Mechanistic study on the therapeutic strategy to enhance glioma cell death through inhibition of proteasome and lysosome

Abstract

Malignant gliomas, the most common brain tumors with high mortality, remain largely incurable despite multimodal treatments including surgical resection, radiotherapy, and chemotherapy. Thus, the researchers are currently attempting to develop novel therapeutic strategies for malignant gliomas. Recent studies show that dysregulation of protein maintenance has been considerably linked to many diseases. Therefore, protein degradation system has become a platform for drug targeting, and mechanism-based drugs are currently developed. Bortezomib (BZ), a proteasome inhibitor, and chloroquine (CQ), a lysosomotropic agent, can disrupt cellular protein homeostasis (proteostasis) via interruption of protein degradation systems, including proteasome and lysosome. In part I, we investigate the role of eIF2a in BZ-induced cell death. We found that ER-derived dilation preceded bortezomib induced cell death. eIF2α was initially phosphorylated but it was dephosphorylated in parallel with the ER dilation. Overexpression of eIF2a WT markedly increased rate of phosphorylated eIF2a (exogenous) and blocked bortezomib-induced ER vacoulation and cell death. In contrast, overexpression of dominant negative eIF2a S51A does not affect rate of phosphorylated eIF2a and cell death. These results suggests that dephosphorylation of eIF2a may be important for the ER dilation and cytotoxic effect of bortezomib. These results suggest that eIF2a dephosphorylation rather than its phosphorylation may be important for the cytotoxic effect of bortezomib. In addition, knockdown of eIF2a sensitized bortezomib-induced cell death via increasing caspase-dependent apoptosis. Collectively, regulation of eIF2a phosphorylation and expression may provide a therapeutic strategy to sensitize glioma cell to bortezomib-mediated cell death. We found that combined treatment with bortezomib and salubrinal, an inhibitor of GADD34-PPiC phosphatase complex, very effectively induced the dilation of the endoplasmic reticulum and subsequent cell death. Interestingly, either salubrinal or bortezomib increased the phosphorylation levels of eIF2α, but combined treatment markedly reduced them. In addition, co-treatment with salubrinal decreased expression levels of eIF2a. Taken together, these results suggest that salubrinal effectively overcomes the resistance of malignant glioma cells to BZ, via dependently- and independent-manner of eIF2a phosphorylation.

In part II, we investigate whether co-treated phytochemicals sensitized CQ-induced cell death in glioma cells. We show that treatment with subtoxic doses of CQ, when combined with kaempferol, a flavonoid, effectively induces cell death in various glioma cells, but not in normal astrocytes, suggesting that the combined regimen using kaempferol and CQ may provide a safe therapeutic strategy to selectively kill resistant glioma cells. The cell death induced by kaempferol and CQ in U251MG cells was partially dependent on caspase-mediated apoptosis and accompanied by mitochondrial dysfunction, ER stress, and DNA damage. We found that kaempferol treatment increased the numbers of lysosome, whereas CQ treatment increased the lysosomal masses leading to their swelling. Combined treatment with kaempferol and CQ further increased the lysosomal masses, but culminated in the disruption of lysosomal compartments. While kaemfperol treatment increased the active mature forms of cathepsin D, CQ treatment markedly blocked the processing and activity of cathepsin D. Interestingly, combined treatment induced the release of unprocessed cathepsin D into the cytosol and the recovery of its activity. Knockdown of cathepsin D significantly attenuated the cell death induced by kaempferol and CQ, suggesting the functional involvement of these released cathepsin D proteins in this cell death. Taken together, our results suggest that the membrane destabilization of accumulated lysosomes and the resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by kaempferol plus CQ.

In this study, we show that salubrinal effectively overcomes the resistance of malignant glioma cells to BZ, via dependently- and independent-manner of eIF2a phosphorylation. Also, we show that combined treatment with kaempferol and CQ effectively induces cell death in glioma cells, via lysosomal overloading and rupture. Collectively, these results suggest that proteostasis perturbation by targeting of UPR components or lysosome may provide potential therapeutic effect for targeting malignant glioma cells over normal cells.