Effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma (TSCC).@*METHODS@#Forty-eight TSCC cases were selected from the Maxillofacial Surgery Center of Qingdao Municipal Hospital from 2017 to 2019. The expression of RhoE in the specimens (TSCC and adjacent tissues) was detected by immunohistochemistry, and RhoE mRNA and protein were extracted to further detect the expression of RhoE. SCC-4 and CAL-27 cells were selected for @*RESULTS@#The expression level of RhoE in TSCC was significantly lower than that in adjacent tissues (@*CONCLUSIONS@#RhoE expression is low in TSCC. Over expression RhoE in TSCC can significantly decrease its migration and invasion abilities. Hence, RhoE may play an important role in regulating the metastasis and invasion of TSCC and provide a new target for gene therapy.

Evaluation of pathogen P21 protein as a potential modulator of the protective immunity induced by Trypanosoma cruzi attenuated parasites

BACKGROUND TcP21 is a ubiquitous secreted protein of Trypanosoma cruzi and its recombinant form (rP21) promotes parasite cell invasion and acts as a phagocytosis inducer by activating actin polymerisation in the host cell. OBJECTIVE Our goal was to evaluate if the additional supplementation of rP21 during a prime/boost/challenge scheme with T. cruzi TCC attenuated parasites could modify the well-known protective behavior conferred by these parasites. METHODS The humoral immune response was evaluated through the assessment of total anti-T. cruzi antibodies as well as IgG subtypes. IFN-γ, TNF-α and IL-10 were measured in supernatants of splenic cells stimulated with total parasite homogenate or rP21. FINDINGS Our results demonstrated that, when comparing TCC+rP21 vs. TCC vaccinated animals, the levels of IFN-γ were significantly higher in the former group, while the levels of IL-10 and TNF-α were significantly lower. Further, the measurement of parasite load after lethal challenge showed an exacerbated infection and parasite load in heart and skeletal muscle after pre-treatment with rP21, suggesting the important role of this protein during parasite natural invasion process. MAIN CONCLUSION Our results demonstrated that rP21 may have adjuvant capacity able to modify the cytokine immune profile elicited by attenuated parasites.

Expression Difference of RhoH Gene in Leukemia and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and clinical significance of RhoH gene in bone marrow cells of leukemia patients.@*METHODS@#31 cases of leukemia and 15 cases of non-tumor as controls were collected. The expression of RhoH in bone marrow cells was detected by real-time quantitative PCR (RQ-PCR). The median expression level of RhoH was used as the cut-off value. The newly diagnosed patients were divided into RhoH high expression group and low expression group. The relationship of different RhoH expression levels with clinical features and prognosis of newly diagnosed patients was analyzed.@*RESULTS@#The mRNA expression of RhoH in the bone marrow cells of 31 cases of leukemia was significantly lower than that in the control group, mRNA expression of RhoH in the ALL group was significantly lower than that in AML group (P＜0.05). Compared with the RhoH high expression group, the proportion of bone marraw blasts and LDH level in the RhoH low expression group was significantly increased (P＜0.05), but there were no significant differences in clinical features such as age, white blood cell count, hemoglobin level, platelets count, PCT and CRP level (P＞0.05). In AML, the recurrence rate after standard chemotherapy in RhoH low expression group was higher than that in high expression group, while the expression of RhoH not correlated with other prognostic genes of AML. In ALL, the recurrence rate in RhoH low expression group was not statistically significant different from that in high expression group.@*CONCLUSION@#RhoH may be involved in the genesis of acute leukemia. In AML, RhoH expression negatively correlates with recurrence rate, which can be used as a prognostic indicator independently. In ALL, RhoH may participate in the disease process through other mechanism.

