(CANCER RESEARCH 48, 3360-3366, June 15,1988|
Photosensitizing Effects of Hematoporphyrin Derivative and Photofrin II on the
Plasma Membrane Enzymes 5'-Nucleotidase, Na^K"1"-ATPase,and
Mg2+-ATPase in R3230AC Mammary Adenocarcinomas1
Scott L. Gibson, Richard S. Murant, and Russell Hilf2
Dépannentof Biochemistry and University of Rochester Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
The sensitivity to photodynamic treatment of three plasma membrane
enzymes in R3230AC mammary adenocarcinomas was assessed. The
activities of Na+K*-ATPase, Mg2+-ATPase and 5'-nucleotidase in iso
lated membranes were measured after exposure of membranes to either
hematoporphyrin derivative or Photofrin II plus light in vitro or in tumor
membranes prepared from animals previously injected with 25 mg/kg
Photofrin II and sacrificed at various times prior to exposure to light (in
vivo-in vitro protocol). The activities of both Na*K*-ATPase and Mg2+ATPase were inhibited at equivalent rates by Photofrin II in vitro;
inhibition was drug dose and light dose related. For S'-nucleotidase in
vitro, a 10-fold higher porphyrin concentration was required to achieve a
similar rate of enzyme inhibition as that for the ion-activated ATPases.
Injection of Photofrin II in vivo followed by preparation of tumor plasma
membranes, which were subsequently exposed to light in vitro, produced
no photosensitization of S'-nucleotidase activity at any time studied (up
to 72 h after Photofrin II administration). Under the same conditions
Na*K*-ATPase activity was reduced by 40-60% from 2 to 72 h after
drug injection, whereas Mg2+-ATPase activity was inhibited by 10-25%
over the same time course. The differential sensitivity of these three
enzymes observed in this in vivo-in vitro protocol suggests that each
enzyme may possess different characteristics, such as three-dimensional
configuration or membrane location, that afford varying susceptibility to
porphyrin photosensitization. The data also suggest that photosensitivityinduced damage to these ion-activated plasma membrane ATPases could
have deleterious effects on tumor cell survival.
INTRODUCTION
PDT3 is a promising new therapeutic modality for the man
agement of malignancy (1-5). This treatment regimen consists
of the systemic administration of a preparation termed hema
toporphyrin derivative (commercially available as Photofrin II,
PII) followed by the exposure of the malignant lesions to visible
light. Tumor regression in vivo and cell cytotoxicity in vitro are
attributed to the production of the highly reactive oxygen
species, singlet oxygen, "O2 (6-11). Many cellular sites have
been implicated as the targets for the damage caused by 'O2.
including the plasma membrane (12, 13), microsomes (14, 15),
the nucleus (16-19), and mitochondria (20-23). Although a
consensus has not been reached regarding the chronology of
events that lead to tumor cell death nor to the cellular site(s)
where the initial event is lethal, there is general agreement that
mitochondria are important targets of porphyrin photosensiti
zation in vitro (24-28).
In our laboratory, we examined the porphyrin-induced photosensitivity of selected enzymes in tumor mitochondria using
Received 11/17/87; revised 3/7/88; accepted 3/15/88.
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 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by USPHS Grant CA36856, NIH.
2To whom requests for reprints should be addressed, at Department of
Biochemistry, P. O. Box 607, University of Rochester School of Medicine and
Dentistry, 601 1,1mwood Avenue, Rochester, NY 14642.
' The abbreviations used are: PDT, photodynamic therapy; Hpd, hematopor
phyrin derivative; PII, Photofrin II; RNO, p-nitrosodimethylaniline; DHE, dihematoporphyrin ether or ester, /V inorganic phosphate.
both in vitro and in vivo-in vitro protocols. Enzymes located
within or attached to the inner mitochondria! membrane, such
as cytochrome c oxidase, succinate dehydrogenase, and the
proton translocating I-"0F|ATPase, were significantly inhibited
by porphyrin photosensitization (20, 21, 24). However, one of
the earliest reports on the action of hematoporphyrin derivative
was from Kessel (12), who observed significant reductions in
transport of cycloleucine (or aminoisobutyric acid) into LI210
cells. He concluded that the plasma membrane was functionally
altered by this treatment. Others have confirmed this in cultured
cells based on the appearance of lactate dehydrogenase in the
medium, interpreted as indicative of physical damage, i.e.,
leakiness, to the plasma membrane (29). Since we suggested
that mitochondria! membranes are sites of photosensitization,
we undertook a study of the tumor plasma membrane, using
similar experimental models. We investigated the effects of
porphyrin photosensitization on selected enzymes known to be
attached to the plasma membrane to determine whether photosensitized-induced enzyme inhibition would occur at this
membrane site. The results presented here indicate that the
activities of Na+K+-ATPase, Mg2+-ATPase, and S'-nucleotid
ase in plasma membrane preparations from tumors display
differences in photosensitivity to Hpd or PH.
MATERIALS
AND METHODS
Materials. Photofrin II was obtained from Photomedica Inc. (Raritan, NJ). From frozen 1.0-nil aliquots (2.5 mg/ml), dilutions of PII
were freshly prepared with phosphate buffered saline (pH 7.4) every
few days and kept refrigerated. Hematoporphyrin derivative was pre
pared according to the method of Lipson et al. (30) from hematopor
phyrin dihydrochloride (Sigma Chemical Co., St. Louis, MO). Unless
otherwise stated, all other chemicals and reagents were purchased from
Sigma.
Immobilization of Photofrin II on AH Sepharose-4B Beads. Photofrin
II was immobilized on AH Sepharose-4B beads (Pharmacia Inc., Piscataway, NJ), using the method described earlier for Hpd (31). Produc
tion of '(): by PII immobilized on Sepharose beads was measured by
the RNO assay (31). Under the same conditions of sensitizer concen
tration and photoradiation, the same rates of RNO bleaching were
obtained for immobilized PII and for immobilized Hpd.
