Parallel damage in mitochondria and lysosomes is an efficient way to photoinduce cell death

• Published in: Autophagy Vol. 15; no. 2; pp. 259 - 279
• Main Authors: Martins, Waleska K., Santos, Nayra Fernandes, Rocha, Cleidiane de Sousa, Bacellar, Isabel O. L., Tsubone, Tayana Mazin, Viotto, Ana Cláudia, Matsukuma, Adriana Yamaguti, Abrantes, Aline B. de P., Siani, Paulo, Dias, Luís Gustavo, Baptista, Mauricio S.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01-02-2019
• Subjects: Autophagy - drug effects; Autophagy - radiation effects; Cell death; Cell Death - radiation effects; Cell Line; Cell Survival - drug effects; Cell Survival - radiation effects; Humans; Light; Lysosomes - drug effects; Lysosomes - metabolism; Lysosomes - pathology; Lysosomes - radiation effects; membrane damage; methylene blue; Methylene Blue - analogs & derivatives; Methylene Blue - pharmacology; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - pathology; Mitochondria - radiation effects; mitophagy; Models, Biological; phenothiazines; photodynamic efficiency; Research Paper - Basic Science; methylene blue; mitophagy; photodynamic efficiency; membrane damage; phenothiazines; Cell death
• ISSN: 1554-8627; 1554-8635
• DOI: 10.1080/15548627.2018.1515609

Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to improve the efficiency of photosensitizers, in terms of inducing cell death, remains unexplored. Here, we addressed the concept that a parallel damage in the membranes of mitochondria and lysosomes leads to a scenario of autophagy malfunction that can greatly improve the efficiency of the photosensitizer to cause cell death. Specific damage to these organelles was induced by irradiation of cells pretreated with 2 phenothiazinium salts, methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB). At a low concentration level (10 nM), only DMMB could induce mitochondrial damage, leading to mitophagy activation, which did not progress to completion because of the parallel damage in lysosome, triggering cell death. MB-induced photodamage was perceived almost instantaneously after irradiation, in response to a massive and nonspecific oxidative stress at a higher concentration range (2 µM). We showed that the parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a late and more efficient cell death, offering significant advantage (2 orders of magnitude) over photosensitizers that cause unspecific oxidative stress. We are confident that this concept can be used to develop better light-activated drugs.
Abbreviations: ΔΨm: mitochondrial transmembrane inner potential; AAU: autophagy arbitrary units; ATG5, autophagy related 5; ATG7: autophagy related 7; BAF: bafilomycin A
1
; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH
2
: 2ʹ,7ʹ-dichlorodihydrofluorescein; DMMB: 1,9-dimethyl methylene blue; ER: endoplasmic reticulum; HaCaT: non-malignant immortal keratinocyte cell line from adult human skin; HP: hydrogen peroxide; LC3B-II: microtubule associated protein 1 light chain 3 beta-II; LMP: lysosomal membrane permeabilization; LTG: LysoTracker™ Green DND-26; LTR: LysoTracker™ Red DND-99; 3-MA: 3-methyladenine; MB: methylene blue; mtDNA: mitochondrial DNA; MitoSOX™: red mitochondrial superoxide probe; MTDR: MitoTracker™ Deep Red FM; MTO: MitoTracker™ Orange CMTMRos; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTT: methylthiazolyldiphenyl-tetrazolium bromide;
1
O
2
: singlet oxygen; OH
.
hydroxil radical; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PBS: phosphate-buffered saline; PI: propidium iodide; PDT: photodynamic therapy; PS: photosensitizer; QPCR: gene-specific quantitative PCR-based; Rh123: rhodamine 123; ROS: reactive oxygen species RTN: rotenone; SQSTM1/p62: sequestosome 1; SUVs: small unilamellar vesicles; TBS: Tris-buffered saline