Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death

Abstract

Increasing evidence suggests that induction of lethal autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7-dependent type of cell death that is significantly inhibited with the cathepsin inhibitors E64D/Pepstatin A and the lipid ROS scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by AKT1 (AKT serine/threonine kinase 1) inhibition and a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of acid sphingomyelinase (ASM), increased lipid-ROS levels and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of cathepsin B into the cytosol in MZ-54 wt cells. While LMP and cell death were significantly attenuated in ATG5/7 KO cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and lysosomal cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide

“…HEK293T (ATCC #CRL-3216) was cultured in Dulbecco’s modified Eagle’s medium (DMEM GlutaMAX) supplied with heat-inactivated 10% FBS and P/S (all from Gibco). GFP-positive NCH644 and NCH421k were created by lentiviral transduction (see Section 4.7 ), as described previously [ 68 ].…”

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases their Susceptibility to Temozolomide

“…HEK293T (ATCC #CRL-3216) were cultured in Dulbecco's modified Eagle's medium (DMEM GlutaMAX) supplied with heatinactivated 10% FBS and 100 U/ml Penicillin 100 µg/ml Streptomycin (all from Gibco). GFP positive NCH644 and NCH421k were created by lentiviral transduction as described previously [68].…”

Autophagy in health and disease: From molecular mechanisms to therapeutic target

“…Several Ca 2+ channel blockers, including fluspirilene, verapamil, loperamide, and amiodarone, have been found to induce autophagy potently 273,547–556 . Although the exact mechanisms are not clearly defined, there are reports indicating that the Ca 2+ channel antagonists‐induced autophagosome formation relates to calpain‐mediated cleavage of ATG5 273,552 …”

“…539,540 Several Ca 2+ channel blockers, including fluspirilene, verapamil, loperamide, and amiodarone, have been found to induce autophagy potently. 273,[547][548][549][550][551][552][553][554][555][556] Although the exact mechanisms are not clearly defined, there are reports indicating that the Ca 2+ channel antagonists-induced autophagosome formation relates to calpain-mediated cleavage of ATG5. 273,552 Inositol monophosphatase (IMPase) inhibitors, including the approved mood-stabilizing agents, have been reported to perform neuroprotective activity via induction of autophagy.…”