Photoactivation Enhances the Mitochondrial Toxicity of the Cationic Rhoadcyanine MKT-077 Josephine S. Modica-Napolitano, Brian T. Brunelli, Keizo Koya, et al. Cancer Res 1998;58:71-75.
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ICANCER RESEARCH58, 71-75, January 1. 19981
Photoactivation Enhances the Mitochondrial Toxicity of the Cationic Rhodacyanine Josephine Department
MKT-077
S. Modica-Napolitano,' of Biology,
Merrimack
Brian T. Brunelli,
College, North Andover,
Massachusetts
Keizo Koya, and Lan Bo Chen 01845 If. S. M-N.J; Department
of Biology.
Tufts University.
Medford,
Massachusetts
02155
If. S. M-N.,B. T.B.];and Divisionof CellularandMolecularBiology.Dana-FarberCancerInstitute,HarvardMedicalSchool.Boston.Massachusetts02!15(K. K., L B. C.J ABSTRACT In this study, the mitochondrial phototoxicity of the cationic rhodacya nine MKT-077 was investigated by comparing its effects on the inhibition of mitochondrial respiration and the structural integrity of mitochondrial DNA (mtDNA) in the presence and absence ofadded high-intensity visible light (7.5 i/cm2). Results indicate that photoirradiation significantly en hances the mitochondrial toxicity of MKT-077 at both the biochemical and DNA levels. For example, the concentration of MKT-077 required to achieve one-half maximal inhibition of ADP-stimulated respiration was observed to be 6-fold lower in the presence versus absence of high intensity light (one-half maximal inhibition at 2.5 versus 15 gag MKT-077/
mg, respectively). In addition, photoirradiation produced a 25-fold in crease in inhibition ofsuccinate-cytochrome c reductase activity by MKT 077 (one-half maximal inhibition at 2 versus 50 gig MKT-077/ml, ±light, respectively) and a 6-fold increase in inhibition of cytochrome oxidase activity (one-half maximal inhibition at S versus 30 @agMKT-077/
ml, ±light, respectively). Furthermore, MKT-077
and 7.5 Jkm2
the combination of 25 g.tg/ml
visible light caused significant
degradation
of
mtDNA in isolated rat liver initochondria, whereas the same concentra tion of dye in the absence of light had only a modest effect on mtDNA. Evaluation
oflight-induced
MKT-077 lipid peroxidation
MATERIALS
in mltochondnal
Materials.
membrane fragments by the thiobarbituric acid test and by measurement of nonrespiratory-linked oxygen uptake suggests that mitochondrial pho totoxicity by MKT-077 may be the result oflipid peroxidation via reactive oxygen species. These results have important
implications
carcinoma (17). We recently demonstrated that MKT-077 selectively inhibits mitochondrial respiration in carcinoma cells as a result of a general perturbation of mitochondrial membranes and consequent nonspecific damage to membrane-bound enzymes (18). MKT-077 also produced a mild-to-moderately degradative effect on mtDNA, but not on nuclear DNA, of various cancer cell types (18). In this study, the mitochondrial phototoxicity of MKT-077 was investigated by measuring the inhibitory effect of the compound on mitochondrial respiratory activity and its effect on the structural integrity of mtDNA in the presence and absence of added high-intensity visible light. Our data demonstrate that the selective mitochondrial toxicity exhibited by MKT-077 is significantly enhanced by photoactivation of the com pound and that this effect is most likely the result of lipid peroxidation via reactive oxygen species. These results have important implications with regard to the use of MKT-077 in alternative strategies for treatment of carcinoma.
with regard
AND METhODS MKT-077
was provided
by Fuji Pharmaceuticals
and was dis
solved in water at a stock concentration of I mg/ml. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Cell Cultures. The monkeykidneyepithelialcell line CV-l was grown in DMEM (Life Technologies, Inc.) supplemented with 10% FCS (Hyclone) and
to
the potential use of MKT-077 in photochemotherapy.
