Expression of Fas antigen and apoptosis caused by 5,10,15,20-tetra(4-methoxyphenyl)porphyrin (TMP) on carcinoma cells: implication for photodynamic therapy

Article abstract of DOI:10.1016/S0300-483X(00)00221-3

The photodynamic effects of 5,10,15,20-tetra(4-methoxyphenyl)porphyrin (TMP) on a Hep-2 cell line were investigated. TMP toxicity in the dark and in relation to illumination with visible light was examined. Hep-2 cells were treated with different TMP conc

Full text of DOI:10.1016/S0300-483X(00)00221-3

Toxicology 149 (2000) 69–74
www.elsevier.com/locate/toxicol
Expression of Fas antigen and apoptosis caused by
,10,15,20-tetra(4-methoxyphenyl)porphyrin (TMP) on
5
carcinoma cells: implication for photodynamic therapy
I. Yslas, M.G. Alvarez, C. Marty, G. Mori, E.N. Durantini, V. Rivarola *
Facultad de Ciencias Exactas, F ´ı sico-Qu ´ı micas y Naturales, Uni6ersidad Nacional de R ´ı o Cuarto, Agencia Postal No. 3,
5800 R ´ı o Cuarto, C o´ rdoba, Argentina
Received 4 January 2000; accepted 10 April 2000
Abstract
The photodynamic effects of 5,10,15,20-tetra(4-methoxyphenyl)porphyrin (TMP) on a Hep-2 cell line were
investigated. TMP toxicity in the dark and in relation to illumination with visible light was examined. Hep-2 cells
were treated with different TMP concentrations (1, 5 and 10 mM). The uptake of TMP by Hep-2 cells increased with
TMP concentration and an increase of the initial uptake rate was observed with increasing TMP concentrations.
However, after 24 h of incubation, a similar value of intracellular TMP concentration was reached at all three
concentrations of TMP added. Cell toxicity induced by TMP was analyzed in the dark at different concentrations of
the photosensitizer and at several incubation periods. The cell mortality obtained after exposure of the cell cultures
to visible light was exclusively due to the photosensitization effect of TMP produced by light irradiation. Staining
with the hematoxylin–eosin method demonstrated that treatment with TMP, followed by exposure to visible light,
notably increased the apoptotic figures. Fas antigen was only expressed in these conditions. The results contribute to
the understanding of the photodynamic therapy (PDT) mechanism produced by TMP on Hep-2 carcinoma cell line.
©
2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Apoptosis; Fas; Porphyrins; Photodynamic therapy
concentrated in tumor cells, and upon subsequent
irradiation with visible light in the presence of
oxygen specifically destroys the cells (Dougherty,
1
. Introduction
Photodynamic therapy (PDT) is based on the
1
987; Penning and Dubbelman, 1994). The treat-
administration of a photosensitizer that becomes
ment is effective for cancerous tissues and malig-
nant hemopoietic cells (Chen et al., 1998). Among
phototherapeutic agents applied to the treatment
of tumors, porphyrins and their analogs, in com-
bination with visible light, have attracted much
Abbre6iations: mM, micromolar; mM, millimolar; TMP,
5
3
,10,15,20-tetra(4-methoxyphenyl)porphyrin.
*
Corresponding author. Tel.: +54-358-4676114; fax: +54-
58-4676232.
E-mail address: vrivarola@exa.unrc.edu.ar (V. Rivarola).
0
300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(00)00221-3

