Phototoxic effects of pyropheophorbide-a from chlorophyll-a on cervical cancer cells

Article abstract of DOI:10.1142/S1088424613501034

Photodynamic therapy (PDT) is a promising modality in both the curative and palliative treatment against a variety of experimental and naturally occurring human cancers. At present, chlorophyll a derivatives are extensively used for the synthesis of photosensitizers (PSs) for PDT of tumors. In the present study, chlorophyll-a was extracted from the blue-green algae Spirulina platensis by refluxing with acetone. The extract was further acid treated to obtain methylpheophorbide-a (MPa), which was then refluxed in collidine and methylpyropheophorbide-a (Mppa) was obtained. After that, Mppa was converted to pyropheophorbide-a (Ppa) by treatment with 50% sulfuric acid. Finally, phototoxicity and dark toxicity of purified Ppa in two different cell lines, TC-1 and CaSki, were examined by MTT assay. The results suggest that Ppa is more toxic to TC-1 cell line than CaSki cell line. In vivo, the photosensitizing efficiency of Ppa was also higher than those of unloaded PS. These results indicate the potential of Ppa in PDT.

Full text of DOI:10.1142/S1088424613501034

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2014; 18: 182–187
DOI: 10.1142/S1088424613501034
Published at http://www.worldscinet.com/jpp/
Phototoxic effects of pyropheophorbide-a from
chlorophyll-a on cervical cancer cells
a
a
b
Pankaj Kumar Chaturvedi , Yong-Wan Kim , Sang Soo Kim
and Woong Shick Ahn*
c
a
Catholic Research Institute of Medical Science, The Catholic University of Korea, 505 Banpo-dong,
Seocho-gu, Seoul 137-701, Republic of Korea
Wonkwang Institute of Integrative Biomedical Science, Wonkwang Digital University, 437 Dorimcheon-ro,
b
Youngdeungpo-gu, Seoul 150-827, Republic of Korea
c
Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea,
5
05 Banpo-dong, Seocho-gu, Seoul 137-701, Republic of Korea
Received 1 April 2013
Accepted 11 September 2013
ABSTRACT: Photodynamic therapy (PDT) is a promising modality in both the curative and palliative
treatment against a variety of experimental and naturally occurring human cancers.At present, chlorophyll
a derivatives are extensively used for the synthesis of photosensitizers (PSs) for PDT of tumors. In the
present study, chlorophyll-a was extracted from the blue-green algae Spirulina platensis by refluxing
with acetone. The extract was further acid treated to obtain methylpheophorbide-a (MPa), which was
then refluxed in collidine and methylpyropheophorbide-a (Mppa) was obtained. After that, Mppa was
converted to pyropheophorbide-a (Ppa) by treatment with 50% sulfuric acid. Finally, phototoxicity
and dark toxicity of purified Ppa in two different cell lines, TC-1 and CaSki, were examined by MTT
assay. The results suggest that Ppa is more toxic to TC-1 cell line than CaSki cell line. In vivo, the
photosensitizing efficiency of Ppa was also higher than those of unloaded PS. These results indicate the
potential of Ppa in PDT.
KEYWORDS: photodynamic therapy, photosensitizer, pyropheophorbide-a, algae.
oxidizer that causes necrosis of the tumor tissue) via
INTRODUCTION
excitation energy transfer from photosensitizer [6].