antibiotics (10,000 lU/mI penicillin and 10,000 p@g/mlst.reptomycin). All cells
were maintained at 37°Cin a 5% CO2 atmosphere. Isolation
INTRODUCTION PCT2 is an investigational cancer treatment involving light activa tion of a photoreactive drug, or photosensitizer, that is selectively taken up or retained by malignant cells (1—3).Whereas ideally the photosensitizer is relatively innocuous to the surrounding normal tissue, local irradiation of the tumor site converts the drug to a toxic or reactive species capable of destroying the malignant cells. PCT, therefore, has the potential to provide a means of highly specific tumor cell killing without injury to normal cells. The mitochondrion has been implicated as an important, perhaps primary, subcellular site of damage by several photosensitizers that show promise for use in PCT (4—8).Like other lipophilic cations, the cationic photosensitizers are concentrated by cells and into mitochon dna in response to negative inside membrane potentials (9—14).The higher plasma and/or mitochondrial membrane potentials of carci noma versus normal cells account for the greater uptake of cationic photosensitizers in carcinoma cells and are responsible for the selec tive anticarcinoma activity exhibited by these compounds (15, 16). The cationic rhodacyanine dye MKT-077 has been shown to exhibit selective cytotoxicity to carcinoma cells in vitro and in vivo and is currently undergoing testing in Phase I clinical trials for treatment of
centrifuged at 8000 x g for 10 mm at 4°C.The resulting mitochondrial
for
reprints
should
be addressed.
Phone:
Tris-HC1 (pH 7.4) to reach a final concentration of approximately 20 mg protein/ml.
Mitochondria were isolated from cultured cells by a modification of the procedure
abbreviations
used are: PC'f,
photochemotherapy;
mtDNA,
of Maltese
harvested in Dulbecco's
and Aprille
(20).
Typically,
5—10 X l0@ cells
were
modified Eagle's medium. Cells were pelleted and
washed once with homogenization buffer [250 mMsucrose, I mMTris-HCI, I mM EDTA, and 1 mg/mI BSA (pH 7.4)] at low speed in a tabletop clinical centrifuge.
The final pellet of cells was resuspended
to a volume of 7 ml and homogenized
in homogenization
buffer
in a Dounce tissue grinder with a tight
pestle until at least 95% of the cells were disrupted (approximately 125 up/down strokes). The homogenate was centrifuged at 800 X g for 10 mm at 4°C.The supematant was removed and saved, and the pellet was resuspended and centrifuged
again at 800
X g for 10 mm. The supernatants
were then
pooled and centrifuged at 9400 X g (10 mm, 4°C);the pellet was resuspended in homogenization buffer and centrifuged pellet was suspended in homogenization protein/ml.
Protein
concentration
again at 9400 X g, and the final buffer to a volume of about 5 mg
was determined
by the method
of Lowry
et
a!. (21), using BSA as the standard. Respiration. Oxygen consumption was measured polarographically with a Clark electrode in a I-mi water-jacketed chamber maintained at 30°C(22). The basic respiratory
assay medium
consisted
of 225 mM sucrose,
10 mM KCI, I
837-5000,
exten
mM EDTA, 10 msi K,HPO4-KH2PO4, 5 mM MgC12, and 10 mM Tris-HCI (pH 7.4); 1 mg/mI BSA was included to stabilize the membranes. An initial rate of
mitochondrial
DNA;
oxygen consumption (state 2 rate) was recorded after the addition of substrate (5 mM each glutamate plus malate), MKT-077 (0—35@tg/mgmitochondrial
(508)
sion 4459. 2 The
pellet
was washed twice in STE and then washed once in 250 mM sucrose and 1 mM
18 U.S.C. Section 1734 solely to indicate this fact. requests
were isolated from male
STE [250 mM sucrose, 1 mM Tris-HCI, and I msi EDTA (pH 7.4)J and then
Received 6/13/97; accepted 10/23/97.
whom
Liver mitochondria
centrifuged at 600 X g for 10 mm at 4°C.The supernatant was collected and
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 1 To
of Mitochondria.
Sprague Dawley rats by differential centrifugation as described previously (19). Briefly, approximately 5 g of tissue were minced and homogenized in
ThA, thiobarbituric acid; MDA, malondialdehyde. 71
Downloaded from on April 28, 2014. © 1998 American Association for Cancer Research.