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I. Yslas et al. / Toxicology 149 (2000) 69–74
attention
Grosseweiner, 1994).
(Milgrom
and
O’Neill,
1993;
purchased from ABAC (Buenos Aires, Argentina)
and kept in liquid nitrogen, was used. The cells
were grown as a monolayer employing Dulbecco’s
modified Eagle’s medium (DMEM) containing
10% fetal calf serum (FCS) and 50 mg/ml gen-
tamycin. The cells were incubated at 37°C in a
It is generally accepted that singlet oxygen
O ), produced after exposure of the sensitizer to
2
1
(
light, is the main species responsible for cell inac-
tivation. Nevertheless, the photodynamic process
of the sensitizers on neoplastic tissues is still not
well understood (Jori et al., 1996). It is believed
that PDT-induced cell death may be divided into
two main types, namely apoptosis and necrosis
humidified 5% CO atmosphere and the medium
2
was changed daily. The cell line was routinely
checked for the absence of mycoplasma
contamination.
(
Agarwal et al., 1991). Apoptosis is characterized
by cell shrinkage, chromatin condensation, the
expression of surface antigens like Fas and forma-
tion of apoptotic bodies. Fas is a type I mem-
brane protein with a molecular mass of 45 kD
that mediates apoptosis. Its cytoplasmic domain
2.2. Chemicals
4-Methoxybenzaldehyde, pyrrole, trifluoro-
acetic acid and 2,3-dichloro-5,6-dicyano-1,4-ben-
zoquinone from Aldrich were used without fur-
ther purification. All solvents were high
performance liquid chromatography (HPLC)
quality. Dipalmitoyl phosphatidylethanolamine
from Sigma was used in liposome preparation.
Silica gel 230–400 mesh for column chromatogra-
phy from Aldrich was used.
(
70 aminoacids), named the ‘death-domain’, is
necessary and enough to translate apoptotic sig-
nals. Inappropriate expression of this receptor
leads to disorders and/or autoimmune diseases. In
many experimental systems Fas is utilized as an
apoptosis marker (Chinnaiyan et al., 1995; Fu-
rukawa et al., 1996).
An important element of a successful PDT drug
may be the ability to induce a rapid apoptotic
response (Luo et al., 1996). The search for alter-
native photosensitizers seeks to find one that ex-
hibits good selectivity, not so much in their
affinity for, malignant cells but rather in the ulti-
mate photokilling. The mode of cell death should
also be investigated once their potency is estab-
lished (Chen et al., 1998).
In this paper, we describe the photodynamic
effects of 5,10,15,20-tetra(4-methoxyphenyl)-por-
phyrin (TMP) on a Hep-2 cell line. The goal of
this research was to establish the toxicity of TMP
in the dark, and in relation to the illumination
with visible light. We also determined which kind
of death TMP produces (necrosis or apoptosis)
and whether Fas expression is an indicator of an
apoptotic event.
2.3. Photosensitizer
5,10,15,20 - Tetra(4 - methoxyphenyl)porphyrin
was synthesized using the Lindsey et al. (1987)
method. A solution of 4-methoxybenzaldehyde
(0.152 ml, 1.5 mmol) and pyrrole (0.10 ml, 1.5
mmol) in 100 ml of dichloromethane was purged
with argon for 15 min. Trifluoroacetic acid (35 ml,
0.45 mmol) was added. The solution was stirred
for 60 min at room temperature. After that, 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (0.34 g,
1.5 mmol) was added and the mixture was stirred
for an additional 60 min, open to the atmosphere.
The solvent was removed under reduced pressure
and flash column chromatography (silica gel,
dichloromethane) yielded 98 mg (35%) of pure
+
TMP. MS [m/z] 734.3 (M ) (734.2895 calculated
for C H N O ).
48
38
4
4
2.4. Liposome preparation
2. Material and methods
The incorporation of TMP into the phospho-
2
.1. Cell culture
lipid bilayer of the dipalmitoyl phospha-
tidylethanolamine was done by a modification of
the ethanol injection procedure of Kremer et
A human larynx-carcinoma cell line (Hep-2),

I. Yslas et al. / Toxicology 149 (2000) 69–74
71
al. (1977). Typically, 2 ml of a solution bearing
.60 mM of phospholipid, 1.91 mM of cholesterol
2.7. Cell photosensitization studies
9
and 0.27 mM of TMP in ethanol–tetrahydrofuran
binary mixture (1:1, v/v) was injected into 10 ml
of phosphate-buffered saline solution (PBS) at
Cells were seeded, incubated and treated with
TMP in 25 cm culture flasks for the selected time
2
in the dark, as described above. Afterwards, the
medium containing the photosensitizer was re-
moved. Cells were washed three times with
medium and kept in 5 ml of it. The dishes were
exposed for different time intervals to visible light.
8
5
0°C. The injection was performed at a speed of
0 ml/min with magnetic stirring.
2.5. Irradiation
The visible light source used was a slide projec-
2
.8. Cell 6iability
tor equipped with a 150 W lamp. The light was
filtered through a 3 cm water layer to absorb heat.
The light intensity at the treatment site was 18
After each irradiation time, the viability of the
cells was estimated by microscopy using TB. The
same procedure without irradiation was carried
out for determining dark toxicity. Four culture
flasks were used for each incubation time. Every
experiment was compared with a culture control
without TMP.
2
mW/cm (Radiometer Laser Mate-Q, Coherent).
2
.6. TMP uptake and quantification
The uptake of TMP by Hep-2 cells was deter-
mined by fluorescence spectroscopy. About 1×
6
2
1
0 cells were inoculated in 25 cm culture flasks
2.9. Apoptosis determination
and incubated to obtain nearly confluent cell lay-
ers. An appropriate volume of the TMP incorpo-
rated into liposomes was added to the culture
flask containing 5 ml of medium. Cells were
treated with different TMP concentration for sev-
eral incubation times. The medium containing the
photosensitizer was then discharged and the cells
were washed three times with PBS and suspended
in 1 ml of PBS. The number of cells in each
suspension was estimated by trypan blue (TB)
exclusion test using a Neubauer chamber counter.
Next, 1.0 ml of 4% sodium dodecyl sulphate
To differentiate among necrosis and apoptosis,
the hematoxylin–eosin method was utilized. Re-
sults were corroborated with a specific kit (TdT
Frag EL™ DNA Fragmentation Detection Kit,
Oncogene Research Products).
2.10. Fas-Ag determination
The Fas-Ag expression was determined in con-
trol cells grown as monolayers. The cells were
incubated with TMP for 24 h in the dark. Other
cultures were treated with TMP for 24 h, illumi-
(
SDS, Merck) was added to the cellular suspen-
sion, which was incubated further for 15 min (in
the dark and at room temperature) and cen-
trifuged at 9000 rpm for 30 min. The concentra-
tion of the sensitizer in the supernatant was
measured by spectrofluorimetry (uexc=420 nm,
uem=658 nm). The fluorescence values obtained
from each sample were referred to the total num-
ber of cells contained in the suspension. The
concentration of TMP in this sample was esti-
mated by comparison with a calibration curve
obtained with standard solutions of TMP in 2%
SDS ([TMP] ꢀ0.5–10 mM). Each experiment was
repeated three times and four culture flasks were
used for each incubation time.
2
nated with 18 mW/cm and maintained during 5 h
at 37°C in the dark. The expression of Fas was
analyzed by Western Blot. Cell monolayers were
washed with PBS buffer, released by using a
rubber policeman, collected in Eppendorf tubes
and centrifuged at room temperature at 10 000
rpm for 2 min. The pellet was collected and the
supernatant discarded. An extraction buffer (150
mM NaCl, 1% NP-40, 0.5% deoxicolate, 0.1%
SDS, 50 mM Tris, pH 8) was added, and after a
25 min incubation at 0°C, the cell suspension was
centrifuged at 150 000×g, the supernatant col-
lected and the protein content was determined.

