ICANCER RESEARCH 56. 339-344. Januaiy 15. 19961
Induction of Tumor Necrosis by 5-Aminolevulinic Acid and 1,10-Phenanthroline
Photodynamic Therapy'
Natalie
Rebeiz, Sean Arkins,
Constantin
A. Rebeiz, Joseph Simon, James F. Zachary,
and Keith
W. Kelle?
Laboratort of Immunophysiology, Department of Animal Sciences (N. R., S. A., K. W. K.J, Laborato,y of Plant Pigment Riochemistiy and Photobiology, Department of
Horticulture (C. A. RI, and Department of Veterinary Pathobiology, College of Veterinary Medicine fJ. S., J. F. ii, University oflllinois at Urbana-Champaign, Urbana, Illinois
61801
ABSTRACT
8.Aminolevulinic
acid (ALA)
causes cells to accumulate
protoporphy.
nfl IX (Proto) and heme. Exposure to light in vitro causes Intracellular
Proto to initiate formation of slngjet oxygen molecules,leading to self.
destruction. This photoactivateddestruction by ALA in vitro is enhanced
by addition of the tetrapyrrole modulator 1,10-phenanthroline(Oph),
which increases cellular
accumulation
of Proto. Here we significantly
extendthis Ideaby evaluatingthe efficacyofALA and Oph photodynamic
therapy of solid tumors in vivo. Methylcholanthrene-induced fibrosar.
coma (Meth.A) cells were used, which lead to the formation of solid
tumors when implanted Into syngeneicredplents. Initially, suspensionsof
Meth-A cells were treated in vitro with combinations of ALA and Oph.
Meth-A cells in suspension accumulated 6-fold greater amounts of Proto
(P < 0.05) after 3.h Incubation with ALA and Oph than when Incubated
with
ALA
tivated
alone, and were also more susceptIble
cell lysis in vitro. Similarly,
to subsequent
solid Meth.A
tumors
grown
photoac
In synge
neic BALB/C mice accumulatedsignificant (P < 0.05)amountsof Proto 3
h after in vivo treatment with ALA, and Oph synergized wIth ALA to
significantly
(P < 0.05) enhance
the Induction
of Proto
In these tumors.
ALA and Oph.based phototreatment of mice bearing Meth.A solid hi
mom resulted in necrosis of tumors, as determined by a significant reduc.
tion in both size and histopathology, with little damageto surrounding
normal tissue.Thesedata directly demonstratethe experImental useful
ness of Proto modulators
for ALA-based
photodynamic
In addition, ALA and Proto are rapidly cleared from the circulation
within 16—48h after treatment (15—17).
ALA is a 5-carbon amino acid that serves as the biological precur
sor of heme in mammalian systems. Once taken up by transformed
cells, ALA induces overproduction of endogenous Proto by avoiding
the inherent negative feedback mechanisms involved in cellular heme
biosynthesis (17). ALA induces Proto accumulation in vitro in a
number of transformed cell lines (18—22).Upon light activation,
endogenously accumulated Proto causescell death by generating toxic
oxygen
intermediates
(23).
We haveadvancedtheideathattetrapyrrolemodulators,suchas
Oph, can act in concert with ALA by showing that ALA and Oph
treatment in vitro leads to significantly enhanced accumulation of
Proto and photoactivated cell death in a T-cell lymphoma (24). How
ever, this important concept of enhancing the efficacy of ALA treat
ment by cotreatment with Oph to induce more Proto accumulation has
not been reported in vivo. This investigation focused on combined
treatment with ALA and Oph in a murine syngeneic solid tumor
model using Meth-A cells. We show that the combination of ALA and
Oph induces accumulation of porphyrins in Meth-A cells and, upon
exposureto light, significantly reducesthe surfaceareaof the tumors
and induces their destruction, as determined by their disappearanceat
both the gross and the histological levels. These data support the
possibility that the accumulation of Proto and subsequent photode
struction of tumor cells can be significantly enhanced in vivo by the
use of tetrapyrrole modulators such as Oph.
therapy In the
treatment of solid tumors in vivo and provide a rationale for their poten
tial application in a multitude of tumor types.
