TGF-ß acts as a dual regulator of COX-2/PGE2 tumor promotion depending of its cross-interaction with H-Ras and Wnt/ß-catenin pathways in colorectal cancer cells.

Transforming growth factor-ß (TGF-ß) plays a dual role acting as tumor promoter or suppressor. Along with cyclooxygenase-2 (COX-2) and oncogenic Ras, this multifunctional cytokine is deregulated in colorectal cancer. Despite their individual abilities to promote tumor growth and invasion, the mechanisms of cross regulation between these pathways is still unclear. Here, we investigate the effects of TGF-ß, Ras oncogene and COX-2 in the colorectal cancer context. We used colon adenocarcinoma cell line HT-29 and Ras-transformed IEC-6 cells, both treated with prostaglandin E2 (PGE2 ), TGF-ß or a combined treatment with these agents. We demonstrated that PGE2 alters the subcellular localization of E-cadherin and ß-catenin and enhanced the tumorigenic potential in HT-29 cells. This effect was inhibited by TGF-ß, indicating a tumor suppressor role. Conversely, in Ras-transformed IEC-6 cells, TGF-ß induced COX-2 expression and increased invasiveness, acting as a tumor promoter. In IEC-6 Ras-transformed cells, TGF-ß increased nuclear ß-catenin and Wnt/ß-catenin activation, opposite to what was seen in the PGE2 and TGF-ß joint treatment in HT-29 cells. Together, our findings show that TGF-ß increases COX-2 levels and induces invasiveness cooperating with Ras in a Wnt/ß-catenin activation-dependent manner. This shows TGF-ß dual regulation over COX-2/PGE2 tumor promotion depending on the H-Ras and Wnt/ß-catenin pathways activation status in intestinal cancer cells.

Kopsanone inhibits proliferation and migration of invasive colon cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer-related death globally. In spite of the increasing knowledge on molecular characteristics of different cancer types including CRC, there is limitation in the development of an effective treatment. The present study aimed to verify the antitumor effect of kopsanone, an indole alkaloid. To achieve this, we treated human colon cancer cells (Caco-2 and HCT-116) with kopsanone and analyzed its effects on cell viability, cell-cell adhesion, and actin cytoskeleton organization. In addition, functional assays including micronuclei formation, colony formation, cell migration, and invasiveness were performed. We observed that kopsanone reduced viability and proliferation and induced micronuclei formation of HCT-116 cells. Also, kopsanone inhibited anchorage-dependent colony formation and modulated adherens junctions (AJs), thus increasing the localization of E-cadherin and ß-catenin in the cytosol of the invasive cells. Finally, fluorescence assays showed that kopsanone decreased stress fibers formation and reduced migration but not invasion of HCT-116 cells. Taken together, these findings indicate that kopsanone reduces proliferation and migration of HCT-116 cells via modulation of AJs and can therefore be considered for future in vivo and clinical investigation as potential therapeutic agent for treatment of CRC.

Effect of Toxoplasma gondii infection on the junctional complex of retinal pigment epithelial cells.

Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis.

Progeny from irradiated colorectal cancer cells acquire an EMT-like phenotype and activate Wnt/ß-catenin pathway.

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer, however, the fractionation schedules frequently allow for the repopulation of surviving tumors cells, neoplastic progression, and subsequent metastasis. The aim of the present study was to analyze the transgenerational effects induced by radiation and evaluate whether it could increase the malignant features on the progeny derived from irradiated parental colorectal cancer cells, Caco-2, HT-29, and HCT-116. The progeny of these cells displayed a differential radioresistance as seen by clonogenic and caspase activation assay and had a direct correlation with survivin expression as observed by immunoblotting. Immunofluorescence showed that the most radioresistant progenies had an aberrant morphology, disturbance of the cell-cell adhesion contacts, disorganization of the actin cytoskeleton, and vimentin filaments. Only the progeny derived from intermediary radioresistant cells, HT-29, reduced the E-cadherin expression and overexpressed ß-catenin and vimentin with increased cell migration, invasion, and metalloprotease activation as seen by immunoblotting, wound healing, invasion, and metalloprotease activity assay. We also observed that this most aggressive progeny increased the Wnt/ß-catenin-dependent TCF/LEF activity and underwent an upregulation of mesenchymal markers and downregulation of E-cadherin, as determined by qRT-PCR. Our results showed that the intermediate radioresistant cells can generate more aggressive cellular progeny with the EMT-like phenotype. The Wnt/ß-catenin pathway may constitute an important target for new adjuvant treatment schedules with radiotherapy, with the goal of reducing the migratory and invasive potential of the remaining cells after treatment.

Rho GTPase signaling in the development of colorectal cancer.