7
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I. Yslas et al. / Toxicology 149 (2000) 69–74
Proteins were separated by sodium dodecyl sul-
of dipalmitoyl phosphatidylethanolamine and
cholesterol. This technique optimizes the release
of lipophilic sensitizer to low density lipoproteins
(LDL). Hep-2 cells were treated with TMP (1
mM). Fig. 1 shows the behavior of TMP, in
relation to its uptake by Hep-2 cells, after differ-
ent periods of incubation with the sensitizer. The
uptake increased with TMP concentrations up to
the highest concentration studied (10 mM). In all
cases, TMP incorporation was initially very rapid
with short incubation times (B5 h), and tended
to reach a saturation value after long incubations
(]24 h). An increase of the initial uptake rate
was observed with increasing TMP concentra-
tions. However, after 24 h of incubation, a similar
value for total uptake was reached. It is interest-
ing to note that by comparison with a calibration
curve obtained with standard solutions of TMP in
2% SDS ([TMP] ꢀ0.5–10 mM), a value of TMP
fate-polyacrylamide gel electrophoresis (SDS-
PAGE) (Laemmli, 1970), in slab 10% gels.
Proteins were transferred to a nitrocellulose mem-
brane previously stabilized in TBS buffer (5 mM
NaCl, 10% albumin, 1 M Tris, pH 7.6) added
with 3%-decreamed skim milk. The membrane
was incubated 45 min with the mouse anti-Fas
antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) diluted 1:250, followed by incubation with
anti-mouse-IgG (1:1000) labeled with peroxidase.
Fas expression was visualized by a final incuba-
tion with 4-Cl-a-naphtol and hydrogen peroxide.
The reaction was stopped by washing with water.
3
. Results and discussion
Since TMP is a hydrophobic photosensitizer, it
was added to the medium in a liposomal solution
6
uptake ꢀ0.018 mmol/10 cells was calculated af-
ter 24 h of incubation.
Cell toxicity induced by TMP was examined in
the dark at different concentrations of the photo-
sensitizer and after several incubation periods.
The cells were treated with 1 mM TMP for 24 h at
3
7°C. Cell survival was determinate by mi-
croscopy in the presence of TB at 0, 4, 8, 12 and
4 h. In all irradiation experiments, a control kept
2
in darkness containing TMP was analyzed at each
time. No toxicity, in terms of cell survival, was
detected at any evaluated time. The same oc-
curred when cell cultures not treated with TMP
were irradiated under similar conditions. There-
fore, the cell mortality obtained after the exposure
of the cell cultures to visible light was exclusively
due to the photosensitization effect of TMP pro-
duced by light irradiation. (Fig. 2).
Fig. 3 shows the results obtained with control
cultures (a), incubated with TMP 1 mM for 24 h,
maintained in the dark (b), illuminated for 30 min
2
with 18 mW cm (c), and stained with hema-
toxylin–eosin. It can be seen that the treatment
with TMP followed by exposure to visible light
notably increased the apoptotic figures. This re-
sult was corroborated by utilizing a specific kit
Fig. 1. Uptake of TMP into Hep-2 cells as a function of
incubation time for different TMP concentrations, (ꢀ) 1 mM;
(ꢁ) 5 mM; (ꢂ) 10 mM of TMP. The amount of the drug taken
(
data not shown).
up by the cells was measured by fluorescence spectroscopy
uexc=420 nm, uem=658 nm). Values represent mean9S.D.
of three separate experiments.
Fas antigen expression was investigated under
three conditions (control, TMP treated cultures in
(

