Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

C-MYC and C-FOS expression changes and cellular aspects of the

photodynamic reaction with photosensitizers TMPyP and ClAlPcS2
Klara Pizova a,b,⇑, Robert Bajgar a,b, Regina Fillerova c, Eva Kriegova c, Vera Cenklova a,b, Katerina Langova a,
Petr Konecny b, Hana Kolarova a,b
a
Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
b
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 5, 77900 Olomouc, Czech Republic
c
Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: Photodynamic therapy (PDT) is based on the tumor-selective accumulation of photosensitizer followed
Received 3 June 2014 by irradiation with light of an appropriate wavelength. After irradiation and in the presence of oxygen,
Received in revised form 25 November 2014 photosensitizer induces cellular damage. The aim of this study was to evaluate effects of two photosen-
Accepted 1 December 2014
sitizers TMPyP and ClAlPcS2 on cell lines to obtain better insight into their mechanisms of action. We
Available online 9 December 2014
determined cell viability, reactive oxygen species (ROS) generation and changes in expression levels of
two important early response genes, C-MYC and C-FOS, on tumor MCF7 (human breast adenocarcinoma)
and G361 (human melanoma) cell lines and non-tumor BJ cell line (human fibroblast) after photody-
namic reaction with TMPyP and ClAlPcS2 as photosensitizers. In addition TMPyP and ClAlPcS2 cellular
uptake and clearance and antioxidant capacity of the mentioned cell lines were investigated. We found
appropriate therapeutic doses and confirmed that both tested photosensitizers are photodynamically
efficient in treatment used cells in vitro. TMPyP is more efficient; it had higher ROS production and tox-
icity after irradiation by intermediate therapeutic doses than ClAlPcS2. We revealed that both TMPyP and
ClAlPcS2-PDT increased C-FOS expression on tumor cell lines (G361 and MCF7), but not on non-tumor BJ
cell line. Conversely, both TMPyP and ClAlPcS2-PDT decreased C-MYC expression on non-tumor BJ cell
line but not on tumor cell lines. As first we tested these photosensitizers in such extent and we believe
that it can help to better understand mechanisms of PDT and increase its efficiency and applicability.
Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction
Photodynamic therapy (PDT) is a clinically approved, minimally
invasive therapeutic procedure for treating cancer with less toxic
adverse effects. This framework involves administration of a
photosensitizing agent (called photosensitizer – PS) and irradiation
with light of appropriate wavelength corresponding to maximum
absorbance of the PS. In the presence of oxygen, PS induces a series
of events leading to direct tumor cell death by generating cytotoxic
reactive oxygen species (ROS), damage to the tumor microvascula-
ture and induction of a local inflammatory reaction [1–3]. Origi-
nally it was developed as a tumor therapy [4] and hence current
clinical applications of PDT mainly include the treatment of various
tumors such as lung, bladder, gastric, cervical, prostate, bronchial,
esophageal, liver, breast, skin and gastrointestinal cancers [5]. PDT
⇑ Corresponding author at: Department of Medical Biophysics, Faculty of
Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech
Republic. Tel.: +420 585632110.
E-mail address: pizova.klara@seznam.cz (K. Pizova).
has also proved successful for some nonmalignant diseases such as
age-related macular degeneration, endometriosis, atherosclerosis
and even bacterial and fungal infections [4–6]. On the other hand,
also PDT enables detection of early stage cancer by recognition of
specifically emitted light from the PS [2]. The use of PDT as a cancer
therapy is particularly attractive because of its exceptional selec-
tivity and specificity – PS concentrates preferentially within the
malignant tissue and also the light can be directly focused on the
lesion [4]. For these reasons, PDT can maximally preserve sur-
rounding normal tissues [2].
