Imunoexpressão de clic4, α-sma, e-caderina e vimentina em lesões odontogênicas epitelias benígnas / Immunoexpression of clic4, α-sma, e-cadherin and vimentin in benign epithelial odontogenic lesions

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).

Decursin affects proliferation, apoptosis, and migration of colorectal cancer cells through PI3K/Akt signaling pathway / 中国中药杂志

We investigated the effects of decursin on the proliferation, apoptosis, and migration of colorectal cancer HT29 and HCT116 cells through the phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway. Decursin(10, 30, 60, and 90 μmol·L~(-1)) was used to treat HT29 and HCT116 cells. The survival, colony formation ability, proliferation, apoptosis, wound hea-ling area, and migration of the HT29 and HCT116 cells exposed to decursin were examined by cell counting kit-8(CCK8), cloning formation experiments, Ki67 immunofluorescence staining, flow cytometry, wound healing assay, and Transwell assay, respectively. Western blot was employed to determine the expression levels of epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, B-cell lymphoma/leukemia-2(Bcl-2), Bcl-2-associated X protein(Bax), tumor suppressor protein p53, PI3K, and Akt. Compared with the control group, decursin significantly inhibited the proliferation and colony number and promoted the apoptosis of HT29 and HCT116 cells, and it significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. Decursin inhibited the wound healing and migration of the cells, significantly down-regulated the expression of N-cadherin and vimentin, and up-regulated the expression of E-cadherin. In addition, it significantly down-regulated the expression of PI3K and Akt and up-regulated that of p53. In summary, decursin may regulate epithelial-mesenchymal transition(EMT) via the PI3K/Akt signaling pathway, thereby affecting the proliferation, apoptosis, and migration of colorectal cancer cells.

miR-497 inhibits the growth and metastasis of SGC-7901 human gastric cancer anoikis resistant cells via blocking Wnt/β-catenin signaling pathway / 细胞与分子免疫学杂志

Objective To investigate the effects of microRNA497 (miR-497) on the metastasis of gastric cancer and its possible molecular mechanism. Methods SGC-7901 gastric cancer parent cells were cultured in an ultra-low adhesion environment, and the anoikis resistance model of SGC-7901 cells was created after re-adhesion. Clone formation assay, flow cytometry, TranswellTM test and scratch healing test were used to detect the differences of biological behavior compared with their parent cells. Fluorescence quantitative PCR was performed to detect the expression of miR-497. Western blot analysis was used to detect the changes of key proteins of Wnt/β-catenin signaling pathway and epithelial mesenchymal transformation (EMT) related proteins such as vimentin and E-cadherin. Parent cells and anoikis resistant SGC-7901 cells were transfected with miR-497 inhibitor or miR-497 mimic, and CCK-8 assay was used to detect the proliferation activity. TranswellTM invasion assay was performed to detect the invasion ability of cells. TranswellTM migration test and scratch healing assay was used to determine the migration ability. Western blot analysis was used to detect the expressions of Wnt1, β-catenin, vimentin and E-cadherin. By transfecting miR-497 mimic into the anoikis resistance SGC-7901 cells and inoculating them subcutaneously in nude mice, the changes in the volume and mass of tumor tissues were measured and recorded. Western blot analysis was used to determine the expressions of Wnt1, β-catenin, vimentin and E-cadherin of tumor tissues. Results Compared with the parent cells, the anoikis resistance SGC-7901 gastric cancer cells had faster proliferation rate, stronger colony formation, lower apoptosis rate, stronger invasion and migration ability. The expression of miR-497 was significantly decreased. After down-regulation of miR-497, the proliferation ability, invasion and migration ability were significantly enhanced. The expressions of Wnt1, β-catenin and vimentin increased significantly, while E-cadherin decreased notably. The results of up-regulation miR-497 were the opposite. The tumor growth rate, tumor volume and mass of miR-497 overexpression group were significantly lower than those of control group. The expressions of Wnt1, β-catenin and vimentin decreased significantly, while the expression of E-cadherin increased significantly. Conclusion The expression of miR-497 is low in the anoikis resistance SGC-7901 cells. miR-497 can inhibit the growth and metastasis of gastric cancer cells by blocking Wnt/β-catenin signaling pathway and EMT.

Effects of miR-9-5p on the migration, invasion and epithelial-mesenchymal transition process in cervical squamous cell carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.

