Transforming growth factor‐β (TGF‐β) 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‐β, 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 E 2 (PGE 2 ), TGF‐β or a combined treatment with these agents. We demonstrated that PGE 2 alters the subcellular localization of E‐cadherin and β‐catenin and enhanced the tumorigenic potential in HT‐29 cells. This effect was inhibited by TGF‐β, indicating a tumor suppressor role. Conversely, in Ras ‐transformed IEC‐6 cells, TGF‐β induced COX‐2 expression and increased invasiveness, acting as a tumor promoter. In IEC‐6 Ras‐transformed cells, TGF‐β increased nuclear β‐catenin and Wnt/β‐catenin activation, opposite to what was seen in the PGE 2 and TGF‐β joint treatment in HT‐29 cells. Together, our findings show that TGF‐β increases COX‐2 levels and induces invasiveness cooperating with Ras in a Wnt/β‐catenin activation‐dependent manner. This shows TGF‐β dual regulation over COX‐2/PGE 2 tumor promotion depending on the H‐ Ras and Wnt/β‐catenin pathways activation status in intestinal 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 β‐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.

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.

Introdução: Avanços na pesquisa científica baseiam-se nas descobertas previamente publicadas. Entretanto, há preocupação com a falta de reprodutibilidade nas pesquisas biológicas das áreas básica e pré-clínica, em função da repercussão na saúde da população. Como células cultivadas in vitro constituem a base para muitos estudos toxicológicos e terapêuticos, a preocupação com a qualidade destas torna-se primordial. Com relação aos contaminantes microbiológicos, embora bactérias e fungos sejam facilmente reconhecidos, vírus e micoplasmas são invisíveis na microscopia óptica. Outro problema delicado seriam os resultados gerados com células com identidade modificada. Objetivo: Discutir as principais metodologias para a garantia da qualidade de células utilizadas em ensaios in vitro e demonstrar como algumas coleções mundiais estão estruturadas para tratar esta questão. Método: Levantamento da literatura científica nas bases de dados PubMed e Scielo e na página da web de diferentes coleções biológicas até dezembro de 2017. Resultados: Recomenda-se a aplicação das seguintes técnicas para detecção de contaminantes em cultivos celulares: 1) vírus: o PCR e o isolamento viral; 2) micoplasmas: o PCR, a bioluminescência e a coloração das células com fluoróforo com afinidade ao DNA; 3) identidade de células humanas: o STR; 4) identidade de células não humanas: o Barcode. Conclusões: Considerando todo o investimento aplicado em pesquisa científica em âmbito mundial, o desenvolvimento de novas metodologias alternativas ao uso de animais e o consenso crítico do conceito de qualidade, conclui-se que qualquer laboratório deve garantir o controle de pureza e autenticidade de suas linhagens.

Introduction: Advances in scientific research are based on previously published findings. However, there is concern about the lack of reproducibility in the biological researches in basic and preclinical areas, due to the repercussion on the population ' s health. Because in vitro cultured cells are the basis for many toxicological and therapeutic studies, concern about their quality becomes paramount. Regarding microbiological contaminants, although bacteria and fungi are easily recognized, viruses and mycoplasmas are invisible under light microscopy. Another delicate issue would be the results generated with cells with modified identity. Objective: To discuss the main methodologies for assuring the quality of cells used in in vitro assays and to demonstrate how some world collections are structured to address this issue. Method: The scientific literature in the PubMed and Scielo databases and the webpage of different biological collections until December 2017. Results: It is recommended to apply the following techniques to detect contaminants in cell cultures: 1) virus : PCR and viral isolation; 2) mycoplasmas: PCR, bioluminescence and staining of cells with DNA affinity fluorophore; 3) human cell identity: the STR; 4) non-human cell identity: the Barcode. Conclusions: Considering all the investment applied in scientific research worldwide, the development of new methodologies alternatives to the use of animals and the critical consensus of the concept of quality, it is concluded that any laboratory should guarantee the control of purity and authenticity of its lineages.

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.

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 β-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.

Journal of Cellular BiochemistryVolume 115, Issue 12 p. fm i-fm iii ContentsFree Access Table of Contents: Volume 115, Number 12 First published: 15 October 2014 https://doi.org/10.1002/jcb.24980AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume115, Issue12December 2014Pages fm i-fm iii RelatedInformation

ABSTRACTRadiotherapy 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 β‐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/β‐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/β‐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. J. Cell. Biochem. 115: 2175–2187, 2014. © 2014 Wiley Periodicals, Inc.

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 G(2)/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.

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. J. Cell. Biochem. 113: 2549–2559, 2012. © 2012 Wiley Periodicals, Inc.

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.

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH)2D3 are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53. However, the exact mechanism by which 24R,25(OH)2D3 and LPA exert their effects is not fully understood. It was found that both 24R,25(OH)2D3 and LPA attenuate Pi-induced caspase-3 activity. The actions of 24R,25(OH)2D3 and LPA were dependent upon Gαi, LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI3K) signaling, and nuclear export. 24R,25(OH)2D3 decreased both p53 abundance and p53-medaited transcription and inhibited Pi-induced cytochrome c translocation. Moreover, LPA induced increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these data show that 24R,25(OH)2D3 inhibits Pi-induced apoptosis through Ca2+, PLD, and PLC signaling and through LPA-LPA1/3-Gαi-PI3K-mdm2-mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity.

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.

We examined the participation of MAPK and PKA in the Golgi complex disassembly caused by light-activated Calphostin C in HT-29 cells. When these cells were incubated with Calphostin C, fragmentation and dispersal of the Golgi complex was observed as assessed by immunofluorescence microscopy. Electron microscopy analysis showed that clusters of vesicles and large tubule-vesicular membrane structures, resembling the Golgi remnants present in mitotic cells, substituted the Golgi stacks. In addition, Calphostin C treatment caused inhibition of the endocytic route. We confirmed that the Golgi disassembly was not due to PKC inhibition, and suggested, based on the use of specific inhibitors, that other kinases are involved. It was shown that pretreatment with PD98059 and H-89, both inhibitors of MAPK and PKA, respectively, prior to incubation with Calphostin C, caused blockade of the Golgi disassembly, as well as the inhibition of the endocytic pathway caused by this drug. This finding supports the existence of a novel mechanism by which MAPK and PKA may regulate the Golgi breakdown caused by Calphostin C in HT-29 cells.