The involvement of Rho GTPases in major aspects of cancer development, such as cell proliferation, apoptosis, cell polarity, adhesion, migration, and invasion, have recently been attracting increasing attention. In this review, we have summarized the current findings in the literature, and we discuss the participation of the Rho GTPase members RhoA, Rac1, and Cdc42 in the development of colorectal cancer, the second most lethal neoplasia worldwide. First, we present an overview of the mechanisms of Rho GTPase regulation and the impact that regulator proteins exert on GTPase signaling. Second, we focus on the participation of Rho GTPases as modulators of colorectal cancer development. Third, we emphasize the involvement of activation and expression alterations of Rho GTPases in events associated with cancer progression, such as loss of cell-cell adhesion, proliferation, migration, and invasion. Finally, we highlight the potential use of novel anticancer drugs targeting specific components of the Rho GTPase signaling pathway with antineoplastic activity in this cancer type.

Gold nanoparticles regulate tight junctions and improve cetuximab effect in colon cancer cells.

Aim: Colon cancer (CC) is the second cause of cancer death worldwide. The use of nanoparticles for drug delivery has been increasing in cancer clinical trials over recent years. Materials & methods: We evaluated cytotoxicity of citrate-capped gold nanoparticles (GNPs) and the role they play on cell-cell adhesion. We also used GNP for delivery of cetuximab into different CC cell lines. Results: CC cells with well-formed tight junctions impair GNP uptake. Noncytotoxic concentration of GNP increases paracellular permeability in Caco-2 cells in a reversible way, concomitantly to tight junctions proteins CLDN1 and ZO-1 redistribution. GNP functionalized with cetuximab increases death of invasive HCT-116 CC cells. Conclusion: GNP can be used for drug delivery and can improve efficiency of CC therapy.

A cross-link between protein kinase A and Rho-family GTPases signaling mediates cell-cell adhesion and actin cytoskeleton organization in epithelial cancer cells.

Disassembly of the apical junctional complex (AJC) together with actin cytoskeleton alterations are among the initial events for the development of epithelial cancer. The cell signaling pathways for these processes have been analyzed separately. However, the existence of a link between these two events has not been defined. In this study, using the extracellular calcium depletion model, we analyzed the signaling pathways regulating AJC disassembly together with actin cytoskeleton organization in colon adenocarcinoma cells (Caco-2). Changes in the location of AJC proteins were examined by immunofluorescence and immunoblotting, and tight junction (TJ) functionality was observed by measuring the transepithelial electrical resistance and permeation to ruthenium red. The actin cytoskeleton was stained with rhodamine-phalloidin and analyzed by confocal microscopy. Rho-GTPase activation was assessed by its translocation to the membrane (a hallmark of RhoA activation) and immunoblotting. Pharmacological inhibition of protein kinase A (PKA) with H-89 [N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)] prevented AJC disassembly and actin disorganization at the apical and medial regions caused by calcium depletion. Rho inhibition using toxin A induced AJC disassembly and actin cytoskeleton reorganization. Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-ciclohexanecarboxamide], a Rho-associated kinase inhibitor, reversed redistribution of E-cadherin, but not of TJ proteins and actin disorganization caused by calcium depletion. Calcium depletion and forskolin treatment caused activation of Rho, as evidenced by their translocation to the membrane, an event concurrent to Rac and RhoGDI translocation, and this effect was also reverted by H-89. Thus, our findings demonstrate a central role of a regulatory cascade that integrates PKA and Rho-family GTPases in the AJC disassembly and actin organization in tumor epithelial cells.

LPA Induces Colon Cancer Cell Proliferation through a Cooperation between the ROCK and STAT-3 Pathways.

Lysophosphatidic acid (LPA) plays a critical role in the proliferation and migration of colon cancer cells; however, the downstream signaling events underlying these processes remain poorly characterized. The aim of this study was to investigate the signaling pathways triggered by LPA to regulate the mechanisms involved in the progression of colorectal cancer (CRC). We have used three cell line models of CRC, and initially analyzed the expression profile of LPA receptors (LPAR). Then, we treated the cells with LPA and events related to their tumorigenic potential, such as migration, invasion, anchorage-independent growth, proliferation as well as apoptosis and cell cycle were evaluated. We used the Chip array technique to analyze the global gene expression profiling that occurs after LPA treatment, and we identified cell signaling pathways related to the cell cycle. The inhibition of these pathways verified the conclusions of the transcriptomic analysis. We found that the cell lines expressed LPAR1, -2 and -3 in a differential manner and that 10 µM LPA did not affect cell migration, invasion and anchorage-independent growth, but it did induce proliferation and cell cycle progression in HCT-116 cells. Although LPA in this concentration did not induce transcriptional activity of ß-catenin, it promoted the activation of Rho and STAT-3. Moreover, ROCK and STAT-3 inhibitors prevented LPA-induced proliferation, but ROCK inhibition did not prevent STAT-3 activation. Finally, we observed that LPA regulates the expression of genes related to the cell cycle and that the combined inhibition of ROCK and STAT-3 prevented cell cycle progression and increased the LPA-induced expression of cyclins E1, A2 and B1 to a greater degree than either inhibitor alone. Overall, these results demonstrate that LPA increases the proliferative potential of colon adenocarcinoma HCT-116 cells through a mechanism involving cooperation between the Rho-ROCK and STAT3 pathways involved in cell cycle control.

