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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| 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 |
| Sumario: | 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. |
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