Animals and Tumors. The R3230AC mammary adenocarcinoma was
maintained by s.c. transplantation in the axillary region of 60-80-g
female Fischer rats, using the sterile trochar procedure described pre
viously (32).
Plasma Membrane Preparation. Animals were sacrificed 17 to 24
days after tumor transplantation; tumors averaged 1 cm in diameter.
The tumors were excised, placed in cold homogenization buffer (see
below), and minced finely with scissors. In a threefold excess of buffer,
the tissue was washed twice and homogenized using a Polytron homogenizer (PCU2-110; Brinkman Industries, Westbury, NY) for two 30-s
periods at a setting of 5. Plasma membranes, isolated by two different
methods, yielded membranes of similar purity. The buffer used in the
first method (Buffer A) contained 250 HIMsucrose, 20 HIMTris-HCl
(pH 7.4), and 0.1 niM phenylmethylsulfonyl fluoride; in the second
method (Buffer B), the buffer contained 250 HIMsucrose, 30 HIMTrisHCl (pH 7.4), 40 mM NaCl, 1.0 mM MgCI2, 1.0 mM dithiothreitol, and
0.1 mM phenylmethylsulfonyl fluoride.
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PHOTOSENSITIZATION
OF PLASMA MEMBRANE ENZYMES
The initial experiments of Hpd photosensitization on S'-nucleotidase
activity utilized membranes prepared by the method of Shin et at. (33)
using Buffer A. This method required initial removal of nuclei by
centrifugation at 700 x g for 15 min and then mitochondria by centrifugation at 27,000 x g for 10 min. These and all subsequent steps were
carried out at 4°C.The plasma membrane fraction was isolated from
the 27,000 x g supernatant by centrifugation at 73,000 x g for 60 min.
The plasma membrane pellet was resuspended in 60% sucrose-30 mM
Tris-HCl (pH 7.4) and adjusted to 40% sucrose, as measured by optical
density, with 30 mM Tris-HCl. Into 30-ml centrifuge tubes and over a
7-ml plasma membrane suspension, 7 ml each of 37%, 33%, and 25%
sucrose-30 mM Tris-HCl were sequentially layered. These discontin
uous gradients were then centrifuged at 96,000 x g for 6-8 h in a
Beckman SW-27 rotor. The membranes located between the uppermost
two interfaces were collected, diluted 4-fold in 30 mM Tris-HCl buffer,
washed twice and pelleted by two 75-min 73,000 x g centrifugations,
and then frozen at —70°C
until use for studying the effects of photosen
sitization on 5'-nucleotidase activity.
A simpler and more rapid method, using the second homogenizing
buffer (Buffer B, see above), was adapted from Maeda et al. (34). Onto
15 ml of 37% sucrose (prepared in Buffer B lacking 250 mM sucrose)
were placed 15 ml of the supernatant collected after a slow speed
centrifugation (700 x g) of the total homogenate. This mixture was
then centrifuged for 75 min at 96,000 x g. The membranes distributing
at the interface were removed, diluted 4-fold with homogenizing buffer,
and then pelleted at 73,000 x g for 75 min. The membranes were
resuspended, washed once in 4 volumes of 30 mM Tris-HCl and pelleted
at 73,000 x g. The membrane pellet was resuspended in 30 mM TrisHCl, using 1.0 ml of buffer/1.0 g of original tumor weight. These
suspensions were stored at -70"C until use for study of photosensiti
zation of Na+K+-ATPase and Mg2+-ATPase in vitro. Membranes ob
tained for the in vivo-in vitro protocol were prepared using this proce
dure.
Treatment of Plasma Membrane Preparations with Hpd or Photofrin
II in Vitro. Frozen plasma membranes were thawed on ice and diluted
to approximately 4.0 mg protein/ml with 30 mM Tris-HCl (pH 7.4),
determined by the method of Lowry et al. (35). Hpd or PII at the
desired concentration in the dark was added either as an aqueous
solution or as a suspension when immobilized on AH Sepharose-4B
beads.
Treatment with Photofrin II in Vivo. The in vivo-in vitro protocol
consisted of isolating plasma membranes from tumors in animals that
had received a single dose of PII (25 mg/kg, administered i.p.) at 30
min, 2, 24, 48, or 72 h prior to sacrifice. All procedures following
administration of PII to the animals were performed in the dark.
Photoradiation Conditions. Photoradiation of plasma membranes was
conducted using two different light sources: fluorescent and quartz
halogen. The initial studies on 5'-nucleotidase in vitro employed flu
orescent light. The incident light energy emitted by the lamps was
measured by an RK5200 power radiometer connected to an RK545
radiometer probe (Laser Precision Corp., Utica, NY).
Fluorescent light (300-900 nm) was used for photoradiation of
plasma membranes that had been treated with Hpd in vitro only in
initial experiments measuring 5'-nucleotidase activity. In 16 x 100mm borosilicate test tubes, 0.2-ml aliquots of membrane samples were
illuminated with a 14-W fluorescent lamp at a distance of 6 cm; incident
light energy was 0.2 mW/cm2. Samples were obtained at selected times
and 5'-nucleotidase activity was assayed.
For plasma membranes treated either in vitro or obtained from
tumors after treatment with PII in vivo, a 1.0-cm diameter filtered beam
of broad band light (570-700 nm) emitted from a 500-W quartz halogen
lamp was focused on a 1.0-ml sample of plasma membranes placed in
a 3.0-ml quartz cuvet. Incident light energy was adjusted with neutral
density filters (NG Series, Schott Optical Glass) to 150 mW/cm2. The
membrane suspensions were stirred magnetically throughout photora
diation, during which portions of the sample (30-50 ^1) were taken at
selected times for measurement of enzyme activities. Temperature of
the plasma membrane suspensions was monitored during photoradiation and did not rise above ambient (22-25°C).
