Insights into the molecular mechanisms of apoptosis induced by glucose deprivation

Abstract

[eng] INTRODUCTION. Tumor cells undergo a complete metabolic reprogramming which allows them to grow and proliferate fast. The metabolic changes are driven by oncogenes and tumor suppressors and are aimed to increase glycolysis and biosynthetic pathways and to reduce oxidative phosphorylation. Cancer cells rely on glucose not only to produce energy but also to synthesize all the biomolecules necessary for their proliferation. They prefer to use glycolysis rather than oxidative phosphorylation, even in presence of oxygen. This metabolic reprogramming can be a disadvantage for tumors when nutrients are limiting. Treatment with anti-glycolytic drugs, such as 2-deoxyglucose, or glucose withdrawal have been shown to sensitize to radiotherapy or chemotherapy or to TRAIL stimulation, making these treatments good candidates for cancer therapy. Glucose deprivation can induce different types of cell death, such as apoptosis, usually through the mitochondrial pathway, or necrosis. OBJECTIVE. This thesis focuses on understanding the molecular mechanisms of apoptosis induced by glucose deprivation in different cell lines. The objective was to determine which platform is activating caspase-8 under glucose deprivation, which seems to be different from the canonical DISC (death-inducing signaling complex). RESULTS. We have described that HeLa cells die in a caspase-dependent manner when subjected to glucose deprivation, as the co-treatment with the caspase inhibitor Q-VD protects from cell death. We also show that Q-VD co-treatment and the silencing of caspase-8 protects HCT116 deficient for Bax and Bak from cell death due to glucose deprivation. Then, we studied the role of the Ripoptosome in caspase-8 activation in HeLa cells subjected to glucose removal. As we already described in Bax/Bak deficient MEFs, in HeLa the knockdown of RIPK1 did not prevent cell death. However, we have demonstrated that FADD is essential for the execution of cell death under glucose deprivation. Recently, it has been shown that an intracellular DISC (iDISC) can be formed upon proteasome inhibition or endoplasmic reticulum stress on autophagosomal membranes, independently of death ligands. We demonstrated by immunoprecipitation and immunofluorescence that caspase-8 interacts with p62, LC3-II and ATG5, proteins that associate with autophagosome, under glucose deprivation. However, we were unable to show significant translocation of caspase-8 to these organelles, or an essential role of p62 to activate caspase-8 and induce apoptosis under glucose deprivation. Furthermore, we observed that glucose deprivation induces the endoplasmic reticulum stress in different cell lines, as demonstrated by the induction of ATF4 and CHOP. Moreover, glucose withdrawal results in the induction of death receptors of TRAIL. We have shown that ATF4, but not CHOP, is responsible for the induction of TRAIL-R2 (DR5) after glucose removal. We show by immunoprecipitation that DR5 interacts with caspase-8 and localizes mostly at Golgi apparatus before and after the treatment, where maybe it could be accumulating and recruiting caspase-8. Moreover, the knockdown of DR5 in HeLa cells protects from apoptosis due to glucose deprivation, an effect that is more significant when Bcl-xL is stably expressed in these cells, suggesting that a component of death upon glucose deprivation is dependent on the mitochondrial pathway. Finally, we describe that caspase-8 is located to filament-like structures in Bax/Bak deficient MEFs subjected to glucose deprivation. We show that those filaments co-localize with β-tubulin, suggesting that a cytoskeleton scaffold could be involved in caspase-8 aggregation and activation under glucose deprivation. CONCLUSIONS. These data suggest that glucose deprivation could be used as potential therapeutic strategy against tumors with impaired mitochondrial pathway of apoptosis.

[spa] INTRODUCCIÓN. Las células cancerosas dependen de la glucosa, no sólo para producir energía, sino también para sintetizar bio-moléculas necesarias para su proliferación, lo que las hace más sensibles a la escasez de glucosa. La privación de glucosa puede inducir diferentes tipos de muerte celular, tales como la apoptosis, generalmente a través de la ruta mitocondrial, o la necrosis. OBJETIVO. Esta tesis se centra en la comprensión de los mecanismos moleculares de la apoptosis inducida por la privación de glucosa en diferentes líneas celulares. El objetivo fue definir qué plataforma está activando la caspasa-8 bajo privación de glucosa. RESULTADOS. En primer lugar, hemos descrito que las células HeLa sometidas a la privación de glucosa mueren por apoptosis dependiente de caspasas ya que el co-tratamiento con el inhibidor de caspasas Q-VD protege de esta muerte. También mostramos que el Q-VD y el silenciamiento de la caspasa-8 protegen a las células HCT116 deficientes en Bax y Bak de la muerte causada por la retirada de glucosa. Entonces, se estudió el papel del Ripoptosoma en la activación de la caspasa-8. El silenciamiento de RIPK1 no impidió la muerte celular. Sin embargo, hemos demostrado que FADD es esencial para la ejecución de la muerte celular bajo la privación de glucosa. Demostramos por inmunoprecipitación e inmunofluorescencia que bajo privación de glucosa la caspasa-8 interactúa con p62, LC3-II y ATG5, proteínas que se asocian con el autofagosoma. Sin embargo, no hemos podido demostrar translocación significativa de la caspasa-8 a estos orgánulos, o un papel esencial de p62 en activar la caspasa-8 e inducir la apoptosis bajo privación de glucosa. Además, se observó que la privación de glucosa induce por un lado, estrés del retículo endoplásmico en diferentes líneas celulares, como demuestra la inducción de ATF4 y CHOP, y por otro lado la inducción de los receptores de muerte de TRAIL. Hemos demostrado que ATF4, pero no CHOP, es responsable de la inducción de TRAIL-R2 (DR5) después de la retirada de glucosa. CONCLUSIONES. Estos datos sugieren que la privación de glucosa podría ser utilizada como posible estrategia terapéutica contra tumores resistentes a la ruta mitocondrial de apoptosis.