Estudo funcional da proteína MRCK-ß em adenocarcinomas colorretais metastáticos ou não / Functional study of the MRCK-ß protein in metastatic or non-metastatic colorectal adenocarcinoma

Introdução: O câncer colorretal (CCR) é uma neoplasia de origem epitelial e abrange tumores de cólon e reto em homens e mulheres. É uma doença tratável e frequentemente curável quando na ausência de extensão para outros órgãos. No entanto, aproximadamente 50% a 60% dos pacientes diagnosticados com CCR irão desenvolver metástases que são mais comumente encontradas em fígado e pulmão. MRCK-ß mediada por Cdc42 tem um papel importante na migração e metástase devido a sua capacidade de promover rearranjos do citoesqueleto. E ROCK2, mediada por RhoA também esta relacionado à capacidade de invasão e migração. Objetivo: Avaliar a importância das proteínas MRCK-ß e Cdc42 no processo de migração celular e formação de metástases em adenocarcinomas colorretais através estudos de expressão gênica, expressão proteína e estudos funcionais em linhagens celulares. Materiais e Métodos: 60 casos com CCR que desenvolveram metástases e 48 casos que não desenvolveram metástase foram selecionados e seu material parafinado foi resgatado para avaliação proteica. Imunoistoquímica foi realizada para os marcadores anti-MRCK-ß, anti-Cdc42 e anti-ROCK2, as reações foram analisadas de forma manual através do sistema Envision FLEX/HRP (DAKO®). A partir de amostras do banco de tumores, presentes no Biobanco da Patologia Investigativa do AC Camargo Cancer Center, foram extraídos RNAs para avaliar a expressão gênica de Cdc42 e MRCK-ß. A superexpressão proteica de MRCK-ß foi avaliada através da linhagem celular SW480 e foram realizados ensaios de migração por transwell e a inibição da expressão foi realizada na linhagem HCT116. Resultados: Cdc42 e ROCK2 mostraram maior expressão nos tumores não metastáticos e MRCK-ß nos tumores metastáticos, todos com um valor p < 0,001. Entre os tumores primários e suas metástases, ROCK2 mostrou estar mais expresso no tumor primário (p = 0,042). Os marcadores também foram avaliados através do qRT-PCR e não mostram correlação com a expressão imunoistoquímica. Os ensaios de migração mostraram que quando ocorre a superexpressão de MRCK-ß as células apresentam maior capacidade de migração (p = 0,017). Conclusões: MRCK-ß mostrou expressão apenas entre os tumores metastáticos e maior expressão na linhagem HCT116, que possui um maior potencial metastático. Quando a superexpressão de MRCK-ß foi ampliada na linhagem SW480, foi avaliado que a capacidade de migração celular é aumentada indicando ser um potencial marcador de metástase em câncer colorretal (AU)

Introduction: Colorectal cancer (CRC) is a neoplasia of epithelial origin and covers tumors of the colon and rectum tumors in men and women. It is a treatable and often curable disease when in the absence of extension to other organs. However, approximately 50% to 60% of patients diagnosed with CRC will develop metastases that are most commonly found in the liver and lung. MRCK-ß mediated by Cdc42 plays a role in migration and metastasis due to its ability to rearrangements of the cytoskeleton. And ROCK2, mediated by RhoA is also related to the capacity of invasion and migration Aim: To evaluate the importance of MRCK-ß and Cdc42 proteins in the process of cell migration and formation of metastases in colorectal adenocarcinomas through studies of gene expression, protein expression and functional studies in cell lines. Materials and Methods: 60 cases of CRC that developed metastases and 48 cases that did not develop metastasis were selected and their paraffin material was rescued. Immunohistochemistry was performed for the anti-MRCK-ß, anti-Cdc42 and anti-ROCK2 markers, reactions were analyzed manually using the Envision FLEX / HRP (DAKO®) protocol. RNAs samples from the tumor bank, presents on Biobank of Investigative Pathology of AC Camargo Cancer Center, were extracted to evaluate the gene expression of Cdc42 and MRCK-ß. The overexpression of the protein of MRCK-ß was evaluated through the SW480 cell line and transwell migration assays were performed and the inhibition of expression was performed in the HCT116 Results: Cdc42 and ROCK2 showed higher expression in non-metastatic tumors and MRCK-ß in metastatic tumors, all with a p-value of <0.001. Among primary tumors and their metastases, ROCK2 showed to be more expressed in the primary tumor (p = 0.042). The markers were also evaluated through qRT-PCR and did not show correlation with immunohistochemical expression. The migration assays showed that when MRCK-ß overexpression occurs the cells have a higher migration capacity (p = 0.017). Conclusions: MRCK-ß showed expression only among metastatic tumors and greater expression in the HCT116 cell line, which has a higher metastatic potential. When overexpression of MRCK-ß was amplified in the SW480 cell line, it was evaluated that the cell migration capacity was increased indicating to be a potential marker of metastasis in colorectal cancer (AU)