Enzyme Assays. Plasma membrane suspensions were assayed for
Na+/K+-ATPase, Mg2*-ATPase, and 5'-nucleotidase activities. Na+/
K+-ATPase activity requires the presence of Na* and K* ions, and is
inhibited by ouabain (36). Mg2+-ATPase activity is defined as the
ouabain-insensitive activity that is stimulated by Mg2+ and ATP (37).
The assays for Na*/K*-ATPase and Mg2+-ATPase employed conditions
described by Quigley and Gotterer (37), where quantitation of P\ re
leased was used as the endpoint. For total plasma membrane ATPase
activity, 50 /ul of sample were added to 0.95 ml of substrate buffer
solution, containing 30 mM Tris-HCl (pH 7.4), 120 mM NaCl, 20 mM
KC1, 5.0 mM ATP, 7.5 mM MgCI2, and 0.5 mM EGTA. For samples
added to a substrate-buffer solution that lacked KC1 and NaCl (buffer
with only Mg2+ and ATP), or to the complete substrate-buffer solution
plus 1.0 mM ouabain, hydrolysis of ATP, i.e., formation of P\ was
attributed to Mg2*-ATPase (ouabain-insensitive ATPase) activity. The
reaction mixtures were incubated at 37*C in a shaking water bath (New
Brunswick Scientific, New Brunswick, NJ) for 60 min, after which 1.0
ml of ice-cold 10% trichloroacetic acid was added to terminate the
reactions. After centrifugation at 700 x g for 10 min, inorganic phos
phate was determined on 1.0 ml of the supernatant according to the
method of Harris and Popat (38). Na+/K+-ATPase activity was calcu
lated by subtracting the amount of ouabain-insensitive ATPase, i.e.,
Mg2+-ATPase, activity from the total ATPase activity. Activity for
these ATPases was expressed in units (1 unit = 1 nmo\ Pi released/
min/mg protein).
Measurement of 5'-nucleotidase activity was performed using the
method of Lesko et al. (39). The plasma membrane sample (100-150
/ig protein) was added to 1.0 ml of an assay buffer solution containing
50 mM Tris-HCl (pH 7.5), 5 mM S'-AMP, and 10 mM MgCU. After
incubating for 30 min at 37'C in a shaking water bath, the reaction was
stopped by addition of 1.0 ml 10% trichloroacetic acid. The P¡concen
tration was determined (38) and enzyme activity was expressed in units,
as noted above.
RESULTS
Photosensitization
of Na*K+-ATPase and Mg2+-ATPase in
Plasma Membranes by Photofrin II in Vitro. Plasma membrane
suspensions were exposed to either 0.7 or 7.0 ng/m\ PII in vitro
and enzyme activities were determined after selected periods of
illumination. The results (Fig. 1, A and It) show a drug- and
light-dose-related inhibition of both ATPases. Neither ATPase
activity was altered in membrane samples exposed to PII but
not illuminated (dark control) nor in membrane samples illu
minated in the absence of drug treatment (light control) over
the range of drug and fluence used. Rates of enzyme inhibition
were calculated from the linear portion of each curve and are
expressed as percentage of enzyme inhibition/J •
cm"2 (control
= 100% activity at zero time prior to photoradiation, which
was 2.5 x IO"2 U for Na+K+-ATPase and 1.3 x 10~' for Mg2+ATPase). At 0.7 ng/ml PII, Na+K+-ATPase activity was inhib
ited at a somewhat higher rate than Mg2+-ATPase, 0.14 versus
0.07% inhibition/J-cm"2, respectively. However, at the higher
7.0 Mg/ml PH dose, no differences, 0.24 versus 0.28% inhibi
tion/J •
cm~2, respectively, were seen for these rates of inhibi
tion. These data demonstrate that there was little difference in
the sensitivity of Na+K+-ATPase and Mg2*-ATPase to porphyrin-induced photosensitization in vitro.
Photosensitization of Na*K*-ATPase and Mg2*-ATPase by
Immobilized Photofrin II in Vitro. The effect of PII-induced
photosensitization on Na*K+-ATPase and Mg2+-ATPase activ
ities in vitro was further investigated to determine whether a
differential sensitivity would be seen if entry of the sensitizer
into the plasma membrane was prevented. To assess this, we
employed our recently published procedure (31), which was
used to immobilize Hpd, to immobilize Photofrin II on AH
Sepharose-4B beads (see "Materials and Methods"). With this
reagent, one can produce equivalent levels of 'Oz (as measured
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PHOTOSENSITIZATION OF PLASMA MEMBRANE ENZYMES
100-
100
300
J/cm2
300
J/cm2
600
600
100-
B
50
f
-
10100
J/cm¿
Fig. 1. Effects of PII-induced photosensitization on the activities of Na*K*ATPase (A) and Mg2*-ATPase (/') in plasma membrane preparations in vitro. A,
0.7 /ig/ml PII, D, 7.0 fig/ml PII. Dala, percentage of initial enzyme activity (zero
time prior to photoradiation; 2.5 x 10~2U for Na*K*-ATPase and 1.3 x 10"' U
for Mg2*-ATPase). Points, mean of at least three experiments; error bars, SEM.