MITOCHONDRIAL PHOTOTOXICITY OF
protein), and approximately 0.15 mg of isolated CV-l mitochondria. Two mm
of the sample was recorded. The sample was then incubated at room temper
after addition of mitochondria, ADP (120 nmol) was added, and a state 3 rate was obtained. After recording a measurable state 4 rate (i.e. , rate after ADP is
ature in the presence
phosphorylated),
80
@tM2,4-dinitrophenol
were added
to obtain
a respiratory
rate in the absence of coupled oxidative phosphorylation. Enzyme
Assays.
Succinate-cytochrome
mined spectrophotometrically
c reductase
activity
or absence
of high-intensity
light (25 mW/cm2)
for 2.5
mm. The sample was immediately returned to the water-jacketed chamber, and oxygen consumption was again recorded. The amount of oxygen consumed as a result oflipid peroxidation was determined for each sample by calculation of the difference in oxygen content as measured before and after incubation (and
was deter
after subtraction of any basal level of oxygen consumption).
by measuring the rate of increase in absorbance
at 550 nm due to the reduction of cytochrome c (23). Freeze-thawed sonicated rat liver mitochondria
were added to a cuveue containing 2 nmi KCN, 50 mivi
potassium
(Kl-12P041K2HP04;
phosphate
pH 7.4),
and 20 mr@i succinate
RESULTS
at
37°C.The reaction was initiated by adding 1 mg of oxidized cytochrome c to the cuvette, and a change in the absorbance reaction
was terminated
by the addition
Mitochondrial Respiration. The effect of photoactivation of MKT-077 on the inhibition of mitochondrial respiration was deter mined polarographically, using functionally intact CV-! mitochondria and glutamate/malate as the respiratory substrate. As shown in Fig. 1, MKT-077 inhibited mitochondrial respiration in a dose-dependent manner in both the presence and absence of added high-intensity light, with one-half maximal inhibition of respiration achieved at concen trations of 2.5 and 15 @tgMKT-077/mg mitochondrial protein, respec tively. These data indicate a 6-fold potentiation of inhibition of mitochondrial respiration by photirradiated MKT-077 as compared to the nonirradiated control. Enzyme Assays. MKT-077 was previously shown to inhibit dcc tron transfer activity in freeze-thawed mitochondrial membrane frag ments. In this study, the effect of light activation of MKT-077 on electron transfer reactions was examined via determination of succi nate-cytochrome c reductase activity (which is a measure of electron transfer from complex II through coenzyme Q to complex ifi) and cytochrome c oxidase activity (which is a measure of the complex IV enzyme) in freeze-thawed mitochondrial membrane fragments. Ac cording to Fig. 2, without photoirradiation, mitochondrial membrane preparations exposed to varying concentrations of MKT-077 exhib ited one-half maximal inhibition of succinate-cytochrome c reductase activity at a concentration of 50 @gMKT-077/ml. When photoirra diated, however, one-half maximal inhibition of activity was achieved at 2 j.ag MKT-077/ml, indicating a 25-fold potentiation of the inhib itory effect of the drug by photoactivation. Fig. 3 shows that photoir radiation of MKT-077 produced a 6-fold increase in the inhibition of cytochrome oxidase activity over that which was produced in the absence of added light, with one-half maximal inhibition obtained at a concentration of 5 and 30 @g/ml,respectively. Photoirradiation of MKT-077 had a minimal effect on citrate synthetase activity, produc ing only 35% inhibition at a concentration of 100 @ag/ml,whereas in the absence oflight, the compound had no effect on the activity of this enzyme, even at the highest concentration of MKT-077 tested (0—100 @g/ml;data not shown).