Porphyrins are classified as tetrapyrroles (which comprise a
major component of hemoglobin and myoglobin). These molecules
contain a highly conjugated, heterocyclic macrocycle and may also
contain a central metallic atom. Hence porphyrins are effective as
PSs in medicine [7]. One of the most studied and very effective cat-
ionic porphyrin is a,b,c,d-Tetrakis(1-methylpyridinium-4-yl)por-
phyrin p-toluenesulfonate (TMPyP) with maximum absorption at
422 nm [8,9]. Another very attractive and effective PS is chloroalu-
minium phthalocyanine disulfonate (ClAlPcS2). The phthalocya-
nines are structurally related to porphyrins but exhibit maximum

http://dx.doi.org/10.1016/j.jphotobiol.2014.12.003
1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
absorption at longer wavelengths [7]. Chelated metals enhance
their phototoxicity and ring substitution with sulphonated groups
will render them water solubility. ClAlPcS2 has maximum absorp-
tion at 670 nm [10].
PDT can elicit many cellular and molecular changes but its main
goal is to induce cell death by both apoptosis and necrosis [11,12].
Analysis of RNA expression level is promising for gaining insight
into mechanism of cellular damage after application of PDT [13].
Many experiments with gene expression profiling after PDT have
been published [13–25]. For our study, we selected two early
response genes involved in modulating cell cycle, proliferation,
stress response and apoptosis: transcription factors C-FOS and C-
MYC, which have been reported in many publications [13–21]
but have not yet been studied after PDT with aforementioned PSs
(TMPyP and ClAlPcS2) on the tested cell lines (BJ, MCF7 and G361).
In our in vitro study, we evaluated the effect of photodynamic
reaction with PSs TMPyP and ClAlPcS2 on tumor MCF7 (human
breast adenocarcinoma) and G361 (human melanoma) cell lines
and non-tumor BJ cell line (human fibroblast). We determined cell
viability, ROS generation and changes in expression levels of C-FOS
and C-MYC after treatment and we determined TMPyP and ClA-
lPcS2 cellular uptake and clearance and antioxidant capacity of
the mentioned cell lines.
2. Materials and methods
2.1. Preparation of cell culture
The MCF7 (human breast adenocarcinoma), G361 (human mel-
anoma) and BJ (human fibroblast) cells were grown on a sterile 96-
well microplates 30,000 cells/well) or on sterile 6-well microplates
(100,000 cells/well), (TPP Techno Plastic Products AG, Switzerland)
in the presence of cultivation medium DMEM (Dulbecco’s Modified
Eagle Medium, with 10% fetal bovine serum and supplied by pen-
icillin, streptomycin and glutamine), (Sigma–Aldrich, USA). Cell
culture was incubated at 37 °C and 5% CO2 in CO2 incubator (Shel-
don Manufacturing, Inc., USA).
2.2. Photodynamic treatment
After 24 h incubation in DMEM cells in 96-well microplate were
loaded with 0, 0.5, 1, 2.5, 5 or 10 lM photosensitizer TMPyP
(Sigma–Aldrich; Fig. 1) or ClAlPcS2 (prepared by Jan Rakušan at
the Research Institute for Organic Syntheses in Rybitví, Czech
Republic; Fig. 2) and incubated for the next 24 h. The cells were
then rinsed with PBS and then PBS was replaced by 5 lM 5-(and
6-)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate (CM-
H2DCFDA), (Life Technologies, USA) dissolved in PBS. After
30 min incubation at 37 °C in darkness, samples were continuously
irradiated by light emitting diodes (LEDs), (excitation wavelength:
414 and 660 nm; full width at half maximum [FWHM]: 15 nm;
Fig. 1. a,b,c,d-Tetrakis(1-methylpyridinium-4-yl)porphyrin p-toluenesulfonate
(TMPyP); (http://www.sigmaaldrich.com/catalog/product/fluka/87639?lang=en
&region=CZ).
187
Fig. 2. Chloroaluminium phthalocyanine disulfonate (ClAlPcS2); (modified accord-
ing to https://www.google.com/patents/US6513921).
intensity: 10 mW/cm2) at a total light dose 0, 1, 5 or 10 J/cm2,
respectively. Irradiance was measured by the radiometer system
IL 1705 (International Light Technologies, USA).
2.3. Measurement of reactive oxygen species (ROS)
Fluorescence of 5-(-6)-chloromethyl-20 ,70 -dichlorofluorescein
(CM-DCF) within cells on 96-well microplate after photodynamic
treatment (Section 2.2) was recorded within 10 min after irradia-
tion as a kinetic measurement by microplate reader Synergy HT
(BioTek, USA). An excitation wavelength of 485 nm and emission
wavelength of 540 nm were used. After ROS measurement, the
excess probe was washed out with PBS, PBS was replaced with
fresh DMEM medium and then the cells were incubated for the
next 24 h at 37 °C and 5% CO2.
