Abstract
The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca2+ was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca2+ from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca2+ levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.
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Abbreviations
- AM:
-
acetoxymethyl ester
- BAPTA-AM:
-
(acetyoxymethyl)-1,2-bis(o-amino phenoxy)ethane N,N,N′,N′-tetra (acetoxymethyl)ester
- CPO:
-
9-capronyloxytetrakis (methoxyethyl) porphycene
- DCF:
-
2′,7′-dichlorofluorescein
- DEVD-R110:
-
asp-glu-val-asp-rhodamine 110
- ER:
-
endoplasmic reticulum
- ERTr:
-
endoplasmic reticulum tracker
- FHS:
-
Fischer's medium with 20 mM HEPES buffer pH 7.2 replacing NaHCO3
- H2DCF:
-
dichlorohydrofluorescein
- H2DCFDA:
-
dichlorodihydrofluorescein diacetate
- LTB:
-
lysotracker blue
- NAO:
-
nonyl acridine orange
- NPe6:
-
N-aspartyl chlorin e6
- PDT:
-
photodynamic therapy
- ROS:
-
reactive oxygen species
- RR:
-
ruthenium red
- Ru360:
-
ruthenium amino binuclear complex
- SnET2:
-
tin etiopurpurin
- THP:
-
thapsigargin
- TMRM:
-
tetramethylrhodamine methyl ester
- Trolox:
-
6-Hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (water-soluble derivative of Vitamin E)
- ΔΨm:
-
mitochondrial membrane potential
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Acknowledgements
We thank Ann Marie Santiago, Brendan Leeson and Patricia Mathieu for excellent technical assistance, and Dr H-R Kim for helpful discussions. This work was supported by grants CA23378, CA92618 (DK) and ES009392 (JJR) awarded by the National Institutes of Health and aided by the Cell Culture and Imaging & Cytometry Facility Cores supported by Grant P30 ES06639.
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Kessel, D., Castelli, M. & Reiners, J. Ruthenium red-mediated suppression of Bcl-2 loss and Ca2+ release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species. Cell Death Differ 12, 502–511 (2005). https://doi.org/10.1038/sj.cdd.4401579
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DOI: https://doi.org/10.1038/sj.cdd.4401579
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