Abstract
We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs. necrotic cell death as a function of subcellular location, including the nucleus, the cytosol, the endoplasmic reticulum, the mitochondria, and the membrane. We find that different subcellular origins of singlet oxygen have different potencies in cytotoxic response and the pathways of cell death, and we observed that CT26 and HEK293 cell lines are differentially sensitive to mitochondrially localized singlet oxygen stresses. This work provides new insight into the function of type II reactive oxygen generating photosensitizing processes in inducing targeted cell death and raises interesting mechanistic questions about tolerance and survival mechanisms in studies of oxidative stress in clonal cell populations.
Highlights
Singlet oxygen (1O2) is the lowest electronic excited state of molecular oxygen that can be generated by biochemical, photochemical, and chemical processes
All the dL5∗∗-expressing HEK293 (HEK) cells, including membrane-anchored fluorogen-activating protein (FAP), cytoplasmically localized FAP, endoplasmic reticulum-retained FAP, mitochondria-anchored FAP, and nucleus-retained FAP were transfected as pcDNA3.1 plasmids (Table 1) using Lipofectamine 2000 (Invitrogen) and selected by G418, sorted for stable cell line generation and cultured in fresh DMEM (Dulbecco’s Modified Eagle’s Medium, Thermo Fisher), containing 1% double resistant, 1% G418, and 10% inactivated FBS at 37◦C under a humidified 5% CO2 and 95% air atmosphere
By comparing the sensitivity of a colorectal tumor cell line to these HEK cells, we showed that the tumor cells showed remarkable resistance to photodynamic singlet oxygen generated in the mitochondria
Summary
Singlet oxygen (1O2) is the lowest electronic excited state of molecular oxygen that can be generated by biochemical, photochemical, and chemical processes. Singlet oxygen production by photosensitizers (PS) has been used for photoactive antiviral drugs (Vigant et al, 2013), for induction of apoptosis or necrosis (Bauer, 2016; Kessel, 2019a), and for generation of cytotoxic or immune-inducing signals in photodynamic tumor ablation (Turan et al, 2016; Kobayashi et al, 2020). The drawbacks of traditional PSs [e.g. porphyrins, chlorin e6, and methylene blue, are low water solubility (Yu et al, 2015) and no (or limited control of) target or cell-type selectivity (Kim et al, 2018) and reactive oxygen species selectivity, resulting in off-target cytotoxicity (Castaneda-Gill et al, 2017)]. We require water-soluble 1O2-specific PSs with stringent targeting and activation properties to improve the selectivity and impact of photodynamic therapeutic approaches
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