[CANCER RESEARCH 49, 1682-1686, April 1,1989]
Role of Lipid Peroxidation in Hematoporphyrin Derivative-sensitized
of Tumor Cells: Protective Effects of Glutathione Peroxidase1
Photokilling
James P. Thomas2 and Albert W. Girotti3
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
ABSTRACT
The ability of cells to detoxify lipid hydroperoxides (LOOHs) gener
ated by hematoporphyrin derivative (HPD)-sensitized photooxidation
was investigated for the first time. The general importance of glutathione
in cytoprotection was confirmed by showing that murine L1210 cells were
more sensitive to the lethal effects of HPD plus red light after being
treated with buthionine sulfoximine. The specific role of Se-dependent
glutathione peroxidase was investigated by using LI 210 cells that were
grown in Se-defìcient
media. Glutathione peroxidase activity of such cells
was typically <5% ofthat exhibited by Se-replete cells. When examined
by means of dye exclusion or clonogenic assay, Se-deficient cells were
dramatically more sensitive to HPD-mediated photokilling than normal
counterparts. Impaired metabolism of hydrogen peroxide was ruled out
as a possible cause of enhanced photokilling, since added catalase had
no protective effect on Se-deficient cells. lodometric analysis of lipid
extracts from photooxidized cells indicated a significantly greater rate of
LOOK accumulation as a result of Se depletion. Moreover, when de
pleted cells were incubated in the dark after a short period of photoperoxidation, LOOM decay was markedly slower than in controls. Similar
results were obtained with human CaSki cells derived from cervical
carcinoma. It is apparent from these results that lipid peroxidation plays
an important role in tumor cell eradication by HPD/phototherapy, and
that glutathione peroxidase serves as a natural protectant against pho
tokilling by catalyzing the reduction of LOOHs.
INTRODUCTION
HPD4 is a complex mixture of photosensitizing
porphyrins
that has the ability to localize in malignant tumors (1,2). PDT
with HPD is a promising new approach for selective eradication
of solid tumor cells (2). This treatment modality has been under
intensive investigation since being proposed more than a decade
ago (3) and is now in Phase III clinical trial (2), yet little is
known about the molecular basis for cytolethality. The active
components of HPD are ether- and/or ester-linked hematopor
phyrin units which exist as large heterogeneous aggregates
under aqueous conditions (4). Being Hydrophobie in character,
these porphyrins tend to localize in plasma membranes and
subcellular membranes, making these structures especially sen
sitive to toxic photodamage (5-8). Important targets of mem
brane photodamage are unsaturated phospholipids and choles
terol, which undergo oxidative degradation commonly termed
Received 8/17/88; revised 11/16/88; accepted 12/29/88.
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.
1This project was supported by grants from the National Science Foundation
(DCB-8501894), the St. Luke's Foundation (Milwaukee), and the Cancer Center
of the Medical College of Wisconsin.
2 These studies are based on a dissertation submitted by J. P. Thomas in partial
fuIfiIImoit of the requirements for the PhD degree in Biochemistry at the Medical
College of Wisconsin.
3To whom requests for reprints should be addressed, at Department of
Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226.
4 The abbreviations used are: HPD, hematoporphyrin derivative; HPD-A,
rapidly eluting fraction of HPD on Bio Gel P-10 chromatography; PBS, phos
phate buffered saline (25 mM sodium phosphate, 125 min NaCl, pH 7.4); HEPES,
4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid; CTAB, cetyltrimethylammonium bromide; GSH, reduced glutathione; GSSG, oxidized glutathione; GSHperoxidase, glutathione peroxidase; GSSG-reductase, glutathione reductase;
LOOH, lipid hydroperoxide; BSO, DL-buthionine-S./f-sulfoximine; PDT, photodynamic therapy.
lipid peroxidation (9). In earlier model studies, we (10, 11) and
others (12) speculated that lipid peroxidation plays an impor
tant role in the cytolethal effects of PDT with HPD. We now
provide direct evidence for this relationship by showing that
selenium-deficient cells, which are severely limited in their
ability to carry out glutathione peroxidase-catalyzed detoxifi
cation of lipid-derived hydroperoxides, are much more suscep
tible to HPD-sensitized photokilling than normal cells.