I. Yslas et al. / Toxicology 149 (2000) 69–74
73
Fig. 2. Inactivation of the cells incubated with 1 mM of TMP
exposed to different irradiation times with visible light. Incu-
bation times with TMP, (ꢂ) 24 h. Irradiated controls (ꢃ).
Values represent mean9S.D. of three separate experiments.
darkness, and illuminated). In Fig. 4 it can be seen
that Fas was only expressed in the last group, when
cultures were treated with the porphyrin following
illumination.
Noodt et al. (1999) studied the induction of
apoptosis by exposing V79 Chinese hamster
fibroblasts to PDT with various porphyrins and
light. They found different kinetics for the induc-
tion of DNA strand breaks characteristic of apop-
totic cells. PDT-induced damage to membranes
resulted in an increasing number of apoptotic cells
for about 12 h after PDT.
In the present study, the uptake kinetics of TMP
on Hep-2 cells was initially very fast,
depending on TMP concentration. Saturation of
TMP intracellular concentration was seen at ꢀ24
h of incubation. No toxicity was observed for
either cells containing TMP under dark conditions
or by irradiating the cells without the photosensi-
tizer. The survival of the irradiated cells incubated
with TMP was dependent on the light dose. The
kind of death produced is apoptosis, probably
through the expression of Fas. These results
Fig. 3. Apoptotic figures as seen by the hematoxylin–eosin
method in, (a) control cultures; (b) cultures incubated with
TMP 1 mM for 24 h and maintained in the darkness; (c)
cultures incubated with TMP 1 mM for 24 h and illuminated
2
for 30 min with 18 mW/cm .

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I. Yslas et al. / Toxicology 149 (2000) 69–74
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Fung, M.C., Cheung, N.H., 1998. A comparison of the
photodynamic effects of temoporfin (mTHPC) and MC540
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FADD a novel death domain-containing protein, interacts
with the death domain of Fas and initiates apoptosis. Cell
Fig. 4. Fas antigen expression in, (A) control cultures; (B)
8
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Dougherty, T.J., 1987. Photosensitization: therapy and detec-
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for 30 min with 18 mW/cm ; (C) cultures incubated with TMP
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Furukawa, Y., Iwase, S., Terui, Y., Kikuchi, J., Takao, S.,
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contribute to the knowledge of the PDT mechanism
produced by TMP on Hep-2 carcinoma cell line.
5,10,15,20-Tetra(4-methoxyphenyl)porphyrin
might be a candidate for studying its effects on
laboratory animals, since it is a non-toxic molecule
in the dark that causes cell death upon illumination
within a short time (30 min). Photodynamic cell
death is by apoptosis and not necrosis, which could
trigger other reactions and inflammation. Further
studies are being planned to study the effects of
TMP on experimental animals.
Grosseweiner, L.I., 1994. The science of phototherapy. In:
Photodynamic Therapy. CRC Press, London, pp. 139–
1
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Wiersema, P.H., 1977. Vesicles of variable diameter pre-
pared by a modified injection method. Biochemistry 16,
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Lindsey, S.J., Schreiman, I.C., Hsu, H.C., Kearney, P.C.,
Marguerettaz, A.M., 1987. Rothemund and Adler–Longo
reactions revisited: synthesis of tetraphenylporphyrins un-
der equilibrium conditions. J. Org. Chem. 52, 827–836.
Luo, Y., Chang, C., Kessel, D., 1996. Rapid initiation of
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biol. 63, 528–534.
Milgrom, L.R., O’Neill, F., 1993. The chemistry of natural
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Acknowledgements
The authors are grateful to SECYT of the
Universidad Nacional de R ´ı o Cuarto for financial
support. V.R. and E.N.D. hold positions as re-
searchers at CONICET. M.G.A. thanks CON-
ICET for a research fellowship.
Noodt, B., Berg, K., Stokke, T., Peng, Q., Nesland, J., 1999.
Different apoptotic pathways are induced from various
intracellular sites by tetraphenylporphyrins and light. Br. J.
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Penning, L.C., Dubbelman, T.M., 1994. Fundamentals of
photodynamic therapy: cellular and biochemical aspects.
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