Photodynamic therapy (PDT) of cancer has emerged
Since first clinically approved PS (photosensitizer),
as a highly effective treatment for oncological diseases
Photofrin, was known to have some drawbacks such as
[
1–3] and it has been successful in the management of
prolonged skin phototoxicity, low absorption at longer
wavelength, and not single compound, etc, researchers
faced challenges to find new photosensitizers [7]. To find
optimal photosensitizers, numerous compounds have
been synthesized and examined including porphyrins,
chlorins, phthalocyanines and purpurins [8, 9]. Among
them, some chlorin derivatives, called second generation
PS with promising physicochemical properties and high
PDT efficiency were found [10–12]. Some of them such
as Foscan, LS11 and Radachlorin, were approved for
clinicaluse[13].3-Devinyl-3-(1-hexyloxyethyl)pyropheo-
phorbide-a (HPPH), photochlor, is one promising deriv-
ativeofpyropheophorbide-a(Ppa),whichisunderphaseII
a wide variety of solid, malignant tumors [4]. The dual
selectivity of PDT is produced by both a preferential
uptake of the photosensitizer by the diseased tissue and
the ability to confine activation of the photosensitizer
to this diseased tissue by restricting the illumination
to that specific region [5]. Basic principle of PDT is a
photogeneration of highly reactive singlet oxygen (strong


SPP student member in good standing
*
Correspondence to: email: woongshickahn@gmail.com,
ahnlab1@catholic.ac.kr, tel: +82 2-2258-7488, fax: +82 2-599-4120
Copyright © 2014 World Scientific Publishing Company

PHOTOTOXICITY EFFECTS OF PYROPHEOPHORBIDE-A FROM CHLOROPHYLL-A ON CERVICAL CANCER CELLS
183
1
clinical trial [14]. It showed a superior PDT effect in some
tumor of animal [15]. Also, many studies were performed
on Ppa-conjugates with other molecules [16, 17], but
almost no studies for PDT efficacy of free Ppa have been
reported. In addition, Ppa is a potential candidate as PS
for PDT because it can be prepared easily as a single
compound that generates singlet oxygen in high yield
and has high absorption intensity at longer wavelength
(80694.4). H NMR (500 MHz, CDCl , TMS int): d, ppm
3
9.43 (s, 1H, 5-meso-H), 9.32 (s, 1H, 10-meso-H), 8.52 (s,
1
1H, 20-meso-H), 7.95 (dd, 1H, 3 -CH, J = 11.5, 11.5 Hz),
2
6.20 (dd, H, 3 -CH , J = 17.9, 11.5 Hz,), 5.17 (dd, 2H,
2
2
13 -CH , J = 19, 19 Hz), 4.46 (q, 1H, 18-CH, J = 7.3 Hz),
2
1
4.29 (d, 1H, 17-CH, J = 9 Hz), 3.64 (q, 2H, 8 -CH , J =
2
1
1
7.5 Hz), 3.61 (s, 3H, 12 -CH ), 3.38 (s, 3H, 2 -CH ), 3.20
3
3
1
2
(s, 3H, 7 -CH ), 2.71–2.59 (m, 2H, 17 -CH ), 2.38–2.23
3
2
1
1
(
665 nm) [18].
(m, 2H, 17 -CH ), 1.80 (3H, d, 18 -CH , J = 7.3 Hz), 1.66
2
3
2
A few cervical cancer cell lines such as TC-1, HeLa,
(t, 3H, 8 -CH J = 7.5 Hz), 0.48 and -1.70 (brs, each 1H,
3
+
and SiHa cells were included for evaluating PDT on the
inhibition of cell growth after Radachlorin treatment on
the cells [19]. While the MTT assay results of SiHa were
consistent with TC-1, HeLa showed a cell line-dependent
Radachlorine/PDT-resistant trend compared to the other
cells. It was also demonstrated that the mechanisms
involved in the death of human cervical cancer cells
triggered by PDT with Photogem exerts its antitumor
activity primarily by inducing necrosis rather than by
inducing apoptosis [20]. While Ppa is a well-known
photosensitizer, no application for TC-1 and CaSki cell
lines yet. In this study, we report the relationship of the
enhanced cytotoxicity of Ppa in the cervical cancer cells
and its potential relationship to the clinical outcome
following PDT. We examined in vitro phototoxic effects
of Ppa that is prepared from Mppa obtained from
Spirulina platensis (a blue-green algae) with and without
light on two different cervical cancer cell lines. We also
evaluated antitumor PDT effects in TC-1 cell-bearing
mice including treatment variables.
21-, 23-NH). EIS-MS: m/z 535 [M] .
