RESUMO
The effects of radiation are known to be potentiated by N-3 polyunsaturated fatty acids, which modulate several signaling pathways, but the molecular mechanisms through which these fatty acids enhance the anticancer effects of irradiation in colorectal cancer (CRC) treatment remain poorly elucidated. Here, we aimed to ascertain whether the fatty acid docosahexaenoic acid (DHA) exerts a modulating effect on the response elicited by radiation treatment (RT). Two CRC cell lines, Caco-2 and HT-29, were exposed to RT, DHA, or both (DHA + RT) for various times, and then cell viability, proliferation, and clonogenicity were assessed. Moreover, cell cycle, apoptosis, and necrosis were analyzed using flow cytometry, and the involvement of WNT/ß-catenin signaling was investigated by immunofluorescence to determine nuclear ß-catenin, GSK3ß phosphorylation status, and TCF/LEF-activity reporter. DHA and RT applied separately diminished the viability of both HT-29 and Caco-2 cells, and DHA + RT caused a further reduction in proliferation mainly in HT-29 cells, particularly in terms of colony formation. Concomitantly, our results verified cell cycle arrest in G0/G1 phase, a reduction of cyclin D1 expression, and a decrease in GSK3ß phosphorylation after the combined treatment. Furthermore, immunofluorescence quantification revealed that nuclear ß-catenin was increased in RT-exposed cells, but this effect was abrogated in cells exposed to DHA + RT, and the results of TCF/LEF-activity assays confirmed that DHA attenuated the increase in nuclear ß-catenin activity induced by irradiation. Our finding shows that DHA applied in combination with RT enhanced the antitumor effects of irradiation on CRC cells, and that the underlying mechanism involved the WNT/ß-catenin pathway. © 2018 BioFactors, 45(1):24-34, 2019.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Raios gama , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , beta Catenina/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células CACO-2 , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Ciclina D1/genética , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HT29 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Radiotherapy is widely used for advanced rectal tumors. However, tumor recurrence after this treatment tends to be more aggressive and is associated with a poor prognosis. Uncovering the molecular mechanism that controls this recurrence is essential for developing new therapeutic applications. In the present study, we demonstrated that radiation increases the EphA4 activation level of the survivor progeny of colorectal cancer cells submitted to this treatment and that such activation promoted the internalization of a complex E-cadherin-EphA4, inducing cell-cell adhesion disruption. Moreover, EphA4 knockdown in the progeny of irradiated cells reduced the migratory and invasive potentials and metalloprotease activity induced by irradiation. Finally, we demonstrated that the cell migration and invasion potential were regulated by AKT and ERK1/2 signaling, with the ERK1/2 activity being dependent on EphA4. In summary, our study demonstrates that these signaling pathways could be responsible for the therapeutic failure, thereby promoting local invasion and metastasis in rectal cancer after radiotherapy. We also postulate that EphA4 is a potential therapeutic target for colorectal cancer treatment.
Assuntos
Neoplasias Colorretais/radioterapia , Receptor EphA4/fisiologia , Transdução de Sinais/fisiologia , Antígenos CD , Caderinas/análise , Neoplasias Colorretais/patologia , Doxazossina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Células HT29 , Humanos , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/fisiologiaRESUMO
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.
Assuntos
Movimento Celular/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos da radiação , Via de Sinalização Wnt , Citoesqueleto de Actina/metabolismo , Antígenos CD , Apoptose , Células CACO-2 , Caderinas/metabolismo , Caspases/metabolismo , Forma Celular , Neoplasias Colorretais , Células HT29 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Invasividade Neoplásica , Tolerância a Radiação , Survivina , Vimentina/metabolismo , beta Catenina/metabolismoRESUMO
A radioterapia constitui uma das principais terapias adjuvantes para o tratamento de câncer colorretal em pacientes com estadiamento avançado. Porém, o esquema de fracionamento dedoses pode permitir a repopulação de células tumorais sobreviventes ao tratamento e o reaparecimento do tumor, a progressão tumoral e a ocorrência de metástases tardias. Nesteestudo, foram analisados os efeitos transgeneracionais induzidos pela radioterapia na progênie derivadas de células de câncer colorretal sobreviventes ao tratamento e avaliado o quanto a radiação poderia induzir um fenótipo mais agressivo nessas progênies. Os resultados mostraram que essas células apresentam uma radiorresistência diferencial entre si, e que as células mais radiorresistentes formam progênies com aumento da expressão da proteína survivina, aquisição de uma morfologia aberrante, desorganização das junções celulares e do citoesqueleto de actina. Também foi observado que a progênie derivada de células HT-29 sobreviventes à radiação apresentam um aumento do potencial migratório invasivo, bem como um aumento da atividade de TCF-LEF, seguido do aumento da expressão demarcadores mesenquimais e redução de E-caderina...
Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer. However, radiotherapy fractionation schedules frequently allow for the repopulation of surviving tumors cells and tumor recurrence, neoplastic progression andsubsequent metastasis. In this study, we analyzed the transgenerational effects induced by radiation on the progeny derived from irradiated parental cells of colorectal cancer andevaluated whether the radiation could increase the malignant features of radiotherapysurvivors cells. Our results showed that these cells displayed a differential radioresistance andthat the most radioresistant progeny had a direct correlation with increased survivin expression, acquisition of an aberrant morphology, disturbance of the cell-cell adhesion contacts and actin cytoskeleton disorganization. We also observed that the progeny that werederived from irradiated HT-29 cells displayed an increased migratory and invasiveness potential as well as increased Wnt/β-catenin-dependent TCF/LEF activity followed by anupregulation of mesenchymal markers and downregulation of E-cadherin...
Assuntos
Humanos , Masculino , Feminino , Neoplasias Colorretais/genética , Neoplasias Colorretais/radioterapia , GlicosilaçãoRESUMO
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.
Assuntos
Autofagia/efeitos da radiação , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Animais , Apoptose/efeitos da radiação , Bovinos , Processos de Crescimento Celular/efeitos da radiação , Linhagem Celular Tumoral , Células HCT116 , HumanosRESUMO
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.