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Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation
Biochemical and Biophysical Research Communications 471 (2016) 109e116
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Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation Daisuke Yamada a, Shinji Saiki a, *, Norihiko Furuya a, b, Kei-Ichi Ishikawa a, Yoko Imamichi a, Taiho Kambe c, Tsutomu Fujimura d, Takashi Ueno d, Masato Koike e, Katsuhiko Sumiyoshi f, Nobutaka Hattori a, b, ** a
Department of Neurology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan Department of Research and Therapeutics for Movement Disorders, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan Laboratory of Biosignals and Response, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan d Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan e Department of Cell Biology and Neuroscience, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan f Department of Health and Nutrion, Tokiwa University, Ibaraki, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2016 Accepted 28 January 2016 Available online 4 February 2016
Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn2þ accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn2þ accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Ethambutol Autophagy Zinc Lysosome
1. Introduction Ethambutol (EB) is widely used as an anti-mycobacterial agent and has a diverse range of pharmacological effects [1]. The World Health Organization (WHO) estimates that approximately 55% of patients with TB are taking EB as a treatment for TB or for Mycobacterium avium infection (Global Tuberculosis Report 2014) [2]. The WHO also estimates that there are around 8.6 million new cases of TB each year. Patients with TB who are being treated with EB occasionally develop serious side effects, such as liver
Abbreviations: EB, ethambutol; TB, tuberculosis. * Corresponding author. Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. ** Corresponding author. Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan E-mail addresses: [email protected] (S. Saiki), [email protected] (N. Hattori).
dysfunction, optic neuropathy and visual field abnormalities [3]. Multiple types of EB-induced cell damage have been reported, including Zn2þ mislocalization to lysosomes, mitochondrial protein synthesis blockade, lysosomal membrane permeabilization and the generation of reactive oxygen species [4,5]. Very recently, EB was shown to induce cell death of the rat retinal cells due to impaired autophagy [6]; however, the precise molecular mechanism was not fully elucidated. Autophagy is a key mechanism for cellular homeostasis and stress adaptation, which facilitates the lysosomal degradation of intracellular proteins or organelles sequestered within autophagosomes [7e9]. Normal lysosomal acidification is essential for autophagosome-lysosome fusion and the activities of lysosomal hydrolytic enzymes, both of which are indispensable for autophagy termination [10e12]. Herein, we reveal that EB is capable of retarding autophagic flux via inhibition of autophagosomelysosome fusion. We further demonstrate that EB induces enlarged lysosomes to lose their characteristic acidic environment and form Zn2þ accumulations, suggesting of a novel molecular mechanism of EB toxicity.
http://dx.doi.org/10.1016/j.bbrc.2016.01.171 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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2. Materials and methods 2.1. Cell lines Human hepatocarcinoma Huh-7 cells (RIKEN Cell Bank) were maintained in RPMI1640 medium (SigmaeAldrich) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich) and 100 U/ml penicillin/streptomycin (SigmaeAldrich) at 37 C and 5% CO2. Human-derived HeLa and SH-SY5Y cells were maintained in Dulbecco's-modified Eagle's medium (DMEM) supplemented with 10% FBS (SigmaeAldrich) and 100 U/ml penicillin/streptomycin (Sigma Aldrich) at 37 C and 5% CO2. A HeLa GFP-LC3 stable cell line was established as previously described [13]. 2.2. Cell culture in zinc-deficient medium FBS was mixed with 10% Chelex-100 resin (Bio-Rad) at 4 C overnight. After centrifugation at 3500 rpm for 5 min, the supernatant was sterilized using a 0.22-mm filter. The chelated FBS was added to culture medium to a final concentration of 10% (DMEM10% chelated FBS). Huh-7 cells were cultured in the zinc-deficient medium for 9 days. Meanwhile, cell passaging was performed once every 2 days.
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E4630), E64d (SigmaeAldrich, E8640), pepstatin A (SigmaeAldrich, P5318), bafilomycin A1 (SigmaeAldrich, B1793), pyrithione (2mercaptopyridine N-oxide sodium salt; SigmaeAldrich, H3261), ZnCl2 (SigmaeAldrich, 229997). 2.6. Plasmid DNAs tfLC3, encoding mRFP-EGFP-LC3, which was originally generated by the Yoshimori group, was purchased from Origene (Plasmid 21075). Mouse lgp120 was cloned into the mCherry vector. 2.7. Western blotting Cell pellets were lysed on ice in RIPA buffer (25 mM TriseHCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) in the presence of protease inhibitors (Roche, Basel, Switzerland) for 20 min. Western blotting was performed according to previously published reports [13]. The antibodies used were as follows: anti-actin (Millipore, clone C4), anti-LC3 (MBL, clone 4E12), anti-p62 (Progen Biotechnik, GP62-C). Antibody signals were enhanced with chemifluorescent methods from GE HealthCare as previously described [13]. 2.8. Immunofluorescence
2.3. Lysosomal pH assay To observe lysosomal alkalinization, we incubated Huh-7 cells with 1 mM LysoSensor Green DND-153 (Life Technologies) for 30 min or 5 mg/ml of LysoSensor Yellow/Blue DND-160 (Life Technologies) for 5 min or 50 nM of LysoTracker Red DND-99 (Life Technologies) for 30 min in RPMI1640. Cells were washed twice with phosphate-buffered saline (PBS) and immediately analyzed using a flow cytometer (FACS Fortessa, BD Biosciences). To quantify lysosomal pH, we incubated Huh-7 cells with 5 mg/ml of LysoSensor Yellow/Blue DND-160 for 5 min and assayed with In Cell Analyzer 1000 (GE Healthcare) according to the manufacturer's protocol. Fluorescent intensities of each emission (460 nm, 535 nm) per visual field were automatically calculated according to the pH standard curve. 2.4. Zn
2þ
accumulation assay
To observe Zn2þ accumulation in lysosomes, we incubated Huh7 cells with 5 mM of FluoZin3 for 30 min. The fluorescence of FluoZin-3 was analyzed using a flow cytometer (FACS Fortessa BD Biosciences), and images were acquired on a Zeiss LSM510 META confocal microscope (63 1.4 NA), at room temperature using Zeiss LSM510 v.3.2 software. Adobe Photoshop 7.0 (Adobe Systems, Inc.) was used for subsequent image processing. 2.5. Compounds Compounds used in this study included EB (SigmaeAldrich,