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

RHO GTPasas como blancos moleculares en cáncer: Estrategias y oportunidades terapéuticas / Rho GTPases as molecular targets in cancer. Strategies and therapeutic opportunities

Las Rho GTPasas son una familia de proteínas que actúan como interruptores moleculares en diversas vías de señalización coordinando la regulación de distintos procesos celulares. La desregulación de dichas proteínas se vincula con transformación maligna y progresión tumoral en distintos tipos de cáncer. Por estos motivos, en los últimos años las Rho GTPasas fueron postuladas como blancos moleculares interesantes. En este trabajo describimos las distintas estrategias estudiadas utilizando a las Rho GTPasas como blanco y su grado de avance, mostrando una estrategia novedosa para el tratamiento del cáncer.

Rho GTPases are molecular switches that control the different cellular processes. Deregulation of these proteins is associated to transformation and malignant progression in several cancer types. Given the evidence available of the role of Rho GTPases in cancer it is suggested that these proteins can serve as potential therapeutic targets. This review focuses on the strategies used to develop Rho GTPases modulators and their potential use in therapeutic settings.

Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.

Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.

Exenatide promotes chemotactic migration of adipose-derived stem cells through SDF-1/CXCR-4/Rho GTPase pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of exenatide on chemotactic migration of adipose-derived stem cells (ADSCs) and confirm that Rho GTPase is the downstream effector protein of SDF-1/CXCR-4 migration pathway.</p><p><b>METHODS</b>ADSCs were isolated, cultured, identified by flow cytometry, and induced to differentiate in vitro. RTCA xCELLigence system was used to analyze the effect of exenatide on ADSC proliferation. The effects of exenatide at different concentrations, AMD3100 (CXCR-4 antagonist), and CCG-1423 (Rho GTPase antagonist) on chemotactic migration of ADSCs were tested using Transwell assay. The expression of CXCR-4 in exenatide-treated ADSCs was measured by flow cytometry and Western blotting. Active Rho pull-down detection kit was used to detect the expression of Rho GTPase. Laser confocal microscopy was used to observe the formation of stress fibers in ADSCs with different treatments.</p><p><b>RESULTS</b>Exenatide treatment for 24 h had no significant effect on ADSC proliferation. Exenatide obviously promoted chemotactic migration of ADSCs in a concentration-dependent manner, and this effect was blocked by either AMD3100 or CCG-1423. Both flow cytometry and Western blotting showed that exenatide dose-dependently up-regulated CXCR-4 expression in ADSCs. Western blotting showed that the expression of Rho GTPase was related to SDF-1/CXCR-4 pathway, and laser confocal microscopy revealed that the formation of stress fibers in ADSCs was related to SDF-1/CXCR-4/ Rho GTPase pathway.</p><p><b>CONCLUSION</b>Exenatide promotes chemotactic migration of ADSCs, and Rho GTPase is the downstream effector protein of SDF-1/CXCR-4 pathway.</p>

Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK / 中国天然药物

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

Influência do cálcio e das proteínas Miro na mobilidade mitocondrial anteriormente e durante a agregação de proteínas envolvidas em neurodegeneração / Influence of calcium and Miro proteins on mitochondrial mobility before and during protein aggregation involved in neurodegeneration

A inibição do transporte axonal é um evento que ocorre prematuramente no curso das doenças neurodegenerativas, inclusive antes da formação dos agregados proteicos, os quais estariam envolvidos no processo fisiopatológico das doenças neurodegenerativas. No presente estudo avaliou-se a hipótese de que alterações no transporte de mitocôndrias ocorrem antes da formação dos agregados proteicos envolvidos em neurodegeneração, devido a desregulação dos níveis citoplasmáticos de Ca2+ e o envolvimento da modulação do transporte mitocondrial provido pela proteína Miro neste cenário. Utilizaram-se dois modelos experimentais: o primeiro utilizando a exposição à rotenona em culturas primárias de neurônios do locus coeruleus, hipocampo e substância negra de ratos, e o segundo utilizando neurônios derivados de células tronco de pluripotência induzida (iPSC), isogênicas humanas contendo mutações que levam à deleção do exon 9 da (deltaE9) no gene da presenilina 1 (PS1), o qual apresenta aumento da síntese do peptídeo beta-amiloide com 42 aminoácidos (Abeta42), sem a formação de agregados proteicos. Os resultados mostram disfunções nos níveis citoplasmáticos de Ca2+ em ambos modelos. A mobilidade mitocondrial alterou-se no hipocampo, locus coeruleus e substância negra após exposição à rotenona. No entanto, a direção das alterações observadas não se correlacionaram com os níveis de Ca2+, de acordo com o já descrito na literatura. Não houve alteração da mobilidade mitocondrial, nem nos níveis de Miro1, nos neurônios derivados de iPSC. Em conclusão, o presente estudo demonstrou que alterações nos níveis citoplasmáticos de Ca2+ ocorrem antes e durante a formação de agregados proteicos, o que pode ser importante para a etiologia de doenças neurodegenerativas. Foi também demonstrado que mudanças na mobilidade mitocondrial, acompanhadas por alterações nas concentrações intracelulares de Ca2+, em níveis fisiológicos, ocorrem de forma independente dos níveis da proteína...

The axonal transport impairment occurs early in neurodegenerative diseases, even before the formation of protein aggregates, which are related with the neuropathophysiology mechanism in neurodegenerative diseases. In this study, we evaluate the hypothesis that disruptions in mitochondria transport occurs before the formation of protein aggregate related with neurodegeneration, triggered by dysregulations in cytosolic Ca2+ levels and the involvement of Miro Ca2+ dependent mechanism of mitochondria trafficking modulation. We employed two experimental models, first using rotenone exposure in primary neuronal cell cultures from locus coeruleus, substantia nigra and hippocampus of newborn rats. Second, using isogenic human neurons derived from induced pluripotent stem cells (iPSCs), harboring mutations, those induce exon 9 deletion (deltaE9) in Presenilin 1 (PS1) gene, and showing increased synthesis of amyloid beta peptide with 42 amino acids (betaA42) without the formation of protein aggregates. We found abnormalities in cytosolic Ca2+ levels in both experimental models, mitochondria trafficking were altered in hippocampus, substantia nigra and locus coeruleus. However, the pattern of mitochondria trafficking alterations did not correlate with cytosolic Ca2+ levels, accordingly with the data that was already published. We did not find alterations in mitochondria trafficking or Miro1 levels in neurons derived from iPSC. In conclusion, our finds demonstrated aberrant cytosolic Ca2+ levels before and during protein aggregation, which may be important for the etiology of neurodegenerative diseases. In addition, this dysfunction in mitochondria trafficking happens after changes in cytosolic Ca2+ levels, in physiological range, independent of Miro1 levels in primary neurons cell cultures. Therefore, new studies need to be done, aiming to elucidate the relation between mitochondria trafficking dysfunctions and the induction of neurodegeneration process.