was recorded over time. The
of 5
@.tgof antimycin
A, which
provided a background rate. The assay was repeated in triplicate. Cytochrome
c oxidase
activity
was determined
spectrophotometrically
by
measuring the decrease in absorbance at 550 nm due to the oxidation of cytochrome c (24). Freeze-thawed
sonicated rat liver mitochondria were added
to a cuvette containing 40 mM potassium phosphate (KH2PO4/K2HPO4;pH 7.0) at 37°C.The reaction was initiated by adding 0.7 mg of reduced cyto
chrome C,and a linear rate was recorded. The assay was repeated in triplicate. Citrate synthetase was recorded spectrophotometrically at 412 nan (25). A background rate was obtained by adding freeze-thawed rat liver mitochondria to 0.1 m@i5,5'-dithiobis-2-nitrobenzoate and 0.3 mM acetyl-CoA. This initial rate was subtracted from the rate obtained on addition of the substrate, 0. 1 mM
oxaloacetate. Photoirradlatlon. In the assays requiring photoirradiation, intact mito chondria or freeze-thawed mmtochondrial fragments were added to a 12 X 75-mm
clear
glass
test tube
and incubated
in the appropriate
assay
mmxtureat the desired dye concentration. Photoactivation was accomplished by using a KOdak slide projector continuously delivering 25 mW/cm2 visible light
for a period of 5 mm (7.5 J/cm2), during which there was no measurable change in temperature of the assay mixture. After photoirradiation,
the assay
mixture was transferred to an appropriate vessel for further treatment and/or analysis.
mtDNA Analysis. Nine mg of isolated intactrat liver mmtochondria were resuspended
in a final volume of 1.5 ml of H2O ±75 @.tlof MKT-077 (1
mg/mI) ±photoirradiation. After the 5-mm incubation, mmtochondriawere pelleted
by centrifugation
glucose,
25 mM Tris-Cl
and resuspended
in 0.1 ml of ice-cold
(pH 8.0), and 10 rims EDTA
50 mM
(pH 8.0). mtDNA
was
then extracted using an alkaline lysis mini-prep procedure. Briefly, 0.2 ml of SDS/NaOH was added, and the tubes were inverted to mix. After an additional 5 mmn,0. 15 ml of ice-cold potassium acetate (3 M potassium-5 M acetate) was
added, and the samples were centrifuged. The mtDNA was precipitated from the resulting supernatants by addition of isopropanol, washed once with ethanol, and resuspended in 10 mMTris (pH 8) and 1 m@iEDTA. Ten @g of mtDNA
extracted
from each sample
run out on a 1% agarose ethidium
bromide.
were digested
with BamHI
gel (95 V; 1.5 h), and observed
The mtDNA
was
then transferred
(1 h; 37°C),
by staining
with
to nitrocellulose
by
Southern blotting (26) and probed with [32P]dCTP-labeledtotal rat mtDNA.
TBA Test. TheTBAtestwasusedto measuretheextentof lipidperoxi dation induced by photoactivation
of MKT-077 in isolated rat liver mitochon
120
dria (27). Three mg of freeze-thawed mmtochondrialfragments were resus pended in water to a final volume of 0.5 ml ±25 pAof MKT-077 (I mg/mi) in the presence
or absence
of high-intensity
light for 5 mm. After incubation,
0.25 ml of a solution of 20% trichloroaceticacid containing I mm of FeSO4 and 0.5 ml of a solution of 0.67% (w/v) ThA were added to each sample. The
a, ‘I a, 0 I.. >@4.' ‘. —
100 80
mixture was then incubated at 80°Cfor 10 mm and cooled to room tempera ture, and the precipitated protein was removed by centrifugation. The amount
LC
00
60
of MDA, a by-product of lipid peroxidation,
0.
40
that was present in each sample
U, a, I-
was determined spectrophotometrically by measuring the absorbance of the corresponding
supematants
at a wavelength
of 530 nm. The extinction
ficient of MDA = I .56 x l0@cm2/nmol (27). Polarographic Determination of Lipid Peroxidation. peroxidation
was
also
evaluated
by measurement
coef
0
The extent of lipid
0
of nonrespiratory-linked
oxygen consumption in freeze-thawed rat liver mitochondria (27). Approxi mately 3 mg of mitochondrial protein were added to 0.8 ml of buffer solution containing
175 mM KCI,
077/mg mitochondrial
10 mM Tris-base
5
10
15
20
25
30
35
ug MKT-077/mg mitochondrial protein Fig. 1. Effect of MKT-077 on respiratory activity in isolated CV-l mitochondria. ADP-stimulated respiration was determined polarographically using glutamate/malate as the respiratory substrate in the presence () and absence ( 0 ) of 7.5 J/cm2 visible light. Data presented are the results from a typical experiment.