Huh-7 cells were seeded on glass coverslips. Twenty-four hours later, cells were transfected or treated with the relevant compounds. Cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 50 mg/ml digitonin in PBS for 15 min. To block nonspecific antigenic sites, cells were incubated with 10% FBS and 1% bovine serum albumin in PBS for 30 min. Cells were immunostained with the following antibodies: anti-FLAG (SigmaeAldrich, F7425; 1:500), anti-LC3B (1:500) (SigmaeAldrich, L7543), anti-LAMP1 (1:50) and anti-LAMP2 (1:50) (Development Studies Hybridoma Bank, clone H4B4). Twenty-four hours later, cells were incubated with AlexaFluor 488 anti-rabbit IgG (Life Technologies; 1:500) or AlexaFluor 546 anti-mouse IgG (Life Technologies; 1:500) for 1 h. Cells were embedded with VectaShield containing DAPI (Vector Laboratories, H-1200), and images were acquired on a Zeiss LSM510 META confocal microscope (63 1.4 NA) at room temperature using Zeiss LSM510 v.3.2 software. Adobe Photoshop 7.0 (Adobe Systems Inc.) was used for subsequent image processing. 2.9. Electron microscopy Huh-7 cells were seeded on glass coverslips. Twenty-four hours later, cells were treated with various concentrations of EB for 24 h, then fixed with 4% glutaraldehyde in 0.1 N sodium-cacodylate at room temperature for 1 h, and processed for electron microscopy using a standard protocol. Cells were imaged at 8000 magnification by transmission electron microscope (HT7700; Hitachi, Tokyo, Japan). The volume fraction of organelles
Fig. 1. Ethambutol (EB) blocks autophagosome-lysosome fusion and autophagic flux. A & B. Huh-7 cells treated with various concentrations of EB for 24 h were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin levels was performed. C & D. Huh-7 cells treated with 250 mg/ml EB for various time periods were subjected to western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. E & F. Western blotting analysis of Huh-7 cells treated with 250 mg/ml EB for 24 h with anti-p62 antibodies. Quantification of relative p62 levels relative to actin is shown in the graph. G & H. After treatment with or without a saturating concentration of EB (1000 mg/ml) for 24 h, with or without 10 mg/ml E64d and 10 mg/ml pepstatin A for 24 h, Huh-7 cells were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. I & J. After treatment with or without a saturating concentration of EB (1000 mg/ml) for 24 h, with or without 400 nM bafilomycin A1 for 3 h, Huh-7 cells were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. K. 24 h after treatment with 250 mg/ml EB, Huh-7 cells were stained with anti-LAMP1 (red) and anti-LC3 (green) antibodies and analyzed by confocal microscopy. The inset shows higher magnification of the boxed area. Scale bar: 20 mm. L. 24 h after transfection of mRFP-GFPLC3 plasmid DNAs, Huh-7 cells were treated with various concentrations of EB for 24 h. Cells were analyzed using live-cell imaging. The upper insets show higher magnifications of the boxed areas. Scale bar: 10 mm. NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
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in each cell was calculated using ImageJ software (National Institutes for Health, USA). 2.10. Statistical analysis When only two groups were compared, data were analyzed using unpaired two-tailed Student's t test. Multiple data sets were analyzed using one-way analysis of variance with Tukey's post-hoc test. 3. Results and discussion 3.1. EB blocks autophagosome-lysosome fusion and autophagic flux Based on a previous report showing that EB blocks autophagic flux in rat retinal cells, we examined whether this effect is ubiquitous and independent of cell type. First, we examined levels of the microtubule-associated protein 1 light chain 3 (LC3)-II, a LC3-
phosphatidyl-ethanolamine conjugate and a marker of autophagosomes in various cell lines [12]. After 24 h treatment with EB, levels of LC3-II increased with 100e1000 mg/ml EB in Huh-7 cells (Fig. 1A, B), 100e1000 mg/ml EB in HeLa cells and 250 mg/ml EB in SH-SY5Y cells (Supplementary Fig. S1AeD). EB also increased the LC3-II/actin ratio in a time-dependent manner in Huh-7 (Fig. 1C, D) and HeLa cells (Supplementary Fig. S1E, F). This was also seen in HeLa cells stably expressing GFP-LC3 (Supplementary Fig. S1G) [14,15]. The expression levels of p62, a well-known autophagic substrate, were also increased by EB treatment in Huh-7 (Fig. 1E, F), HeLa and SH-SY5Y cells (Supplementary Fig. S1HeK). LC3-II accumulates following increased upstream autophagosome formation or impaired downstream autophagosome-lysosome fusion. To distinguish between these two possibilities, we assayed LC3-II in the presence of E64D plus pepstatin A (pepA) or bafilomycin A1, which inhibit lysosomal proteases or blocks downstream autophagosome-lysosome fusion and activation of lysosomal proteases, respectively [16e18]. At 1000 mg/ml EB, a saturation
Fig. 2. Ethambutol (EB) increased the number of enlarged lysosomes and enhanced Zn2þ accumulation in lysosomes. A. Huh-7 cells treated with or without 250 mg/ml EB for 24 h were visualized directly by differential interference contrast (DIC) and stained with anti-LAMP1 antibodies and analyzed by confocal microscopy. Scale bar: 10 mm. BeG. Electron micrographs of Huh-7 cells treated with various concentrations of EB for 24 h. Lower panels (C, E, G) show higher magnifications of the boxed areas in the upper panels (B, D, F). Arrowhead: lysosome; black vertical arrow: endosome; asterisk: enlarged lysosome; white arrow: autolysosome; black horizontal arrow: autophagosome. White scale bar: 5 mm; black scale bar: 1 mm. H. Percentage total volume of each organelle compared with the total cell volume in Huh-7 cells treated with various concentrations of EB for 24 h are shown in the bar chart. I. Huh-7 cells were transfected with lgp120-mCherry and treated with EB for 24 h. These cells were stained with FluoZin3 and analyzed by live-cell imaging. Insets show higher magnification of the boxed area. Scale bar: 10 mm.
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concentration sufficient to block autophagic flux, the presence of E64d (10 mg/ml) plus pepstatin A (10 mg/ml) or bafilomycin (400 nM) in Huh-7 cells (Fig. 1GeJ) did not significantly increase the ratios of LC3-II/actin. Taken together, EB-induced LC3-II accumulation results from impaired downstream autophagosomelysosome fusion. Immunocytochemical analysis revealed that EB increases the number of autophagosomes positive for LC3-II near LAMP1-positive enlarged vesicles (Fig. 1K). To further investigate autophagosome-lysosome fusion activity and consequent insufficient autophagic flux, we analyzed cells transiently expressing a tandem fluorescent-tagged LC3 (tfLC3) plasmid DNA, according to a well-established protocol [19]. This plasmid was tagged with monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP). After EB treatment, there was no significant difference in the numbers of autolysosomes positive for mRFP only; however, the numbers of autophagosomes positive for both mRFP and GFP were significantly increased in a dose-dependent manner (Fig. 1L). These results indicate that high concentrations of EB treatment blocked autophagic flux because autophagosomes were unable to fuse with lysosomes, thus resulting in the accumulation of autophagosomes.