INTRODUCTION
ALA3 has been widely studied as a possible photodynamic chemo
therapeutic agent for various cancers (1—6).Topical administration of
ALA to various skin lesions has, in particular, been very successful in
clinical trials. For instance, Kennedy et a!. (1) reported on clinical
experiences with ALA in treatment of basal cell carcinomas, super
ficial squamous cell carcinomas, and actinic keratoses; they found a
complete response rate of 90% for basal cell carcinomas. Overall,
topical, oral, or systemic administration of ALA and subsequent
photodynamic therapy has been successful in a variety of tumor
models, including amelanotic melanomas (7), pancreatic cancer (8),
and colonic tumors (9). Furthermore, the ALA molecule has a number
of properties that make it more effective than other traditional pho
tosensitizing drugs (10). ALA is a small, water soluble molecule, can
easily penetrate cells ( 11, 12), and is rapidly taken up by transformed
cells (13—15).Photoactivation of ALA-induced Proto requires less
light energy than commonly used photosensitizing drugs require (16).
Received 6/6/95; accepted I I/I 3/95.
The costsof publicationof this article weredefrayedin part by the paymentof page
charges.This article mustthereforebe herebymarkedadvertisementin accordancewith
18 U.S.C. Section 1734 solely to indicate this fact.
I This
research
was
supported
in
part
by
the
J.
P.
Trebellas
Photobiotechnology
research endowment (to C. A. R.) and by NIH Grant AG-06246 (to K. W. K.).
2 To
whom
requests
for
reprints
should
be
addressed,
at
Laboratory
of
Immunophysi
ology, 207 Plant and Animal Biotechnology Laboratory, 1201 West Gregory Drive,
University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-5142;
Fax: (217) 244-5617.
3 The
abbreviations
used
are:
ALA,
8-aminolevulinic
acid;
Meth-A,
methylcholan
threne-induced fibrosarcoma; Oph, l,lO-phenanthroline; Proto, protoporphyrin IX.
MATERIALS
AND METHODS
Reagentsand Chemicals. RPMI 1640tissueculturemediumwassupple
mentedwith 2 g/liter sodiumbicarbonate,100units/ml penicii1h@
100p@g/m1
streptomycin,and 25 mi,@HEPES (Sigma ChemicalCo., St. Louis, MO).
Triton
(Aldrich
X-lOO (Sigma),
Chemical
ALA
(Biosynth
International,
Chicago,
IL), and Oph
Co., St. Louis, MO) were used at the indicated concentra
tions. Solutionsof ALA and Oph were made in RPMI 1640 immediately
before use and were heated to 50°Cin a water bath to dissolve the Oph, stirred
for 5 mm, cooled,andadjustedto pH 7.2.
Cells. Murine Meth-A cells were kindly provided by Dr. Robert M.
Lorence(RushPresbyterianSt. Luke's Hospital,Chicago,IL).
Cellular
Cytotoxicity
Studies.
Cytotoxicity
was measured by 51Cr release
as describedby Rebeizet al. (24). Chromium-labeledcells (100 s.d)were
placedin microtiterplates(3 X 10' cells/well) andtreatedwith the indicated
concentrationsof ALA, Oph,Triton X-lOO,or mediumalone(100 @tl)
for 3 h
in darkness at 37°C.To initiate photodynamic damage, designated plates were
exposedto a uniform sourceof light, as measuredby a spectroradiometer
(Model SR. InstrumentationSpecialtiesCo., Lincoln, NE), generatedby a
metal halide lamp that emits from 400 to 700 am (1000-W Multi-Vapor lamp,
GeneralElectric,Cleveland,OH; 2.11 mW/cm2)for 30 mm for a total light
doseof 3.798J/cm2.The releaseof 51Crwas determinedimmediatelyafter
irradiationfrom 100 @.d
of supernatantby mixing with 3 ml of liquid scmtil
lation
cocktail
(Ready-solv-MP,
Beclcman Instruments,
Fullerton,
CA)
and
radioactivitywascountedin a BeckmanLS58O1scintillationcounter.
Proto Analysis.