(pH 7.4), and 0—100 @.tgMKT
protein and maintained in a water-jacketed
20
chamber at
37°C.Using a Clark oxygen electrode, the basal rate of oxygen consumption 72
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M1TOCHONDRIALPHOTOTOXICITY OF MKT-077
ration in cancer cells and has a mild-to-moderately degradative effect on mDNA, but not nuclear DNA, of various cancer cell types (18). As has been suggested for several other lipophilic cations, this selective mitochondrial toxicity may provide the basis for the significant anti tumor activity exhibited by MKT-077 in vitro and in vivo. Rhodacyanine dyes are well known in the photographic industry as photosensitizers for silver halides (28). It was reasonable to hypoth esize, therefore, that MKT-077 may also act as a biological photo sensitizer. In this study, we examined the mitochondrial phototoxicity of MKT-077 by comparing its effect on mitochondrial structure and function in the presence and absence of high-intensity visible light. Our data demonstrate a 6-fold increase in inhibition of both ADP stimulated respiration and mitochondrial cytochrome oxidase activity and a 25-fold increase in inhibition of succinate-cytochrome c reduc tase on photoactivation of MKT-077 with 7.5 J/cm2 visible light. Although a direct comparison between the effect of photoirradiated MKT-077 on respiratory rates and electron transfer activities is not valid due to vastly different assay conditions, a comparison of the effect of photoirradiated MKT-077 on the two measured electron transfer reactions indicates a 4-fold greater sensitivity of succinate cytochrome c reductase activity as compared to mitochondrial cyto chrome oxidase activity. Interestingly, succinate dehydrogenase ac tivity has previously been found to be much more sensitive than
U
05.
MKT-077
(ug/mI)
Fig. 2. Effect of MKT-077 on succinate-cytochrome c reductase activity measured in freeze-thawed mitochondria in the presence (0) and absence (0) of high-intensity light. Data presented are the average of three separate experiments
±SE.
mtDNA Analysis. Electrophoresis and Southern blot analysis were used to investigate the effect of light-activated MKT-077 on the structural integrity of mtDNA. According to Fig. 4, the BamHI digested, —dye, —light control (A and B, Lane 1) produced, as expected, two distinct bands corresponding to approximately 11 and 5
cytochrome
oxidase
activity
to the effects
of photosensitization
(29).
We previously demonstrated a selective loss of mtDNA, but not nuclear DNA, in cultured carcinoma cells that had been treated with 3 p.g/ml MKT-077 over a period of 3 days. In this study, we examined the immediate effect (after a 5-mn incubation) of a much higher concentration of MKT-077 (25 @g/ml)±photoirradiation on the
kbinlength.A concentration of25p.g/mlMKT-077intheabsenceof light (A and B, Lane 2) had a modest effect on the structural integrity of mtDNA, producing two bands that were slightly less intense than that of the control. Photoirradiation of mtDNA in the absence of MKT-077 was shown to have no effect (A and B, Lane 3), whereas the combination of 25 @.tg/mlMKT-077 and 7.5 J/cm2 visible light (A and
structural
integrity
of mtDNA
extracted
from isolated
intact rat liver
mtDNA. Evaluation of Lipid Peroxidation. The ThA test was used for direct determination of the lipid peroxidation degradation product,
mitochondria. Results showed a modest decrease in the level of mtDNA in +dye/—light samples as compared to —dye/—lightcon trols. This decrease in mtDNA levels was similar in magnitude to that which was observed in cultured carcinoma cells after MKT-077 treatment. A much more pronounced degradation of mDNA was observed for the combination of +dye/+ light, however. These data suggest that MKT-077 alone, in the absence of light, has a primary
MDA,producedin mitochondrial membranefragmentsthathadbeen
and immediate
treated with 25 p,g/ml MKT-077 in the presence or absence of high-intensity light. Our results indicate that photoirradiation of MKT-077-treated mitochondria induced a 5-fold increase in MDA production over that which is produced in nonirradiated MKT-077treated controls (Fig. 5). Furthermore, in the absence of light, there was no significant difference in MDA production in untreated mito chondria versus mitochondria that had been treated with 25 @tg/mi MKT-077 (data not shown). The extent of MKT-077-induced lipid peroxidation in the presence and absence of added light was further assessed by measuring non respiratory-linked oxygen uptake in mitochondrial fragments using a Clark oxygen electrode. As shown in Fig. 6, our data demonstrate that when photoirradiated at a constant light intensity, a range ofO—l00 @.tg MKT-077/mg mitochondrial protein caused a dose-dependent in crease in nonrespiratory-linked oxygen consumption. Furthermore, although there was a slight background level of oxygen consumption under the condition of +MKT-077/—light at any given concentration of MKT-077, more oxygen was consumed in the presence of light
and that this effect can be significantly enhanced by photoirradiation of the compound. On photoirradiation, certain photosensitizers can react with molec ular oxygen to initiate one or more of several types of photochemical processes, each of which can ultimately result in the damage or destruction of important biomolecules. Mitochondria are especially susceptible to the photodamaging effects of lipid peroxidation due to
B, Lane4) produceda smearindicatingsignificantdegradation of
than in its absence.