Knockdown of ACC1 promotes migration of esophageal cancer cell / 中华肿瘤杂志

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

Estudo da imunoexpressão de proteínas envolvidas na transição epitélio-mesênquima em tumores de glândula salivar / Study of the immunoexpression of proteins involved in the epithelial-mesenchymal transition in salivary gland tumors

Os tumores de glândula salivar (TGS) apresentam notável complexidade clínica e biológica, razão para a qual muitos estudos investigam os eventos envolvidos na sua progressão. Uma das dinâmicas envolvidas na invasão tumoral de diversos tipos de carcinomas é a transição epitélio-mesênquima (TEM). Neste processo, as células epiteliais sofrem transição para um estado mesenquimal móvel, favorecendo a invasão e metástase. Sendo assim, esta pesquisa analisou a expressão imuno-histoquímica de E-caderina, Twist1, Snail1, α-SMA, metaloproteinases de matriz 9 (MMP-9) e Vimentina (VM) em 90 casos de TGS, correlacionando-os entre si e com parâmetros clinicopatológicos. Foram selecionados 20 casos de Adenoma pleomórfico (AP), 20 casos de Carcinoma mucoepidermoide (CME), 20 casos de Carcinoma adenoide cístico (CAC), 10 casos de Adenocarcinoma polimorfo (ACP), 10 casos de Carcinoma epitelial-mioepitelial (CEME) e 10 casos de Carcinoma ex-adenoma pleomórfico (CexAP). A análise de E-caderina, Twist1, Snail1 foi realizada em parênquima tumoral sendo observado o percentual de células positivas (PP), com escores variando de 0 a 4, e a intensidade de expressão (IE), cujos escores variaram de 0 a 3. A avaliação de MMP-9 foi realizada em parênquima e estroma tumoral, também avaliando-se a PP e a IE, ambos baseados em escores que variaram de 0 a 3. A marcação para α-SMA e VM foi analisada em região de estroma tumoral. Células positivas para α-SMA foram contabilizadas em 10 campos, obtendo-se, então a média. A VM foi avaliada de forma qualitativa, utilizando-se 4 escores de acordo com a IE e se a marcação é difusa ou focal. Os dados obtidos foram analisados no software Statistical Package for Social Science, GraphPad Prism e STATA. O nível de significância de 5% foi adotado para os testes estatísticos. Foi verificada menor imunomarcação de E-caderina nos APs em relação às neoplasias malignas de glândula salivar (NMGS). Observou-se baixa imunoexpressão de Twist1 e Snail1 em APs. Em relação a expressão nuclear do Twist1, constatou-se maior expressão nas neoplasias malignas quando comparadas aos APs. Ainda, Twist1 em núcleo foi correlacionado à expressão citoplasmática de E-caderina nas NMGS. No que concerne aos parâmetros clinicopatológicos, esta proteína se relacionou estatisticamente com maiores chances de óbito. Foi evidenciada baixa imunoexpressão de Snail1 entre as NMGS. No entanto, na análise dos CACs, foi verificada maior expressão nuclear na variante sólida em relação às demais. A expressão de MMP-9 em parênquima demonstrou correlação positiva com Twist1 citoplasmático e Snail1nuclear nas NMGS. A MMP-9 também apresentou correlação positiva na comparação da sua imunoexpressão em região de parênquima e de estroma. A VM se apresentou como um biomarcador a ser considerado na avaliação clínica dos pacientes, já que esta apresentou relação significativa com tamanho do tumor (T3-T4) e maior frequência de óbito. Ademais, a alta expressão desta proteína se apresentou como um fator preditivo independente para piores taxas de sobrevida global (SG). A avaliação dos demais fatores clinicopatológicos apresentou estágios clínicos avançados como indicador de valor prognóstico independente para menores taxas de SG, enquanto que para a sobrevida livre da doença, estes foram a localização em glândula salivar menor e presença de metástase à distância. Os resultados deste estudo sugerem que o processo de TEM pode estar relacionado ao estágio de diferenciação celular em APs e à progressão tumoral nas NMGS. Ressalta-se, também, maior participação de Twist1 e MMP-9 no cenário da TEM em tumores malignos de glândula salivar, além da possibilidade de utilização da VM como indicador de valor prognóstico (AU).