MATERIALS AND METHODS
Materials. The following enzymes and chemicals were obtained from
Sigma Chemical Co. (St. Louis, MO): Bovine GSH-peroxidase, cata
lase, Superoxide dismutase, insulin and transferrin, yeast GSSG-reduc
tase, hematoporphyrin dihydrochloride, NADPH, GSH, /-butyl hydroperoxide, buthionine sulfoximine, and sodium selenite. All other chem
icals were of the highest purity available and all aqueous solutions were
prepared with deionized, glass-distilled water.
HPD was prepared according to Gomer and Dougherty (13) and
partially purified by gel exclusion chromatography on Bio Gel P-10
(14). Cells were photosensitized with the rapidly eluting fraction, which
is enriched in tumor-localizing porphyrins. This fraction, designated
HPD-A, was stored in HEPES-buffered saline at -20°C until used.
HPD-A is considered to be equivalent to Photofrin II (Quadra Logic
Technologies, Vancouver, BC). Porphyrin concentrations were deter
mined spectrophotometrically in 10 mM CTAB/50 mM HEPES (pH
7.0), using an extinction coefficient of 220 (mg/ml)"'cm"1 at 399 nm
(10, 14).
Murine L1210 leukemia cells and human CaSki cervical carcinoma
cells were provided respectively by Dr. F. Sieber, Department of Pedi
atrics, and Dr. R. Palliilo, Departmenl of Gynecology/Obslelrics,
Medical College of Wisconsin. The cells were grown in conlinuous
cullure under previously defined condilions (15, 16).
Depletion of Cellular Selenium and Glutathione. For experiments in
which the consequences of Se deprivation were to be examined by
clonal assay, LI210 or CaSki cells were gradually weaned from 10%
fetal calf serum to 1% serum (in 5%, 3%, and 1% decrements) over a
period of 7 days. The cells were then divided into two groups and grown
in defined RPMI media containing insulin (10 /jg/ml) and transferrin
(5 ¿ig/ml),one group lacking Se and the other containing Se in the
form of sodium selenite (5 ng/ml, ~28 nM) (17). The cell groups were
maintained under these conditions for up to 10 days prior to being used
experimentally.
GSH depletion was accomplished by treating cells with 1 mM BSO
for 12 h immediately before HPD photosensitization. GSH was deter
mined by the method of Tietze (18); its concentration in BSO-treated
cells was typically 5-10% of control values.
Enzymatic Assays. GSH-peroxidase activity of Se-depleted and Sereplete cells was measured by coupling the peroxidase-catalyzed oxi
dation of GSH by /-butyl hydroperoxide to the GSSG-reductase-catalyzed oxidation of NADPH by GSSG, and tracking the time course of
A34odecay at 30°C(19). Typical assay components were as follows: ~2
x 10' cells/ml, 0.1% Triton X-100, 0.4 mM EDTA, 0.2 mM NADPH,
2 HIMGSH, 3.5 units/ml GSSG-reductase, and 0.16 mM /-butyl hydroperoxide in 20 mM phosphate buffer (pH 7.4). Specific enzymatic
activities were expressed in terms of units per IO7cells, 1 unit of enzyme
catalyzing the oxidation of 1 nmol NADPH per min. Certain GSH-5transferases can act as hydroperoxidases (20), but are virtually unreactive toward, e.g., hydrogen peroxide or /-butyl hydroperoxide (21).
Therefore, the assay described avoids possible interference by GSH-5transferases. Test assays without cells were carried out with commer-
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LIPID PEROXIDATION
IN HPD-SENSITIZED
dally available GSH-peroxidase (0.03-0.05 units/ml), as described
previously (22).
Glutathione-S-transferase activity was measured according to Habig
et al. (23), using l-chloro-2,4-dinitrobenzene as the GSH acceptor.
Irradiation Conditions. The typical procedure was as follows. Sesufficient or Se-deficient L1210 cells (106/ml) in 1% serum/RPMI
medium were mixed with HPD-A (15 Mg/ml) under sterile conditions,
incubated in the dark for 2 h at 37°C,and transferred to 24-well tissue
culture plates. The plates were placed on a translucent plastic platform
over a twin bank of 20-W daylight fluorescent tubes and irradiated for
different periods as indicated. Except where indicated, incoming light
was filtered through a film of red cellophane (>600 nm transmittance)
to mimic PDT conditions. Light intensity at the platform surface was
measured with a Yellow Springs radiometer (Yellow Springs, OH) and
maintained at ~0.1 mW/cm2 for most of the experiments described.