damaging
effect on the structural
integrity
of mIDNA
400
:E#@ 350 4.' 0
a,
@a.
300 250
XE 0.,,,
200
EE
150
.=
100
0
uE 0@
50 0
DISCUSSION
0
10
20
30
40
50
60
MKT-077 (ug/mL)
The mitochondrion has been identified as the primary site of intracellular localization of MKT-077 (17). In a recent report, we demonstrated that MKT-077 selectively inhibits mitochondrial respi
Fig. 3. Effect of MKT-077 on cytochrome c oxidase activity in freeze-thawed mito
chondria in the presence ( • ) and absence (U) of high-intensity light. Data presented are the average of three separate experiments ±SE. 73
Downloaded from on April 28, 2014. © 1998 American Association for Cancer Research.
MITOCHONDRIALPHOTOTOXICITYOF MKT-077
A
B
1234
1234 @:
—
—
Fig. 4. Ethidium bromide-stained gel electrophoresis (A)
and Southern blot analysis (B) of BamHI-digested mtDNA extracted from rat liver mitochondria that had been treated ±25 p@g/mlMKT-077 ±7.5 J/cm2 visible light. Lanes I, —dye/—lightcontrol; Lanes 2, +dye/—light sample; Lanes 3, —dye/+Iight sample; Lanes 4, +dye/+light sample.
nonrespiratory-linked oxygen consumption and by measurement of the lipid peroxidation by-product MDA in mitochondria exposed to various concentration of dye plus light. Under these conditions, cvi dence of lipid peroxidation was detected using both methods. These data suggest that mitochondrial phototoxicity by MKT-077 involves, at least in part, lipid peroxidation via reactive oxygen species. How ever, because most photodynamic actions are quite complex and can proceed simultaneously by more than one pathway, additional photo sensitizing reactions cannot be ruled out. Photoirradiation of MKT 077 did in fact induce a modest inhibition of activity of the mitochon drial matrix enzyme, citrate synthetase, an effect that was not observed in the absence of light (18). These data may also implicate
the high proportion of polyunsaturated fatty acids comprising their membrane phospholipids. Lipid peroxidation has been previously implicated in the impairment of membrane function and inactivation of membrane-bound enzyme activity (30). There is evidence to sug gest that lipid peroxidation induces damage to mtDNA as well (31). The possibility that lipid peroxidation may be involved in MKT-077induced mitochondrial photodamage was explored by monitoring
@0 a, 0@@.,
@
I@:
@
cc U,
type II photosensitization
.!@3 o.s 0
reactions
in the phototoxicity
induced
by
MKT-077. Studies investigating the mechanism(s) of photooxidative damage to mitochondrial proteins by this compound are currently underway. There recently has been considerable interest in PCT as a form of treatment for locally invasive tumors of the skin, lung, breast, bladder,
;@L@1@;@
Fig. 5. MDA production in rat liver mtDNA fragments treated with 25 @g/ml MKT 077 in the presence and absence of high-intensity light. Results presented are the average of three separate experiments ±SE.
and brain. Cationic
photosensitizers
are especially
promising
as PCT
agents for the treatment of carcinoma, because they offer the benefit 74
Downloaded from on April 28, 2014. © 1998 American Association for Cancer Research.
