Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines

Harmful algal blooms (HABs) are natural events that occurs in seas worldwide, HABs are a result of a high density of microalgal in oceans. Many species of phytoplankton, under certain circumstances, are capable of produce phycotoxins, which are accumulated in shellfishes, bivalve mollusk and gastrop...

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Autor: Jiménez-Carcamo, Danae Francisca
Tipo de recurso: tesis de maestría
Estado:Versión publicada
Fecha de publicación:2019
País:Chile
OAI Identifier:oai:repositorio.anid.cl:10533/236318
Acceso en línea:https://hdl.handle.net/10533/236318
Access Level:acceso abierto
Palabra clave:Medicina y Ciencias de la Salud
Medicina Básica
Otros Temas de Medicina Básica
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dc.title.es_CL.fl_str_mv Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
Efecto protumoral del Ácido Okadaico y sus análogos DTX-1 y DTX-2 en líneas celulares de epitelio de colon
title Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
spellingShingle Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
Jiménez-Carcamo, Danae Francisca
Medicina y Ciencias de la Salud
Medicina Básica
Otros Temas de Medicina Básica
title_short Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
title_full Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
title_fullStr Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
title_full_unstemmed Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
title_sort Protumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell lines
dc.creator.none.fl_str_mv Jiménez-Carcamo, Danae Francisca
author Jiménez-Carcamo, Danae Francisca
author_facet Jiménez-Carcamo, Danae Francisca
author_role author
dc.contributor.advisor.none.fl_str_mv Contreras-Muñoz, Hector Ruberly
Garcia-Mansilla, Carlos
dc.contributor.institution.es_CL.fl_str_mv UNIVERSIDAD DE CHILE
dc.subject.oecd1n.es_CL.fl_str_mv Medicina y Ciencias de la Salud
topic Medicina y Ciencias de la Salud
Medicina Básica
Otros Temas de Medicina Básica
dc.subject.oecd2n.es_CL.fl_str_mv Medicina Básica
dc.subject.oecd3n.es_CL.fl_str_mv Otros Temas de Medicina Básica
description Harmful algal blooms (HABs) are natural events that occurs in seas worldwide, HABs are a result of a high density of microalgal in oceans. Many species of phytoplankton, under certain circumstances, are capable of produce phycotoxins, which are accumulated in shellfishes, bivalve mollusk and gastropods. These toxins may cause syndromes that impacts public health, ecology and generate severe economic losses. Diarrheic Shellfish Poisoning (DSP) is a gastrointestinal syndrome caused by ingestion of contaminated shellfish, symptoms include: diarrhea, nausea, vomits and abdominal pain. These groups of toxins are characterized by a fat-soluble character and thermostability. Okadaic acid (OA) is the main compound of DSP. Other toxins of this group are acyl derivates of OA: dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2) and dinophysistoxin-3 (DTX-3). Toxins of OA group are potent Ser/Thr protein phosphatases inhibitors, mainly protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), the direct consequences of this inhibition produces hyperphosphorylation of cellular proteins and deregulation of many cellular processes. Many cellular effects of this toxins on different cellular systems are reported in literature, these changes are at cellular, molecular and genetic level. Cytotoxic, genotoxic and carcinogenic effects are reported for some groups due to AO and DTXs toxins. Although toxins of OA group are not classified as neurotoxins, some studies report neurotoxic effects induced by OA including neuronal apoptosis and cytoskeleton reorganization. The hypothesis of this project was: “OA and its analogues DTX-1 y DTX-2 enhance the proliferative and invasive capacity of normal and tumoral colon epithelial cell lines”. The general objective was to determine phenotypical changes on colon epithelial cell lines (normal and tumoral) induced by OA, DTX-1 and DTX-2. To do it we choose CCD 841 CoN cell line as a non-tumoral model, Sw480 cell line as a primary tumor model and Sw620 cell line as a metastatic tumor model. Cell were exposed to commercial toxins OA, DTX-1 and DTX-2. Viability was evaluated by MTT assay, also PP2A activity was quantified, PCNA expression was quantified by western blot, caspase-8 and activated caspase-3/7 were evaluated as cellular death marks, apoptosis by Annexin V and PI was evaluated by flow cytometry, LDH release as a necrosis marker, migration and invasion were evaluated by transwell chambers, finally, expression of FAK and ILK proteins were evaluated by western blot because of their role in migration process. MTT assay results show that DTX-2 was less toxic than OA and DTX-1 comparing IC50 values. This coincides with literature reports because OA and DTX-1 are equally toxic and DTX-2 is 40% less toxic. Furthermore, comparing non tumoral cell line with tumoral cell lines, non-tumoral cells were more sensitive to all toxin effects. IC50 values were smaller than Sw480 and Sw620 IC50 values (IC50 OA Con841: 54,4 nM; IC50 DTX-1 Con841: 43,5 nM; IC50 DTX-2 Con841: 81,2 nM; IC50 OA Sw480: 89,3 nM; IC50 DTX-1 Sw480: 113,8 nM; IC50 DTX-2 Sw480: 187,2 nM; IC50 OA Sw620: 137,2 nM; IC50 DTX-1 Sw620: 192,9 nM; IC50 DTX-2 Sw620: 202,9 nM).. We could see morphological changes after all toxin incubations. Cells took round shaped, tend to group and detach from the plate, these effects are more notorious in non-tumoral cells. Considering functional assays results, cellular death and expression of protein associated to cellular adhesion and motility, we can see a dual effect of this toxins, in non-tumoral cell line there is cellular death induced by the toxins, probably anoikis, while, in tumoral cell lines we see a more aggressive phenotype induced by toxins, characterized by greater resistance to toxins, migration increased and more activated FAK expression levels, which is related to focal adhesions replacement and migration processes.
