Abstract
In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called “bystander effects”. To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2−∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2−∙ production in these bystander cells. Our results support a key role for ER–mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.
Highlights
PS photoactivation is a well-established therapeutic approach used to promote cell killing based on the interaction between visible light and matter [1,2]
All the results described in this article were obtained in standard stimulation (SS) conditions, except for data shown in Figure 3, Figure 4, and Video 1, where we modulated the delivered optical energy by tuning both laser power and photoactivation flash duration
We performed time-lapse confocal fluorescence microscopy to investigate the occurrence of apoptotic processes using the Polarity Sensitive Indicator of Viability Apoptosis (pSIVA)-IANBD polarity sensitive probe, which binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide (PI), which selectively stains the nuclei of damaged cells
Summary
PS photoactivation is a well-established therapeutic approach used to promote cell killing based on the interaction between visible light and matter [1,2]. In PDT, wavelength-selective light exposure of cells loaded with a PS promotes the inert PS molecule to a relatively long-lived excited state in which it interacts with molecular oxygen (O2 ). Primary products of PS activation are singlet oxygen (1 O2 ) and O2 − ·, which in turn initiate a cascade of secondary reactions, leading to the formation of H2 O2 , hydroxyl radical (OH·), and oxidation of substrates [8,9]. These events are followed by activation of numerous cellular pathways, including signaling by Ca2+ and nitric oxide (NO), terminating with cellular damage or demise [10].
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