How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria

Mitochondrial function generates an important fraction of the heat that contributes to cellular and organismal temperature maintenance, but the actual values of this parameter reached in the organelles is a matter of debate. The studies addressing this issue have reported divergent results: from detecting in the organelles the same temperature as the cell average or the incubation temperature, to increasing differences of up to 10 degrees above the incubation value. Theoretical calculations based on physical laws exclude the possibility of relevant temperature gradients between mitochondria and their surroundings. These facts have given rise to a conundrum or paradox about hot mitochondria. We have examined by Blue-Native electrophoresis, both in intact cells and in isolated organelles, the stability of respiratory complexes and supercomplexes at different temperatures to obtain information about their tolerance to heat stress. We observe that, upon incubation at values above 43 °C and after relatively short periods, respiratory complexes, and especially complex I and its supercomplexes, are unstable even when the respiratory activity is inhibited. These results support the conclusion that high temperatures (>43 °C) cause damage to mitochondrial structure and function and question the proposal that these organelles can physiologically work at close to 50 °C

PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies

PT-112 is a novel pyrophosphate–platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112’s effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929dt cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dtL929 cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization—only in the sensitive cells harboring mitochondrial dysfunction—along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications

Combinación de los inhibidores del proteasoma y TRAIL como tratamiento antitumoral del Mieloma Múltiple

El cáncer es una de las patologías más comunes y con mayor tasa de mortalidad, entre las cuales encontramos el mieloma múltiple (MM), un tipo de cáncer hematológico que representa el 10% de neoplasias sanguíneas y afecta principalmente a mujeres de raza negra con una edad media de diagnosis de entre 65 y 70 años. Durante años, los tratamientos clásicos utilizados para tratar esta patología han sido los agentes alquilantes, que inhiben la replicación del ADN, los glucocorticoides, que aumentan la expresión de genes proapoptóticos como Bim , o el trasplante de células autólogas. Actualmente, los fármacos inhibidores del proteasoma son el tratamiento estándar para tratar el MM, entre los cuales se encuentra carfilzomib. Este fármaco de segunda generación parece tener un efecto positivo en la regresión de las células mielomatosas gracias a un mecanismo de acción bloqueando el proteasoma. En estudios realizados con carfilzomib se ha constatado que además de activar la muerte celular a través de vía intrínseca, esta droga podría tener un efecto sensibilizante a la terapia con ligandos mortales, en concreto con Apo2L/TRAIL. Este ligando mortal, utilizado previamente en ensayos para el tratamiento de otros cánceres, es capaz de activar la vía extrínseca de la apoptosis en células que expresan sus receptores mortales DR4 y DR5. En la última década, la inmunoterapia basada en el uso de ligandos mortales ha evolucionado hasta el punto de desarrollar diferentes formulaciones de ligando mortal TRAIL (bajo una forma soluble recombinante, asociado a liposomas, etc.) con el objetivo de potenciar su citotoxicidad en una gran variedad de células cancerosas. En este trabajo se plantea el estudio del mecanismo de inducción de apoptosis de una terapia que combina carfilzomib con diferentes formulaciones de TRAIL en líneas celulares tumorales humanas de MM

Efecto del DCA y de inhibidores de tirosín quinasas sobre células tumorales con mutaciones en el DNA mitocondrial

En trabajos anteriores se había demostrado mediante el estudio de su quinoma que la línea celular L929dt, la cual se generó de forma espontánea en nuestro laboratorio hace años, tenía una actividad exacerbada de las actividades proteín tirosín quinasa (PTK) respecto a sus parentales L929 (fibroblastos de ratón). Las células L929dt, a diferencia de las células L929 parentales, exhiben mutaciones en el DNA mitocondrial, crecen en suspensión y tienen un metabolismo de tipo fermentativo. La dependencia de la actividad PTK en este modelo in vitro de células metastásicas (L929dt) se demostró por su sensibilidad a la genisteína, un inhibidor general de PTK. Uno de los sustratos que exhibían una mayor diferencia en su fosforilación entre las células L929 y L929dt era la quinasa de adhesión focal (FAK). En este trabajo se ha estudiado si su inhibición farmacológica con defactinib podría afectar de forma diferencial a las células L929dt. Los resultados obtenidos demuestran la localización nuclear de FAK en las células L929dt y sugieren que la inhibición de la actividad quinasa de FAK no es especialmente relevante para eliminar estas células, ya sea como monoterapia o en combinación con el fármaco antimetabólico dicloroacetato (DCA).<br /

A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism