Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells

Abstract

Mitochondrial autophagy eliminates damaged mitochondria and decreases reactive oxygen species (ROS). The autophagy inhibitor chloroquine (CQ) potentiates temozolomide (TMZ) cytotoxicity in glioma cells, but it is not known whether CQ does this by inhibiting mitochondrial autophagy. The effects of CQ and TMZ on MitoSOX Red fluorescence, a mitochondrial ROS indicator, and cell death were examined in rat C6 glioma cells. Mitochondrial autophagy was monitored by the colocalization of MitoTracker Red fluorescence and EGFP-LC3 dots. Mitochondrial content was measured by MitoTracker Green fluorescence and immunoblotting for a mitochondrial protein. Finally, CQ's effects on tumor cells derived from a glioblastoma patient and human U87-MG glioblastoma cells were assessed. TMZ (100-1,000 μM) alone did not affect mitochondrial ROS or cell death in C6 cells, but when administered with CQ (10 μM), it increased mitochondrial ROS and cell death. Antioxidants significantly suppressed the CQ-augmented cell death in TMZ-treated cells, indicating that mitochondrial ROS were involved in this cell death. TMZ treatment reduced MitoTracker Green fluorescence and mitochondrial protein levels, and these effects were inhibited by CQ. TMZ also increased the colocalization of EGFP-LC3 dots with mitochondria, and CQ enhanced this effect. CQ potentiated TMZ-induced cytotoxicity in patient-derived glioblastoma cells as well as human U87-MG glioblastoma cells. These results suggest that CQ increases cellular ROS and augments TMZ cytotoxicity in glioma cells by inhibiting mitochondrial autophagy.

Tris (1, 3-dichloro-2-propyl) phosphate induces apoptosis and autophagy in SH-SY5Y cells: Involvement of ROS-mediated AMPK/mTOR/ULK1 pathways

“…However, in the CQ-pretreated cells, the LC3II expression was markedly increased compared to that following TDCIPP treatment alone. CQ prevents endosomal acidification, thereby inhibiting lysosomal enzymes and causing the accumulation of LC3II (Hori et al, 2015). Thus, 3-MA and CQ can inhibit autophagy and increase apoptotic cell death in SH-SY5Y cells.…”

“…The SH-SY5Y cells were treated in the presence of the autophagy inhibitor CQ. CQ is a lysosomotropic agent that prevents endosomal acidification, thereby inhibiting lysosomal enzymes and causing the accumulation of LC3II (Hori et al, 2015). CQ (10 mM) increased LC3II accumulation (Fig.…”

“…CQ is generally used to block autophagosome combination with lysosomes and eventually obstructs degradation at the late stage (Hori et al, 2015). Rapa is widely used as a canonical inhibitor of mTOR complex 1 (mTORC1) and also acts as an inducer of autophagy (Pan et al, 2009).…”

Chloroquine Sensitizes Nasopharyngeal Carcinoma Cells but Not Nasoepithelial Cells to Irradiation by Blocking Autophagy

“…It can be cytoprotective, helping the cell to survive or it can be cytotoxic, leading to cell death [15–16]. Autophagy has been shown in various tumor cell systems, including hepatocellular carcinoma and glioblastoma to protect cells against the cytotoxic effect of radiation or chemotherapy [42–44]. In nasopharyngeal carcinoma, several studies have demonstrated the induction of autophagy by radiation mostly using one cell line [31–32, 45].…”

Re-purposing Chloroquine for Glioblastoma: Potential Merits and Confounding Variables

“…Most of the existing studies addressing the effects of CHQ in glioma cells have used conventional serum-dependent cell lines that lack stemness properties and/or poorly recapitulate characteristic features of human GBs. For example, the human glioma cell line U87MG, which has been widely used as an experimental model for investigating biological responses mediated by CHQ (21, 22, 29, 30, 81, 82) does not reproduce certain characteristic traits of GBs such as an invasive tumor phenotype, intra-tumoral heterogeneity and high degree of intrinsic radio-resistance. Considering that GSCs are fundamentally distinct from non-stem glioma cells, it is conceivable that their responses to CHQ might also differ from those operating in the latter ones.…”