2.4. Cell viability test
The standard MTT test was carried out on photodynamically
treated cells in 96-well microplate (Section 2.2). Briefly, DMEM
was replaced by 2 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT solution), (Sigma–Aldrich) dis-
solved in PBS and then the mixture was incubated for 3.5 h at
37 °C and 5% CO2. After incubation, the MTT solution was removed
and 100 ll DMSO (Sigma–Aldrich) was added to liquefy a violet
film. The absorbance of the prepared solution was measured by
microplate reader Synergy HT at 570 nm and 690 nm. Percent of
cell viability of the test samples was related to control samples
(100 times the average of test samples per average of control
samples).
2.5. Gene expression analysis
Photodynamically treated cells in 96-well microplate (Sec-
tion 2.2) were centrifugated for 1, 3 and 5 h after irradiation and
total RNA was immediately isolated from cells using the Total
RNA Purification Kit (Norgen, Canada) according to the manufac-
turer’s protocol. Total RNA was converted to double-stranded
cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit
(Roche Applied Science, USA) in a 20 ll reaction volume according
to the manufacturer’s protocol. Reverse transcription was per-
formed on Mastercycler pro (Eppendorf, Germany). cDNA was
stored at 20 °C before further use.
PCR mixes were prepared as follows: 5 ll of cDNA was added to
20 ll PCR-Mix (FastStart Taq DNA Polymerase, dNTPack, Roche
188 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
Applied Science, USA) with primers (Table 1). The final concentra-
tions of each component were: 900 nM of each sense and antisense
primers and 100 nM probe, 3.5 mM MgCl2, 200 lM each dNTPs,
1U FastStart Taq DNA Polymerase, 1  PCR reaction Buffer. After
initial denaturation (one cycle at 94 °C for 15 min), 40 cycles
amplification (94 °C for 45 s, 60 °C for 30 s) were performed on
RotorGene Q (Quiagen, Netherlands). Relative expression was
calculated using the second derivative method (Rotor Gene
software Q Series v.2.0.2, Quiagen) as follows: Expression =
average amplification(CTtcalibratorCTtsample). The GAPDH gene was
used as a reference gene and human universal reference RNA
(Stratagene, La Jolla, USA) was used as calibrator (in triplicate) on
a concentration of 1.25 ng/reaction.
2.6. Measurement of PS cellular uptake and clearance
After 24 h incubation in DMEM cells in 96-well microplate were
exposed to 10 lM TMPyP or ClAlPcS2 and after 0, 6, 12 and 24 h
cells were 3 times well rinsed with PBS and fluorescence intensi-
ties in range of the photosensitizers’ emission maxima was mea-
sured by microplate reader Tecan Infinite 200 PRO (Tecan,
Switzerland). An excitation wavelength of 424 nm and the range
of emission wavelengths from 708 to 717 nm were used for TMPyP
and an excitation wavelength of 645 nm and the range of emission
wavelengths from 675 to 684 nm were used for ClAlPcS2 uptake
measurements. To measure their cellular clearance, cells incubated
24 h with PS were well rinsed with PBS and left in fresh DMEM
medium for next 6, 12 and 24 h. After each period the medium
was replaced by PBS and fluorescence intensities of the cellular
photosensitizers were determined by microplate reader Tecan
Infinite 200 PRO, similarly as for the uptake measurements.
2.7. Measurement of the cell antioxidant capacity
The antioxidant capacity was determined using 2,2-diphenyl-1-
picrylhydrazyl (DPPH), (Sigma–Aldrich) as a free radical. After 24 h
incubation in DMEM cells in 6-well microplate were rinsed with
PBS and than PBS was replaced by 0.1% Triton X-100 (Serva Electro-
phoresis GmbH, Germany) in 96% ethanol. After 15 min incubation
at 20 °C samples were transferred to 1.5 ml Eppendorf tubes and
centrifugated for 30 min at 10,000 rpm and 5 °C (centrifuge HER-
MLE Z 300 K, rotor 220.87 VO3/4, Hermle Machine Company,
Germany). 294 ll of supernatant was transferred into 96-well
microplate and 6 ll of 5  103 mol/l DPPH solution in 96% ethanol
was added (thus final DPPH concentration was 1  104 mol/l).