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3.2. EB increases the number of enlarged lysosomes containing increased concentrations of Zn2þ Because EB induced multiple large structures positive for the lysosomal marker LAMP1 (Supplementary Fig. S2A), we investigated lysosomal morphological changes in more detail by electron microscopy. As shown in Fig. 2A-F, the number of normal lysosomes (Fig. 2A, B: highly-dense organelles indicated by the arrowhead) around the nucleus gradually decreased in an EB dose-dependent manner. In contrast, the nucleus was surrounded by low-density enlarged lysosomes (Fig. 2D, F: asterisk), which were similar to those seen with chloroquine treatment, an anti-malarial agent that inhibits lysosomal acidification [20]. Although the number of normal lysosomes was significantly decreased, that of enlarged lysosomes was markedly increased in a dose-dependent manner, as revealed by stereotaxic methods (Fig. 2G). A previous study revealed that lysosomal Zn2þ accumulation was enhanced by EB [5], so, we checked if EB changes the intracellular distribution and concentration of Zn2þ using FluoZin3® reagent, which accumulates in organelles in a Zn2þ concentration-dependent manner in cells overexpressing mCherrylgp120, a mouse homolog of human LAMP1. We observed that
Fig. 3. Ethambutol (EB) treatment inhibits lysosomal acidification via enhancement of Zn2þ uptake into lysosomes A & B. Huh-7 cells treated with 250 mg/ml EB for 24 h were stained with 1 mM LysoSensor Green DND-153 for 30 min, and were analyzed by flow cytometry. **p < 0.01. C. Huh-7 cells treated with various concentrations of EB for 24 h were stained with 5 mg/ml LysoSensor Yellow/Blue for 5 min. Lysosomal pH was measured using In Cell Analyzer 1000. **p < 0.01, ***p < 0.001. D. Huh-7 cells treated with various concentrations of EB for 24 h were stained with 50 nM LysoTracker Red (Red) for 30 min and 5 mM FluoZin3 (Green) for 30 min, and analyzed by live-cell imaging. Scale bar: 5 mm.
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enlarged vesicles surrounded by lgp120-mCherry contained increased FluoZin3 fluorescence in cells treated with EB (Fig. 2H). In addition, flow cytometry analysis revealed that intracellular FluoZin3 intensity was significantly increased by EB in a dose-dependent manner (Fig. 2I). We confirmed that EB induced FluoZin3 accumulation in cells cultured in either plain DMEM or Zn2þ-deficient medium, suggesting that lysosomal Zn2þ accumulation was caused by
intracellular redistribution (Supplementary Fig. S2B). 3.3. EB treatment inhibits lysosomal acidification via enhanced lysosomal uptake of Zn2þ We next investigated whether lysosomal pH was affected by EB treatment in Huh-7 cells using various LysoSensor agents [21]. EB
Fig. 4. Pyrithione-induced Zn2þ influx to the cytoplasm results in lysosomal neutralization associated with lysosomal Zn2þ accumulation. A. Huh-7 cells were transfected with lgp120-mCherry and treated with or without 10 mM pyrithione (PT, Zn ionophore) and with or without 500 mM ZnCl2. These cells were stained with FluoZin3 and analyzed by live-cell imaging. Scale bar: 10 mm. B & C. After 1 h treatment with or without 10 mM PT and with or without 500 mM ZnCl2, Huh-7 cells were stained with LysoTracker Red and FluoZin3 and analyzed by live-cell imaging. Time-lapse images were obtained every 10 min (C). Scale bar: 20 mm.
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treatment significantly increased the mean of fluorescent intensity of LysoSensor DND-153, which only fluoresces green in neutral organelles (Fig. 3A, B). The numbers of cells positive for LysoSensor Blue, which fluoresces blue in neutral organelles, were increased by EB treatment (Supplementary Fig. S3A, B). Similarly, using LysoSensor Yellow/Blue with the IN Cell Analyzer 1000®, we confirmed that the mean lysosomal pH was significantly increased in a dosedependent manner by up to 250 mg/ml EB (Fig. 3C). We also found that 50 or 100 mg/ml EB treatment for 24 h increased the number of lysosomes positive for both FluoZin3 and LysoTracker Red, which exhibited decreased intensity in lysosomes with higher pHs (Fig. 3D). Although enlarged vacuoles were observed following 250 mg/ml EB treatment, most were only positive for FluoZin3, but not for LysoTracker Red, indicating that EB induced Zn2þ accumulated in lysosomes, while simultaneously elevating their luminal pH. 3.4. Zn2þ ionophore-induced intralysosomal Zn2þ accumulation and lysosomal neutralization To further investigate the association of lysosomal neutralization and Zn2þ storage in lysosomes, we examined the phenomena following the forced upregulation of the cytoplasmic Zn2þ concentration using pyrithione (PT), which activates Zn2þ uptake into the cytoplasm [22], in cells overexpressing lgp120-mCherry. No apparent FluoZin3 fluorescence was detected in cells without PT treatment; however, vesicles positive for FluoZin3 and mCherry were detected, implying that Zn2þ accumulated in the lysosomes (Fig. 4A). Furthermore, lysosomes in cells treated with PT and cultured in Zn2þ-enriched DMEM were much more enlarged than cells treated only with PT. To monitor whether the lysosomal accumulation of Zn2þ was associated with lysosomal neutralization, we analyzed changes in the fluorescent intensity of LysoTracker Red. As expected, PT treatment in cells cultured in Zn2þenriched DMEM significantly reduced lysosomal LysoTracker Red intensity compared with PT treatment alone (Fig. 4B). Finally, to confirm this phenomenon under continuous observation, we performed live-cell imaging experiments. Although cells cultured in Zn2þ-enriched DMEM without PT showed static LysoTracker Red fluorescence, those cultured the same medium with PT caused rapid infiltration of FluoZin3 into lysosomes, with simultaneous loss of LysoTracker fluorescence within 10 min (Fig. 4C). These results indicate that high concentrations of intracellular Zn2þ induced lysosomal neutralization, in accordance with their function as zincbuffering