A Selenopyrylium Photosensitizer
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4497
Photodynamic Effect of meso-Tetra(hydroxyphenyl)porphine and
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solution. Final concentrations of the dye were determined
using absorbance. Ten microliters of the dye/ethanol solution
was transferred to 1.0 mL of mitochondrial preparation buffer
and the absorbance determined using a diode array spectro-
photometer (HP8452A, Hewlett-Packard, Palo Alto, CA). The
dye in mitochondrial preparation buffer, at a final concentra-
tion that gave an OD of 0.2, was then added to mitochondrial
suspensions (1.0 mL) and allowed to incubate in the dark on
ice for 15 min. The dye/mitochondrial suspension was then
centrifuged at 8000g for 3 min, the supernatant was aspirated
with a Pasteur pipet, and the pellet was resuspended in 1.0
mL of mitochondrial preparation buffer. The suspension was
then transferred to a 3.0-mL quartz cuvette which was
positioned in a focused, 1.0-cm diameter, filtered (570-750 nm)
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Cincotta, L.; Foley, J .; Bonventre, J . V.; Oseroff, A. R. Mecha-
nisms of Mitochondrial Photosensitization by the Cationic Dye,
N,N′-Bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC): Prefer-
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light beam emitted from
a 750-W tungsten source. The
intensity of the beam was uniform over the wavelength band
used and adjusted to a fluence rate of 100 mW cm-2 using
neutral density filters. Beam intensity was measured using a
radiometer (model 210, Coherent Inc., Palo Alto, CA). The light
was cooled by passing it through a water filter eliminating
thermal effects as the sample temperature did not rise above
25 °C. The mitochondrial suspensions were magnetically
stirred continuously during the irradiation period, 1.0 h.
Aliquots, 10 µL, were removed at various times during
irradiation for determination of cytochrome c oxidase activity.
A portion of the mitochondria/dye suspension was maintained
in the dark and determinations of cytochrome c oxidase activity
were performed on aliquots from these samples as dark
controls. Measurement of cytochrome c oxidase activity was
performed according to a method described earlier.21 Initial
enzyme activity was adjusted to obtain a decrease in the
reduced cytochrome c oxidase absorbance at 550 nm of 0.4-
0.6 OD units/min. Data are expressed as the percent (%) of
initial, preirradiation cytochrome c oxidase activity.
E xp osu r e of Mit och on d r ia t o Dye 5-Se a n d Oxygen
Ra d ica l Qu en ch er s or a Red u ced Oxygen En vir on m en t.
To determine whether singlet oxygen was the major factor
involved in the inhibition of cytochrome c oxidase when tumor
mitochondria were exposed to dye 5-Se and light, we per-
formed experiments using superoxide dismutase to remove
superoxide. Superoxide dismutase was added to mitochondria/
dye suspensions at 30 enzyme units/mL. An oxygen-free
environment was established by nitrogen purging of the
sample in a specifically designed chamber described earlier.22
Control samples containing dye but not exposed to light were
maintained in the nitrogen purged atmosphere in the dark for
1 h and no inhibition of mitochondrial cytochrome c oxidase
activity was observed.
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Mitochondria by the Rhodacyanine MKT-077. Cancer Res. 1996,
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Related in Structure to the Antitumor Agent AA1 as in Vitro
Sensitizers for the Photodynamic Therapy of Cancer. J . Med.
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Simard, T. P.; Davies, S. R.; Gollnick, S. O.; Oseroff, A. R.;
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L. B. Mitochondrial and Plasma Membrane Potentials Cause
Unusual Accumulation and Retention of Rhodamine 123 by
Breast Adenocarcinoma-Derived MCF-7 Cells. J . Biol. Chem.
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Summerhayes, I. C.; Chen, L. B. Selective Toxicity of Rhodamine-
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720. (c) Bernal, S. D.; Lampidis, T. J .; McIsaac, R. M.; Chen, L.
B. Anticarcinoma Activity in vivo of Rhodamine 123, a Mito-
chondrial-Specific Dye. Science 1986, 222, 169-172. (d) Modica-
Napolitano, J . S.; Aprille, J . R. Basis for the Selective Cytotox-
icity of Rh123. Cancer Res. 1987, 47, 4361-4365. (e) Nadaka-
Sta tistica l An a lyses. All statistical analyses were per-
formed using the Student’s t-test for pairwise comparisons. A
P value of < 0.05 was considered significant.
Ack n ow led gm en t. The authors thank the National
Institutes of Health (Grant CA69155 to M.R.D., Grant
CA55791 to A.R.O., and Grant CA36856 to R.H.) and
the shared resources of the Roswell Park Cancer Center
support grant (P30 CA16056) for partial support of this
work.
Su p p or tin g In for m a tion Ava ila ble: Figure S1b showing
the dark and phototoxicity of 5-Se against the murine T-cell
line Molt-4; Figure S3 showing the effect of 5-Se photosensi-
tization on mitochondrial cytochrome c oxidase activity in
cultured whole R3230AC tumor cells; Figure S4 showing the
effect of 5-Se photosensitization on isolated mitochondrial
suspensions; and Table S2 showing the distribution of 1-Se
and 5-Se in female Fischer rats bearing R3230AC adenocar-
cinomas. This information is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
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