Salivary gland tumors (SGTs) present remarkable clinical and biological complexity; therefore, many studies investigate the events involved in their progression. One of the dynamics involved in the tumor invasion of different types of carcinomas is the epithelial-mesenchymal transition (EMT). In this process, epithelial cells undergo a transition to a mobile mesenchymal state, favoring invasion and metastasis. Therefore, this research analyzed the immunohistochemical expression of E-cadherin, Twist1, Snail1, α-SMA, vimentin (VM) and matrix metalloproteinase 9 (MMP-9) in 90 SGTs cases; correlations among the biomarkers, as well as between the biomarkers and clinicopathological parameters were made. We selected 20 cases of pleomorphic adenoma (PA), 20 cases of mucoepidermoid carcinoma (MEC), 20 cases of adenoid cystic carcinoma (ACC), 10 cases of polymorphous adenocarcinoma (PAC), 10 cases of epithelial-myoepithelial carcinoma (EMC) and 10 cases of carcinoma ex-pleomorphic adenoma (CXPA). E-cadherin, Twist1, and Snail1 were analyzed in tumor parenchyma, observing the percentage of positive cells (PP) using scores ranging from 0 to 4, and the expression intensity (EI), whose scores were ranged from 0 to 3. The evaluation of MMP-9 was performed in tumor parenchyma and stroma, also evaluating PP and IE, both based on scores that ranged from 0 to 3. The labeling for α-SMA and VM was analyzed in stromal cells. Positive cells for α-SMA were counted in 10 fields and the mean was calculated. VM was evaluated qualitatively, using 4 scores according to EI and whether the labeling was diffuse or focal. Obtained data were analyzed using Statistical Package for Social Science, GraphPad Prism, and STATA software. The significance level of 5% was adopted for the statistical tests. Patients were mostly female, with a mean age of 49.8 years; the major salivary glands were the most affected anatomical site, mainly the parotid gland. A lower E-cadherin immunostaining was verified in PAs in comparison to malignant neoplasms of salivary glands (MNSGs). Low immunoexpression of Twist1 and Snail1 was observed in PAs. Regarding the nuclear expression of Twist1, it was found greater expression in malignant neoplasms than in PAs. Furthermore, Twist1 in the nucleus was correlated with cytoplasmic expression of E-cadherin in MNSGs. Regarding clinicopathological parameters, this protein was statistically related to higher chances of death. Low immunoexpression of Snail1 was evidenced among the MNSGs. However, in the analysis of CACs, greater nuclear expression was observed in the solid variant compared to the others. Expression of MMP-9 in parenchyma showed a positive correlation with cytoplasmic Twist1 and Snail1nuclear in MNSGs. MMP-9 also showed a positive correlation when comparing its immunoexpression in the parenchyma and the stroma. VM was presented as a biomarker to be considered in the clinical evaluation of patients since it showed a significant correlation between greater tumor size and a higher frequency of death. Furthermore, the high expression of this protein appeared as an independent predictive factor for worse overall survival (OS) rates. The evaluation of the rest of the clinicopathological factors showed advanced clinical stages as an indicator of independent prognostic value for lower rates of OS. For disease-free survival, these indicators were the location in the minor salivary gland and the presence of distant metastasis. Our results suggest that the EMT may be related to myoepithelial differentiation in PAs and tumor progression in MNSGs. Also, Twist1 and MMP-9 appear to play a greater role in the scenario of EMT in MNSGs; finally, VM might be used as a prognostic value indicator (AU).

Perfil imuno-histoquímico de marcadores associados à transição epitelial-mesenquimal em lesões odontogênicas epiteliais benignas / Immunohistochemical profile of markers associated with epithelial-mesenchymal transition in benign epithelial odontogenic lesions