Control Meth-A
cells (1.5 X l0@cells/ad)
were suspended
in 50 ml of RPM! 1640containing25 m@iHEPESin 100-mmCorningplates
(ComingGlassWorks,Coming,NY), whereastreatedcellsweresuspended
in
RPM! 1640with 25 mMHEPESandtheindicatedconcentrationsof ALA and
339
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ALA AND Opli INDUCE TUMOR NECROSIS
Oph. After 3.5 h of dark incubation, treated and control cells were centrifuged
at 1000 X g for 10 mm. PeHetedcells were homogenized(45 5; Brinkman
Instruments,Inc.,Westbury,NY) in 12 ml acetone:0.1Nammoniumhydroxide
(9:1, v/v; almost 100% recovery) and centrifuged for 12 mm at 25,000 X g.
The acetonesupernatantcontainingextractedporphyrinswasdelipidatedby
extractionwith hexane,andthehexane-extracted
acetonefractionwasusedfor
Treatrnent@@
Proto determination at room temperature (25). Spectrofluorometric emission
ALA 1'4@
waselicitedby excitationat400 nm.Amountsof Protoequalto or higherthan
100pmol/l00 mg proteinwere readily detectableusingan SLM 8000spec
trofluorometer
@
(SLM Instruments,
Treatment,
mice were bred in our specific
and Measurement
pathogen-free
of Tumors.
facilities
Oph
BALB/c
at the University
of
Illinois. The animals were kept in micro-isolator cages and had food and water
available ad libitum. All proceduresinvolving experimentalanimals were
approved
by the University
of Illinois
Laboratory
Animal
Medium@ b
Care Committee.
Control
After harvestingof ascites,I ml of 1 X l0@cells were injecteds.c.into the
right flank of female 6—8-week-old
BALB/c mice. Solid tumors 1—2
cm
0
long X 0.5—1.0cm wide developed 7 days after injection (27). A 25-gauge
needle was used to inject 0.5 ml of medium, ALA, and/or Oph directly into the
tumor. For analysis of Proto accumulation
cervical
dislocation
at different
s.c. Meth-A
ALA
time points. Tumors
tumors
and Oph efficacy
were treated with
50
100
200
250
IX
300
350
400
450
accumulation
(nmoles/100 mg protein)
were excised and pro
in photodynamic
ALA
150
Protoporphyrin
in tumors, mice were euthanized by
Fig. 1. Proto accumulation is synergistically induced by ALA and Oph. Meth-A cell
suspensionswere treated for 3 h in vitro with I msi ALA and/or 0.75 msi Oph. Columns,
mean; bars, SEM (n = 2). Means with different letters denote a significant difference
cessedin darknessfor Protodetermination,asdescribedabove.
To determine
b
Rochester, NY). The protein concentrations
of treatedand control cell pellets were determinedusing bicinchonic acid
(PierceChemicalCo., Rockford,IL; Ref. 26).
In Vivo Culture,
,a
therapy, mice with
and Oph for 3 h in darkness.
betweenmeans(P < 0.05).
Briefly, the skin over the tumor wasshaved,and the tumor area(lengthand
width) was measured in cm2 at its widest points with vernier calipers (Fisher
Scientific, Pittsburgh, PA) in a blind test. After intratumor administration
of
ALA and Oph, the mice were maintainedfor 3 h in darkness.Control mice
were given injections
of a similar
volume
of medium.
The mice were then
anesthetizedwith 250 pi of ketamine(108.75 mg/kg) and xylazine (16.25
mg/kg) administered
i.m. Light-treated
animals were placed in lateral recum
bency on a 1-cm Perspex sheet and received 30 mm of illumination from a
quartzfilamentlamp(141.2i/cm2).All micewerereturnedto a 12-hlight/dark
cycle after recovery
minimal,
from anesthesia. Light intensity
approximately
0.014 i/cm2,
in the animal cages was
as measured by a foot-candle
the additionof Oph to ALA-treated Meth-A cells inducedmore Proto
accumulationthan what was seenpreviouslyin MLA 144 cells (24).