organelles, which is also consistent with our EB treatment results. In this study, we revealed that autophagy was inhibited in various cell lines, including Huh-7 (hepatocellular carcinoma), HeLa (uterus cervical carcinoma) and SH-SY5Y (human neuroblastoma) cells, indicating that this phenomenon could be ubiquitous, resulting in systemic side effects. Furthermore, we determined the mechanism of EB-induced lysosomal neutralization and Zn2þ accumulation, which leads to autophagic insufficiency. Zn2þ transport across the plasma membrane and limiting membranes of intracellular organelles is mediated by two separate families of integral membrane proteins: the Zrt and Irt-like proteins (Zip), also known as solute-linked carrier 39A (SLC39A) family members, and the zinc transporter (ZnT)/cation diffusion facilitator/SLC30 family members [23,24]. Zip proteins accelerate Zn2þ uptake into the cytosol from the extracellular space or from the luminal compartments of cytoplasmic vesicles and organelles. In contrast, most ZnT family members facilitate the removal of Zn2þ from the cytosol, by either transporting it out of the cell or into the lumen of vesicles and organelles. However, no apparent ZnTs have been identified that regulate lysosomal acidification. Therefore,
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future experiments should be performed focusing on ZnTs localized in lysosomes to determine the overall mechanism of lysosomal pH control. Competing financial interests All authors declared that they have no competing financial interests. Acknowledgments We thank Prof. Yoshinori Ohsumi, Drs. Tomoko Kawamata, Yoshio Fujitani, Ayako Fukunaka, and Junzo Kinoshita for critical comments. We are grateful for a Juntendo University Young Investigator Award 2014 (D. Y.), Grant-in-Aid for Scientific Research on Priority Areas (S. S. (25111007, 25111001), N. H. (23111003)) for Young Scientists (A) (S. S. (23689046)), a Challenging Exploratory Research Grant (S. S. (24659435)), a Grant-in-Aid for Scientific Research (C) (N. F. (15K09325)), a Grant-in-Aid for Research Activity Start-up (K. I. (26893263)) from the Japan Society for the Promotion of Science, and grants from the Takeda Scientific Foundation, the Cell Science Research Foundation and the Nakajima Foundation (S. S). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.171. References [1] A.M. Bode, Z. Dong, The enigmatic effects of caffeine in cell cycle and cancer, Cancer Lett. 247 (2007) 26e39. [2] WHO, WHO Global Tuberculosis Report 2015, 2015. Geneva. [3] R.K. Tsai, Y.H. Lee, Reversibility of ethambutol optic neuropathy, J. Ocul. Pharmacol. Ther. 13 (1997) 473e477. [4] E.J. Lee, S.J. Kim, H.K. Choung, J.H. Kim, Y.S. Yu, Incidence and clinical features of ethambutol-induced optic neuropathy in Korea, J. Neuroophthalmol. 28 (2008) 269e277. [5] H. Chung, Y.H. Yoon, J.J. Hwang, K.S. Cho, J.Y. Koh, J.G. Kim, Ethambutolinduced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells, Toxicol. Appl. Pharmacol. 235 (2009) 163e170. [6] S.P. Huang, J.Y. Chien, R.K. Tsai, Ethambutol induces impaired autophagic flux and apoptosis in the rat retina, Dis. Models Mech. 8 (2015) 977e987. [7] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through cellular self-digestion, Nature 451 (2008) 1069e1075. [8] D.C. Rubinsztein, The roles of intracellular protein-degradation pathways in neurodegeneration, Nature 443 (2006) 780e786. [9] L. Galluzzi, F. Pietrocola, J.M. Bravo-San Pedro, R.K. Amaravadi, E.H. Baehrecke, F. Cecconi, P. Codogno, J. Debnath, D.A. Gewirtz, V. Karantza, A. Kimmelman, S. Kumar, B. Levine, M.C. Maiuri, S.J. Martin, J. Penninger, M. Piacentini, D.C. Rubinsztein, H.U. Simon, A. Simonsen, A.M. Thorburn, G. Velasco, K.M. Ryan, G. Kroemer, Autophagy in malignant transformation and cancer progression, EMBO J. (2015), http://dx.doi.org/10.15252/embj.201490784. [10] C. De Duve, The Lysosome 208, Scientific American, 1963, pp. 64e72. [11] C. De Duve, R. Wattiaux, Functions of lysosomes, Annu. Rev. Physiol. 28 (1966) 435e492. [12] N. Mizushima, M. Komatsu, Autophagy: renovation of cells and tissues, Cell 147 (2011) 728e741. [13] S. Saiki, Y. Sasazawa, Y. Imamichi, S. Kawajiri, T. Fujimaki, I. Tanida, H. Kobayashi, F. Sato, S. Sato, K. Ishikawa, M. Imoto, N. Hattori, Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition, Autophagy 7 (2011) 176e187. [14] D.C. Rubinsztein, A.M. Cuervo, B. Ravikumar, S. Sarkar, V. Korolchuk, S. Kaushik, D.J. Klionsky, In search of an “autophagomometer”, Autophagy 5 (2009) 585e589. [15] I. Tanida, T. Ueno, E. Kominami, LC3 and autophagy, Methods Mol. Biol. 445 (2008) 77e88. [16] A. Yamamoto, Y. Tagawa, T. Yoshimori, Y. Moriyama, R. Masaki, Y. Tashiro, Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H4-II-E cells, Cell Struct. Funct. 23 (1998) 33e42. [17] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell, 140 313e326.
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D. Yamada et al. / Biochemical and Biophysical Research Communications 471 (2016) 109e116
[18] T. Yoshimori, A. Yamamoto, Y. Moriyama, M. Futai, Y. Tashiro, Bafilomycin A1, a specific inhibitor of vacuolar-type H(þ)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells, J. Biol. Chem. 266 (1991) 17707e17712. [19] S. Kimura, T. Noda, T. Yoshimori, Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3, Autophagy 3 (2007) 452e460. [20] N. Zheng, X. Zhang, G.R. Rosania, Effect of phospholipidosis on the cellular pharmacokinetics of chloroquine, J. Pharmacol. Exp. Ther. 336 (2011) 661e671.
[21] J. Han, K. Burgess, Fluorescent indicators for intracellular pH, Chem. Rev. 110 (2010) 2709e2728. [22] B.M. Krenn, E. Gaudernak, B. Holzer, K. Lanke, F.J. Van Kuppeveld, J. Seipelt, Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections, J. Virol. 83 (2009) 58e64. [23] T. Kambe, Y. Yamaguchi-Iwai, R. Sasaki, M. Nagao, Overview of mammalian zinc transporters, Cell Mol. Life Sci. 61 (2004) 49e68. [24] J.P. Liuzzi, R.J. Cousins, Mammalian zinc transporters, Annu. Rev. Nutr. 24 (2004) 151e172.