Cell cultures
TC-1 cell line positive for HPV E6/E7 was
generously provided by Dr. T.C. Wu, at the Johns
Hopkins University, MD, USA. TC-1 and CaSki (a
new epidermoid cervical cancer cell line) cells were
routinely propagated in monolayer cultures in RPMI-
1
640 medium, supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 0.22% sodium bicarbonate and
penicillin/streptomycin. The cells were cultured in a 5%
CO incubator at 37 °C. Additionally, 400 mg/L G418
2
was added to TC-1 culture media.
Phototoxicity
3
For viable cell counting, CaSki and TC-1 cells (3 × 10
cells) per well (96 well plate) were treated with Ppa, at
concentration range of 0.03, 0.06, 0.125, 0.25, 0.5, 1 and
2 mM, after incubation for 12 h (37 °C, 5% CO ) in RPMI
2
1
640 containing 10% FBS. Following 24 h incubation,
2
laser irradiation (662 ± 3 nm, 6.25 J/cm ) was performed.
Cell growth inhibition was determined by MTT
MATERIALS AND METHODS
(
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) 24 h after irradiation. For the MTT assay,
0 mL of 5 mg/mL MTT solution was added to each
Chemical reagents
2
All reagents and solvents used in this study were
purchased from Sigma-Aldrich. Structures of PS were
well and incubated for 4 h. 100 mL of dimethylsulfoxide
(DMSO) was added to each well, shaken for 10 s, and
the absorbance was measured with an ELISA-reader
(Spectra Max 340/Molecular Devices, USA) at 570 nm.
Each group consisted of three wells; the means of their
values were used as the measured values.
1
characterized by H NMR, MS, and UV-vis spectroscopy
at NCIRF, Seoul National University and at Integrative
Research Support Center, The Catholic University of
Korea. Mpa was prepared from Spirulina plantesis
algae (purchased from DIC LIFETEC Co., Ltd, Japan)
by extraction with acetone. Mppa was obtained from
Mpa by refluxing in collidine for 2.5 h. At last Ppa was
prepared as reported previously [21]. Briefly, Mppa (1 g)
was dissolved in 20 mL of sulfuric acid (50%) and stirred
at room temperature for 2.5 h. The reaction mixture
was diluted with cold distilled-water and extracted with
dichloromethane (200 mL). The organic layer was washed
with distilled water two times and evaporated by rotary
evaporator. The residue was purified on silica gel (60–
In vivo photosensitizing efficacy
The in vivo PDT efficacies of Ppa were investigated
in 6-week-old C57BL/6 mice with TC-1 tumor implanted
in the peritoneum. Groups of five mice were given
intravenous injections of several type of Ppa (1.25 or
2.5 mg/kg) or PBS as control when the tumors grew to a
3
volume of 450–500 mm . After 6 h of Ppa administration,
the mice were anesthetized by an intraperitoneal injection
of 50 mg/kg Zoletil 50 (Virbac, France), and the tumors
were laser irradiated (UPL-PDT) at a wavelength of 670 ±
10 nm and an interstitial total impact energy of 150 J/
2
30 mesh, Merck, Germany) by column chromatography
using 2–5% methanol in dichloromethane. Finally, Ppa
was obtained in yield of 88%. R : 0.25 (5% methanol in
f
-
1
-1
2
dichloromethane). UV-vis (CH Cl ): l , nm (M .cm )
67.7 (43162.1), 539.5 (22223.3), 511.7 (18963.6), 414.2
cm . The mice were kept in an optimal condition, and the
2
2
max
6
tumor volumes were measured every 2 days with calipers.
Copyright © 2014 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2014; 18: 183–187

184
P.K. CHATURVEDI ET AL.
3
2
Tumor volume [V (mm )] was calculated as (L × W )
×
0.524 (L = length of the longest dimension) and W =
width (perpendicular to the long axis).