Indeed, there may be some cell type specificity becausethe efficacy
of ALA and Oph in stimulating Proto accumulation seems to vary
significantly between cell types.
Sensitivity of Meth-A Cells to ALA and Oph Treatment. Our
previous
results
amount
meter
(24) have shown
of Proto
accumulation
a positive
and
specific
correlation
cell
lysis
between
upon
the
illumina
tion in several transformed cell lines treated with ALA and Oph.
Therefore,
Meth-A cells were screened for sensitivity to light
Pathology. Tumor-bearingmice weresacrificedfor serialhistopathologi
activatedcell
lysis after ALA and Oph treatment.
cal examination of tumor sites. At days 1, 4, 6, and 7 after tumor treatments,
There was no apparent cell lysis in darkness (3 h) in Meth-A cells
mice were sacrificed, and the sites of the original tumors and surrounding
in medium, or in cells treated with 1 msi ALA or 0.75 m@iOph alone
tissue were excised, sliced, placed in 10% neutral buffered formalin, embedded
in paraffin,sectionedat 5 @.tm,
stainedwith hematoxylinandeosinaccording (Table I ). However, there was an increase in cell lysis when cells
to standard procedures,
and evaluated
by light microscopy.
Evaluation
of
were treated with both compounds in darkness (P < 0.05). Upon light
tumorhistopathologywasperformedin a blind test.Presenceof tumortissue, activation (30 mm, 3.798 J/cm2), 16.8% of the 1 m@iALA-treated
tumor necrosis,and panniculitiswasdocumented.Other histologicalcharac Meth-A cells were lysed (P < 0.05). When ALA and Oph were used
teristics such as lymphocytic
infiltration or dermal damage were also noted.
jointly, specific cell lysis increased over 7-fold (P < 0.05) compared
Statistical Analysis. Statistical analyses were performed using a SAS
to ALA-treated cells after light treatment.
ANOVA program (SAS Institute, Inc., Carey, NC). Duncan's multiple range
Meth-A Tumors Accumulate Proto after Treatment with ALA
test was used to partition all multiple pairwise differences between means.
and Oph in Vivo. Preliminary experiments on Proto accumulation in
Meth-A tumors in vivo were conducted using a range of ALA (1—10
(Model 756, Weston Electrical Corp., Newark, Ni).
mM)
RESULTS
tration
Proto Accumulation in ALA- and Oph-treated Meth-A Ascites
Cell Suspensions.We determinedwhethertreatmentof Meth-A cells
in vitro with ALA and Oph would result in Proto accumulation.
Meth-A ascites cells were treated with 1 mr@iALA and 0.75 mr@iOph
for 3.5 h in darkness (Fig. 1). The combination of ALA and Oph
induced very significant increases in Proto accumulation (338 nmoll
100 mg protein; P < 0.05). Treatment with ALA alone also induced
measurable accumulation of Proto (61.73 nmol/lOO mg protein) in
comparison
with
control
cells,
but this was not statistically
consistent
with
the specific
cell
lysis
data
in which
ALA
from treatment
with ALA alone (Table
(0.75—7.5
Proto
mM) doses.
accumulation
In general,
levels
>30
the
highest
nmol/l00
concen
mg protein,
Table I AlA and Oph inducephotoactivatedcell h'sis in Met/i-A cells
Cells (3 X 106/well) were treated for 3 h in suspension with the indicated concentra
tions of ALA and Oph and either irradiated for 30 mm (3.798 i/cm2) or maintained in
darkness for an additional 30 mm. The percent specific lysis in the dark and light was then
immediately determined by measuring 5tCr in the supematant. Results are presented as
mean ±SEM.
Specific
(mM)No.%
ALAlysis0.00.04Øf@*aØ@*a1.00.04—0.2
(mM)OPH
1). It is interesting
2.2c0.00.7546.3
dark lysis%
±1.6a16.8
±l.la b12.9
±1.6―
c87.3
c1.00.7549.6
and
Oph-induced cell lysis was approximately 7 times higher than that
arising
Oph
yielded
Specific
light
significant.
This level of Proto accumulation is approximately 6-fold lower than
the levels that accumulate with ALA and Oph treatment. This finding
is also
and
that
* Background
cell
lysis
a Means with different
was
±
±32b.