After 30 min incubation at room temperature, absorbance of sam-
ples and controls was measured by microplate reader Tecan Infi-
nite 200 PRO at 520 nm. As negative control we used DPPH
solution alone in concentration 1  104 mol/l and as positive con-
trol and evaluation standard we used known antioxidant ascorbic
acid (Sigma–Aldrich) in concentrations 0, 0.2, 2, 20, 40, 100, 200
and 400 lmol/l.
Moreover we tested total protein amount in samples by Bio-Rad
Protein Assay (Bio-Rad Laboratories, Inc., USA) according to the
Table 1
Used primers.
Gene Gene name (synonyms) GenBank
abbreviation accession nb
FOS FBJ murine osteosarcoma viral oncogene NM_005252.3
homolog
MYC v-myc myelocytomatosis viral oncogene NM_002467.4
homolog (avian)
GAPDH Glyceraldehyde-3-phosphate dehydrogenase NM_002046
(human)
manufacturer’s protocol. Briefly, 10, 20 and 30 ll of supernatant
after centrifugation (in Section 2.7) were transferred to 96-well
microplate and supplemented by distillated water to overall
amount 90 ll. Than 10 ll of Protein Assay Dye Reagent (Bio-Rad
Laboratories, Inc.) was added and absorbance was measured by
microplate reader Tecan Infinite 200 PRO at 595 nm.
2.8. Statistical analysis
The data from ROS and cell viability measurement are pre-
sented as the medians and interquartile range of three indepen-
dent experiments. Groups of different PSs (for the same cell line)
were compared by the non-parametric Mann–Whitney U-test.
Groups of different cell lines (for the same PS) were compared by
the Kruskal–Wallis test and if differences were significant, multiple
comparisons by Mann–Whitney U-test with Bonferroni correction
were performed. Data from real-time PCR, PSs fluorescence and
antioxidant capacity are presented as the means and standard
deviations from three independent measurements. SPSS version
15 (SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis
of the data. A p value of <0.05 was considered as statistically
significant.
3. Results
3.1. Influence of PDT on cell viability
In order to determine PSs efficiency on the cell destruction, we
evaluated and compared cell viability after treatment with various
PDT doses for two different PSs.
As expected, PS alone in the absence of irradiation (both TMPyP
and ClAlPcS2) in any concentration used did not significantly
decrease cell viability (cell viabilities did not decrease below
95%). However, the higher the total irradiation dose and the higher
the PS concentration the sample was subjected to, the lower was
the cell viability. The highest differences between cell viability
after TMPyP and ClAlPcS2 treatment were found mainly for lower
irradiation dose (1 J/cm2) of the appropriate light in combination
with 5 or 10 lmol/l of PS), when TMPyP-PDT induced significantly
lower cell viability compared to ClAlPcS2-PDT. The optimal photo-
toxic effect was found for total light dose of 5 J/cm2 in combination
with 1 lmol/l of TMPyP or 5 lmol/l of ClAlPcS2. In the cases of this
and higher therapeutic doses, almost all cells was eradicated (cell
viability counts decreased below 10%) for all tested cell lines. More
detailed informations about cell viabilities after TMPyP and ClA-
lPcS2-PDT on BJ, MCF7 and G361 cells and statistical analyses are
shown in Fig. 3 and in Table E1 (On-line Supplement).
3.2. Influence of PDT on reactive oxygen species (ROS) production
To determine the effect of PSs on the cell destruction, we eval-
uated ROS generation after treatment with various PDT doses with
the PSs mentioned.
Amplicon size Sense, antisense primers/ Manufacturer
(bp) assay ID
77 Hs00170630_m1 Life technologies
87 Hs00905030_m1 Life technologies
143 Hs.PT.39a.22214836 Integrated DNA
technologies, USA
 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
Fig. 3. Percentage of living cells in the case of using various concentrations of photosensitizer TMPyP (left graphs) or ClAlPcS2 (right graphs) in combination with various total
light doses on different cell lines. ⁄Statistically significant differences between TMPyP and ClAlPcS2-PDT (p value < 0.05).