Blockade of irradiation-induced autophagosome formation impairs proliferation but does not enhance cell death in HCT-116 human colorectal carcinoma cells.

This work was undertaken to gain further information on the molecular mechanisms underlying autophagosome formation and its relation with tumor cell survival in response to radiation in colon cancer. A human colon cancer cell line, HCT-116, was examined with respect to cell survival after blockade of irradiation-induced autophagosome formation by pharmacological interference. Autophagosome formation was confirmed using a kinetic study with incorporated bovine serum albumin gold-conjugate (BSA-Au) analyzed by electron microscopy and an autophagosome-associated LC3B antibody measured by immunofluorescence and Western blotting. Annexin V/PI double staining was used to monitor cell death by apoptosis, and cell cycle profiles by flow cytometry. Ionizing radiation (IR) promoted autophagosome formation in the HCT-116 IR-surviving cells. Pharmacological interference showed that PI3K/Akt and Src were involved in early stages of autophagosome formation. IR alone decreased cell proliferation by arresting cells in the G2/M phase, and pharmacological interference of autophagosome formation decreased proliferation, but did not affect cell survival. Also, our data suggest that decreased proliferation caused by PI3K and Src inhibitors could be through S phase cell cycle delay. Our results clearly indicate that blockade of IR-induced autophagosome formation impairs proliferation but does not enhance cell death in colon cancer cells.

Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells.

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.

Participação da GTPase Rho na dinâmica do complexo juncional apical e do citoesqueleto de actina na progressão do câncer colorretal

A desmontagem do complexo juncional apical (CJA), que compreende as junções aderentes (JAs) e junções tight (JTs), e a reorganização do citoesqueleto de actina são etapas iniciais da progressão do câncer colorretal (CCR). Subsequentemente, as células adquirem um fenótipo migratório e invasivo, no entanto, os mecanismos moleculares que modulam esses eventos iniciais permanecem desconhecidos. O objetivo do presente estudo foi identificar as vias de sinalização envolvidas na regulação da dinâmica do CJA e do citoesqueleto de actina durante a progressão do CCR. Células de adenocarcinoma de cólon humano Caco-2, utilizadas como modelo de CCR, foram submetidas ao ensaio de retirada do cálcio do meio extracelular e ao tratamento com ácido lisofosfatídico (LPA), um ativador da GTPase Rho. Pré-tratamentos com inibidores específicos de vias de sinalização foram também utilizados nos diferentes ensaios. Na primeira etapa do estudo, verificamos que a retirada do cálcio extracelular ocasionou redistribuição das proteínas do CJA, conforme mostrado por imunofluorescência e immunoblotting usando frações solúveis e insolúveis em Triton X-100; perda da funcionalidade das JTs, como visto pelo decréscimo na resistência elétrica transepitelial (RET) e microscopia eletrônica de transmissão utilizando o traçador vermelho de rutênio; alterações no citoesqueleto de actina na região perijuncional e na distribuição das fibras de estresse, observados por microscopia confocal; e ativação localizada das GTPases Rho e Rac. Todos esses efeitos foram mediados por uma via de sinalização envolvendo a ativação de PKA e Rho-ROCK. Na segunda parte do estudo, observamos que o tratamento com LPA ocasionou redistribuição das proteínas do CJA e redução da expressão de E-caderina bem como reorganização do citoesqueleto de actina perijuncional, e aumento na formação de fibras de estresse. Além disso, o LPA causou: aumento da migração, mas não da invasão celular, como mostrado pelas técnicas do wound healing e de invasão em matrigel, respectivamente; formação de adesões focais, analisada pelo aumento da fosforilação da quinase de adesão focal (FAK) e imunofluorescência; ativação da GTPase Rho, mas não de Rac, conforme o ensaio de pull-down; e aumento do crescimento independente de ancoragem. Esses resultados mostram que o LPA modula as JAs, o citoesqueleto de actina e a migração celular através de uma via de sinalização que integra Rho-ROCK e Src-FAK. Em conclusão, juntos esses resultados mostram um papel central da via de sinalização Rho-ROCK na modulação do CJA e do citoesqueleto de actina durante a progressão do CCR, via que pode constituir um novo e alternativo alvo terapêutico no tratamento deste tipo de câncer