±6.2―
22%.
superscript letters indicate that the means are significantly
different from each other (P < 0.05).
340
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
ALA ANDOph INDUCETUMORNECROSIS
@
(5.4-fold) in size compared to control tumors by day 1 and at all
subsequent time points (P < 0.05). By days 4 and 5, there were no
palpable tumors in S of the 6 treated mice. In contrast, the control
bc
-i@&-—-————----——————-H
a
tumors doubled in size by days 4 and 5 (P < 0.05). On day 1, one of
the treated mice sacrificed for histopathology appeared to have a
palpable tumor, but histopathological examination showed only
edema and epidermal necrosis with no evidence of sarcoma tissue
ab
present. Subsequent histopathology confirmed that in ALA and Oph
phototreatedmice, the Meth-A tumor tissue,which is nonmetastasiz
ing, was eradicated, except for a small amount of sarcoma tissue in
one mouse. In 5 of 6 mice in which the tumor tissue was eradicated,
. Medium
control
only necrotic sarcoma tissue remained, indicating a high level of
U 2.5mM ALA + 1.8mM Oph
tumor necrosis. In addition, one-half of the ALA and Oph-treated
n 10mMALA+7.5Oph
mice had panniculitis and epidermal and dermal damage. This is
consistent with a local inflammatory responseoccurring at the site of
3.0
U)
1@
@
a
0
.u
6.0
E
I-
@I
12.0
@119bc
0
10
20
Protoporphyrin
(nmoles/100
30
40
50
ALA
IX accumulation
mg protein)
and Oph injection
tumor
Fig. 2. Kinetics of Proto accumulation in solid Meth-A tumors in mice treated with
@
different concentrationsof ALA andOph at 3, 6, and
12h. Mice were injectedwith
ALA and Oph directly into s.c. tumors for the indicated time periods, after which the mice
were killed, and the tumors were excised and processed for analysis of Proto accumula
tion. Columns, mean; bars, SEM (n = 2). Means with different letters denote a significant
difference between means (P < 0.05).
whereas 1 mM ALA and 0.75 mr@i
Oph yielded Proto levels of 1.1
nmol/100 mg. Therefore, for the next series of experiments, we used
dosesrangingfrom 2.5 to 10 mr@i
ALA (Fig. 2) at differenttime points.
Untreated tumors did not accumulate Proto, whereas significant
amounts of Proto accumulated after 3 and 6 h (31 and 17 nmol/l00 mg
protein, respectively) at the highest dosage of ALA and Oph tested.
Twelve h or more after injection, the levels of accumulated Proto were
lower, probably due to degradation by intracellular enzymes (28). We
have also observed a similar pattern of decline in Proto accumulation
in single-cell suspensions of a T-cell lymphoma (MLA 144) incubated
with 0.5 mM ALA and 0.38 mr@iOph for 18 h. The amount of
accumulated Proto fell over 10-fold at 18 versus 3 h (1.7 ±0.16
versus 19.6 ±9.90 nmol/lOO mg protein; n 2). Because substantial
Proto had accumulated 3 h after injection with ALA and Oph, this
time point was usedin subsequentexperiments.
The next series of experiments focused on the enhancement of
ALA-induced Proto accumulation by Oph in Meth-A solid tumors
(Fig. 3). Previous studies with Oph have shown that the optimal
concentration is about 75% of the ALA concentration (29). As before,
medium-treated tumors or tumors treated with Oph alone at 1.8, 3.75,
or 7.5 mM did not accumulate
@
@
any Proto. Oph at two concentrations,
7.50 and 3.75 mM, enhanced Proto accumulation in the presence of 10
(2.7-fold) and 5 (4.7-fold) m@ ALA, respectively (P < 0.05). How
ever, at 1.8 mM, Oph did not enhance Proto accumulation when used
in conjunction with I mr@iALA.