The suspensions were hand agitated at regular intervals to assure
uniform light exposure. Ambient temperature (initially 25-27°C)rose
by <5°Cover a 20-min illumination period, the longest used. At various
time points, cells were collected for assessment of viability (see below).
All subsequent manipulations were done under minimal room illumi
nation. BSO-treated LI210 cells were sensitized with HPD-A and
irradiated in similar fashion.
A different procedure was used for CaSki cells (16), which attach to
culture plates during proliferation. Attached cells (IO6 per 20 cm2
culture flask) were dark-incubated with 15 Mg/ml HPD-A for 24 h, then
washed with fresh medium (4 h), and irradiated as described above. 24
h after a given light dose, the cells were trypsinized from the flask and
examined for viability by means of dye exclusion.
Determination of Cellular Porphyrin. L1210 cells were incubated with
HPD-A as described above. Prior to irradiation, samples containing 5
x IO6cells were centrifuged and washed twice with 1 volume of PBS.
The pellets (~0.1 ml) were mixed with 0.9 ml of 2% sodium dodecyl
sulfate in 5 itiM sodium phosphate (pH 8), heated at 100°Cfor 15 min,
and the absorbance values of the solutions at 395 nm were recorded.
Porphyrin concentrations were calculated by using an extinction coef
ficient of 0.163 (mg/ml)~'cm~'. Specific values were expressed in terms
of Mg porphyrin/mg cellular protein. Protein was determined by the
conventional Lowry method, using bovine serum albumin as the stand
ard (24).
Biological Assays. Cell viability was monitored (a) by the conven
tional Trypan Blue assay, cells taking up the dye being counted as
nonviable; and (b) by the more rigorous measurement of colony for
mation, using the procedure of Sieber et al. (15). For clonal assays,
200-500 cells were plated on 35-mm Petri dishes containing 0.9%
methylcellulose and 20% fetal bovine serum in a medium. The cells
were incubated at 36°Cin a humidified atmosphere of 5% CO2/95%
CELLS
(26). As shown in Fig. 1, irradiation of HPD-A-sensitized
control cells (non-BSO-treated) resulted in a gradual decrease
in the viable cell count with increasing porphyrin concentration
above 0.5 Mg/ml. Significantly, the BSO-treated cells were much
more photosensitive; note the >threefold increase in log kill at
a sensitizer concentration of 0.75 Mg/ml. BSO on its own, i.e.,
in the absence of HPD-A, was relatively nontoxic in the dark
or light, causing <40% cell kill at the concentration used.
Although BSO produced a >90% reduction in GSH concentra
tion, it had no significant effect on porphyrin uptake by the
cells (data not shown), ruling out greater uptake as a possible
reason for the enhanced photokilling. Similar effects of BSO in
conjunction with photodynamic action have been reported for
other cell lines (27).
Cytological Effects of Se Depletion. There are several possible
explanations for the observed protective effects of GSH against
photodynamic cell killing. These include (a) direct scavenging
of activated oxygen species such as singlet oxygen ('02), superoxide (O2~), hydrogen peroxide (H2O2) and hydroxyl radical
(OH-) or organic radicals such lipid peroxyl (LOO-) and lipid
oxyl (LO-) (28); (b) involvement in the peroxidatic activity of
certain Se-independent GSH-5-transferases (20); (c) involve
ment in the peroxidatic activity of Se-dependent GSH-peroxi
dase (29). To focus directly and unequivocally on the possible
antioxidant role of GSH-peroxidase, we prepared Se-deficient
LI210 cells by growing in defined media (with or without 1%
serum) that lacked Se. As shown in Fig. 2, deficient cells were
markedly more sensitive to photokilling by HPD-A than Sereplete counterparts. Note that at a light dose of ~0.05 J/cm2
(9 min), cell survival, as measured by colony formation, was
decreased by three orders of magnitude as a result of Se depri
vation. Significantly, the GSH-peroxidase activity of these cells
was only -2% of normal, i.e., 0.9 U/107 cells vs. 48.8 U/107
cells. However, the GSH content of the Se-deprived cells (5.2
±0.4 nmol/106 cells) was only slightly different from that of
the controls (4.5 ±0.2 nmol/106 cells). Similar results were
obtained with CaSki cells (Fig. 3). Thus, there was a strong
positive correlation between the selective incapacitation of
GSH-peroxidase and increased susceptibility to photokilling.