Os cistos e tumores odontogênicos, lesões que acometem o complexo maxilomandibular, podem exibir comportamento clínico-biológico mais agressivo. E a transição epitelialmesenquimal (TEM), processo pelo qual as células epiteliais perdem propriedades fenotípicas e adquirem características de células mesenquimais, incluindo maior motilidade e capacidade de invasão, através da regulação de fatores centrais de transcrição e suas vias associadas, podem fazer parte de características associadas às lesões odontogênicas. Dessa forma, o presente trabalho buscou analisar e comparar a expressão imuno-histoquímica de proteínas (Zeb1, Ecaderina, N-caderina e vimentina) envolvidas no processo de TEM, em lesões odontogênicas epiteliais benignas. A amostra consistiu em 88 casos de lesões odontogênicas, das quais compreendem 28 casos de ameloblastoma (AB), 30 de ceratocisto odontogênico (CO) e 30 de cisto dentígero (CD). Todos os espécimes submetidos à técnica imuno-histoquímica foram avaliados por microscopia de luz, e submetidos à escolha aleatória de 5 (cinco) campos, os quais foram fotografados em um aumento de 400x. A avaliação da expressão de cada marcador, a partir da análise em seu compartimento celular específico, foi feita de forma semiquantitativa, através da multiplicação dos escores associados à porcentagem de células imunomarcadas pelos escores relacionados à intensidade da coloração, sendo feita uma média dos cinco campos e o resultado definido como baixa expressão ou alta expressão, conforme metodologia utilizada. As associações foram feitas através do teste de Qui-quadrado e as correlações através do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Os resultados mostraram um pico de prevalência entre a 2ª e 3ª décadas de vida, em todas as lesões estudadas, com um acometimento maior em região posterior de mandíbula, e os ABs foram as lesões de maiores tamanhos, com 65% medindo acima de 2,5cm. A imuno-histoquímica evidenciou baixa expressão de Zeb1 em epitélio odontogênico das lesões estudadas, alta expressão de E-caderina e N-caderina, e uma expressão intermediária de vimentina. Quando realizada a correlação entre os marcadores, observou-se nos casos de AB uma correlação positiva e moderada entre Zeb1 nuclear e E-caderina membranar, Zeb1 citoplasmática e E-caderina membranar e entre E-caderina e vimentina citoplasmáticas. Como também uma correlação positiva moderada, nos casos de CD, entre Zeb1 nuclear e vimentina citoplasmática, e entre Zeb1 e vimentina citoplasmáticas. Logo, podemos concluir que Zeb1 pode estar atuando indiretamente nas vias responsáveis pelo crescimento e características morfológicas dessas lesões estudadas. Além disso, a expressão diferencial de E-caderina, Ncaderina e vimentina demonstraram fazer parte de um processo de TEM parcial nas lesões odontogênicas epiteliais benignas estudadas (AU).

Odontogenic cysts and tumors, lesions that affect the maxillomandibular complex, may exhibit a more aggressive clinical-biological behavior. And the epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose phenotypic properties and acquire characteristics of mesenchymal cells, including increased motility and invasiveness, through the regulation of central transcription factors and their associated pathways, may be part of characteristics associated with odontogenic lesions. Thus, the present work sought to analyze and compare the immunohistochemical expression of proteins (Zeb1, E-cadherin, N-cadherin and vimentin) involved in the MET process in benign epithelial odontogenic lesions. The sample consisted of 88 cases of odontogenic lesions, comprising 28 cases of ameloblastoma (AB), 30 of odontogenic keratocyst (CO) and 30 of dentigerous cyst (CD). All specimens submitted to the immunohistochemical technique were evaluated by light microscopy and submitted to the random choice of 5 (five) fields, which were photographed at a magnification of 400x. The evaluation of the expression of each marker, based on the analysis in its specific cellular compartment, was carried out in a semi-quantitative manner, through the multiplication of the scores associated with the percentage of immunostained cells by the scores related to the intensity of staining, with an average of the five fields and the result defined as low expression or high expression, according to the methodology used. The associations were made using the chi-square test and the correlations using the Spearman correlation test. The significance level was set at 5% (p < 0.05). The results showed a prevalence peak between the 2nd and 3rd decades of life, in all the lesions studied, with a greater involvement in the posterior region of the mandible, and the ABs were the largest lesions, with 65% measuring above 2, 5cm. Immunohistochemistry showed low expression of Zeb1 in the odontogenic epithelium of the lesions studied, high expression of E-cadherin, high expression of N-cadherin and an intermediate expression of vimentin. When the correlation between the markers was performed, a positive and moderate correlation was observed in the cases of AB between nuclear Zeb1 and membrane E-cadherin, cytoplasmic Zeb1 and membrane E-cadherin and between cytoplasmic E-cadherin and vimentin. As well as a moderate positive correlation, in CD cases, between nuclear Zeb1 and cytoplasmic vimentin, and between cytoplasmic Zeb1 and vimentin. Therefore, we can conclude that Zeb1 may be acting indirectly on the pathways responsible for the growth and morphological characteristics of these lesions studied. Furthermore, the differential expression of E-cadherin, N-cadherin and vimentin was shown to be part of a partial TEM process in the benign epithelial odontogenic lesions studied (AU).

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice / 南方医科大学学报

OBJECTIVE@#To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.@*METHODS@#The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.@*RESULTS@#We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.@*CONCLUSION@#The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.

Pseudolaric Acid B Inhibits Proliferation, Invasion and Epithelial-to-Mesenchymal Transition in Human Pancreatic Cancer Cell

PURPOSE: This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism. MATERIALS AND METHODS: The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB. RESULTS: PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups. CONCLUSION: PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.