As expected, ROS production was negligible in the control sam-
ples. However in majority of cases, the higher the PS concentration
and the higher the total exposure dose the sample was subjected
to, the higher the ROS production was observed. Only at the appli-
cation of the highest therapeutic doses of ClAlPcS2-PDT, ROS pro-
duction slightly decreased, however it can be caused by an
alteration in cellular integrity accompanying release of PSs from
cells. Almost at all of used therapeutic doses TMPyP-PDT induced
markedly and significantly higher ROS production in all tested cell
lines when compared to ClAlPcS2-PDT. Moreover the highest ROS
production was observed in MCF7 cell line, while the lowest ROS
production was detected in BJ cell line after both TMPyP and ClA-
lPcS2-PDT and at all therapeutic doses. More detailed informations
about ROS production after TMPyP and ClAlPcS2-PDT on BJ, MCF7
and G361 cells and statistical analysis are shown in Fig. 4 and in
Table E2 (On-line Supplement).
The increment in ROS production within the first 10 min after
treatment showed very similar results when it was measured
immediately after treatment. In the control samples, we found
negligible ROS production increment and no significant differences
between ROS increment after TMPyP and ClAlPcS2 treatment. The
higher the PS concentration and the higher the total exposure dose
the sample was subjected to, the faster the ROS production, except
some highest therapeutic doses of ClAlPcS2-PDT, when the rate of
ROS production slightly decreased. We also observed that
TMPyP-PDT induced significantly faster ROS production than
ClAlPcS2-PDT in all cell lines where the fastest ROS production
189
was detected in MCF7 cell line and the slowest ROS production
in BJ cell line considering both TMPyP and ClAlPcS2-PDT and all
therapeutic doses. More detailed informations about ROS produc-
tion increments after TMPyP and ClAlPcS2-PDT on BJ, MCF7 and
G361 cells and appropriate statistical analysis are shown in Fig. 5
and in Table E3 (On-line Supplement).
3.3. Influence of PDT on C-MYC and C-FOS expression
Based on the results of MTT experiments (Section 3.1, Fig. 3) one
photosensitizer concentration and one irradiation dose were cho-
sen for TMPyP and ClAlPcS2-PDT, which induced approximately
50% mortality of cells. For TMPyP-PDT it was 5 lmol/l of TMPyP
in combination with 1 J/cm2 of blue light (414 nm) and for ClA-
lPcS2-PDT it was 1 lmol/l of ClAlPcS2 in combination with 5 J/
cm2 of red light (660 nm). For the selected PDT doses we evaluated
C-MYC and C-FOS expression.
Generally C-MYC expression slightly increased in BJ and G361
cells during their growth, while MCF7 cells showed a decrease in
C-MYC expression. After PDT of BJ cells in the presence of both
PSs C-MYC expression was lower and did not markedly increase
during the first 5 h after treatment when compared to control sam-
ples. Similarly C-MYC expression in G361 cells after both TMPyP
and ClAlPcS2-PDT as well as in MCF7 cells after TMPyP-PDT did
not significantly change in comparison to control samples. Only
1 h after ClAlPcS2-PDT of MCF7 cells C-MYC expression slightly
190 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
Fig. 4. ROS production in the case of using various concentrations of photosensitizer TMPyP (left graphs) or ClAlPcS2 (right graphs) in combination with various total light
doses immediately after treatment on different cell lines. ⁄Statistically significant differences between TMPyP and ClAlPcS2-PDT (p value < 0.05).
increased in contrast to control samples, however 4 h later its
expression did not differ from the control levels (Fig. 6).
Generally, C-FOS expression decreased during time in control
samples of BJ and MCF7 cells, however increased in G361 cells.