publishDate 2019
dc.date.accessioned.none.fl_str_mv 2019-07-17T13:34:53Z
2022-08-17T18:20:06Z
dc.date.available.none.fl_str_mv 2019-07-17T13:34:53Z
2022-08-17T18:20:06Z
dc.date.issued.es_CL.fl_str_mv 2019
dc.type.none.fl_str_mv Tesis Magíster
dc.type.driver.es_CL.fl_str_mv info:eu-repo/semantics/masterThesis
dc.type.openaire.none.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.tesis.none.fl_str_mv Tesis
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dc.identifier.folio.es_CL.fl_str_mv 22160169
dc.identifier.uri.none.fl_str_mv https://hdl.handle.net/10533/236318
identifier_str_mv 22160169
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spelling UNIVERSIDAD DE CHILEJiménez-Carcamo, Danae Francisca2019https://hdl.handle.net/10533/236318http://purl.org/coar/access_right/c_abf2Otros Temas de Medicina BásicaMedicina BásicaMedicina y Ciencias de la SaludProtumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell linesContreras-Muñoz, Hector RuberlyGarcia-Mansilla, CarlosUNIVERSIDAD DE CHILEChileJiménez-Carcamo, Danae Francisca2019-07-17T13:34:53Z2022-08-17T18:20:06Z2019-07-17T13:34:53Z2022-08-17T18:20:06Z2019Harmful algal blooms (HABs) are natural events that occurs in seas worldwide, HABs are a result of a high density of microalgal in oceans. Many species of phytoplankton, under certain circumstances, are capable of produce phycotoxins, which are accumulated in shellfishes, bivalve mollusk and gastropods. These toxins may cause syndromes that impacts public health, ecology and generate severe economic losses. Diarrheic Shellfish Poisoning (DSP) is a gastrointestinal syndrome caused by ingestion of contaminated shellfish, symptoms include: diarrhea, nausea, vomits and abdominal pain. These groups of toxins are characterized by a fat-soluble character and thermostability. Okadaic acid (OA) is the main compound of DSP. Other toxins of this group are acyl derivates of OA: dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2) and dinophysistoxin-3 (DTX-3). Toxins of OA group are potent Ser/Thr protein phosphatases inhibitors, mainly protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), the direct consequences of this inhibition produces hyperphosphorylation of cellular proteins and deregulation of many cellular processes. Many cellular effects of this toxins on different cellular systems are reported in literature, these changes are at cellular, molecular and genetic level. Cytotoxic, genotoxic and carcinogenic effects are reported for some groups due to AO and DTXs toxins. Although toxins of OA group are not classified as neurotoxins, some studies report neurotoxic effects induced by OA including neuronal apoptosis and cytoskeleton reorganization. The hypothesis of this project was: “OA and its analogues DTX-1 y DTX-2 enhance the proliferative and invasive capacity of normal and tumoral colon epithelial cell lines”. The general objective was to determine phenotypical changes on colon epithelial cell lines (normal and tumoral) induced by OA, DTX-1 and DTX-2. To do it we choose CCD 841 CoN cell line as a non-tumoral model, Sw480 cell line as a primary tumor model and Sw620 cell line as a metastatic tumor model. Cell were exposed to commercial toxins OA, DTX-1 and DTX-2. Viability was evaluated by MTT assay, also PP2A activity was quantified, PCNA expression was quantified by western blot, caspase-8 and activated caspase-3/7 were evaluated as cellular death marks, apoptosis by Annexin V and PI was evaluated by flow cytometry, LDH release as a necrosis marker, migration and invasion were evaluated by transwell chambers, finally, expression of FAK and ILK proteins were evaluated by western