Differentiation inducing factor 3 mediates its anti-leukemic effect through ROS-dependent DRP1-mediated mitochondrial fission and induction of caspase-independent cell death

Abstract

Differentiation-inducing factor (DIF) defines a group of chlorinated hexaphenones that orchestrate stalk-cell differentiation in the slime mold Dictyostelium discoideum (DD). DIF-1 and 3 have also been reported to have tumor inhibiting properties; however, the mechanisms that underlie the effects of these compounds remain poorly defined. Herein, we show that DIF-3 rapidly triggers Ca2+ release and a loss of mitochondrial membrane potential (MMP) in the absence of cytochrome c and Smac release and without caspase activation. Consistently with these findings, we also detected no evidence of apoptosis in cells treated with DIF-3 but instead found that this compound induced autophagy. In addition, DIF-3 promoted mitochondrial fission in K562 and HeLa cells, as assessed by electron and confocal microscopy analysis. Importantly, DIF-3 mediated the phosphorylation and redistribution of dynamin-related protein 1 (DRP1) from the cytoplasmic to the microsomal fraction of K562 cells. Pharmacological inhibition or siRNA silencing of DRP1 not only inhibited mitochondrial fission but also protected K562 cells from DIF-3-mediated cell death. Furthermore, DIF-3 potently inhibited the growth of imatinib-sensitive and imatinib-resistant K562 cells. It also inhibited tumor formation in athymic mice engrafted with an imatinib-resistant CML cell line. Finally, DIF-3 exhibited a clear selectivity toward CD34+ leukemic cells from CML patients, compared with CD34− cells. In conclusion, we show that the potent anti-leukemic effect of DIF-3 is mediated through the induction of mitochondrial fission and caspase-independent cell death. Our findings may have important therapeutic implications, especially in the treatment of tumors that exhibit defects in apoptosis regulation.

Autophagy-dependent cell death – where, how and why a cell eats itself to death

“…Interestingly, DIF family morphogens have anti-tumorigenic properties in mammalian cells. For instance, when DIF-3 is introduced to human cancer cells, it induces mitochondrial depolarization and fission, autophagy and caspase-independent cell death (Dubois et al, 2016). During Drosophila metamorphosis, developmental cell death of the obsolete larval midgut and salivary glands requires autophagy (see Box 2).…”

Mitochondrial network structure homeostasis and cell death

“…Drp1 activation can disrupt the homeostasis of the mitochondrial network structure and impair mitochondrial function by inducing oxidative stress through increased production of reactive oxygen species (ROS). ROS induces damage of mitochondria and is correlated with an increase in the phosphorylation of Drp1 on Ser616 and mitochondrial fragmentation, activating autophagic cell death . In addition, knocking down Drp1 expression interrupts the balance of the mitochondrial network structure, blocking mitophagy, and affects mitochondrial quality, leading to ischemia/reperfusion injury .…”

“…ROS induces damage of mitochondria and is correlated with an increase in the phosphorylation of Drp1 on Ser616 and mitochondrial fragmentation, activating autophagic cell death. 30 In addition, knocking down Drp1 expression interrupts the balance of the mitochondrial network structure, blocking mitophagy, and affects mitochondrial quality, leading to ischemia/reperfusion injury. 31 However, when faced with starvation or severe energy depletioninduced cell autophagy, lack of ATP can potentially activate fission through the elevation of ADP and AMP levels.…”

The Dictyostelium model for mitochondrial biology and disease

“…This had significant deleterious effects on ATP production, induced superoxide production and depleted the mitochondrial membrane potential. DIF-3 induces autophagy in K562 cells by rapid inhibition of mTOR pathway signalling as a result of dephosphorylation of mTOR at Ser2481 (Dubois et al, 2016). DIF-3 triggered caspaseindependent cell death through the induction of Ca 2+ influx into the cytoplasm, and the subsequent recruitment of DRP1 to the mitochondria.…”