C-FOS expression in BJ cells slightly increased after both TMPyP
and ClAlPcS2-PDT, but these levels were still too low. Conversely,
C-FOS was markedly upregulated after both TMPyP and ClAlPcS2-
PDT in MCF7 and G361 cells in contrast to control samples. In par-
ticular, C-FOS expression in MCF7 cells was strongly upregulated
1 h after TMPyP-PDT and its expression further increased. The
highest expression was found 3 h after treatment. Although after
next 2 h C-FOS expression decreased, so its level was still relatively
high compared to control samples. In the case of ClAlPcS2-PDT C-
FOS was also strongly upregulated 1 h after treatment, but in con-
trast to TMPyP-PDT, its expression decreased over time. On the
other hand C-FOS expression in G361 cells increased 3 h after both
TMPyP and ClAlPcS2-PDT and continued with the increase for the
next 2 h (Fig. 7).
3.4. PS uptake and clearance
We determined the PSs uptake and clearance for various cell
lines.
We observed the highest concentration of both TMPyP and ClA-
lPcS2 in tumor G361 cells, which accumulated much more PS than
MCF7 cells and non-tumor BJ cells. Moreover various cell lines
expressed various kinetics of TMPyP uptake, G361 and BJ cells
showed the highest TMPyP concentration 12 h after PSs adminis-
tration however MCF7 cells reached the highest TMPyP concentra-
tion 24 h after PS administration and probably they are able to
accumulate more TMPyP. In the case of ClAlPcS2 uptake, the kinetic
study showed similar behavior for all three tested cell lines when
they were treated by TMPyP (Fig. 8).
In the absence of PS in the growth media, cells started to clear
accumulated PS, markedly at the higher rate for G361 cells. In
the case of all three tested cell lines, fluorescence of accumulated
TMPyP and ClAlPcS2 was on similar values 12 and 24 h after PS
removal, respectively. Rates of PSs clearance by MCF7 and BJ cells
seems to be similar (Fig. 8).
3.5. Cell antioxidant capacity
We determined the antioxidant capacity of various cell lines.
The lowest antioxidant capacity showed BJ cell line and the
highest MCF7 cell line (Fig. 9A). Based on the dose–response of
the ascorbic acid (Fig. 9B) we can demonstrate that difference
between DPPH absorbance values 0.651 (achieved for BJ cells)
and 0.475 (achieved for G361 cells) can represent more than 10-
fold antioxidant capacity.
When we measured protein amount in our samples for antiox-
idant activity measurements, we found no significant differences
among cell lines and among individual samples (data not shown).
Thus we presume that our results of the antioxidant capacity mea-
surement are not affected by potentially different response of the
 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
Fig. 5. ROS production increment in the case of using various concentrations of photosensitizer TMPyP (left graphs) or ClAlPcS2 (right graphs) in combination with various
total light doses within 10 min after treatment on different cell lines. ⁄Statistically significant differences between TMPyP and ClAlPcS2-PDT (p value < 0.05).
different cells to our cell membrane permeabilization procedure,
and therefore we assume that results (Fig. 9A) are comparable.
4. Discussion
In this in vitro study, we evaluated the efficiency of two PSs,
TMPyP and ClAlPcS2, on MCF7 (human breast adenocarcinoma),
G361 (human melanoma) and BJ (human fibroblast) cells. We also
assessed the effect of PDT with these PSs on the expression of early
response genes C-MYC and C-FOS. Our research group is focused on
in vitro PDT experiments predominantly and we tested a number of
potentional PSs [10,26–37] but we had never tested TMPyP and
ClAlPcS2 to such an extent.
As expected, generally the higher the photosensitizer concen-
tration and the higher the total exposure dose the sample was sub-
jected to, the higher and faster the ROS production and conversely
the lower the cell viability. The results showed that both tested PSs
are efficient for photodynamic treatment, 1 lmol/l of TMPyP in
combination with 5 J/cm2 of blue light (414 nm) and 5 lmol/l of
ClAlPcS2 in combination with 5 J/cm2 of red light (660 nm) eradi-
cated almost all cells in all tested cell lines. TMPyP is more effi-
cient, it exerts higher and faster ROS production and higher
toxicity after irradiation than ClAlPcS2 on all three tested cell lines.
Although TMPyP seems to be more efficient compared to CAlPcS2,
for some medical applications, ClAlPcS2 may have greater advanta-
ges because the penetration of light into tissue increases with
wavelength [1,5] as we have also shown in our earlier study [38].