by RNO bleaching) after exposure of immobilized or aqueous
PII to light. We exposed plasma membranes to immobilized
PII to determine the extent of inhibition of Na+K'f-ATPase and
Mg2+-ATPase. The data (Fig. 2, A and B) demonstrate that
inhibition of these two ion activated ATPase enzymes was
similar. The rates of inhibition (percentage of inhibition/J
em'2) were: 0.07 and 0.16 for Na+K+-ATPase at 0.7 and 7.0
//g/ml immobilized PII concentrations, respectively, and 0.06
and 0.16, respectively, for Mg2+-ATPase at the same concen
trations of sensitizer. The concordance of results for inhibition
of Na+K+-ATPase and Mg2+-ATPase, using either free or im
mobilized PII, suggests that these plasma membrane enzymes
in R3230AC tumors may reside in similar topographical loca
tions and/or are equally susceptible to 'O2. Although the degree
of inhibition for both ATPases was consistently greater for
photosensitization with PII in aqueous solution than with the
immobilized preparation, the data, nevertheless, suggest that
'().- produced exterior to the plasma membrane had lifetimes
300
J/cm'
600
Fig. 2. Effects of immobilized PII to photosensitize Na*K*-ATPase (A) or
Mg2*-ATPase (B) in plasma membrane preparations. Preparation of immobilized
PII is detailed in "Materials and Methods." A, Data obtained with 0.7 /ig/ml and
squares with 7.0 mi/ml of the immobilized PH. Data, percentage of initial enzyme
activity (zero time prior to photoradiation); 2.5 x IO"2 U for Na*K*-ATPase and
1.3 x 10"' U for Mg2*-ATPase). Points, mean of at least three experiments; error
bars, SEM.
of sufficient duration to inflict damage on these membrane
enzymes.
Effects of Porphyrin-induced Photosensitization on the Activ
ity of Plasma Membrane 5'-Nucleotidase. Earlier, we investi
gated the effects of Hpd photosensitization in vitro on another
plasma membrane enzyme, 5'-nucleotidase. UUderthe experi
mental conditions used (Hpd 0.7, 7.0, 35, or 70 ng/ml and
photoradiation by a 14-W fluorescent lamp), concentrations of
0.7 or 7.0 Mg/ml Hpd in vitro plus light had only a minimal
effect on 5'-nucleotidase activity (<20% inhibition) (see Fig.
3). Even at 35 ng/ml Hpd, photoradiation-induced
inhibition
of 5'-nucleotidase activity was less than that observed for
Na+K+-ATPase or Mg2+-ATPase exposed to a photosensitizer
concentration of 7.0 ¿ig/ml(Figs. IA and 2/1)- At the highest
Hpd concentration in vitro, 70 Mg/ml, however, inhibition of
5'-nucleotidase activity approached that observed for the
ATPases studied.
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PHOTOSENSmZATlON
OF PLASMA MEMBRANE ENZYMES
100 -,
50W
i
2
10
(540)
0.1
DOS
0.7
J/cm''
Fig. 3. Effects of various concentrations of Hpd on S'-nucleotidase activity
(Militi lines) during photoradiation of plasma membranes with fluorescent light
in vitro (see "Materials and Methods"). The concentrations of Hpd used were: A,
0.7 Mg/nil: O, 7.0 Mg/ml; •.35 pg/ml; •.70 MB'ml. Data, percentage of initial
5'-nucleotidase activity (100% = 4.05 - 6.25 x 10~2U). Also shown is effect of
7.0 xg/ml Hpd plus broad band (570-700 nm) light at 150 mW/cm2 on 5'nucleotidase activity (A, broken line). Points, mean of at least three separate
determinations; hars. SEM.
OÕS
03
036
J/cm2
Fig. 4. Effects of Hpd photosensitization on purified 5'-nucleotidase exposed
in aqueous solution to fluorescent light (see "Materials and Methods"). The
activity of purified S'-nucleotidase was adjusted by dilution to approximate that
observed in plasma membrane preparations (4-6 x I(I ' U). Data, percentage of
initial activity (activity measured prior to photoradiation in the presence of Hpd).
The two concentrations of Hpd employed were: A, 0.7 *ig/ml; O, 7.0
Points, mean of at least three separate experiments; bars, SEM.
To determine whether this weaker response of S'-nucleotid
ase was due to the lower intensity of light emitted from the
fluorescent source (0.2 mW/cm2), plasma membranes were
exposed to 7.0 ng/m\ PII followed by photoradiation with 150
mW/cm2 of 570-700 nm light (see "Materials and Methods").
This treatment produced <20% inhibition of 5'-nucleotidase
activity after 1 h photoradiation (Fig. 3), an inhibition much
lower than seen for either Na+K+-ATPase or Mg2+-ATPase
under the same experimental conditions (Fig. 1). To ascertain
whether the lack of inhibition of 5'-nucleotidase was due to
some intrinsic property of the enzyme, we treated a purified
preparation of the enzyme (activity adjusted to the same level
found in membrane preparations) with 0.7 or 7.0 iig/m\ Hpd
plus fluorescent light (Fig. 4). Exposure of purified S'-nucleo
tidase to 7.0 ne/ml Hpd and 0.36 J/cm2 photoradiation pro
duced a 60% inhibition of activity. Since the purified prepara
tion of S'-nucleotidase was photosensitized by Hpd, we infer
that either the enzyme's location and/or environment in the
membrane afforded protection against photosensitization by
either hematoporphyrin preparation.
Photosensitization of Plasma Membrane Enzymes following
Administration of Photofrin II in Vivo. Plasma membranes were
prepared from R3230AC tumors borne on animals sacrificed
at selected times after a single i.p. administration of 25 mg/kg
PII and subsequently exposed to light in vitro. The data (Fig.