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Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation Daisuke Yamada a, Shinji Saiki a, *, Norihiko Furuya a, b, Kei-Ichi Ishikawa a, Yoko Imamichi a, Taiho Kambe c, Tsutomu Fujimura d, Takashi Ueno d, Masato Koike e, Katsuhiko Sumiyoshi f, Nobutaka Hattori a, b, ** a
Department of Neurology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan Department of Research and Therapeutics for Movement Disorders, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan Laboratory of Biosignals and Response, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan d Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan e Department of Cell Biology and Neuroscience, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan f Department of Health and Nutrion, Tokiwa University, Ibaraki, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2016 Accepted 28 January 2016 Available online 4 February 2016
Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn2þ accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn2þ accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Ethambutol Autophagy Zinc Lysosome
1. Introduction Ethambutol (EB) is widely used as an anti-mycobacterial agent and has a diverse range of pharmacological effects [1]. The World Health Organization (WHO) estimates that approximately 55% of patients with TB are taking EB as a treatment for TB or for Mycobacterium avium infection (Global Tuberculosis Report 2014) [2]. The WHO also estimates that there are around 8.6 million new cases of TB each year. Patients with TB who are being treated with EB occasionally develop serious side effects, such as liver
Abbreviations: EB, ethambutol; TB, tuberculosis. * Corresponding author. Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. ** Corresponding author. Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan E-mail addresses: [email protected] (S. Saiki), [email protected] (N. Hattori).
dysfunction, optic neuropathy and visual field abnormalities [3]. Multiple types of EB-induced cell damage have been reported, including Zn2þ mislocalization to lysosomes, mitochondrial protein synthesis blockade, lysosomal membrane permeabilization and the generation of reactive oxygen species [4,5]. Very recently, EB was shown to induce cell death of the rat retinal cells due to impaired autophagy [6]; however, the precise molecular mechanism was not fully elucidated. Autophagy is a key mechanism for cellular homeostasis and stress adaptation, which facilitates the lysosomal degradation of intracellular proteins or organelles sequestered within autophagosomes [7e9]. Normal lysosomal acidification is essential for autophagosome-lysosome fusion and the activities of lysosomal hydrolytic enzymes, both of which are indispensable for autophagy termination [10e12]. Herein, we reveal that EB is capable of retarding autophagic flux via inhibition of autophagosomelysosome fusion. We further demonstrate that EB induces enlarged lysosomes to lose their characteristic acidic environment and form Zn2þ accumulations, suggesting of a novel molecular mechanism of EB toxicity.
http://dx.doi.org/10.1016/j.bbrc.2016.01.171 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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2. Materials and methods 2.1. Cell lines Human hepatocarcinoma Huh-7 cells (RIKEN Cell Bank) were maintained in RPMI1640 medium (SigmaeAldrich) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich) and 100 U/ml penicillin/streptomycin (SigmaeAldrich) at 37 C and 5% CO2. Human-derived HeLa and SH-SY5Y cells were maintained in Dulbecco's-modified Eagle's medium (DMEM) supplemented with 10% FBS (SigmaeAldrich) and 100 U/ml penicillin/streptomycin (Sigma Aldrich) at 37 C and 5% CO2. A HeLa GFP-LC3 stable cell line was established as previously described [13]. 2.2. Cell culture in zinc-deficient medium FBS was mixed with 10% Chelex-100 resin (Bio-Rad) at 4 C overnight. After centrifugation at 3500 rpm for 5 min, the supernatant was sterilized using a 0.22-mm filter. The chelated FBS was added to culture medium to a final concentration of 10% (DMEM10% chelated FBS). Huh-7 cells were cultured in the zinc-deficient medium for 9 days. Meanwhile, cell passaging was performed once every 2 days.
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E4630), E64d (SigmaeAldrich, E8640), pepstatin A (SigmaeAldrich, P5318), bafilomycin A1 (SigmaeAldrich, B1793), pyrithione (2mercaptopyridine N-oxide sodium salt; SigmaeAldrich, H3261), ZnCl2 (SigmaeAldrich, 229997). 2.6. Plasmid DNAs tfLC3, encoding mRFP-EGFP-LC3, which was originally generated by the Yoshimori group, was purchased from Origene (Plasmid 21075). Mouse lgp120 was cloned into the mCherry vector. 2.7. Western blotting Cell pellets were lysed on ice in RIPA buffer (25 mM TriseHCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) in the presence of protease inhibitors (Roche, Basel, Switzerland) for 20 min. Western blotting was performed according to previously published reports [13]. The antibodies used were as follows: anti-actin (Millipore, clone C4), anti-LC3 (MBL, clone 4E12), anti-p62 (Progen Biotechnik, GP62-C). Antibody signals were enhanced with chemifluorescent methods from GE HealthCare as previously described [13]. 2.8. Immunofluorescence
2.3. Lysosomal pH assay To observe lysosomal alkalinization, we incubated Huh-7 cells with 1 mM LysoSensor Green DND-153 (Life Technologies) for 30 min or 5 mg/ml of LysoSensor Yellow/Blue DND-160 (Life Technologies) for 5 min or 50 nM of LysoTracker Red DND-99 (Life Technologies) for 30 min in RPMI1640. Cells were washed twice with phosphate-buffered saline (PBS) and immediately analyzed using a flow cytometer (FACS Fortessa, BD Biosciences). To quantify lysosomal pH, we incubated Huh-7 cells with 5 mg/ml of LysoSensor Yellow/Blue DND-160 for 5 min and assayed with In Cell Analyzer 1000 (GE Healthcare) according to the manufacturer's protocol. Fluorescent intensities of each emission (460 nm, 535 nm) per visual field were automatically calculated according to the pH standard curve. 2.4. Zn
2þ
accumulation assay
To observe Zn2þ accumulation in lysosomes, we incubated Huh7 cells with 5 mM of FluoZin3 for 30 min. The fluorescence of FluoZin-3 was analyzed using a flow cytometer (FACS Fortessa BD Biosciences), and images were acquired on a Zeiss LSM510 META confocal microscope (63 1.4 NA), at room temperature using Zeiss LSM510 v.3.2 software. Adobe Photoshop 7.0 (Adobe Systems, Inc.) was used for subsequent image processing. 2.5. Compounds Compounds used in this study included EB (SigmaeAldrich,