Effect of ALA and Oph on Meth-A Tumors: Size and Histopa.
thology. Having established that Meth-A tumors accumulate Proto
after in vivo injection of ALA and Oph, we next determined the effects
of ALA and Oph phototreatment on the tumors by histopathological
examination of the tumor and the surrounding normal tissue. Treat
ment groups consisted of mice with tumors treated with medium or
ALA and Oph for 3 h in darkness, followed by illumination for 30
mm. Tumor
area measurements
from three separate
and is commonly
observed
in patients
treated
with ALA photodynamic therapy (1, 3, 6). Neither panniculitis nor
experiments
necrosis
was
evident
in control
mice.
Histopathological sections of control tumors and lesions 3 days
after ALA and Oph photodynamic therapy are shown in Fig. 4.
Meth-A tumors are undifferentiated, as reported earlier (30). In the
control mice, there is some necrosis of adjacent dermis related to
expansion of the tumor, and the tumor shows muscle invasion, which
is typical of established tumors. The inset in Fig. 4 shows in more
detail the numerous mitotic figures, also typical of tumorous tissues.
Fig. 4b is a photomicrograph from an ALA and Oph-phototreated
mouse in which the tumor is eradicated, leaving only necrotic tumor
tissue. Some panniculitis is apparent, along with necrosis of the
epidermis and inflammation of the dermis, sloughing of the skin, and
keratinization,
occurring
at
which
the
are all indicators
site
of
injection.
of a photodynamic
The
inset
shows
in
reaction
detail
the
pyknotic nuclei, karyolysis, and karyorrhesis that occur during necro
sis of this tumor.
Treatment
a
ALA + Oph, 10.0 + 7.5 mM
a
ALA + Oph, 5.0 + 3.75 mM
ALA+Oph,2.5+1.8mM
ALA 10.0mM
ALA 5.0 mM•
ALA 2.5 mM
Oph 7.5 mM
Oph 3.75 mM
Ophl.SmM
b
Medium control
b
0.0
5.0
10.0
Protoporphyrin
15.0
IX
20.0
accumulation
(nmoles/100 mg protein)
are
shown in Table 2, along with histopathology results from two exper
iments.The initial mean tumor size in both control and treatedmice
was similar. Mice were allocated randomly to treatment groups.
Tumors that received ALA and Oph photodynamic therapy (3 h
incubation before a 30-mm illumination) were significantly reduced
Fig. 3. Induction
of Proto accumulation
in mice with established
Meth-A
tumors by
treatment
withALA,Oph,or ALA andOphfor 3.5h in darkness.
ALA andOphwere
injecteddirectly into s.c.tumorsin mice;3.5h later,themicewerekilled, andthetumors
were excised and processed for analysis of Proto accumulation. Columns, mean; bars,
SEM (n = 3). Meanswith differentlettersdenotea significantdifferencebetweenmeans
(P < 0.05).
341
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
@
@
@
@
I@'
b
.
@!
:@@;
@4b@
@.
@,
‘@
ALA AND Oph INDUCE TUMOR NECROSIS
2 Surfacearea and histopathologyscoresof establishedMeth-A tumorsafter in vivo ALA and Ophphotodynamictherapy
Results
areTa
presented asble
means ±SEM from three separateexperiments. Six of eight mice treated with ALA and Oph and two of four control mice were usedfor
histopathology.ALAOphSurface
characteristicsLight
area (cm2)
Lesion
Tumor(mM)(mM)(mm)
Sarcoma
atprimary
necrosisPanniculitis0.00.030
Day 0
@
Days 1—3
0.50 ±0.l0I@@
n4
0.49 ±0.06@
n40/210.07.530
0.59 ±0.21 lab
n3
0.1 1 ±00828
n8
I Within
each
day,
means
with
different
superscript
Days 4—5
indicate
0/2
o.o@i±0.0428
n6
numbers
tumor site
1.19 ±0.401@@
1/6
6/6
n73/6
a significant
difference
between
means
(paired
I test;
P
a. A Within each treatment, means with different superscript letters indicate a significant difference between means (Duncan's
<
0.05).
multiple range test; P < 0.05).