As was the case for GSH (Fig. 1), Se depletion in LI210 cells
had no significant effect on plating efficiency or subsequent
uptake of HPD-A. In a representative experiment, porphyrin
air. Colonies were counted after 7 days. Plating efficiency was typically
65% for both Se-deficient and Se-replete cells.
Determination of Lipid Hydroperoxides. For determination of LOOH
values, experimental conditions were similar to those already described,
except that larger numbers of cells were required. In a typical experi
ment, ~200 ml of HPD-A-sensitized cells (10"/ml) in 175-cm2 culture
flasks were either analyzed during the course of continuous irradiation,
or exposed to a given light dose and analyzed during subsequent dark
incubation. At various time points, 30-ml samples (3 x IO7cells) were
withdrawn from the flasks and immediately centrifuged (1000 x g, 3
min). Subsequent steps were done under minimal room lighting. Each
pellet of cells (~0.2 ml) was resuspended with 0.3 ml of cold PBS,
mixed with 10 ^1 of 0.2 M EDTA, and transferred to a l .5-ml microfuge
tube. The sample was extracted with 0.8 ml of chloroform/methanol
(2:1, v/v), centrifuged, and 0.4 ml of the organic phase was transferred
to a second tube and evaporated under a stream of argon at 50°C.The
lipid extract was analyzed iodometrically as described previously (25).
1.9
0.9
HPD (ug/ml)
RESULTS
Cytological Effects of GSH Depletion. To learn initially
whether GSH can protect against HPD-sensitized photokilling,
we pretreated LI210 cells with BSO, which is known to block
GSH synthesis by inhibiting 7-glutamylcysteine synthetase
Fig. 1. Effect of GSH depletion on HPD-sensitized photokilling of L1210
cells. The cells (lO'/ml in RPMI medium) were incubated for 12 h in the absence
(O) or presence (A) of 1 mM BSO, sensitized with increasing concentrations of
HPD up to 1.5 Mg/ml, and then irradiated for 3 min with broad band visible light
(intensity —¿l
mW/cm2). Viable cell numbers 24 h after irradiation were deter
mined by means of Trypan Blue exclusion. Each data point is an average of four
experimental values, which differed by <10%.
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LIPID PEROXIDATION
IN HPD-SENSITIZED
100
C
O
10 --
o
•¿o
1.0 -0)
E
co
'ew
E
_o
o
o
0.1 --
0.01
5
10
Irradiation
15
Time (min)
Fig. 2. Effect of selenium depletion on HPD-sensitizedphotokillingof
L1210
cells. Cells were grown for 10 days in 1% serum/RPMI medium that either lacked
(O) or contained (A) sodium selenite (4 ng/ml). The resulting cells (106/ml) were
incubated for 2 h with HPD-A (15 Mg/ml) and then irradiated with red light (0.1
mW/cm2) for different periods, as shown. Survival was determined by clonogenic
assay, the colonies being counted 7 days after irradiation. Data points are means
±deviation of values from duplicate assays. GSH-peroxidase activity of the Sesufficient and Se-deficient cells was 48.8 ±4.1 U/107 cells and 0.9 ±0.1 U/107
cells, respectively.
CELLS
antioxidant enzymes, e.g., Superoxide dismutase or catalase,
might respond.
A crucial question relating to the protective role of GSHperoxidase (Fig. 2) is whether detoxification of H2O2, LOOHs,
or both is involved. To examine possible H2O2 intermediacy in
HPD-sensitized photokill ing, we irradiated Se deficient LI 210
cells in the presence of added catalase. As shown in Table 1,
viable cell counts after illumination in the presence of HPD-A
at several different concentrations were essentially the same in
the absence or presence of catalase (40 fig/ml). By way of
contrast, this level of catalase was found to be totally inhibitory
toward cytodamage induced by an oxygen radical source, xanthine oxidase acting on xanthine (30). Similar results were
obtained with Se-replete cells (Table 1), although in this case
endogenous GSH-peroxidase could conceivably have been more
important in destroying toxic H2O2 (if it had been formed),
making catalase less effective as a diagnostic. Based on these
results, cytoprotection of HPD-A/light-treated
cells by GSHperoxidase is not attributed to H2O2 removal, but rather to the
removal of organic hydroperoxides, presumably LOOHs.