blot because of their role in migration process. MTT assay results show that DTX-2 was less toxic than OA and DTX-1 comparing IC50 values. This coincides with literature reports because OA and DTX-1 are equally toxic and DTX-2 is 40% less toxic. Furthermore, comparing non tumoral cell line with tumoral cell lines, non-tumoral cells were more sensitive to all toxin effects. IC50 values were smaller than Sw480 and Sw620 IC50 values (IC50 OA Con841: 54,4 nM; IC50 DTX-1 Con841: 43,5 nM; IC50 DTX-2 Con841: 81,2 nM; IC50 OA Sw480: 89,3 nM; IC50 DTX-1 Sw480: 113,8 nM; IC50 DTX-2 Sw480: 187,2 nM; IC50 OA Sw620: 137,2 nM; IC50 DTX-1 Sw620: 192,9 nM; IC50 DTX-2 Sw620: 202,9 nM).. We could see morphological changes after all toxin incubations. Cells took round shaped, tend to group and detach from the plate, these effects are more notorious in non-tumoral cells. Considering functional assays results, cellular death and expression of protein associated to cellular adhesion and motility, we can see a dual effect of this toxins, in non-tumoral cell line there is cellular death induced by the toxins, probably anoikis, while, in tumoral cell lines we see a more aggressive phenotype induced by toxins, characterized by greater resistance to toxins, migration increased and more activated FAK expression levels, which is related to focal adhesions replacement and migration processes.Los florecimientos algales nocivos (FANs) o “Harmful algal blooms” son condiciones generadas por un aumento de la densidad de microalgas en los océanos. Distintas especies de fitoplancton, bajo ciertas circunstancias, son capaces de producir toxinas, que son acumuladas por los mariscos y gastrópodos, pudiendo ocasionar diversos cuadros tóxicos en quienes los consumen, así como ocasionar problemas ecológicos y económicos. El veneno diarreico de mariscos (VDM) es el síndrome provocado por la ingesta de mariscos contaminados que genera síntomas que incluyen: diarrea, náusea, vómito y dolor abdominal. Este grupo de toxinas que se caracterizan por ser de carácter lipofílico y estables a variaciones de temperatura. Ácido okadaico (AO) es el principal compuesto del VDM. Otras toxinas dentro de este grupo son compuestos derivados del AO: dinofisistoxina-1 (DTX-1), dinofisistoxina-2 (DTX-2) y dinofisistoxina-3 (DTX-3), entre otros. Las toxinas del grupo del AO son potentes inhibidores de las Ser/Thr fosfatasas, principalmente fosfatasas del tipo 1 (PP1) y la fosfatasa del tipo 2A (PP2A), lo que produce una hiperfosforilación en las proteínas intracelulares y una desregulación de diferentes procesos célulares. Se han reportan distintos efectos celulares de este grupo de toxinas sobre distintos sistemas celulares, los que incluyen cambios a nivel celular, molecular y genético. Aunque las toxinas del grupo del AO no se clasifican como neurotoxinas, hay estudios que reportan efectos neurotóxicos inducidos por AO que incluyen apoptosis neuronal y reorganización del citoesqueleto. Incluso, se ha reportado que el AO y las DTXs tienen efectos citotóxicos, genotóxicos, capacidad promotora de tumores y carcinogénicos. De acuerdo con los antecedentes presentados, la hipótesis de trabajo en este proyecto es: “el AO y sus análogos DTX-1 y DTX-2 potencian la capacidad proliferativa e invasiva de líneas celulares de epitelio de colon normal y tumoral”. El objetivo general es Determinar cambios del fenotipo de células de epitelio de colon (normales y tumorales) inducidos por Ácido Okadaico, DTX-1 y DTX-2. Para esto, se utilizaron las líneas celulares de epitelio colon, un modelo no tumoral (CCD 841 CoN), una línea de tumor primario (Sw480) y otra de tumor metastásico (Sw620), las que serán expuestas a las toxinas comerciales estandarizadas AO, DTX-1 y DTX-2. Se evaluó viabilidad mediante MTT, actividad PP2A, expresión de PCNA como marcador de proliferación, evaluación de muerte celular mediante la expresión de caspasa-8, actividad de caspasa 3/7 activada, ensayo de AnnexinaV y PI mediante citometría de flujo, liberación de LDH, migración e invasión mediante cámaras transwell y expresión de proteínas FAK e ILK. Los resultados obtenidos mediante el ensayo de MTT muestran que DTX-2 resultó menos tóxico que AO y DTX-1 al comparar los valores de IC50. Esto coincide con lo reportado en literatura, se considera que AO y DTX-1 son igualmente tóxicos, en tanto que DTX-2 es alrededor de un 40% menos tóxico. Además, al comparar las células no tumorales con las líneas tumorales, las primeras resultaron ser mucho más sensibles al efecto de las tres toxinas, obteniendo valores de IC50 (IC50 AO: 54,4 nM; IC50 DTX-1: 43,5 nM; IC50 DTX-2: 81,2 nM) mucho menores en comparación a las líneas Sw480 y Sw620 (IC50 AO Sw480: 89,3 nM; IC50 DTX-1 Sw480: 113,8 nM; IC50 DTX-2 Sw480: 187,2 nM; IC50 AO Sw620: 137,2 nM; IC50 DTX-1 Sw620: 192,9 nM; IC50 DTX-2 Sw620: 202,9 nM). Respecto de los cambios morfológicos posteriores a la incubación con las toxinas, indican que las células se vuelven esféricas y tienden a agruparse, sugiriendo un incremento en la adhesión célula-célula y perdiendo adhesión a la placa, el efecto es mucho más evidente en la línea no tumoral. Considerando los resultados de los ensayos funcionales, de muerte celular y expresión de proteínas asociadas a adhesión celular, es posible observar un efecto dual de las toxinas, ya que en la línea no tumoral hay una mayor inducción de muerte celular presumiblemente anoikis, en tanto que en las líneas tumorales hay una inducción de un fenotipo más agresivo caracterizado por una mayor resistencia a las toxinas, aumento de migración y FAK activado, el cual se relaciona con un recambio de adhesiones focales.PFCHA-BecasPFCHA-Becas22160169https://hdl.handle.net/10533/236318instname: Conicytreponame: Repositorio Digital RI2.0info:eu-repo/grantAgreement//22160169info:eu-repo/semantics/dataset/hdl.handle.net/10533/93488info:eu-repo/semantics/openAccessMedicina y Ciencias de la SaludMedicina BásicaOtros Temas de Medicina BásicaProtumoral effect of okadaic acid DTX-1 and DTX-2 on epithelial colon cell linesEfecto protumoral del Ácido Okadaico y sus análogos DTX-1 y DTX-2 en líneas celulares de epitelio de colonTesis Magísterinfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/publishedVersionTesisTesishttps://hdl.handle.net/10533/236318PFCHA-Becasabef0a45-7f7b-4648-9bce-977b13de51ecvirtual::54408-1abef0a45-7f7b-4648-9bce-977b13de51ecvirtual::54408-1ORIGINALTesis Danae Jimenez.pdfTesis magister Danae Jimenez. beca 22160169application/pdf1149255https://repositorio.anid.cl/bitstreams/8d2fd3d8-8f1c-4d19-b39a-f1b93c6ca160/download99ad2db6f6386b7a9aac5756ed9d7e16MD51LICENSElicense.txttext/plain1779https://repositorio.anid.cl/bitstreams/f3f8a4a2-e946-4bb9-b2b0-93e68ed8ad5c/download593a6e7305c66c56041a9f9e15a649c1MD52TEXTTesis Danae Jimenez.pdf.txtExtracted texttext/plain85855https://repositorio.anid.cl/bitstreams/5ea32c99-9703-4da2-b1d2-a7e0eb3cdf6e/downloadd42014fcd6cd123abf8926b79722fb7dMD53THUMBNAILTesis Danae Jimenez.pdf.jpgIM Thumbnailimage/jpeg2559https://repositorio.anid.cl/bitstreams/fad6320d-64bf-4075-99b1-9166685c4f1f/download267ca0dc0650d9b93affc6ac96f21510MD5410533/236318oai:repositorio.anid.cl:10533/2363182023-07-24 12:50:05.042https://repositorio.anid.clRepositorio ANIDaletelier@anid.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