Estudo de genes diferencialmente expressos em câncer de vulva: validações a partir de resultados de CGH array / Study of differentially expressed genes in vulvar cancer: results from the validation of array CGH

Introdução: O carcinoma de vulva é um tumor de baixa ocorrência, sendo responsável por menos de 3% de todos os tumores malignos que acometem mulheres. Em mulheres jovens a ocorrência da doença está atrelada a fatores de risco como tabagismo e infecção por HPV, entretanto em mulheres acima dos 50 anos ocorre por mecanismos genéticos ainda pouco elucidados. Nos últimos anos poucos autores estudaram alterações genômicas em carcinomas vulvares, contudo nenhum deles determinou um fator prognóstico definitivo, nem tampouco correlacionou esses achados com a expressão gênica, mesmo representando pontos-chave na compreensão do processo de carcinogênese. A partir da análise de arranjos de hibridação genômica comparativa (CGH-array) realizada previamente em nosso laboratório, dois genes candidatos, ROCK1 e RhoD, localizados em regiões com alta frequência de ganhos em nossas amostras de neoplasias vulvares, foram selecionados para este estudo. Objetivo: Validar a expressão dos genes candidatos, ROCK1 e RhoD, que foram identificados em regiões com ganho de cópias nas amostras de carcinoma vulvar pelo método de CGHarray, a fim de determinar melhores e mais acurados valores prognósticos no carcinoma vulvar...

Introduction: The vulvar carcinoma is a low occurrence tumor, accounting for less than 3% of all malignant tumors that affect women. In young women the occurrence of the disease is linked to risk factors such as smoking and HPV infection, but the majority of cases of vulvar cancer occurs in women over 50 years by genetic mechanisms still poorly understood. Recently, few authors studied genomic changes in vulvar carcinomas, however none of them determined on prognostic factor, nor correlate these findings with gene expression, even representing key points in understanding the carcinogenesis process. From the analysis of comparative genomic hybridization arrays (array CGH) previously performed in our laboratory, two candidate genes, ROCK1 and Rhod, located in regions with high frequency gains in our samples of vulvar cancer were selected for this study. Objective: To validate the expression of the candidate genes, RhoD and ROCK1, which have been identified in regions with a gain of copies in vulvar carcinoma samples by CGH-array method, in order to determine the best and most accurate prognostic values in vulvar carcinoma. Methods: 16 cases of vulvar cancer were rescued from AC Camargo Cancer Center’s Biobank...

Lysophosphatidic acid (LPA) stimulates invasion and metastatic colonization of ovarian cancer cells through Rac activation / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms of lysophosphatidic acid (LPA) in stimulating invasion and metastatic colonization of ovarian cancer cells.</p><p><b>METHODS</b>The metastatic ability in vivo of ovarian cancer SK-OV3, HEY, OVCAR3, and IGROV1 cells was determined in tumor-bearing nude mouse models. Matrigel assay was used to detect the changes of response in vitro of ovarian cancer cells to LPA after Rac(-) or Rac(+) adenovirus treatment. LPA-induced Rho GTPase activation was detected by GST-fusion protein binding assay.</p><p><b>RESULTS</b>The peritoneal metastatic colonization assay showed overt metastatic colonization in mice receiving SK-OV3 and HEY cell inoculation, indicating that they are invasive cells. Metastatic colonization was not detected in animals receiving OVCAR3 and IGROV1 cells, indicating that these cells are non-invasive cells. In the matrigel invasion assay, exposure to LPA led to a notably greater migratory response in metastatic SK-OV3 and HEY cells (Optical density: SK-OV3 cells: 0.594±0.023 vs. 1.697±0.049, P<0.01; HEY cells: 0.804±0.070 vs. 1.851±0.095, P<0.01). But LPA did little in the non-metastatic OVCAR3 and IGROV1 cells (Optical density A: OVCAR3 cells: 0.336±0.017 vs. 0.374±0.007, P>0.05; IGROV1 cells: 0.491±0.036 vs. 0.479±0.061, P>0.05). LPA migratory responses of ovarian cancer cells were closely related to their metastatic colonization capabilities (r = 0.983, P<0.05). Rac(-) blocked the LPA response of invasive SK-OV3 and HEY cells (LPA-induced fold increase of cell migration: SK-OV3 cells: 2.988±0.095 vs. 0.997±0.100,P=0.01; HEY cells: 2.404±0.059 vs. 0.901±0.072, P=0.01). But Rac(+) confered the non-invasive cells with LPA response and invasion capability (LPA-induced fold increase of cell migration: OVCAR3 cells: 1.072±0.080 vs. 1.898±0.078, P<0.01; IGROV1 cells: 1.002±0.044 vs. 2.141±0.057, P<0.05). Among Rho GTPases, only Rac activation was different between ovarian cancer cell lines with different metastatic capability after LPA stimulation: Cdc42 could not be activated in both the invasive and non-invasive cell lines. RhoA could be activated in both the invasive and non-invasive cell lines. Rac could be activated by LPA in the invasive ovarian cancer cell lines. However, Rac could not be activated in the non-invasive cell lines.</p><p><b>CONCLUSION</b>Lysophosphatidic acid stimulates invasion and metastasis of ovarian cancer cells through Rac activation.</p>