Huh-7 cells were seeded on glass coverslips. Twenty-four hours later, cells were transfected or treated with the relevant compounds. Cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 50 mg/ml digitonin in PBS for 15 min. To block nonspecific antigenic sites, cells were incubated with 10% FBS and 1% bovine serum albumin in PBS for 30 min. Cells were immunostained with the following antibodies: anti-FLAG (SigmaeAldrich, F7425; 1:500), anti-LC3B (1:500) (SigmaeAldrich, L7543), anti-LAMP1 (1:50) and anti-LAMP2 (1:50) (Development Studies Hybridoma Bank, clone H4B4). Twenty-four hours later, cells were incubated with AlexaFluor 488 anti-rabbit IgG (Life Technologies; 1:500) or AlexaFluor 546 anti-mouse IgG (Life Technologies; 1:500) for 1 h. Cells were embedded with VectaShield containing DAPI (Vector Laboratories, H-1200), and images were acquired on a Zeiss LSM510 META confocal microscope (63 1.4 NA) at room temperature using Zeiss LSM510 v.3.2 software. Adobe Photoshop 7.0 (Adobe Systems Inc.) was used for subsequent image processing. 2.9. Electron microscopy Huh-7 cells were seeded on glass coverslips. Twenty-four hours later, cells were treated with various concentrations of EB for 24 h, then fixed with 4% glutaraldehyde in 0.1 N sodium-cacodylate at room temperature for 1 h, and processed for electron microscopy using a standard protocol. Cells were imaged at 8000 magnification by transmission electron microscope (HT7700; Hitachi, Tokyo, Japan). The volume fraction of organelles
Fig. 1. Ethambutol (EB) blocks autophagosome-lysosome fusion and autophagic flux. A & B. Huh-7 cells treated with various concentrations of EB for 24 h were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin levels was performed. C & D. Huh-7 cells treated with 250 mg/ml EB for various time periods were subjected to western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. E & F. Western blotting analysis of Huh-7 cells treated with 250 mg/ml EB for 24 h with anti-p62 antibodies. Quantification of relative p62 levels relative to actin is shown in the graph. G & H. After treatment with or without a saturating concentration of EB (1000 mg/ml) for 24 h, with or without 10 mg/ml E64d and 10 mg/ml pepstatin A for 24 h, Huh-7 cells were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. I & J. After treatment with or without a saturating concentration of EB (1000 mg/ml) for 24 h, with or without 400 nM bafilomycin A1 for 3 h, Huh-7 cells were analyzed by western blotting with anti-LC3 and anti-actin antibodies. Densitometry analysis of LC3-II levels relative to actin was performed. K. 24 h after treatment with 250 mg/ml EB, Huh-7 cells were stained with anti-LAMP1 (red) and anti-LC3 (green) antibodies and analyzed by confocal microscopy. The inset shows higher magnification of the boxed area. Scale bar: 20 mm. L. 24 h after transfection of mRFP-GFPLC3 plasmid DNAs, Huh-7 cells were treated with various concentrations of EB for 24 h. Cells were analyzed using live-cell imaging. The upper insets show higher magnifications of the boxed areas. Scale bar: 10 mm. NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
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in each cell was calculated using ImageJ software (National Institutes for Health, USA). 2.10. Statistical analysis When only two groups were compared, data were analyzed using unpaired two-tailed Student's t test. Multiple data sets were analyzed using one-way analysis of variance with Tukey's post-hoc test. 3. Results and discussion 3.1. EB blocks autophagosome-lysosome fusion and autophagic flux Based on a previous report showing that EB blocks autophagic flux in rat retinal cells, we examined whether this effect is ubiquitous and independent of cell type. First, we examined levels of the microtubule-associated protein 1 light chain 3 (LC3)-II, a LC3-
phosphatidyl-ethanolamine conjugate and a marker of autophagosomes in various cell lines [12]. After 24 h treatment with EB, levels of LC3-II increased with 100e1000 mg/ml EB in Huh-7 cells (Fig. 1A, B), 100e1000 mg/ml EB in HeLa cells and 250 mg/ml EB in SH-SY5Y cells (Supplementary Fig. S1AeD). EB also increased the LC3-II/actin ratio in a time-dependent manner in Huh-7 (Fig. 1C, D) and HeLa cells (Supplementary Fig. S1E, F). This was also seen in HeLa cells stably expressing GFP-LC3 (Supplementary Fig. S1G) [14,15]. The expression levels of p62, a well-known autophagic substrate, were also increased by EB treatment in Huh-7 (Fig. 1E, F), HeLa and SH-SY5Y cells (Supplementary Fig. S1HeK). LC3-II accumulates following increased upstream autophagosome formation or impaired downstream autophagosome-lysosome fusion. To distinguish between these two possibilities, we assayed LC3-II in the presence of E64D plus pepstatin A (pepA) or bafilomycin A1, which inhibit lysosomal proteases or blocks downstream autophagosome-lysosome fusion and activation of lysosomal proteases, respectively [16e18]. At 1000 mg/ml EB, a saturation
Fig. 2. Ethambutol (EB) increased the number of enlarged lysosomes and enhanced Zn2þ accumulation in lysosomes. A. Huh-7 cells treated with or without 250 mg/ml EB for 24 h were visualized directly by differential interference contrast (DIC) and stained with anti-LAMP1 antibodies and analyzed by confocal microscopy. Scale bar: 10 mm. BeG. Electron micrographs of Huh-7 cells treated with various concentrations of EB for 24 h. Lower panels (C, E, G) show higher magnifications of the boxed areas in the upper panels (B, D, F). Arrowhead: lysosome; black vertical arrow: endosome; asterisk: enlarged lysosome; white arrow: autolysosome; black horizontal arrow: autophagosome. White scale bar: 5 mm; black scale bar: 1 mm. H. Percentage total volume of each organelle compared with the total cell volume in Huh-7 cells treated with various concentrations of EB for 24 h are shown in the bar chart. I. Huh-7 cells were transfected with lgp120-mCherry and treated with EB for 24 h. These cells were stained with FluoZin3 and analyzed by live-cell imaging. Insets show higher magnification of the boxed area. Scale bar: 10 mm.
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concentration sufficient to block autophagic flux, the presence of E64d (10 mg/ml) plus pepstatin A (10 mg/ml) or bafilomycin (400 nM) in Huh-7 cells (Fig. 1GeJ) did not significantly increase the ratios of LC3-II/actin. Taken together, EB-induced LC3-II accumulation results from impaired downstream autophagosomelysosome fusion. Immunocytochemical analysis revealed that EB increases the number of autophagosomes positive for LC3-II near LAMP1-positive enlarged vesicles (Fig. 1K). To further investigate autophagosome-lysosome fusion activity and consequent insufficient autophagic flux, we analyzed cells transiently expressing a tandem fluorescent-tagged LC3 (tfLC3) plasmid DNA, according to a well-established protocol [19]. This plasmid was tagged with monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP). After EB treatment, there was no significant difference in the numbers of autolysosomes positive for mRFP only; however, the numbers of autophagosomes positive for both mRFP and GFP were significantly increased in a dose-dependent manner (Fig. 1L). These results indicate that high concentrations of EB treatment blocked autophagic flux because autophagosomes were unable to fuse with lysosomes, thus resulting in the accumulation of autophagosomes.