Inmunoexpresión de E-cadherina y vimentina en mucosa oral normal, displasia epitelial oral y carcinoma oral de células escamosas / Immunoexpression of E-cadherin and vimentin in normal oral mucosa, oral epithelial displasia and oral squamous cell carcinoma

El objetivo fue evaluar la inmunoexpresión de E-cadherina y Vimentina en mucosa oral normal (MON), displasia epitelial oral (DEO) y carcinoma oral de células escamosas (COCE). Se realizó un estudio descriptivo de una serie de casos analizandolos mediante técnica de inmunohistoquímica contra E-cadherina y Vimentina 16 muestras de MON, 16 de DEO y 19 de COCE. La inmunotinción fue evaluada cualitativamente considerando extensión e intensidad para E-cadherina e intensidad para Vimentina. El análisis de la extensión e intensidad de la inmunotinción de E-cadherina y Vimentina según diagnóstico reveló una asociación estadísticamente significativa (p<0,001). Siendo la expresión de E-cadherina más alta en MON, seguido por DEO y más baja en COCE, inversamente a lo que se observó con Vimentina. El presente estudio reveló la subregulación del marcador molecular E-cadherina junto con la expresión aberrante por parte de células epiteliales del marcador mesenquimal Vimentina en muestras de MON, DEO y COCE.

The aim was to evaluate the expression of E-cadherin and Vimentin in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), in comparison with normal oral mucosa (NOM) in a descriptive case study using immunohistochemistry. A total of fifty-one (N=51) histological samples were included; as follows: n = 16 (NOM), n = 16 (OED) and n = 19 (OSCC). All samples were analyzed using immunohistochemistry against the expression of E-cadherin and Vimentin. Immunostaining was qualitatively evaluated by extent and intensity of its expression for E-cadherin and intensity for Vimentin. Extension and intensity analysis of E-cadherin and Vimentin immunostaining according to group revealed a statistically significant association (r<0.001). E-cadherin expression was found to be highest in NOM followed by OED and lowest in OSCC, inverse to what was observed with Vimentin. The present study revealed the down regulation of the molecular marker E-cadherin, suggestive of reduction in dysplastic cells on comparison to NOM cells, and aberrant expression of the mesenchymal marker Vimentin by epithelial cells in samples of NOM, OED and OSCC; questioning their value as a prognostic marker.

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.

Undifferentiated embryonal sarcoma of the liver in an adult patient

Undifferentiated embryonal sarcoma of the liver (UESL) is rare primary hepatic sarcoma and is known to occur in pediatric patients. This case is the UESL occurred in a 51-year old male patient. Multilocular cystic lesion was composed of primitive spindle cells without specific differentiation. This rare case would help to review differential diagnosis of primary sarcoma in liver and cystic neoplasm of the liver.

Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1alpha

PURPOSE: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1alpha (HIF-1alpha) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1alpha-induced invasive characteristics. MATERIALS AND METHODS: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1alpha, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1alpha and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1alpha, tissue analysis was done. RESULTS: Hypoxia induces HIF-1alpha expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1alpha via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1alpha with RNA interference suppressed hypoxia-induced HIF-1alpha and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1alpha. These were confirmed in the orthotopic FTC model. CONCLUSION: Hypoxia induced HIF-1alpha, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

Sarcomatoid hepatocellular carcinoma with mixed osteoclast-like giant cells and chondroid differentiation

No abstract available.

Histone demethylase retinoblastoma binding protein 2 regulates the expression of -smooth muscle actin and vimentin in cirrhotic livers

Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.

Intramedullary Spinal Cord Metastasis in Renal Cell Carcinoma: A Case Report of the Surgical Experience

Intramedullary spinal cord metastasis (ISCM) from renal cell carcinoma (RCC) is rare manifestation and most of them are treated by adjuvant treatment modalities like radiotherapy. Despite the radio-resistance of RCC itself, focal radiotherapy has been preferred as the first-line treatment modality of ISCM from RCC and only a few cases underwent surgical treatment. We describe a case of ISCM from RCC, which underwent surgical excision and pathologically confirmed. A 44-yr-old man was presented with rapid deterioration of motor weakness during focal radiotherapy for ISCM from RCC. After the surgery for removal of the tumor mass and spinal cord decompression, his motor power was dramatically improved to ambulate by himself. We report the first published Korean case of ISCM from RCC confirmed pathologically and describe our surgical experience and his clinical characteristics.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.