Phthalocyanine has strong absorbance at longer wavelengths (in
191
the red region of the visible spectrum) and should be more effec-
tive in the eradication of deeply seated tumors [1,5]. After both
TMPyP and ClAlPcS2-PDT the highest and the fastest ROS produc-
tion was detected in MCF7 cells, while the lowest ROS production
was observed in BJ cells. This can indicate that after this treatment
BJ cells may die not primarily due to cytotoxicity of ROS species
and moreover our results from antioxidant capacity measurements
indicate that their resistance to oxidative stress (antioxidant
capacity) may be poor when compared to tumor cell lines.
Higher ROS production after TMPyP and ClAlPcS2-PDT treatment
on tumor MCF7 and G361 cells also indicate that tested photody-
namic treatment is effective considering a consequence with the
cell death.
We evaluated expression of two important early response
genes, C-MYC and C-FOS after PDT. C-fos is a leucine zipper which
can dimerize with proteins of the jun family. C-fos and jun com-
bined with ATF (activating transcription factor) form the transcrip-
tion factor complex AP-1. AP-1 may modulate cell proliferation and
differentiation, stress response and stress-induced apoptosis by
signal transduction of growth factors in the cytoplasm to the
nucleus via the MAPK (mitogen-activated protein kinases) signal-
ling pathway. Expression increase of both genes is inducible by
many stimuli including stress and cell damage after PDT. C-myc
is a transcription factor of many cell cycle genes (such as late G1
cyclins and cyclin dependent kinases, essential for transition from
G1 to S phase). C-myc is also major apoptotic inducer and it inter-
venes in the mitochondrial apoptotic pathway by induction of
cytochrome c release or by targeting the anti-apoptotic Bcl2-family
[19,21].
192 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196

Fig. 6. Fold change of C-MYC expression at various times after treatment with photosensitizer TMPyP (left graphs) or ClAlPcS2 (right graphs) in combination with appropriate
irradiation (dark columns) or without irradiation (light columns). Expression level in controls (fold change) = 1, fold change <1 means that expression decreases compared to
control, fold change >1 means that expression increases compared to control.

Expression of these genes has been studied in many cases of
PDT treatments but not on our tested cell lines under our tested
PDT conditions. A number of scientific groups have reported C-
FOS overexpression in response to PDT with various PSs on various
cell lines [13–21]. For C-MYC expression after PDT, the results are
not in agreement. For example, Luna et al. [14] observed increased
mRNA levels of C-MYC on RIF-1 cells after Photofrin-PDT and Verw-
anger et al. [16] also observed transient overexpression of C-MYC
on human fibroblasts after ALA-PDT as well as Sanovic et al. [21]
who found C-MYC upregulation on A-431 cells in response to
hypericin-PDT. Conversely, Cekaite et al. [18] observed C-MYC
downregulation on Jurkat cells in response to HAL-PDT and Ruh-
dorfer et al. [19] also published C-MYC downregulation on A-431
cells after ALA-PDT. Our results confirmed that expression of both
C-MYC and C-FOS has variable dynamics during time and among
different cell lines. We can see that C-MYC expression after both
TMPyP and ClAlPcS2-PDT treatment on both tumor cell lines gener-
ally did not markedly change (as compared to controls) whereas on
non-tumor BJ cells it decreased. On the other hand C-FOS is mark-
edly overexpressed after both TMPyP and ClAlPcS2-PDT treatment
(as compared to controls) on tumor cell lines whereas did not
markedly change after treatment on BJ cells. It can indicate,
that especially tumor cells react to oxidative and other stress
after TMPyP and ClAlPcS2-PDT by a genomic response. Our results
also showed that changes in expression of both early response
genes after TMPyP-PDT were similar than after ClAlPcS2-PDT,
that can be a consequence of the different subcellular target for
these PSs.
 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196 193

Fig. 7. Fold change of C-FOS expression at various times after treatment with photosensitizer TMPyP (left graphs) or ClAlPcS2 (right graphs) in combination with appropriate
irradiation (dark columns) or without irradiation (light columns). Expression level in controls (fold change) = 1, fold change <1 means that expression decreases compared to
control, fold change >1 means that expression increases compared to control.