NH
N
N
Spirulina platensis
a
Algae
HN
Western blot
For immunoblots, confluent monolayers of CaSki,
HeLa, and TC-1 cells were lysed with cell extraction
buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na P O ,
H3COOC
O
H3COOC
Methylpheophorbide-a, 1
b
4
2
7
2
mM Na VO , 1% Triton X-100, 10% glycerol, 0.1%
3 4
SDS, 0.5% deoxycholate) (Biosource, CA) in the
presence of protease inhibitors. Protein concentration
was determined using the BCA Protein Assay (Bio-
Rad, Hercules, CA). Electrophoresis was performed on
NH
N
N
NH
N
N
10–12% SDS-polyacrylamide gels for 1h, and the gels
c
were then transferred onto PVDF membrane (Millipore,
CA). Membranes were blocked in Tris-buffered saline
with 0.1% Tween 20 (TBST) containing 5% skim milk
for 1 h at room temperature. After blocking, membranes
were incubated for 1 h at room temperature or overnight
at 4°C with mouse monoclonal anti-ABCG2 (Santa Cruz,
CA), and mouse monoclonal anti-b-actin (Santa Cruz,
CA) antibodies in a 1:200–500 dilution buffer (5% skim
milk in TBST). Membranes were washed three times
with TBST and then incubated with goat anti-mouse
IgG-HRP (Zymed, San Francisco, CA) antibodies for
HN
HN
O
O
HOOC
H3COOC
Pyropheophorbide-a, 3
Methylpyropheophorbide-a, 2
Fig. 1. Preparation of Ppa from Spirulina platensis algae.
(a) Extraction by acetone three times, treatment of hydrochloric
acid in MeOH for 12 h. (b) Reflux in collidine at 110 °C for
2
(
.5 h, evaporation under high vacuum. (c) Treatment of H SO
2 4
50%) at room temperature for 2 h
1
h at room temperature. Immunoblots were developed
with enhanced chemiluminescence agents according to
the manufacturer’s instructions (SuperSignal West Pico
Chemiluminescent Substrate: PIERCE, Rockford) and
exposed to imaging film.
by refluxing in collidine as reported by Kenner et al.
23], Mpa was obtained from Spiriluna platensis blue
[
green algae according to reports published [8, 24] as
shown in Fig. 1. After preparing these compounds, all
Cellular uptake
1
were characterized by H NMR, mass spectroscopy,
4
CaSkiandTC-1cells(5×10 cells/well)wereincubated
and UV-vis spectrophotometers. As shown in Fig. 2,
UV absorption and fluorescence emission of Ppa in
DMSO were determined by nano-drop and fluorescence
spectrophotometer. Two main absorption peaks of Ppa
were observed at 416 and 669 nm wavelength while
fluorescence emission peak was observed at 685 nm.
Although in vitro studies of Mppa on several cell lines
were documented [25], in case of Ppa, very few reports
were noted.Also, as reported by Bellnier et al. [13], Mppa
can be hydrolyzed in in vitro and in vivo conditions in the
presenceofplasma,probablybyesteraseenzymesbecause
in wells of a 12 well plate overnight at 37 °C with 5%
CO . The medium was replaced with fresh medium
2
containing Ppa (0.0, 0.01, 0.1, and 1.0 mM). After 1, 3,
6, 12 and 24 h of incubation, the cells were washed twice
with 1 mL of PBS and 1 mL of fresh medium was added.
Fluorescence of the cells was measured by fluorescence
microscopy using an Axiovert 200 MAT apparatus (Zeiss,
Gottingen, Germany).
Statistical analysis
3
of its methoxy group at 17 position. Consequently, it is
Every experiment was conducted in at least triplicate,
and the acquired data expressed as the mean ± standard
deviation (SD). The statistical comparisons of the cytotoxic
effects were performed using Student’s t-test. The differ-
ences were considered significant at a P-value <0.05.
considered less stable than Ppa. As well, Ppa can widely
be used for its conjugation with any molecule due to its
3
free reactive carboxylic group at 17 position. In addition,
it is a well-known potential candidate for photosensitizer
due to the photophysical and photochemical properties.
For these reasons, in this study, we proposed to test
photosensitizing activity of Ppa with and without light on
two different cancer cell lines; TC-1 cells, which is a lung
epithelial cell immortalized with HPV-16 E6 and E7 and
transformed with the c-Ha-ras oncogene, CaSki which is
a cervical epidermoid carcinoma cell line.