A desmontagem do complexo juncional apical (CJA), que compreende as junções aderentes (JAs) e junções tight (JTs), e a reorganização do citoesqueleto de actina são etapas iniciais da progressão do câncer colorretal (CCR). Subsequentemente, as células adquirem um fenótipo migratório e invasivo, no entanto, os mecanismos moleculares que modulam esses eventos iniciais permanecem desconhecidos. O objetivo do presente estudo foi identificar as vias de sinalização envolvidas na regulação da dinâmica do CJA e do citoesqueleto de actina durante a progressão do CCR. Células de adenocarcinoma de cólon humano Caco-2, utilizadas como modelo de CCR, foram submetidas ao ensaio de retirada do cálcio do meio extracelular e ao tratamento com ácido lisofosfatídico (LPA), um ativador da GTPase Rho. Pré-tratamentos com inibidores específicos de vias de sinalização foram também utilizados nos diferentes ensaios. Na primeira etapa do estudo, verificamos que a retirada do cálcio extracelular ocasionou redistribuição das proteínas do CJA, conforme mostrado por imunofluorescência e immunoblotting usando frações solúveis e insolúveis em Triton X-100; perda da funcionalidade das JTs, como visto pelo decréscimo na resistência elétrica transepitelial (RET) e microscopia eletrônica de transmissão utilizando o traçador vermelho de rutênio; alterações no citoesqueleto de actina na região perijuncional e na distribuição das fibras de estresse, observados por microscopia confocal; e ativação localizada das GTPases Rho e Rac. Todos esses efeitos foram mediados por uma via de sinalização envolvendo a ativação de PKA e Rho-ROCK. Na segunda parte do estudo, observamos que o tratamento com LPA ocasionou redistribuição das proteínas do CJA e redução da expressão de E-caderina bem como reorganização do citoesqueleto de actina perijuncional, e aumento na formação de fibras de estresse. Além disso, o LPA causou: aumento da migração, mas não da invasão celular, como mostrado pelas técnicas do wound healing e de invasão em matrigel, respectivamente; formação de adesões focais, analisada pelo aumento da fosforilação da quinase de adesão focal (FAK) e imunofluorescência; ativação da GTPase Rho, mas não de Rac, conforme o ensaio de pull-down; e aumento do crescimento independente de ancoragem. Esses resultados mostram que o LPA modula as JAs, o citoesqueleto de actina e a migração celular através de uma via de sinalização que integra Rho-ROCK e Src-FAK. Em conclusão, juntos esses resultados mostram um papel central da via de sinalização Rho-ROCK na modulação do CJA e do citoesqueleto de actina durante a progressão do CCR, via que pode constituir um novo e alternativo alvo terapêutico no tratamento deste tipo de câncer

Regulação da adesão célula-célula e do citoesqueleto de acina em células de adenocarcinoma de cólon humano, CACO-2 / Regulation of the adhesion cell-cell and cytoskeleton of actin in cells of human adenocarcinoma of colon, CACO-2

Células epiteliais adquirem motilidade durante a transição epitelial-mesenquimal, evento importante da tumorigênese. Esta transição é caracterizada por perda da polaridade celular, desorganização da adesão célula-célula, e reorganização do citoesqueleto de acina, porém as vias de sinalização envolvidas nesse processo são pouco conhecidas. Alguns estudos..O objetivo desse trabalho foi analisar... Nesse estudo, verificamos que a retirada do cálcio extracelular ocasionou: a) a redistribuição da proteína de junções aderentes E-caderina, e das proteínas ocludina, claudina-1 e ZO-1, das junções oclusivas, conforme mostrado por imunofluorescência e fracionamento subcelular em Triton X-100 seguido por imunotransferência; b) alteração na funcionalidade das junções oclusivas, de acordo com o decréscimo na resistência elétrica transepitelial; c) alterações morfológicas com a abertura de espaços nas regiões de contatos célula-célula, visualizados por microscopia eletrônica de transmissão; d) alterações no citoesqueleto de actina, na região perijuncional e na distribuição das fibras de estresse, observados por microscopia confocal; e e) redistribuição de proteína Rho-GDI, analisada por imunotransferência. Além disso, foi mostrado que as modificações no complexo juncional e na região apical do citoesqueleto de actina, ocasionadas pela retirada do cálcio extracelular, foram prevenidas pela inibição da PKA com o inibidor H-89, e a ativação de PKA com Forskolina, ocasionou efeitos similares à retirada do cálcio sobre os microfilamentos. As inibições da GTPase Rho com Toxina A, e de sua efetora ROCK com Y-27532, também ocasionaram desorganização do citoesqueleto de actina. Ainda, a ativação de PKA alterou a distribuição de Rho-GDI, sugerindo que PKA poderia inibir Rho. A inibição de ROCK em meio normal causou aumento na permeabilidade paracelular, abertura de espaçõs no complexo uncional, e redistribuição de claudina-1, mas surpreendentemente preveniu a translocação de E-cader.