When we compared PS uptake among cell lines, we observed
the highest concentration of both TMPyP and ClAlPcS2 in tumor
G361 cells and the lowest in non-tumor BJ cells. Previously
reported studies about different PSs uptake and clearance showed
various results, some results showed higher accumulation in tumor
cells compared to non-tumor cells [39,40] and other results
revealed no differences in PS accumulation and retention between
tumor and non-tumor cells [41,42].
It should be considered that our experiments were performed
in vitro. The photodamage and cytotoxicity after PDT in real organ-
isms in vivo are multifactorial and can depend on much more fac-
tors than we can evaluate in vitro [43]. One of the main benefits of
PDT, high selectivity originates from the fact that in living organ-
isms PSs are preferably accumulated in tumor tissues, due to dif-
ferent rate at which PSs are taken up by tumor and health cells,
due to rapid clearance of PSs from healthy tissues, which was con-
firmed by many studies with various PSs [3,44–48]. PDT selectivity
also originates from the fact that the activating light beam can be
targeted with high precision on pathological tissue [1,4,49]. Studies
also showed that PS accumulation and retention in a tissue is prob-
ably due to host factors, i.e., vascular permeability and lymphatic
clearance, coupled with its ability to dissociate from serum pro-
tein-binding sites and bind to cellular sites [46,50,51]. Even if not
all processes and factors can be studied by our in vitro experiments,
194 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196

Fig. 8. TMPyP (left graph) and ClAlPcS2 (right graphs) fluorescence at different times after their addition to various cell lines.

Fig. 9. Graph A represents antioxidant capacity of cell lines BJ, G361 and MCF7 expressed as DPPH absorbance at 520 nm (negative control = DPPH solution alone). Graph B
represents dose-response of DPPH absorbance on the ascorbic acid concentration.

it can be important milestone for further analysis including other 6. Abbreviations

models with a perspective in practical applications.

5. Conclusions
CM-DCF 5-(-6)-chloromethyl-20 ,70 -
dichlorofluorescein
Searching for and developing more efficient antitumor thera-
CM-H2DCFDA 5-(and 6-)-chloromethyl-2́,7́-
pies is of paramount importance. PDT attracts particular attention
dichlorodihydrofluorescein diacetate
because of its exceptional selectivity and specificity. A number of
C-FOS FBJ murine osteosarcoma viral oncogene
successes have been attained the field of PDT and many PS have
homolog
been approved for clinical use. However, PDT is still the subject
ClAlPcS2 chloroaluminium phthalocyanine
of ongoing research and searching for new PSs and more effective
disulfonate
PDT regiment are the main goals.
C-MYC v-myc myelocytomatosis viral oncogene
We confirmed that both TMPyP and ClAlPcS2 are very effective
homolog (avian)
in the photodynamic treatment and we found appropriate thera-
DMEM Dulbecco’s Modified Eagle Medium
peutic doses (the combination of 5 J/cm2 blue light with 1 lmol/l
DPPH 2,2-diphenyl-1-picrylhydrazyl
of TMPyP and 5 J/cm2 red light with 5 lmol/l of ClAlPcS2). We
LEDs light emitting diodes
observed extensive changes on a cellular and molecular level
Pc4 phthalocyanine photosensitizer
(ROS production, cell death induction, changes in C-FOS and C-
PDT photodynamic therapy
MYC expression) after TMPyP and ClAlPcS2-PDT, which confirm
PS photosensitizer
the efficiency of the investigated PSs. We demonstrated markedly
ROS reactive oxygen species
different response of tumor cells to PDT on molecular level as com-
TMPyP a,b,c,d-Tetrakis(1-methylpyridinium-4-
pare to non-tumor cells in vitro. This provides a deeper insight into
yl)porphyrin p-toluenesulfonate
PDT mechanisms and can thus help increase its efficiency and
application. This PSs and PDT regimen deserves further attention.
 K. Pizova et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 186–196
Acknowledgement
This work was supported by grant Ministry of Health of Czech
Republic NT14060-3/2013.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.
2014.12.003.
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