Influência da PGE2 e do TGF-β em eventos relacionados a transição epitéliomesênquima em células de câncer colorretal / [ Influence of PGE2 and TGF-ß on events related to epitheliomasenchyma transition in colorectal cancer cells]

Diversos estudos mostram que a Prostaglandina E2 (PGE2) possui um papel pró-tumoral em diferentes modelos experimentais. Por outro lado, o Fator de Crescimento Transformante-1 (TGF-β1, Transforming Growth Factor-1) é relatado como uma citocina capaz de exercer um papel dual no contexto tumorigênico, ora funcionando como agente supressor, ora como promotor tumoral. Além disso, foi demonstrado que estes agentes alteram a organização do citoesqueleto de actina aumentando o potencial migratório e invasivo. No entanto, o papel destes mediadores pró inflamatórios nesse contexto foi analisado isoladamente e estudos mostrando os efeitos da associação entre eles, particularmente na transição epitélio mesenquima (TEM) em câncer colorretal (CCR), não têm sido relatados. Assim, o objetivo do nosso estudo foi avaliar os efeitos desses agentes em eventos relacionados à aquisição daTEM e determinar quais efeitos são desencadeados pelo tratamento associado entre esses dois agentes. Inicialmente, utilizamos células derivadas de carcinoma de pulmão, A549, como controle positivo de alterações celulares relacionadas a TEM após o tratamento com TGF-β. Nossos resultados mostraram que esse agente foi capaz de: a) induzir alterações morfológicas; b) diminuir os níveis protéicos de E-caderina e aumentar os níveis das proteínas β-catenina eN-caderina; c) alterar a localização subcelular de todas essas proteínas e; d) aumentar a formação de fibras de estresse e o potencial migratório. Em seguida, analisamos os efeitosdesses agentes de forma isolada e em associação usando células HT-29, derivadas de adenocarcinoma colorretal...

Studies have shown that Prostaglandin E2 (PGE2) plays a pro-tumor role in different experimental models. On the other hand, the Transforming Growth Factor-1 (TGF-1) has been reported as a cytokine that plays a dual role in the tumorigenesis context, may act as atumor suppressor or tumor promoter. Moreover, it have been shown that these agents alter the actin cytoskeleton organization and increase the migratory and invasive potential. However, the role of these pro-inflammatory mediators in this context have been evaluated separatelyand studies showing the combined effects, particularly during epithelial-mesenchymal transition (EMT) development in colorectal cancer (CRC), have not been shown. Therefore,the aim of our study was to evaluate the effects of these agents in events related to EMT acquisition and determine which effects are triggered by the associated treatment of these two agents. First, we used A549 cells derived from lung carcinoma, as a positive control of cellular changes related to EMT, after treatment with TGF-β. Our results showed that thisagent was able to: a) induce morphological alterations; b) decrease protein levels of Ecadherin and increase protein levels of β-catenin and N-cadherin; c) alter the subcellularlocalization of these proteins; d) increase the formation of stress fibers and the migratory potential. Next, we analyzed the effects of these agents separately and associated using cellsderived from colon adenocarcinoma, HT-29. We observed that treatment with these agents separately decreased the protein levels of E-cadherin and increased these levels of β-catenin, N-cadherin and vimentin...

Papel das vias de sinalização Rho-ROCK e STAT3 no controle da proliferação celular induzida pelo LPA em células de carcinoma de cólon, HCT-116 / [Role of the Rho-ROCK and STAT3 signaling pathways in the control of LPA-induced cell proliferation in colon carcinoma cells, HCT-116]

O câncer colorretal (CCR) é o segundo tipo de neoplasia mais incidente na população brasileira feminina e o terceiro na masculina. Durante a progressão do CCR, células adquirem capacidades proliferativas, migratórias e invasivas. Diversos estímulos são capazes de modular tais eventos, por exemplo, o ácido lisofosfatídico (LPA). Esse fosfolipídeo se liga em re Palavras-chave: 1. Câncer colorretal 2. Ácido Lisofosfatídico 3.GTPase RhoA 4. Proliferação celular ceptores específicos desencadeando diversas vias de sinalização envolvidas com a progressão tumoral. No entanto, pouco se sabe sobre o papel do LPA na progressão do CCR. O objetivo desse estudo foi investigar o papel do LPA em eventos celulares e moleculares relacionados com a progressão do CCR, assim como identificar as vias de sinalização ativadas por este agente. Inicialmente, células derivadas de câncer de cólon com diferentes potenciais invasivos e metastáticos (Caco-2, HT-29 e HCT-116) tiveram seu perfil de expressão proteica dos três principais receptores de LPA (LPA 1-3) analisados por immunoblotting. Em seguida, avaliamos a capacidade do LPA em mediar migração, invasão e crescimento independente de ancoragem e verificamos que este agente não induziu alteração destes eventos. Verificamos então, se o LPA era capaz de causar mudanças no potencial proliferativo nessas linhagens usando a técnica de cristal violeta e analisando a progressão do ciclo celular por citometria de fluxo. Observamos que o LPA causou um aumento de proliferação apenas nas células HCT-116 e de forma dependente da GTPase Rho e de sua efetora ROCK. Sabendo que as vias de ß-catenina e de STAT 3 podem ser reguladas pela via Rho-ROCK, verificamos a capacidade do LPA modular a atividade transcricional de ß-catenina e os níveis de fosforilação de STAT 3 (pSTAT). Embora os resultados não tenham indicado a ativação de ß-catenina pelo ensaio de luciferase de TCF/Lef, observamos um aumento nos níveis de pSTAT por immunoblotting e de sua localização nuclear por microscopia confocal, indicando ativação desta via. Além disso, essa ativação foi independente da via Rho-ROCK, visto que o inibidor de ROCK, Y27632, não reverteu esse efeito. De forma interessante, observamos que a inibição farmacológica de ambos, STAT 3, com STA21, e ROCK, com Y27632, prevenia o aumento de proliferação induzido pelo LPA em HCT-116 e que a inibição conjunta dessas vias apresentava um efeito ainda maior na prevenção da progressão do ciclo celular causada pelo LPA. Finalmente, a análise de expressão gênica global das células HCT-116 tratadas com LPA por ChipArrray, mostrou um aumento na expressão gênica das ciclinas E1, A2 e B1, efeito este confirmado por immunoblotting. Ainda, nossos resultados mostraram que a inibição concomitante das vias Rho-ROCK e STAT 3 preveniu o aumento da expressão dessas proteínas. Em conclusão, no presente estudo mostramos que o LPA aumenta o potencial proliferativo das células HCT-116, uma linhagem celular com potencial mais invasivo, através de um mecanismo envolvendo uma cooperação das vias Rho-ROCK e STAT3 no controle do ciclo celular.

