Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.
Disclosure: No relevant conflicts of interest to declare.