DISCUSSION
Next, the effect of ALA and Oph on the induction of Proto accumu
This report is the first to demonstrate that ALA can be effectively
combined with Oph photodynamic therapy to eradicate solid tumors
in vivo. Most applications of ALA chemotherapy have used topical
Oph solutions accumulated significant amounts of Proto after 3.5 h.
The addition of Oph significantly enhanced the amount of Proto
administration
Proto than did solid tumors (Fig. 1 versus Figs. 2 and 3), which is
probably due to the fact that single cells are suspended in the incu
bation medium containing ALA and Oph.
Having demonstrated that tumors accumulate porphyrins in vivo,
we next investigated the effects of ALA and Oph treatment on tumor
growth and progression. In three different experiments, ALA and Oph
lationin vivo was investigated.Meth-A tumorsinjectedwith ALA and
in the treatment
of a variety of skin cancers
formation. Single-cell suspensions accumulated significantly more
(I, 3—5).In
this work, we investigated whether ALA, in conjunction with Oph,
could induce necrosis of solid Meth-A tumors in vivo. First, we
established that ALA and Oph acted synergistically to induce accu
mulation of very significant amounts of Proto in Meth-A cells in vitro
(Fig. 1) and subsequent cell lysis upon exposure to light (Table 1).
.@,
.@.‘
@
@%
@
.-\
..@@_4
.%v@ , r:
@;
@.%
@
@
.
t;@54@
Fig. 4. a, photomicrographof control Meth-A tumor.Large
arrowheo4s. outer boundaries of tumor, surrounded by con
nective tissue and normal epidermis; bar, 300 @m.Meth-A
tumors are classified as undifferentiated sarcomas (28). Inset,
4
@;L@4b.
.
bizarrenuclearformsandnumerousmitotic figurescontaining
@
abnormal mitoses; bar. 30
@m.
b, photomicrograph of site of
s_@
Meth-A tumor from an ALA and Oph-phototreatedmouse3
days posttreatment.Small arrowheads,site of epidermalne
@%
crosis overlying the tumor that occurs after a photodynamic
response;large arrowheads,outer boundaryof the necrotic
tissue; bar, 300 @m.
Inset, tumor necrosisin detail, with
pyknotic nuclei, karyolysis,and karyorrhesis;bar, 30 ,.@m.
342
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
ALA AND Oph INDUCE TUMOR NECROSIS
photodynamic
mice.
Only
therapy resulted in tumor disappearance
one
of the
treated
mice
had
residual
ACKNOWLEDGMENTS
in 5 of 6
tumor
tissue
We thank Deborah L. Brunke-Reesefor her technical assistanceand
RichardFrenchfor his help in preparingand evaluatingthe histopathology
remaining after treatment. ALA and Oph treatment caused some
epidermal irritation, including sloughing of the epidermis overly
ing the tumor, and infiltration of lymphocytes to the site of tumors.
This finding is consistent with other reports of inflammatory
responses after ALA treatment in humans ( 1, 3, 10) and in mice
(2). Kennedy and Pottier (10) reported that after topical application
of ALA and light therapy, patients felt sensations of burning,
itching, and throbbing. The lesions became edematous, and a small
zone of erythema similar to a histamine response was observed.
Similarly, Wolf et a!. (3) noted epidermal necrosis and acute
dermal inflammation with no vascular damage in posttreatment
biopsies of patients treated with ALA photodynamic therapy. In
our study, histopathological sections of tumors treated in vivo with
ALA and Oph photodynamic therapy showed extensive tumor loss,
as well as panniculitis, epidermal necrosis, skin keratinization,
sloughing of the epidermis, and local inflammation (Fig. 4b). All
of these lesions subsequently disappear, as has been shown in other
studies with humans and mice (1, 2).
One exciting finding from our studies is that the addition of the
porphyrin
biosynthesis
in solid tumors.
modulator
Because
Oph enhanced
it has been shown
studies.
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worthy
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tissues,
offers
the combination
promise;
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tumor eradication
occurs
within
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Induction of Tumor Necrosis by δ-Aminolevulinic Acid and
1,10-Phenanthroline Photodynamic Therapy
Natalie Rebeiz, Sean Arkins, Constantin A. Rebeiz, et al.
Cancer Res 1996;56:339-344.
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