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.
This is a preview of subscription content,to check access.
Access this article
Sotelo J, Briceno E, Lopez-Gonzalez MA (2006) Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 144(5):337–343
Briceno E, Calderon A, Sotelo J (2007) Institutional experience with chloroquine as an adjuvant to the therapy for glioblastoma multiforme. Surg Neurol 67(4):388–391
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075
Amaravadi RK, Thompson CB (2007) The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 13(24):7271–7279
Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI et al (2007) Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117(2):326–336
Han W, Sun J, Feng L, Wang K, Li D, Pan Q et al (2011) Autophagy inhibition enhances daunorubicin-induced apoptosis in K562 cells. PLoS ONE 6(12):e28491
Gini B, Zanca C, Guo D, Matsutani T, Masui K, Ikegami S et al (2013) The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas. Clin Cancer Res 19(20):5722–5732
Hu YL, DeLay M, Jahangiri A, Molinaro AM, Rose SD, Carbonell WS et al (2012) Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 72(7):1773–1783
Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40(2):280–293
Balaban RS, Nemoto S, Finkel T (2005) Mitochondria, oxidants, and aging. Cell 120(4):483–495
Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12(9):823–830
Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12(1):9–14
Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC et al (2010) Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 12(3):222–230
Zhou R, Yazdi AS, Menu P, Tschopp J (2011) A role for mitochondria in NLRP3 inflammasome activation. Nature 469(7329):221–225
Ducharme J, Farinotti R (1996) Clinical pharmacokinetics and metabolism of chloroquine. Focus on recent advancements. Clin Pharmacokinet 31(4):257–274
Ni HM, Bockus A, Wozniak AL, Jones K, Weinman S, Yin XM et al (2011) Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy 7(2):188–204
Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4):445–544
Zhu J, Dagda RK, Chu CT (2011) Monitoring mitophagy in neuronal cell cultures. Methods Mol Biol 793:325–339
Ding WX, Yin XM (2012) Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 393(7):547–564
Narendra D, Tanaka A, Suen DF, Youle RJ (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183(5):795–803
Zhang C, Lin M, Wu R, Wang X, Yang B, Levine AJ et al (2011) Parkin, a p53 target gene, mediates the role of p53 in glucose metabolism and the Warburg effect. Proc Natl Acad Sci U S A 108(39):16259–16264
Lin CJ, Lee CC, Shih YL, Lin CH, Wang SH, Chen TH et al (2012) Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells. PLoS ONE 7(6):e38706
Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB et al (2008) Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 283(16):10892–10903
Katayama M, Kawaguchi T, Berger MS, Pieper RO (2007) DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 14(3):548–558
White E (2012) Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer 12(6):401–410
Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M et al (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352(10):997–1003
Ozawa A, Kadowaki E, Haga Y, Sekiguchi H, Hemmi N, Kaneko T et al (2013) Acetylcholine esterase is a regulator of GFAP expression and a target of dichlorvos in astrocytic differentiation of rat glioma C6 cells. Brain Res 1537:37–45
Kuo TC, Yang JS, Lin MW, Hsu SC, Lin JJ, Lin HJ et al (2009) Emodin has cytotoxic and protective effects in rat C6 glioma cells: roles of Mdr1a and nuclear factor kappaB in cell survival. J Pharmacol Exp Ther 330(3):736–744
Gilbert MR, Liu Y, Neltner J, Pu H, Morris A, Sunkara M et al (2014) Autophagy and oxidative stress in gliomas with IDH1 mutations. Acta Neuropathol 127(2):221–233
This work was supported in part by grants for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 24791513) and from Adaptive and Seamless Technology Transfer Program through target-driven R&G, JST.
Conflict of interest
The authors declare that they have no conflict of interest.
All experiments were conducted in compliance with the ethics committee of Sapporo Medical University.
Yusuke S Hori and Ryusuke Hosoda have equally contributed to this work.
About this article
Cite this article
Hori, Y.S., Hosoda, R., Akiyama, Y. et al. Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells. J Neurooncol 122, 11–20 (2015). https://doi.org/10.1007/s11060-014-1686-9