title: Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination.
abstract: The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia. daria.s.kuznetsova@gmail.com.|Lobachevsky State University of Nizhny Novgorod, 603950, Gagarin Ave., 23, Nizhny Novgorod, Russia. daria.s.kuznetsova@gmail.com.
authors: Daria S
authors: DS
authors: Kuznetsova
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia.|Lobachevsky State University of Nizhny Novgorod, 603950, Gagarin Ave., 23, Nizhny Novgorod, Russia.
authors: Marina V
authors: MV
authors: Shirmanova
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia.|Lobachevsky State University of Nizhny Novgorod, 603950, Gagarin Ave., 23, Nizhny Novgorod, Russia.
authors: Varvara V
authors: VV
authors: Dudenkova
authors: Institute of Applied Physics RAS, 603950, Ulyanov St., 46, Nizhny Novgorod, Russia.
authors: Pavel V
authors: PV
authors: Subochev
authors: Institute of Applied Physics RAS, 603950, Ulyanov St., 46, Nizhny Novgorod, Russia.
authors: Ilya V
authors: IV
authors: Turchin
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia.|Lobachevsky State University of Nizhny Novgorod, 603950, Gagarin Ave., 23, Nizhny Novgorod, Russia.
authors: Elena V
authors: EV
authors: Zagaynova
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia.|Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, Miklukho-Maklaya St., 16/10, Moscow, Russia.|Pirogov Russian National Research Medical University, 117997, Ostrovitianova St. 1, Moscow, Russia.
authors: Sergey A
authors: SA
authors: Lukyanov
authors: Nizhny Novgorod State Medical Academy, 603005, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia.
authors: Boris E
authors: BE
authors: Shakhov
authors: Institute of Applied Physics RAS, 603950, Ulyanov St., 46, Nizhny Novgorod, Russia.
authors: Vladislav A
authors: VA
authors: Kamensky
mesh_terms: D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D009336:Necrosis; D061848:Optical Imaging; D038761:Photobleaching; D010778:Photochemotherapy / Q000379:methods*; D010789:Phototherapy / Q000295:instrumentation / Q000379:methods*; D054457:Tissue Scaffolds; D002583:Uterine Cervical Neoplasms / Q000628:therapy*
chemical_list: C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins
keywords: KillerRed; photobleaching; photodynamic therapy; phototoxicity; tumor spheroids