S) are expressed as a percentage of control (enzyme activity
prior to photoradiation) for each of the enzymes in plasma
membranes. In these experiments, illumination consisted of 45
min of photoradiation with 150 mW/cm2 broad band light
(570-700 nm). Throughout the time course examined, S'nucleotidase activity was not affected (<5% decrease in activity),
whereas Mg2+-ATPase activity was modestly reduced, reaching
a 25% decrease in plasma membranes prepared from tumors
POST INJECTION (hr)
Fig. 5. Time course of I'll induced photosensitization of selected plasma
membrane enzymes, using the in vivo-in vitro protocol. Plasma membranes were
prepared from animals treated with 25 mg/kg PII, as described in "Materials and
Methods." Dutu, percentage of initial enzyme activity for S'-nucleotidase (•),
Mg2*-ATPase (•),and Na*/K*-ATPase (D). Enzyme activity at each time point
was measured after 45 min photoradiation at 150 mW/cm! (570-700 nm). Points,
mean of several separate experiments; error bars, SEM.
obtained 72 h after PII treatment. In contrast, Na+K+-ATPase
activity was rapidly inhibited reaching greater than 50% reduc
tion by 2 h after administration of PII and remaining approxi
mately at this decreased level throughout the time course stud
ied. These data demonstrate differences in the susceptibility of
these plasma membrane enzymes to porphyrin-induced photo
sensitization when PH was administered systemically to tumorbearing animals.
DISCUSSION
Efficacy of PDT is probably due to hydrophobic "active
components" of Hpd or Photofrin II (either di hematoporphyrin
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PHOTOSENSITIZATION
OF PLASMA MEMBRANE ENZYMES
ether or ester) in the membranes of tumor cells (40-42), with
retention of dihematoporphyrin ether or ester in vivo prolonged
in tumor tissues compared to most normal tissues. Once se
questered in the tumor, exposure to visible light causes produc
tion of '<):, a highly reactive oxygen species that mediates
cellular damage. Questions remain regarding the discrete ac
tions of porphyrin photosensitization, such as identifying crit
ical intracellular target(s) where initial 'O2-induced damage
might occur. Tumor cell mitochondria appear to be an impor
tant target of porphyrin-induced photosensitization, with re
ports demonstrating a decrease in oxidative phosphorylation
(24), ( "a" uptake and respiration (43), and inhibition of key
enzymes associated with these processes (20, 21, 24). However,
functional or morphological disruption of the plasma mem
brane could also be an important factor in cell viability.
Damage to plasma membranes was observed after porphyrin
photosensitization of various cells in culture. Effects such as
protein cross-linking (44), inhibition of transport of substrates
required for DNA (25), and reduced protein and RNA synthesis
( 12) were reported and suggested as contributing to cytotoxicity.
Morphological changes in the plasma membrane were seen,
such as bleb formation (45), membrane swelling, and leakage
of eyloso lie components (46), all indicating disruption of mem
brane structure. However, these responses may relate to the
length of time of exposure of cells to porphyrins in vitro.
Christensen et al. (29) observed láclatedehydrogenase leakage,
bleb formation, membrane swelling and cell inactivation after
a 30-min exposure of cultured NHIK 3025 cells to Hpd and
light, whereas after 24 h of incubation, plasma membrane
damage was minimal, although cytotoxicity was produced. Kes
sel (25) demonstrated a similar pattern of events for L1210
cells in culture. After a 30-min incubation with 10 Mg/ml Hpd
plus light, transport of cycloleucine was decreased but DNA
synthesis, viability, and ATP pool size were minimally affected.
After 18 h, however, it was the latter parameters that were
affected, indicating that the damage was at intracellular sites
rather than the plasma membrane. Dubbelman and Van Steveninck (46), studying L929 fibroblast cells in culture, found
that active transport of Rb+ and a-aminoisobutyric acid were
inhibited by 5 and 10 ng/m\ Hpd and l h illumination, whereas
the passive efflux of K+ and passive influx of Rb+ required
exposure to an Hpd concentration of 25 Mg/ml plus light before
any effect occurred. From these findings, they concluded that
active transport systems were more sensitive to photodynamic
action than the passive membrane barrier function. Results
reported here on several plasma membrane enzymes support
the findings by Dubbelman and Van Steveninck (46), since we
observed both drug-dose- and light-dose-related inhibition of
the activities of Na+K+-ATPase and Mg2+-ATPase, enzymes
that utilize ATP to maintain ion gradients across the plasma
membrane (47). Both ATPases were affected similarly by PII
plus light //; vitro, effects that should impair ion movements
against the gradients.
We also employed an immobilized PII reagent and light,
reasoning that this approach might provide insight into differ
ences in membrane locations or enzyme configurations for the
two ATPases, based on differential sensitivity to 'O2 produced
externally. Na+/K+-ATPase was reported to span the entire
plasma membrane (36). Although rates of enzyme inhibition
were generally lower with the immobilized PII reagent both ion
activated ATPases were affected similarly. These results con
trast to those observed for two inner mitochondrial membrane
enzymes; cytochrome c oxidase activity was inhibited by both
aqueous Hpd and immobilized Hpd whereas inhibition of suc-
cÃ-ñate
dehydrogenase occurred only when soluble Hpd was used
(31). In this earlier report (31), immobilized Hpd did not cause
inhibition of Na+K+-ATPase, but those experiments employed
dissociated cells and fluorescent light at 0.2 mW/cm2 compared
to membrane preparations and broadband illumination, 570700 nm, at 150 mW/cm2, used here. Nevertheless, the similarity
of the porphyrin-induced photoinactivation of Na+K+-ATPase
and Mg2+-ATPase in plasma membrane preparations implies
that these enzymes may be similar in configuration and location
where oxidative damage occurs and/or that the intimate envi
ronment of these enzymes is similar, permitting sufficient
amounts of porphyrin components to accumulate and act as
photosensitizers. While details of the structure and location of
Na+K+-ATPase are being evolved (48), little information on
Mg2+-ATPase has been published.
Our data indicate that 5'-nucleotidase was relatively insen
sitive to Hpd- or PH-induced photosensitization, since only
when membranes were exposed to 70 ^g/ml Hpd and fluores
cent light did we observe rates of inhibition comparable to those
seen for the ion-activated ATPases. Under the same conditions
used to study photoinactivation of Na+K+-ATPase and Mg2+ATPase in vitro (broad band light at 570-700 nm, 150 mW/
cm2 for 1 h, 7.0 tig/ml PII), we observed little or no alteration
in the activity of 5'-nucleotidase. This was probably not due to
intrinsic properties of the enzyme, since the purified enzyme
was significantly inhibited by Hpd plus light in vitro. However,
the three-dimensional structure of 5'-nucleotidase in the plasma
membrane may differ from the purified enzyme in solution.
