Evaluación del potencial de sensores fluorescentes análogos a esfingomielina para identificar interacciones quimioterapéuticas basadas en mecanismos autofágicos
Fecha
2024-08
Tipo
tesis de maestría
Autores
Mesén Porras, Susana
Título de la revista
ISSN de la revista
Título del volumen
Editor
Resumen
La heterogeneidad tumoral conduce a la resistencia a la quimioterapia en el tratamiento del cáncer; así, los esfingolípidos tienen un rol fundamental en el destino celular y la señalización del estrés.
En este proyecto de tesis, se analiza el metabolismo de los esfingolípidos y el flujo autofágico para estudiar las interacciones quimioterapéuticas en el modelo celular de cáncer de pulmón (A549) y de mama (MCF-7).
Se utilizaron células cargadas con un análogo de esfingomielina fluorescente (BODIPY) y una sonda para rastrear el flujo autofágico (mCherry-EGFP-LC3B), con el fin de evaluar la citotoxicidad cuando las células se exponen a quimioterapia (epirrubicina, cisplatino y paclitaxel) junto con inhibidores de la vía de los esfingolípidos y moduladores de la autofagia.
Las células de cáncer de mama MCF-7 no lograron mostrar internalización del análogo fluorescente, por lo que se determinó que los resultados restantes del proyecto se iban a generar únicamente basados en las células A549.
El enfoque de modelo celular se basó en biosensores de esfingolípidos fluorescentes y un modelo matemático de mezclas Gaussianas (GMM) del perfil de heterogeneidad celular para mapear la influencia de la quimioterapia en la vía de los esfingolípidos e inferir posibles interacciones sinérgicas.
Los resultados mostraron sinergias significativas, especialmente cuando se combina la quimioterapia epirrubicina con inductores de autofagia (rapamicina y torin), lo que reduce la viabilidad celular. El cisplatino, otro fármaco quimioterapéutico, también tuvo sinergia con un inhibidor de la ceramidasa. Por su parte, el agente anti-cáncer paclitaxel mostró principalmente efectos antagónicos.
El modelo de mapeo planteado sugiere que la combinación de quimioterapias con inductores de autofagia aumenta la formación de vesículas, posiblemente relacionadas con la acumulación de ceramida, lo que desencadena la muerte celular. Sin embargo, el modelo in silico propuso la acumulación de ceramida en los autofagosomas, y el análisis cinético experimental proporcionó evidencia de colocalización de esfingolípidos en los autofagosomas. Es por lo anterior que, se requieren más estudios para identificar los esfingolípidos específicos que se acumulan en los autofagosomas.
Estos hallazgos ofrecen información sobre estrategias potenciales para superar la resistencia a la quimioterapia dirigidos a la vía de los esfingolípidos.
Tumor heterogeneity leads to drug resistance in cancer treatment with the crucial role of sphingolipids in cell fate and stress signaling. This thesis aimed to analyze the sphingolipid metabolism and autophagic flux to study chemotherapeutic interactions on the A549 lung cancer model and MCF-7 breast cancer model. Loaded cells with fluorescent sphingomyelin analog (BODIPY) and mCherry-EGFPLC3B were used to track autophagic flux and assess cytotoxicity when cells are exposed to chemotherapy (epirubicin, cisplatin, and paclitaxel) together with sphingolipid pathway inhibitors and autophagy modulators. The MCF-7 breast cancer cells failed to show internalization of the fluorescent analog; thus, it was determined that the subsequent results of the project were going to be generated based on the A549 cells only. The cell model approach employed fluorescent sphingolipid biosensors and a Gaussian Mixture Model of cell heterogeneity profiles to map the influence of chemotherapy on the sphingolipid pathway to infer potential synergistic interactions. Results showed significant synergy, especially when combining epirubicin with autophagy inducers (rapamycin and torin), reducing cell viability. Cisplatin also synergized with a ceramidase inhibitor. However, paclitaxel often led to antagonistic effects. The mapping model proposed, suggests that combining chemotherapies with autophagy inducers increases vesicle formation, possibly linked to ceramide accumulation, triggering cell death. However, the in silico model proposed ceramide accumulation in autophagosomes, and kinetic analysis provided evidence of sphingolipid colocalization in autophagosomes. Further research is needed to identify specific sphingolipids accumulating in autophagosomes. These findings offer insights into potential strategies for overcoming chemotherapy resistance by targeting the sphingolipid pathway.
Tumor heterogeneity leads to drug resistance in cancer treatment with the crucial role of sphingolipids in cell fate and stress signaling. This thesis aimed to analyze the sphingolipid metabolism and autophagic flux to study chemotherapeutic interactions on the A549 lung cancer model and MCF-7 breast cancer model. Loaded cells with fluorescent sphingomyelin analog (BODIPY) and mCherry-EGFPLC3B were used to track autophagic flux and assess cytotoxicity when cells are exposed to chemotherapy (epirubicin, cisplatin, and paclitaxel) together with sphingolipid pathway inhibitors and autophagy modulators. The MCF-7 breast cancer cells failed to show internalization of the fluorescent analog; thus, it was determined that the subsequent results of the project were going to be generated based on the A549 cells only. The cell model approach employed fluorescent sphingolipid biosensors and a Gaussian Mixture Model of cell heterogeneity profiles to map the influence of chemotherapy on the sphingolipid pathway to infer potential synergistic interactions. Results showed significant synergy, especially when combining epirubicin with autophagy inducers (rapamycin and torin), reducing cell viability. Cisplatin also synergized with a ceramidase inhibitor. However, paclitaxel often led to antagonistic effects. The mapping model proposed, suggests that combining chemotherapies with autophagy inducers increases vesicle formation, possibly linked to ceramide accumulation, triggering cell death. However, the in silico model proposed ceramide accumulation in autophagosomes, and kinetic analysis provided evidence of sphingolipid colocalization in autophagosomes. Further research is needed to identify specific sphingolipids accumulating in autophagosomes. These findings offer insights into potential strategies for overcoming chemotherapy resistance by targeting the sphingolipid pathway.
Descripción
Palabras clave
Cáncer, Vía de los esfingolípidos, BODIPY, LC3/m-Cherry, Sinergismos, Autofagia, Sphingolipids, Pathway, Synergisms, Autophagy