Metabolism of Lipid Hydroperoxides. To focus more directly
on the question of LOOH toxicity in HPD-sensitized cells and
the role of GSH-peroxidase in its prevention, we compared the
LOOH contents of Se-sufficient and Se-deficient LI210 cells
during the course of continuous irradiation. Under the condi
tions shown in Fig. 4, the deficient cells accumulated total
extractable LOOHs at approximately twice the rate of their
normal counterparts over a 40-min reaction period. No signif
icant peroxidation above background was observed in irradiated
controls that lacked HPD-A (data not shown). The total lipid
content of cultured leukemic cells is ~0.2 mg per IO7cells (31).
Using this number and nominal values of approximately 65%,
19%, 10%, and 6% for phospholipid, glycosphingolipid, cho
lesterol, and triglycéridecontents, respectively (31), one can
calculate that the total lipid content on a molar basis is ~0.27
/umol per IO7 cells. This provides some indication as to the
S
E
D
C
fractional extent of photoperoxidation. For example, after 15
min of irradiation (Fig. 4), approximately 0.3 mol% of the lipid
was in the form of LOOH in control cells and twice this amount
in Se-deficient cells.
Additional information was available when cells were photooxidized for a relatively short period and then allowed to
incubate in the dark while LOOH concentrations were tracked.
In the experiment shown in Fig. 5, a 15-min light pulse pro
duced LOOH values of -17 and ~8 nmol/108 cells in Se
n
O
10
15
20
Irradiation Time (min)
Fig. 3. Photosensitivity of selenium- deficient vs. selenium-replete CaSki cells.
Cells were grown in 1% serum/RPMI medium either without sodium selenite (A)
or with sodium selenite, 5 ng/ml (O). After a 24-h period of dark incubation with
15 Mg/ml HPD-A, the cells (2 x 106/ml) were washed with their respective
starting media, and irradiated with red light for periods up to 20 min. 24 h after
irradiation,
the cells
trypsinized
and assayed for
viability
Trypan
Blue.
Each data point
is anwere
average
of two experimental
values,
with with
•¿MO'1;
deviation.
recovery was 2.14 ±0.15 ¿tg/mgprotein and 2.22 ±0.12 ng/
mg protein in control and Se-deficient cells, respectively.
We were interested in learning whether Se-deprived cells
were able to at least partially compensate for the loss of func
tional GSH-peroxidase by inducing higher levels of GSH-Stransferase. Thus, in one experiment we measured total transferase activity after 10 days of Se deprivation and found it to
be no different from that of control cells, i.e., 121 ±19 U/107
cells vs. 117 ±25 U/107 cells. GSH-peroxidase activity in the
same population of depleted cells was 1.5 U/107 cells, as
compared to 36.9 U/107 cells in the control. We have not yet
deficient and control cells, which compare reasonably well with
Table 1 Effect of added catalase on HPD-sensitized photolysis of Se-deficient
and Se-replete cells
Murine 1.1210 cells (2 x 105/ml) were incubated with the indicated concentra
tions of HPD-A for 2 h. The cells were then irradiated for 16 min in the absence
(-CAT) or presence (+CAT) of catalase (40 Mg/ml). Other conditions are as
described in Fig. 2. The irradiated cells were incubated in the dark for 24 h, after
which viability was determined by Trypan Blue assay. Numbers are means ±
deviation of values from duplicate experiments.
determined whether longer growing periods without Se would
affect transferase levels, nor have we determined how other
Cell Number (%)
-Se
+Se
HPD dig/ml)00.51.02.03.04.05.06.0-CAT100±498
±3104
±4103
±486
±494
±247
±648
±098
±29± ±2102
±396
±510
11
191±
±342
±33±
±0ND"5±23±0+CAT100
±5ND4±
11±0NDND+CAT100
±00±0NDND-CAT100
11
±0
1ND, not determined.
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LIPID PEROXIDATION
IN HPD-SENSITIZED
- Se
30O
O
CO
20--
10--
+ Se
8
20
30
Irradiation
Time (min)
10
40
Fig. 4. Photogeneration of lipid hydroperoxides in LI210 cells. Se-deficient
cells (A) and Se-sufficient cells (O), 106/ml in RPMI medium, were subjected to
continuous red light irradiation (0.1 mW/cm2) in the presence of 15 ^g/ml HPDA. At the indicated time points, 3 X IO7 cells were collected by centrifugation,
resuspended in PBS containing 4 mM EDTA, and extracted with CHC13/CH3OH
(2:1). LOOHs recovered in the organic phase were determined iodometrically.