One possible explanation for the relative lack of sensitivity of
5'-nucleotidase, which is thought to be deeply anchored in the
plasma membrane with its main enzymatic activity located at
the outer surface (49), may be attributed to protection of the
enzyme from oxidative damage by the local membrane environ
ment via components in the membrane that react with '(): to
quench it before it interacts with susceptible sites in the enzyme.
To compare the results on plasma membrane enzymes with
previous studies of certain mitochondrial enzymes (28), initial
rates of inhibition were calculated by linear regression analysis
of data obtained under identical conditions of drug dose and
fluence. Expressed as percentage of inhibition/J •
cm"2, these
values at 7.0 Mg/ml PII and 150 mW/cm2 broad band (570700 nm) illumination are: Na+-K+ ATPase, 0.24; Mg2+ ATPase,
0.28, 5'-nucleotidase, 0.04; and for the mitochondrial enzymes,
cytochrome c oxidase, 0.46; monoamine oxidase, 0.24; and
adenylate kinase, 0.04. These data indicate that the ATPases
were inhibited at approximately half the rate as the inner
mitochondrial enzyme cytochrome c oxidase. Although such
results imply differences in enzyme sensitivity to photosensiti
zation, other factors, i.e., membrane environment, enzyme
structure in situ, affect overall sensitivity. Furthermore, the
relationship between extent of inhibition of enzyme activity and
its impact on cellular metabolism may differ among these
enzymes.
From the in vivo-in vitro protocol, a differential inhibition of
the three plasma membrane enzymes was seen. Over the timecourse, 5'-nucleotidase was unaffected (<5% inhibition), Mg2+ATPase activity was reduced by 15-25% (30 min to 72 h) and
Na+K+-ATPase was inhibited by 40-60% from 2 to 72 h. These
differences in inhibition for Na+K+-ATPase versus Mg2*ATPase were not observed with plasma membrane preparations
exposed to porphyrins in vitro. Assuming that some metabolism
of PII occurred in the animal, such differences in photosensitivity could be due to differences in local environment, i.e.,
concentration of porphyrins and/or presence of components
3364
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PHOTOSENSITIZATION OF PLASMA MEMBRANE ENZYMES
capable of scavenging 'O:, or to differences in enzyme configuration in the membrane. Regardless of these considerations,
inhibition of these enzymes should be detrimental to the cell.
Without the ability to maintain an intracellular ion balance of
high K+ and low Na+, diffusion of these ions across the mem
brane would alter intracellular osmolarity. If Na+ ions that
freely entered the cell could not be pumped out, due to loss of
active transport mechanisms, entry of water would ensue to
maintain constant intracellular osmolarity. This in turn would
cause the cell to swell, as shown by Christensen earlier (29),
and ultimately could cause the cell to burst. Thus, the data from
the in vivo-in vitro protocol implies that porphyrin-induced
photosensitization damaged components of the plasma membrane and that damage might contribute to tumor cell cytotoxicity.
Recently, evidence has been obtained implicating vascular
damage as the critical event that produced cytotoxicity in vitro
(50-52). Using [31P]NMR spectroscopy to monitor directly
phosphate-containing
metabolites in R3230AC tumors that
were treated by PDT, we found marked reductions in ATP
levels, often reaching undetectable levels within 2 h after com
pletion of therapy (53, 54) and morphological evidence of cell
death was observed. In the above studies, we used essentially
the same photoradiation conditions in vivo as those used here
in vitro. Even if inhibition of Na+-K+ ATPase enzyme was
repaired rapidly, the absence of ATP would prevent the enzymecatalyzed maintenance of ion gradients in such cells. Thus, the
studies presented here provide additional insight into the se
quence of metabolic events resulting from PDT.
ACKNOWLEDGMENTS
We acknowledge the continued assistance of Kathy Faro of the
Animal Tumor Research Facility, University of Rochester Cancer Cen
ter (CAI 1198) in maintaining the R3230AC mammary adenocarcinoma.
REFERENCES
1. Dougherty, T. J., Boyle, D. G., Weishaupt, K. R., Henderson, B. A., Potter,
W. R., Bellnier, D. A., and Wityk, K. E. Photoradiation therapy—clinical
and drug advances. In: D. Kessel and T. J. Dougherty (eds.). Porphyrin
Photosensitization, pp. 15-21. New York: Plenum Publishing Corp., 1983.
2. Dahlman, A., Wile, A. G., Burns, R. G., Mason, G. R., Johnson, F. M., and
Berns, M. W. Laser photoradiation therapy of cancer. Cancer Res., 43:430434, 1983.
3. Laws, E. R., Cortese, D. A., Kinsey, T. H., Eagan, R. T., and Anderson, R.
E. Photoradiation therapy in the treatment of malignant brain tumors: a
phase I (feasibility) study. Neurosurgery, 9:672-678, 1981.
4. McCaughan, J. S., Schellhas, H. F., Lomsno, J., and Bethel, B. H. Photodynamic therapy of gynecologic neoplasma after presensitization with hematoporphyrin derivative. Lasers Surg. Med., 5:491-498, 1985.
5. Balchum, O. S., Doiron, D. R., and Huth, G. C. Hpd photodynamic therapy
for obstructing lung cancer. In: D. R. Doiron and C. J. Corner (eds.),
Porphyrin Localization and Treatment of Tumors, pp. 727-745. New York:
Alan R. Liss, 1984.
6. Weishaupt, K. R., Comer, C. J., and Dougherty, T. J. Identification of singlet
oxygen as the cytotoxic agent in photoinactivation of a murine tumor. Cancer
Res., 36: 2322-2329, 1976.