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3.2. EB increases the number of enlarged lysosomes containing increased concentrations of Zn2þ Because EB induced multiple large structures positive for the lysosomal marker LAMP1 (Supplementary Fig. S2A), we investigated lysosomal morphological changes in more detail by electron microscopy. As shown in Fig. 2A-F, the number of normal lysosomes (Fig. 2A, B: highly-dense organelles indicated by the arrowhead) around the nucleus gradually decreased in an EB dose-dependent manner. In contrast, the nucleus was surrounded by low-density enlarged lysosomes (Fig. 2D, F: asterisk), which were similar to those seen with chloroquine treatment, an anti-malarial agent that inhibits lysosomal acidification [20]. Although the number of normal lysosomes was significantly decreased, that of enlarged lysosomes was markedly increased in a dose-dependent manner, as revealed by stereotaxic methods (Fig. 2G). A previous study revealed that lysosomal Zn2þ accumulation was enhanced by EB [5], so, we checked if EB changes the intracellular distribution and concentration of Zn2þ using FluoZin3® reagent, which accumulates in organelles in a Zn2þ concentration-dependent manner in cells overexpressing mCherrylgp120, a mouse homolog of human LAMP1. We observed that
Fig. 3. Ethambutol (EB) treatment inhibits lysosomal acidification via enhancement of Zn2þ uptake into lysosomes A & B. Huh-7 cells treated with 250 mg/ml EB for 24 h were stained with 1 mM LysoSensor Green DND-153 for 30 min, and were analyzed by flow cytometry. **p < 0.01. C. Huh-7 cells treated with various concentrations of EB for 24 h were stained with 5 mg/ml LysoSensor Yellow/Blue for 5 min. Lysosomal pH was measured using In Cell Analyzer 1000. **p < 0.01, ***p < 0.001. D. Huh-7 cells treated with various concentrations of EB for 24 h were stained with 50 nM LysoTracker Red (Red) for 30 min and 5 mM FluoZin3 (Green) for 30 min, and analyzed by live-cell imaging. Scale bar: 5 mm.
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enlarged vesicles surrounded by lgp120-mCherry contained increased FluoZin3 fluorescence in cells treated with EB (Fig. 2H). In addition, flow cytometry analysis revealed that intracellular FluoZin3 intensity was significantly increased by EB in a dose-dependent manner (Fig. 2I). We confirmed that EB induced FluoZin3 accumulation in cells cultured in either plain DMEM or Zn2þ-deficient medium, suggesting that lysosomal Zn2þ accumulation was caused by
intracellular redistribution (Supplementary Fig. S2B). 3.3. EB treatment inhibits lysosomal acidification via enhanced lysosomal uptake of Zn2þ We next investigated whether lysosomal pH was affected by EB treatment in Huh-7 cells using various LysoSensor agents [21]. EB
Fig. 4. Pyrithione-induced Zn2þ influx to the cytoplasm results in lysosomal neutralization associated with lysosomal Zn2þ accumulation. A. Huh-7 cells were transfected with lgp120-mCherry and treated with or without 10 mM pyrithione (PT, Zn ionophore) and with or without 500 mM ZnCl2. These cells were stained with FluoZin3 and analyzed by live-cell imaging. Scale bar: 10 mm. B & C. After 1 h treatment with or without 10 mM PT and with or without 500 mM ZnCl2, Huh-7 cells were stained with LysoTracker Red and FluoZin3 and analyzed by live-cell imaging. Time-lapse images were obtained every 10 min (C). Scale bar: 20 mm.
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treatment significantly increased the mean of fluorescent intensity of LysoSensor DND-153, which only fluoresces green in neutral organelles (Fig. 3A, B). The numbers of cells positive for LysoSensor Blue, which fluoresces blue in neutral organelles, were increased by EB treatment (Supplementary Fig. S3A, B). Similarly, using LysoSensor Yellow/Blue with the IN Cell Analyzer 1000®, we confirmed that the mean lysosomal pH was significantly increased in a dosedependent manner by up to 250 mg/ml EB (Fig. 3C). We also found that 50 or 100 mg/ml EB treatment for 24 h increased the number of lysosomes positive for both FluoZin3 and LysoTracker Red, which exhibited decreased intensity in lysosomes with higher pHs (Fig. 3D). Although enlarged vacuoles were observed following 250 mg/ml EB treatment, most were only positive for FluoZin3, but not for LysoTracker Red, indicating that EB induced Zn2þ accumulated in lysosomes, while simultaneously elevating their luminal pH. 3.4. Zn2þ ionophore-induced intralysosomal Zn2þ accumulation and lysosomal neutralization To further investigate the association of lysosomal neutralization and Zn2þ storage in lysosomes, we examined the phenomena following the forced upregulation of the cytoplasmic Zn2þ concentration using pyrithione (PT), which activates Zn2þ uptake into the cytoplasm [22], in cells overexpressing lgp120-mCherry. No apparent FluoZin3 fluorescence was detected in cells without PT treatment; however, vesicles positive for FluoZin3 and mCherry were detected, implying that Zn2þ accumulated in the lysosomes (Fig. 4A). Furthermore, lysosomes in cells treated with PT and cultured in Zn2þ-enriched DMEM were much more enlarged than cells treated only with PT. To monitor whether the lysosomal accumulation of Zn2þ was associated with lysosomal neutralization, we analyzed changes in the fluorescent intensity of LysoTracker Red. As expected, PT treatment in cells cultured in Zn2þenriched DMEM significantly reduced lysosomal LysoTracker Red intensity compared with PT treatment alone (Fig. 4B). Finally, to confirm this phenomenon under continuous observation, we performed live-cell imaging experiments. Although cells cultured in Zn2þ-enriched DMEM without PT showed static LysoTracker Red fluorescence, those cultured the same medium with PT caused rapid infiltration of FluoZin3 into lysosomes, with simultaneous loss of LysoTracker fluorescence within 10 min (Fig. 4C). These results indicate that high concentrations of intracellular Zn2þ induced lysosomal neutralization, in accordance with their function as zincbuffering