Hori Et Al. Chloroquine Potentiates Temozolomide Cytotoxicity by Inhibiting Mitochondrial

Hori et al. Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells.

Supplementary Materials and Methods

Reagents and antibodies.Hoechst 33342 and N-acetylcysteine (NAC) were obtained from Wako Pure Chemicals (Osaka, Japan); chloroquine diphosphate salt, TMZ (3,4-dihydro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-8-carboxamide), and antimycin A (AntA) from Sigma-Aldrich (St. Louis, MO, USA); MitoSOX Red, MitoTracker Green (MTG), and MitoTracker Red (MTR) from Life Technologies (Carlsbad, CA, USA); bafilomycin A1 (Baf) from LC Laboratories (Woburn, MA, USA); and EUK-134 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following antibodies were used: mouse monoclonal anti-cyclophilin D (ab110324, Abcam, Cambridge, MA, USA), mouse monoclonal anti-GAPDH (G8795, Sigma-Aldrich),guinea pig polyclonal anti-p62 (GP62-C, Progen), rabbit monoclonal anti-LC3A/B (#12741, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-β-actin (017-24573, Wako Pure Chemicals),mouse monoclonal anti-parkin (sc-32282, Santa Cruz Biotechnology), rabbit polyclonal anti-parkin (#2132, Cell Signaling Technology),and mouse monoclonal anti-nestin (M-1385-100, Biosensis, Thebarton, Australia).

Isolation of neurosphere-forming cells from a human glioblastoma tissue and measurement of cellular ATP content.

The tumor tissue was minced in a 50-ml conical tube with phosphate-buffered saline (PBS) at 4ºC. After centrifugation, the tissue pieces were suspended in 5 ml of culture medium (described below) with 1 ml of trypsin-EDTA and incubated for 5 min at 37ºC, after which 1 ml of FBS was added to inhibit trypsin. The tissue was dissociated by gently moving the solution up and down with a P1000 pipette. Cells were collected by centrifugation, and the medium was replaced with fresh medium. The tube was allowed to stand for 3 min for tissue debris to sink to the bottom, and the supernatant was collected and centrifuged. This clearing step was repeated once, and finally cells were cultured in media hormone mix (MHM) medium with EGF (20 ng/ml) and bFGF (20 ng/ml). Neurospheres were dissociated for passage by gentle trituration with a Pasteur pipette. To measure cellular ATP levels, cells were seeded on 96-well plates and incubated with various experimental solutions for 48 h, after which ATP was measured using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol, with an ARVO Light Luminescence Counter (PerkinElmer, Waltham, MA, USA).

Analysis of cell death. Cell death was induced by pretreating C6 cells with CQ or dimethylsulfoxide (vehicle) for 3 h and then incubating the cells with TMZ for 24 h. To analyze the role of ROS in cell death, cells were pretreated with NAC (200 μM) or EUK-134 (50 μM). Cell viability was assessed with a MuseTM Cell Analyzer using a Cell Count and Viability Assay Kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s protocol.To analyze apoptosis, the nuclei were stained with Hoechst 33342, and apoptotic cells were detected by nuclear condensation. At least six fields randomly selected were examined in each experiment, and the data from three independent experiments were compared.

Cell death was also analyzed in U87-MG cells by measuring the release of intracellular lactate dehydrogenase (LDH) into the culture medium. U87-MG cells were incubated with vehicle or TMZ (100 and 300 μM) to induce cell death with or without pretreatment with either CQ (10 μM)for 1 h. In addition, we used another autophagy inhibitor bafilomycin A1 (Baf A1) that blocks it at the late stage. Since 10 and 100 nM Baf A1 alone caused cell death (data not shown), we used Baf A1 at 1 nM. Forty eight hours after starting temozolomide treatment, LDH activity from the supernatant and total cells was determined by using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). To determine LDH activity released from total cells, the cell and medium samples were subjected to freeze-thaw.

Analysis of mitochondrial ROS level.Cells were pretreated with CQ or vehicle for 3 h, incubated with TMZ for 24 h, and incubated with 5 μM MitoSOX Red for 10 min at 37°C.Then the intensity of red MitoSOX fluorescence was imaged by using a FLoid Cell Imaging Station (Life Technologies)and quantified using ImageJ software (NIH, Bethesda, MD, USA).For experiments using cultured glioblastoma cells, cell spheres were dissociated by pipetting, and MitoSOX Red fluorescence in the cells was detected using a FLoid Cell Imaging Station. At least 5 randomly selected fields were examined in each experiment, and data from three independent experiments were analyzed.

Measurement of mitochondrial content.Mitochondria in C6 cells were stained with MTG, which accumulates preferentially in mitochondria regardless of the mitochondrial membrane potential. Cells were loaded with MTG (100 nM) in culture medium for 20 min at 37 ºC, and then were treated with vehicle or TMZ with or without CQ for 6 h. MTG fluorescence was imaged and stored with a FLoid Cell Imaging Station. The MTG fluorescence intensity per cell was quantified by ImageJ software. Five randomly selected fields were examined in each experiment, and three independent experiments were conducted.

Immunocytochemistry.After cells from a patient-derived glioblastoma tissue were fixed with 4% paraformaldehyde, samples were then blocked and permeabilized with PBS containing 3% bovine serum albumin, 1% goat serum and 0.1% Triton X-100 for 30 min. For immunostaining, samples were incubated overnight with an antibody against nestin at 4ºC, and with Alexa Fluor 594 anti-mouse IgG (1:2000) for 1 h at room temperature. The samples were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA, USA). Images were collected by confocal laser microscopy.

Immunoblots.Cells were lysed in CelLytic M Cell LysisReagent (Sigma-Aldrich) with protease inhibitor cocktail and phosphatase inhibitor cocktail (NacalaiTesque). The antibodies used were anti-cyclophilin D (1:2000),anti-p62 (1:1000), anti-LC3A/B (1:2000),anti-β-actin (1:10000),anti-parkin (1:250 for sc-32282 and 1:1000 for #2132), and anti-GAPDH (1:10000).

Transfection.Control siRNAs (100 nM; Sigma-Genosys Japan, Ishikari, Japan) and siRNAs against beclin-1 (100 nM; Cell Signaling Technology) were electroporated into C6 cells twice 24 h apart using a Nucleofector Kit (Lonza, Basel, Switzerland)according to the manufacturer’s protocol; the cells were used for experiments 24 h after the last transfection. For transient expression, C6 cells were transfected with the pEGFP-LC3 construct (Addgene) using the Nucleofector Kit.

Quantitative reverse transcription polymerase chain reaction. Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized using SuperScript III (Life Technologies). DNA amplification was performed in StepOneTM (Applied Biosystems/Life Technologies) using the TaqMan PCR Kit for beta-actin, or Power SYBR Green Master Mix (Applied Biosystems/Life Technologies) for beclin-1. The primer sequences were 5’-CTGACAGACAAATCTAAGGAG-3’ (beclin-1 forward) and 5’-AATAGGAGCCGCCACTGCCTC-3’ (beclin-1 reverse).