Data points with error bars are means ±deviation of values from duplicate
determinations.
CELLS
terms of (a) determinants of HPD localization in solid tumors;
(¿>)
molecular mechanisms of photodamage; (c) crucial cellular
sites of toxic photodamage; and (ti) cellular defense mechanisms
that may be involved in the prevention or repair of photodamage. In the present study, we have been particularly interested
in questions (b) and (d) and have provided what appears to be
the first definitive evidence that membrane lipid peroxidation
plays a crucial role in the photodynamic killing of cells. This
evidence confirms previous speculations along these lines (1012) and adds to our basic understanding of how PDT works at
the cellular and molecular level. An important finding in these
studies is that GSH-depleted LI210 cells (BSO-treated) are
more sensitive to photoinactivation than normal controls. Sim
ilar results have been reported recently by Miller and Henderson
(27) for other cell lines. However, there are several different
ways by which GSH might protect cells against the damaging
effects of oxidative stress. Simple manipulation of GSH levels
does not always allow one to distinguish between these possi
bilities. For example, GSH can scavenge activated oxygen spe
cies such 'O2, O2~, H2O2, or OH-, some of which have been
implicated in HPD-induced photodamage. Recent studies in
this laboratory (10) have shown that HPD-sensitized lipid
peroxidation in a model system, the erythrocyte ghost, occurs
predominantly via the Type II ("O2) mechanism as opposed to
the Type I (free radical) mechanism. Principal supporting evi
dence was the identification of cholesterol 5a-hydroperoxide, a
definitive 'O2 adduci that was generated in high yield. GSH
also serves as a substrate for GSH-peroxidase, the classic selenoenzyme involved in the detoxification of H2O2 and a large
variety of organic hydroperoxides, including lipid hydroperox
ides (29). When acted upon by this enzyme, or by certain GSHS'-transferases (20), a phospholipid-derived
hydroperoxide
(LOOH), e.g., is converted to an alcohol (LOH), with concom
itant oxidation of GSH to GSSG (Eq. A). Natural regeneration
of GSH requires NADPH and is catalyzed by GSSG-reductase
(Eq. B). It is now clear that GSH-
100
Time
150
(min)
Fig. 5. Postillumination decay of lipid hydroperoxides in L1210 cells. Sedepleted cells (A) and Se-replete cells (O), 106/ml in RPMI medium, were
irradiated for 15 min in the presence of 15 /¿g/mlHPD-A and then incubated in
the dark at 37°C.Samples were removed periodically for iodometric LOOH
assay. Points with error limits are means ±deviation of values from duplicate
determinations. Starting GSH-peroxidase activity of the Se-replete and Se-depleted cells was 44.7 ±2.4 U/107 cells and 0.75 ±0.15 U/107 cells, respectively.
values shown in Fig. 4 for similar photooxidation conditions.
During subsequent dark treatment at 37°C,LOOHs in the
deficient cells are seen to decay much more slowly than LOOHs
in the control cells. A first order plot of the data (Fig. 5, inset)
reveals a 9-fold difference in the rates for this particular exper
iment. These results are consistent with the large difference in
GSH-peroxidase activity observed for these cells. We have not
yet determined whether the enzyme undergoes any photoinactivation under the conditions of these experiments. Since por
phyrin uptake and light exposure was identical for control and
Se-deficient cells, it is apparent that the higher initial peroxide
value for deficient cells (Fig. 5,15 min) reflects their impaired
ability to metabolize LOOHs during the course of irradiation.
DISCUSSION
Photodynamic therapy with HPD is one of the most prom
ising antineoplastic modalities to be proposed and tested over
the past 10 years. Despite the intensive efforts of numerous
investigators, we still know very little about this treatment in
LOOH + 2 GSH -»LOH + GSSG + H2O
(A)
NADPH + H+ + GSSG -»NADP+ + 2 GSH
(B)
peroxidase and also the transferases do not act directly on
membrane-bound LOOHs, but rather on fatty acid hydroperoperoxides that are liberated by phospholipase A2-catalyzed hy
drolysis (22, 32).