Tanshinone II a protects against lipopolysaccharides-induced endothelial cell injury via Rho/Rho kinase pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To test whether tanshinone II A (Tan II A), a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin (lipopolysaccharides, LPS)-induced endothelial injury.</p><p><b>METHODS</b>Endothelial cell injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone II A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase (ROCK) pathway-associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray.</p><p><b>RESULTS</b>Tan II A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 (ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, or PIP2 in Western blotting), focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan II A, Y27632 and valsartan.</p><p><b>CONCLUSION</b>Tan II A exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.</p>

miR-128a is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation by targeting RND3 / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of microRNA-128a (miR-128a) and its role in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Nineteen pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-128a expression using qRT-PCR. A miR-128a mimics or inhibitor was transfected into HCC cells, and the cell viability was analyzed by MTT assay. RND3, one of the potential targets of miR-128a, was predicted by bioinformatics software and demonstrated by dual luciferase reporter assay. The expression of RND3 after transfection was detected using qRT-PCR and Western blotting, and the cell cycle-related proteins were determined with Western blotting.</p><p><b>RESULTS</b>The expression of miR-128a were significantly up-regulated in HCC tissues as compared to the adjacent tissues (P<0.05). In cultured HCC cells, miR-128a promoted the cell proliferation and resulted in down-regulated RND3 mRNA and protein expressions by targeting RND3' 3'UTR (P<0.05) and also in the down-regulation of cyclin B1, cyclin D1 and CDK4 protein expressions.</p><p><b>CONCLUSION</b>miR-128a is up-regulated in HCC and promotes HCC cell proliferation by targeting RND3.</p>

Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Melanoma has the highest mortality among all superficial malignant tumors. The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets. As a molecular switch that controls tumor metastasis, Ras homology C (RhoC) has been correlated with tumor progression, especially tumor invasion and metastasis. However, little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma. In this study, we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.</p><p><b>METHODS</b>Based on the RhoC gene encoding information, three pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed. After detecting their silencing effects on the RhoC gene of A375 cells, the most effective pGPU6/GFP/Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC. The lentivirus vector was used to infect A375 cells, and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.</p><p><b>RESULTS</b>The plasmids pGPU6/GFP/Neo-shRNA 336, pGPU6/GFP/Neo-shRNA 453, and pGPU6/GFP/Neo-shRNA 680 were constructed. After they were transfected into A375 cells, the expressions of RhoC mRNA and protein were 1.47 ± 0.26, 1.13 ± 0.16, 1.39 ± 0.11 and 70.98 ± 9.21, 50.67 ± 6.06, 65.77 ± 4.06, respectively. pGPU6/GFP/Neo-shRNA 453 was the most effective sequence, and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. pLenti6.3-EGFP-453 was used to infect A375 cells. The expression of RhoC mRNA and protein were 1.05 ± 0.05 and 62.04 ± 15.86 in the lentivirus group, 4.21 ± 0.24 and 220.86 ± 24.07 in the negative lentivirus control group, and 4.63 ± 0.32 and 257.39 ± 12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant (P < 0.05).</p><p><b>CONCLUSION</b>The successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro, and significantly inhibit the RhoC mRNA and protein expression.</p>

CK2beta promotes Pink1/Parkin-mediated MIRO1 degradation / 生物医学工程学杂志

PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex.

Expression of JMJD2A in infiltrating duct carcinoma was markedly higher than fibroadenoma, and associated with expression of ARHI, p53 and ER in infiltrating duct carcinoma.

Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.