-metastasis of hypopharyngeal cancer patients were significantly negatively correlated with microRNA-29a-3p (pâ¯<â¯0.05). Immunohistochemica results further confirmed that microRNA-29a-3p was negatively correlated with the expression of Ki67 and E-cadherin. The survival time of patients positively related with microRNA-29a-3p expression (pâ¯<â¯0.05). Moreover, ROC curve analysis showed that the area under the curve of the combined detection of miRNA-29a-3p+Ki67+E-cadherin was larger than that of the single detection of the three indexes. CONCLUSIONS: The expression of microRNA-29a-3p is closely related to the occurrence, development and prognosis of hypopharyngeal cancer, and it affects the proliferation and invasion. This indicates that microRNA-29a-3p serves as a therapeutic target for the occurrence and development of hypopharyngeal cancer. The evidence of study designs of this study is IV using "Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence".
Assuntos
Neoplasias Hipofaríngeas , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Hipofaríngeas/genética , Neoplasias Hipofaríngeas/cirurgia , Relevância Clínica , Antígeno Ki-67 , Caderinas/genéticaRESUMO
INTRODUCTION: Cholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. MATERIALS AND METHODS: Cell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-ß-cyclodextrin (MßCD - 7.5, 10 or 15 mM) RESULTS: Decreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein ß-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. CONCLUSION: Cholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Mioma , Neoplasias da Língua , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Caveolina 1/metabolismo , Neoplasias da Língua/patologia , Caderinas/metabolismo , Movimento Celular , Linhagem Celular , Colesterol , Linhagem Celular TumoralRESUMO
ABSTRACT Background Reduced or null expression of E-cadherin protein is a frequent cause of diffuse gastric cancer (DGC). More than 50% of patients with DGC present somatic variants in CDH1 gene. Objectives The objectives of this study were to study E-cadherin expression and identify variants in the CDH1 gene in gastric tumors of patients with DGC. Methods We studied 18 Mexican DGC patients who attended a hospital of the Mexican Social Security Institute; E-cadherin expression was determined by immunohistochemistry, and variants were identified by Sanger sequencing in promoter and coding regions. Predictive analysis was performed using PolyPhen-2 and HOPE software. Results We found that 56% of DGC patients showed reduced expression of E-cadherin. All patients carried CDH1 variants; overall, 12 different CDH1 variants were identified. Predictive analysis revealed that the rs114265540 variant was probably damaging, with a value of 0.985, indicating a functional impact on the E-cadherin protein. Variants rs34939176 and rs33964119 were identified as risk factors for DGC (odds' ratios [OR] = 31.3, 95% CI 6.3-154.0, p < 0.001; OR = 6.1, 95% CI 2.0-19.0, p < 0.001, respectively) given their elevated frequency and by comparing it with those reported for MXL population in the 1000 Genomes Project database. Conclusions In this Mexican population, the percentage of diffuse gastric tumors with reduced expression of E-cadherin was similar to that reported in other populations. All gastric tumors of DGC patients studied had somatic CDH1 gene variants; however, the rs114265540, rs34939176, and rs33964119 variants were importantly related to DGC.
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Background: Reduced or null expression of E-cadherin protein is a frequent cause of diffuse gastric cancer (DGC). More than 50% of patients with DGC present somatic variants in CDH1 gene. Objectives: The objectives of this study were to study E-cadherin expression and identify variants in the CDH1 gene in gastric tumors of patients with DGC. Methods: We studied 18 Mexican DGC patients who attended a hospital of the Mexican Social Security Institute; E-cadherin expression was determined by immunohistochemistry, and variants were identified by Sanger sequencing in promoter and coding regions. Predictive analysis was performed using PolyPhen-2 and HOPE software. Results: We found that 56% of DGC patients showed reduced expression of E-cadherin. All patients carried CDH1 variants; overall, 12 different CDH1 variants were identified. Predictive analysis revealed that the rs114265540 variant was probably damaging, with a value of 0.985, indicating a functional impact on the E-cadherin protein. Variants rs34939176 and rs33964119 were identified as risk factors for DGC (odds' ratios [OR] = 31.3, 95% CI 6.3-154.0, p < 0.001; OR = 6.1, 95% CI 2.0-19.0, p < 0.001, respectively) given their elevated frequency and by comparing it with those reported for MXL population in the 1000 Genomes Project database. Conclusions: In this Mexican population, the percentage of diffuse gastric tumors with reduced expression of E-cadherin was similar to that reported in other populations. All gastric tumors of DGC patients studied had somatic CDH1 gene variants; however, the rs114265540, rs34939176, and rs33964119 variants were importantly related to DGC.
Assuntos
Neoplasias Gástricas , Humanos , Antígenos CD/genética , Caderinas/genética , Predisposição Genética para Doença , México , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.