MITOCHONDRIAL PHOTOTOXICITY OF MKT-077
C 0
4.' 0.
120
transmembrane potential in isolated rat liver mitochondria: spectral and metabolic
100
11. Zanotti, A., and Azzone, G. F. Safranine as membrane potential probe in rat liver
properties. Biochim. Biophys. Acts, 850: 436—448, 1996. mitochondria. Arch. Biochem. Biophys., 201: 255—265,1980. 12. Lana, P. C., Bahr, D. P., and Chaffee, R. R. J. Membrane potentials in mitochondrial
80 UIE Co 04.. C a,
preparations as measured by means of a cyanine dye. Biochim. Biophys. Acts, 376: 415—425,1975.
60 40
13. Akerman, K. E. 0., and Wikstrom, M. K. F. Safranine as a probe ofthe mitochondrial
membrane potential. FEBS Lett., 68: 191—197, 1976.
20 0
14. Bernal, S. D., Lampidis, T. J., Mclsaac, R. M., and Chen, L. B. Anticarcinoma activity in vivo of rhodamine 123, a mitochondrial specific dye. Science (Washington
0 0
20
40
MKT-077
60 (ug/mg
80
100
DC),222:169—172, 1983.
120
15. Davis, S., Weiss, M. J., Wong, J. R., Lampidis, T. J., and Chen, L. B. Mitochondrial
protein)
and plasma membrane potentials cause unusual accumulation and retention of rho damine 123 by human breast adenocarcinoma-derived MCF-7 cells. J. Biol. Chem.,
Fig. 6. Nonrespiratory-linked oxygen consumption in freeze-thawed rat liver mito chondria treated with 25 gsg/ml MKT-077 in the presence and absence of added high intensity light. Results presented are the average values of three separate experi
260: 13844—13850, 1985.
16. Modica-Napolitano, J. S., and Aprille, J. R. Basis for the selective cytotoxicity of rhodamine 123. Cancer Res., 47: 4361—4365, 1987. 17. Koya, K., Li, Y., Wang, H., Ukai, T., Tatsuta, N., Kawakami, M., Shishido, T., and
ments ±SE.
Chen, L. B. MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticar
of dual selectivity based on preferential accumulation in carcinoma mitochondria and localized irradiation of a malignant lesion or surgi cal excision site. Other cationic photosensitizers, including rhodam ines, phthalocyanines, and certain chalcogenapyrylium dyes (32—35), have been evaluated in preclimcal studies for their potential as PCT agents. MKT-077 is the only member of this class of compounds currently undergoing United States Food and Drug Administration approved Phase I clinical trials for the treatment of carcinoma. The
favorable pharmacological and photochemical properties of MKT-077 may make this compound an especially attractive candidate for de velopment as a photochemotherapeutic agent as well.
cinoma activity in preclinical studies based on selective mitochondrial accumulation.
Cancer Res., 56: 538—543,1996. 18. Modica-Napolitano,
J. S., Koya, K., Weisberg, E., Brunelli, B. T., Li, Y., and Chen,
L. B. Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077. Cancer Res., 56: 544—550, 1996.
19. Aprille, J. R., and Austin, J. Regulation of the mitochondrial adenine nucleotide pool size. Arch. Biochem. Biophys., 2312: 689—699, 1981. 20. Maltese, W. A., and Aprille, J. R. Relation of mevalonate synthesis to mitochondrial ubiquinone content and respiratory function in cultured neuroblastoma cells. J. Biol.
Chem.,260:11524—11529, 1985. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L, and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265—275,1951. 22. Aprille, J. R., and Asimakis, G. K. Postnatal development of rat liver mitochondria:
state 3 respiration, adenine nucleotide translocase activity, and the net accumulation of adenine nucleotides. Arch. Biochem. Biophys., 201: 564—575,1980. 23. Singer, T. P. Determination of the activity of succinate, NADH, choline and glycer
ophosphate dehydrogenases. Methods Biochem. Anal., 22: 123—175, 1974. 24. Wharton, D. C., and Tzagoloff, A. Cytochrome oxidase from beef heart mitochondria.
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