In this work, we have focused specifically on the protective
role of GSH-peroxidase by preparing Se-deficient cells and
exposing them to a photodynamic challenge. Two cell lines,
murine LI210 and human CaSki, were dramatically more sen
sitive to photokill in»after growing 7-10 days under Se depri
vation. GSH-peroxidase activity in these cells was typically
<5% of the control activity, whereas GSH-5-transferase activity
and, more importantly, GSH content, was normal. Therefore,
a direct role of GSH-peroxidase in the metabolism of damaging
photoperoxides was identified. We ruled out H2O2 as one of
these species by demonstrating that supplemental catatase did
not protect Se-starved cells against photoinactivation. Superoxide dismutase also had no effect (data not shown), which
argues against O2~ involvement. These results are in agreement
with previous findings on simpler systems (e.g., erythrocyte
ghosts), which indicated that HPD-A-sensitized photodamage
is mediated predominantly by 'O2, partially reduced oxygen
species (O2~, H2O2, or OH •¿
) playing a relatively minor role, if
any (10, 11). Moreover, based on other studies of oxyradical
formation by membrane-bound sensitizers, e.g., protoporphyrin
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LIPID PEROXIDATION
IN HPD-SENSITIZED
(33), HPD-A would be expected to be a relatively poor photogenerator of C>2~or H2O2, even in the presence of electron
donors such as ascorbate or thiols. Thus, the observed effects
of GSH peroxidase are ascribed primarily to the detoxification
of organic hydroperoxides, presumably lipid hydroperoxides
that are generated in plasma membranes and/or subcellular
membranes where HPD-A tends to localize (1, 2). Direct sup
port for this argument was obtained by showing that Se-deficient cells accumulate significantly higher levels of extractable
LOOH than normal cells during HPD-A/light treatment.
Moreover, a dark chase after a brief period of photooxidation
revealed that the LOOH decay rate is markedly decreased as a
result of Se depletion. It is interesting to note for the experiment
shown in Fig. 5, that Se depletion caused nearly a 60-fold
decrease in GSH-peroxidase activity, but only a 9-fold decrease
in the rate of LOOH loss. The discrepancy in these values could
be due to (a) a significant background of nonenzymatic LOOH
loss or (b) the peroxidatic action of one or more cytosolic GSH.S-transferase(s), which are not affected by Se manipulation. An
additional possibility is that the GSH-peroxidase-catalyzed re
action may not necessarily be the rate limiting step of LOOH
metabolism in Se-replete cells (32).
When considered as a whole, this study provides convincing
evidence that lipid peroxidation plays an important role in the
cytotoxic effects of HPD/PDT. This appears to be the first
clear demonstration of this relationship in any type of cellular
photosensitization. Lipid peroxidation is well known to be
deleterious to cell membrane structure and function (9). How
ever, it is not always clear (as in this study, for example) whether
toxicity and/or lethality is due to lipid peroxidation per se, or
whether this process triggers other events that are even more
detrimental, e.g., modification of ion channels or cytoskeletal
proteins (6). Another question relates to the relative importance
of lipid peroxidation at different membrane sites. Cellular mem
brane targets of HPD/PDT may differ depending on whether
the contact time with sensitizer is short or long (8). Relatively
long incubation periods allow for porphyrin redistribution from
the plasma membrane to organellar membranes (e.g., lysosomes, mitochondria), where most of the lethal photodamage
of HPD/PDT has been proposed to take place (7, 8, 34, 35).
In the present study, efficient photokilling was observed after
both short (2 h, Fig. 2) and long (24 h, Fig. 3) incubations with
HPD-A, suggesting that lipid peroxidation is a lethal damaging
event in the plasma membrane as well as in organellar mem
branes. However, rigorous discrimination between different
membrane sites undergoing lipid peroxidation was not at
tempted in the present work. It is clear that future investigation
of this and related questions will provide important new insights
into how tumor cells succumb to PDT.
ACKNOWLEDGMENTS
We thank Fritz Sieber and David Gaffney for their advice and
assistance in the clonogenic assay determinations. Helpful discussions
with Larry Hopwood are also appreciated. James Kurtz provided ex
cellent technical assistance.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1989 American Association for Cancer Research.
Role of Lipid Peroxidation in Hematoporphyrin
Derivative-sensitized Photokilling of Tumor Cells: Protective
Effects of Glutathione Peroxidase
James P. Thomas and Albert W. Girotti
Cancer Res 1989;49:1682-1686.
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