7. Ho, T. Photodynamic action of hematoporphyrin on yeast cells: a kinetic
approach. Photochem. Photobiol., 34: 521-524, 1981.
8. Stenstrom, A. G. K., Moan, J., Brunborg, G., and Eklund, T. Photodynamic
inactivation of yeast cells sensitized by hematoporphyrin. Photochem. Photobiol., 32: 349-352, 1980.
9. Richard, P., Blum, A., and Grossweiner, L. I. Hematoporphyrin photosen
sitization of serum albumin and subtilisin BPN'. Photochem. Photobiol., 37:
287-291, 1983.
10. Gibson, S. L., Cohen, H. J., and Hilf, R. Evidence against the production of
Superoxide by photoirradiation of hematoporphyrin derivative. Photochem.
Photobiol., 40: 441-448,1984.
11. Parker, J. G. Optical monitoring of singlet oxygen generation during photodynamic treatment of tumors. IEEE Circuits and Devices Magazine, pp.
10-2 I.January, 1987.
12
Kessel, D. Effects of photoactivated porphyrins at the cell surface of leukemia
L 1210 cells. Biochemistry, 16: 3443-3449, 1977.
13 Kohn, K., and Kessel, D. On the mode of cytotoxic action of photoactivated
porphyrins. Biochem. Pharmacol., 28: 2465-2470, 1979.
14 Dixit, R., Mukhtar, H., and Bickers, D. R. Destruction of microsomal P-450
by reactive oxygen species generated during photosensitization of hemato
porphyrin derivative. Photochem. Photobiol., 37: 173-176, 1983.
15 Das, M., Dixit, R., Mukhtar, M., and Bickers, D. R. Role of active oxygen
species in the photodestruction of microsomal P-450 and associated monooxgenases by hematoporphyrin derivative in rats. Cancer Res., 45: 608-615,
1985.
l6 Moan, J., Watsvik, H., and Christensen, T. DNA single strand breaks and
sister chromatid exchange induced by treatment with hematoporphyrin and
light or X-rays in human NHIK 3025 cells. Cancer Res., 40: 2915-2918,
1980.
17 Corner, C. J. DNA damage and repair in CHO cells following hematopor
phyrin photoradiation. Cancer Lett., //: 161-167, 1980.
ig. Gutter, G., Speck, W. T., and Rosenkranz, H. S. The photodynamic modi
fication of DNA by hematoporphyrin. Biodi ini. Biophys. Acta, 475: 307314, 1977.
,9 Fiel, R. J., Datta-Gupta, N., Mark, E. H., and Howard, J. C. Induction of
DNA damage by porphyrin photosensitizes. Cancer Res., 41: 3543-3545,
1981.
Gibson, S. L., and Hilf, R. Photosensitization of mitochondria! cytochrome
c oxidase by hematoporphyrin derivative and related porphyrins in vitro and
in vivo. Cancer Res., 43:4191-4197, 1983.
21 Hilf, R., Smail, D. B., Murant, R. S., Leakey, P. B., and Gibson, S. L.
Hematoporphyrin derivative-induced photosensitivity of mitochondria! succinate dehydrogenase and selected cytosolic enzymes of R3230AC mammary
adenocarcinoma of rats. Cancer Res., 44:1483-1488, 1984.
22. Berns, M. W., Dahlman, A., Johnson, F. M., Burns, R. G., Sperling, D.,
Guiltin, G., Siemens, A., Walter, R., Wright, W., Hammer-Wilson, M., and
Wile, A. In vitro cellular effects of hematoporphyrin derivative. Cancer Res.,
Â¥2:2325-2329, 1982.
Sandberg, S., and Romslo, I. Porphyrin-sensitized photodynamic damage of
isolated rat liver mitochondria. Biochim. Biophys. Acta, 593:187-195,1980.
24 Perlin, D. S., Murant, R. S., Gibson, S. L., and Hilf, R. Effects of photosen
sitization by hematoporphyrin derivative on mitochondria! adenosine triphosphate-mediated proton transport and membrane integrity of R3230AC
mammary adenocarcinoma. Cancer Res., 45:653-658, 1985.
25 Kessel, D. Sites of photosensitization by derivatives of hematoporphyrin.
Photochem. Photobiol., 44:489-493, 1986.
26. Atlante, A., Moreno, G., Passarella, S., and Salet, C. Hematoporphyrin
derivative (Photofrin II) photosensitization of isolated mitochondria: impair
ment of anióntranslocation. Biochem. Biophys. Res. Commun., 141: 584590, 1986.
27. Moreno, G., Atlante, A., Salet, C., Santus, R., and Vinzens, F. Photosensi
tivity of DNA replication and respiration to hematoporphyrin derivative
(Photofrin II) in mammalian CV-1 cells. Int. J. Radiât.Biol., 52: 213-222,
1987.
28. Murant, R. S., Gibson, S. L., and Hilf, R. Photosensitizing effects of
Photofrin II on site-selected mitochondria! enzymes: adenylate kinase and
monoamine oxidase. Cancer Res., 47:4323-4328, 1987.
29. Christensen, T., Sandquist, T., Feren, K., Waksvik, H., and Moan, J. Reten
tion and photodynamic effects of hematoporphyrin derivative in cells after
prolonged cultivation in the presence of porphyrin. Br. J. Cancer, 48: 35-43,
1983.
30. Lipson, R. L., Baldes, E. J., and Olsen, A. M. The use of a derivative of
hematoporphyrin in tumor detection. J. Nati. Cancer Inst., 26: 1-8, 1960.
31. Gibson, S. L., Murant, R. S., and Hilf, R. Photosensitizing effects of
hematoporphyrin derivative immobilized on sepharose. Photochem. Photo
biol., ¥5:93-104, 1987.
'-•Hilf, R., Michel, I., Bell, C., Freeman, J. J., and Borman, A. Biochemical
and morphological properties of a new lactating tumor line in the rat. Cancer
Res., 25: 286-299, 1965.
33. Shin, B. C., Ebner, K. E., Hudson, B. G., and Carraway, K. L. Membrane
glycoprotein differences between normal lactating mammary tissue and the
R3230AC mammary tumor. Cancer Res., 35:1135-1140, 1975.
34. Macela, T., Balakrishnan, K., and Mehdi, S. Q. A simple and rapid method
for the preparation of plasma membranes. Biochim. Biophys. Acta, 731:
115-120,1983.
35. Lowry, O. 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.
36. Scheiner-Bobis, G., Zimmermann, M., Kirch, U., and Schoner, W. Ouabainbinding site of (Na* + K*) All'uso in right-side-out vesicles has not an
externally accessible SH group. Eur. J. Biochem., 165:653-656, 1987.
37. Quigley, J. P., and Gotterer, G. S. Distribution of (Na*-K*)-stimulated
ATPase activity in rat intestinal mucosa. Biochim. Biophys. Acta, 173:456468, 1969.
38. Harris, W. D., and Popat, P. Determination of the phosphorus content of
lipids. J. Amer. Oil Chem. Soc., 31:124-127, 1954.
39. Lesko, L., Dondon, M., Marmeln. G. V., and Hare, J. D. A rapid method
for the isolation of rat liver plasma membranes using an aqueous two-phase
polymer system. Biochim. Biophys. Acta, 311: 173-179, 1973.
40. Kessel, D. Transport and binding of hematoporphyrin derivative and related
porphyrins by murine leukemia L1210 cells. Cancer Res., 41: 1318-1323,
1981.
3365
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research.
PHOTOSENSITIZATION
OF PLASMA MEMBRANE ENZYMES
41. Bellnier, D. A., and Lin, C. Photodynamic destruction of cultured human
bladder tumor cells by hematoporphyrin derivative: effects of porphyrin
molecular aggregation. Photobiochem. Photobiophys., 6: 3S7-366, 1983.
42. Sun, C., Duzman, E., Mellott, J., Liaw, L., and Hi-rns. M. W. Spectroscopic,
morphologic, and cytotoxic studies on major fractions of hematoporphyrin
derivative and Photofrin II. Lasers Surg. Med., 7:171-179, 1987.
43. Sale!, C., Moreno, G., and Vinzens, F. Effects of photodynamic action of
energy coupling of C,\:' uptake in liver mitochondria. Biochem. Biophys.
Res. Commun., 115: 76-81, 1983.
44. Dubbelman, T. M. A. R., DeGoeij, A. F. P. M., and Van Steveninck, J.
Photodynamic effects of protoporphyrin on human erythrocytes; nature of
cross-linking of membrane proteins. Biochim. Biophys. Acta, Sil: 141-151,
1978.
45. Volden, G., Christensen, T., and Moan, J. Photodynamic membrane damage
of hematoporphyrin derivative-treated NHIK 3025 cells in vitro. Photobi
ochem. Photobiophys., J: 105-111, 1981.
46. Dubbelman, T. M. A. R., and Van Steveninck, J. Photodynamic effects of
hematoporphyrin-derivative on transmembrane transport systems of murine
L929 fibroblasts. Biochim. Biophys. Acta, 771: 201-207, 1984.
47. Mercer, R. W., and Dunham, P. B. Membrane-bound ATP fuels the Na/K
pump. J. Gen. Physiol., 78: 547-568, 1981.
48. Poupon, R. E., and Evans, W. H. Biochemical evidence that Na*K*-ATPase
is located at the lateral region of the hepatocyte surface membrane. FEBS
Lett., 108: 374-378, 1979.
49. Rohr, G., and Mannbuz, H. G. The activation of actin: DNase I complex
with rat liver plasma membranes; the possible role of 5'-nucleotidase. FEBS
Lett., 99:351-356, 1979.
50. Selman, S. H., Kreimer-Birnbaum, M., Klaunig, J., Goldblatt, P. J., Keck,
R. W., and Britton, S. L. Blood flow in transplantable bladder tumors treated
with hematoporphyrin derivative and light. Cancer Res., 44: 1924-1927,
1984.
51. Henderson, B. W., Waldow, S. M., Mang, T. S., Potter, W. R., Malone, P.
B., and Dougherty, T. J. Tumor destruction and kinetics of tumor cell death
in two experimental mouse tumors following photodynamic therapy. Cancer
Res., «:572-576, 1985.
52. Star, W. M., Marijnissen, H. P. A., van den Berg-Blok, A. E., Versteeg, J.
A. C., Franken, K. A. P., and Reinhold, H. S. Destruction of rat mammary
tumor and normal tissue microcirculation by hematoporphyrin derivative
photoradiation observed in vivo in sandwich observation chambers. Cancer
Res., 46: 2532-2540, 1986.
53. Ceckler, T. C., Bryant, R. G., Penney, D. P., Gibson, S. L., and Hilf, R. "PNMR spectroscopy demonstrates decreased ATP levels in vivo as an early
response to photodynamic therapy. Biochem. Biophys. Res. Commun., I4II:
273-279, 1986.
54. Hilf, R., Gibson, S. L., Penney, D. P., Ceckler, T. C., and Bryant, R. G.
Early biochemical responses to photodynamic therapy monitored by NMR
spectroscopy. Photochem. Photobiol., 46:809-817, 1987.
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Photosensitizing Effects of Hematoporphyrin Derivative and
Photofrin II on the Plasma Membrane Enzymes 5 ′-Nucleotidase,
Na +K+-ATPase, and Mg2+-ATPase in R3230AC Mammary
Adenocarcinomas
Scott L. Gibson, Richard S. Murant and Russell Hilf
Cancer Res 1988;48:3360-3366.
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