organelles, which is also consistent with our EB treatment results. In this study, we revealed that autophagy was inhibited in various cell lines, including Huh-7 (hepatocellular carcinoma), HeLa (uterus cervical carcinoma) and SH-SY5Y (human neuroblastoma) cells, indicating that this phenomenon could be ubiquitous, resulting in systemic side effects. Furthermore, we determined the mechanism of EB-induced lysosomal neutralization and Zn2þ accumulation, which leads to autophagic insufficiency. Zn2þ transport across the plasma membrane and limiting membranes of intracellular organelles is mediated by two separate families of integral membrane proteins: the Zrt and Irt-like proteins (Zip), also known as solute-linked carrier 39A (SLC39A) family members, and the zinc transporter (ZnT)/cation diffusion facilitator/SLC30 family members [23,24]. Zip proteins accelerate Zn2þ uptake into the cytosol from the extracellular space or from the luminal compartments of cytoplasmic vesicles and organelles. In contrast, most ZnT family members facilitate the removal of Zn2þ from the cytosol, by either transporting it out of the cell or into the lumen of vesicles and organelles. However, no apparent ZnTs have been identified that regulate lysosomal acidification. Therefore,
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future experiments should be performed focusing on ZnTs localized in lysosomes to determine the overall mechanism of lysosomal pH control. Competing financial interests All authors declared that they have no competing financial interests. Acknowledgments We thank Prof. Yoshinori Ohsumi, Drs. Tomoko Kawamata, Yoshio Fujitani, Ayako Fukunaka, and Junzo Kinoshita for critical comments. We are grateful for a Juntendo University Young Investigator Award 2014 (D. Y.), Grant-in-Aid for Scientific Research on Priority Areas (S. S. (25111007, 25111001), N. H. (23111003)) for Young Scientists (A) (S. S. (23689046)), a Challenging Exploratory Research Grant (S. S. (24659435)), a Grant-in-Aid for Scientific Research (C) (N. F. (15K09325)), a Grant-in-Aid for Research Activity Start-up (K. I. (26893263)) from the Japan Society for the Promotion of Science, and grants from the Takeda Scientific Foundation, the Cell Science Research Foundation and the Nakajima Foundation (S. S). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.171. References [1] A.M. Bode, Z. Dong, The enigmatic effects of caffeine in cell cycle and cancer, Cancer Lett. 247 (2007) 26e39. [2] WHO, WHO Global Tuberculosis Report 2015, 2015. Geneva. [3] R.K. Tsai, Y.H. Lee, Reversibility of ethambutol optic neuropathy, J. Ocul. Pharmacol. Ther. 13 (1997) 473e477. [4] E.J. Lee, S.J. Kim, H.K. Choung, J.H. Kim, Y.S. Yu, Incidence and clinical features of ethambutol-induced optic neuropathy in Korea, J. Neuroophthalmol. 28 (2008) 269e277. [5] H. Chung, Y.H. Yoon, J.J. Hwang, K.S. Cho, J.Y. Koh, J.G. Kim, Ethambutolinduced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells, Toxicol. Appl. Pharmacol. 235 (2009) 163e170. [6] S.P. Huang, J.Y. Chien, R.K. Tsai, Ethambutol induces impaired autophagic flux and apoptosis in the rat retina, Dis. Models Mech. 8 (2015) 977e987. [7] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights disease through cellular self-digestion, Nature 451 (2008) 1069e1075. [8] D.C. Rubinsztein, The roles of intracellular protein-degradation pathways in neurodegeneration, Nature 443 (2006) 780e786. [9] L. Galluzzi, F. Pietrocola, J.M. Bravo-San Pedro, R.K. Amaravadi, E.H. Baehrecke, F. Cecconi, P. Codogno, J. Debnath, D.A. Gewirtz, V. Karantza, A. Kimmelman, S. Kumar, B. Levine, M.C. Maiuri, S.J. Martin, J. Penninger, M. Piacentini, D.C. Rubinsztein, H.U. Simon, A. Simonsen, A.M. Thorburn, G. Velasco, K.M. Ryan, G. Kroemer, Autophagy in malignant transformation and cancer progression, EMBO J. (2015), http://dx.doi.org/10.15252/embj.201490784. [10] C. De Duve, The Lysosome 208, Scientific American, 1963, pp. 64e72. [11] C. De Duve, R. Wattiaux, Functions of lysosomes, Annu. Rev. Physiol. 28 (1966) 435e492. [12] N. Mizushima, M. Komatsu, Autophagy: renovation of cells and tissues, Cell 147 (2011) 728e741. [13] S. Saiki, Y. Sasazawa, Y. Imamichi, S. Kawajiri, T. Fujimaki, I. Tanida, H. Kobayashi, F. Sato, S. Sato, K. Ishikawa, M. Imoto, N. Hattori, Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition, Autophagy 7 (2011) 176e187. [14] D.C. Rubinsztein, A.M. Cuervo, B. Ravikumar, S. Sarkar, V. Korolchuk, S. Kaushik, D.J. Klionsky, In search of an “autophagomometer”, Autophagy 5 (2009) 585e589. [15] I. Tanida, T. Ueno, E. Kominami, LC3 and autophagy, Methods Mol. Biol. 445 (2008) 77e88. [16] A. Yamamoto, Y. Tagawa, T. Yoshimori, Y. Moriyama, R. Masaki, Y. Tashiro, Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H4-II-E cells, Cell Struct. Funct. 23 (1998) 33e42. [17] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell, 140 313e326.
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D. Yamada et al. / Biochemical and Biophysical Research Communications 471 (2016) 109e116
[18] T. Yoshimori, A. Yamamoto, Y. Moriyama, M. Futai, Y. Tashiro, Bafilomycin A1, a specific inhibitor of vacuolar-type H(þ)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells, J. Biol. Chem. 266 (1991) 17707e17712. [19] S. Kimura, T. Noda, T. Yoshimori, Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3, Autophagy 3 (2007) 452e460. [20] N. Zheng, X. Zhang, G.R. Rosania, Effect of phospholipidosis on the cellular pharmacokinetics of chloroquine, J. Pharmacol. Exp. Ther. 336 (2011) 661e671.
[21] J. Han, K. Burgess, Fluorescent indicators for intracellular pH, Chem. Rev. 110 (2010) 2709e2728. [22] B.M. Krenn, E. Gaudernak, B. Holzer, K. Lanke, F.J. Van Kuppeveld, J. Seipelt, Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections, J. Virol. 83 (2009) 58e64. [23] T. Kambe, Y. Yamaguchi-Iwai, R. Sasaki, M. Nagao, Overview of mammalian zinc transporters, Cell Mol. Life Sci. 61 (2004) 49e68. [24] J.P. Liuzzi, R.J. Cousins, Mammalian zinc transporters, Annu. Rev. Nutr. 24 (2004) 151e172.