Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Early downregulation of Mcl-1 regulates apoptosis triggered by cardiac glycoside UNBS1450.

Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na(+)/K(+))-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid.

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10(-9) vs. 10(-5)M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10(-5)M, but drops to 10(-9)M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism.

We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca(2+)-independent, but not Ca(2+)-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, as occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca(2+)-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.

Hyperpolarization of plasma membrane of tumor cells sensitive to antiapoptotic effects of magnetic fields.

Chemical/physical agents able to prevent apoptosis are receiving much attention for their potential health hazard as tumor promoters. Magnetic fields (MFs), which have been shown to increase the occurrence of some tumors, reduce damage-induced apoptosis by a mechanism involving Ca2+ entry into cells. In order to discover the mechanism of such effect of MFs, we investigated the interference of MFs on cell metabolism and analyzed cell parameters that are involved in apoptotic signaling and regulation of Ca2+ fluxes. Here we show that different types (static and extremely low-frequency, ELF pulsating) of MFs of different intensities alter plasma membrane potential. Interestingly, MFs induce plasma membrane hyperpolarization in cells sensitive to the antiapoptotic effect of MFs, whereas cells that are insensitive showed a plasma membrane depolarization. These opposite effects suggest that protection against apoptosis and membrane potential modulation are correlated, plasma membrane hyperpolarization possibly being part of the signal transduction chain determining MFs' antiapoptotic effect.