RESULTS AND DISCUSSION
In this study, we prepared pyropheophorbide-a from
Mppa by treatment of sulfuric acid based on Burke
et al.’s protocol [22]. Mppa was prepared from Mpa
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PHOTOTOXICITY EFFECTS OF PYROPHEOPHORBIDE-A FROM CHLOROPHYLL-A ON CERVICAL CANCER CELLS
185
Fig. 2. UV-vis spectrum (a) and fluorescence spectrum (b) of Ppa in DMSO; the highest peak (Sorat band) is at 416 nm and second
highest peak (Q-band) is at 669 nm, while the highest fluorescence emission is at 685 nm
Fig. 3. Phototoxicity and dark toxicity results of Ppa at various concentration on TC-1 and CaSki cell lines. MTT assay was
performed under dark and cells were incubated at 37 °C with 5% CO for 24 h. Error bars represent standard deviation
2
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide, a yellow tetrazole) assay on two cell lines
was performed in the presence of light and in the absence
of light. In both cases, after seeding in 96 well plates the
0.06 and 0.03 mM, Ppa revealed negligible phototoxicity.
In addition, there was no dark toxicity of Ppa in both
cell lines as shown in Fig. 3. Thus, Ppa on TC-1 cell line
was over 10 fold more phototoxic as compared to CaSki
cell line. This photosensitizing activity may be attributed
to the localization difference of Ppa in each cell or the
over-expression of ABCG2 protein in CaSki cells that
can efflux Ppa derivatives. To evaluate whether ABCG2
protein expression can lead to photosensitizing resistance,
we performed western blot analyses as shown in Fig. 4.
cells were incubated at 37 °C under 5% CO for 24 h, cells
2
were treated with Ppa in DMSO and medium solution at
concentrations 0.03, 0.06, 0.125, 0.25, 0.5, 1 and 2 mM.
2
The light at 665 nm with 6.25 J/cm was given to the
cell-containing plate at 24 h after treatment of Ppa under
darkness. Further, the cells were incubated for 24 h and
20 mL of MTT reagent was added into each well. Following
additional 4 h incubation, absorption of the cells at 570 nm
was recorded by ELISA reader. Figure 3 shows the PDT
and dark toxicity effect on the two cell lines.
Phototoxicity of Ppa on the two cell lines was
observed to be different as shown in Fig. 3. In case of
TC-1 cell line, around 100% efficiency was observed
at the concentration range of 0.06–2 mM. At 0.03 mM
concentration, Ppa revealed around 75% efficiency. In
case of CaSki cell line, 100% efficiency was observed at
Fig. 4. In vitro expression of ABCG2 protein using western
blotting. After 24 h of incubation, the cells were collected and
proteins were extracted from TC-1, CaSki, and HeLa cells
0.25–2.0 mM while at 0.125 mM it exhibited only around
30% efficiency. It was noted that at concentration of
Copyright © 2014 World Scientific Publishing Company
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186
P.K. CHATURVEDI ET AL.
Fig. 5. Cellular uptake of Ppa in TC-1 and CaSki cells. The cells were incubated with Ppa in medium containing 10% FBS at 37 °C,
and their concentrations in the cells after incubation for 1–24 h were measured by fluorescence microscopy
In CaSki, HeLA, and TC-1 cells, the levels of ABCG2
protein expressions were similar as compared with b-actin.
These data suggest that ABCG2 protein expression did
not regulate the phototoxicity in the cervical cancer cells.
We examined the uptake of Ppa (0.0, 0.01, 0.1 and
1
.0 mM) into these cells by fluorescence microscopy as
shown in Fig. 5. Only 1.0 mM of Ppa began to penetrate
into the cells after incubation for 1 h. 0.1 mM of Ppa started
showing higher uptake into TC-1 cells than CaSki cells
after incubation for 12 h. And 0.1 mM of Ppa exhibited
much higher accumulation in TC-1 cells compared to
CaSki cells at 24 h. High accumulation of 1.0 mM of
Ppa in these cells was observed at all time points, while
0
.01 mM of Ppa did not show any accumulation for 24 h
(
Supplementary material). We observed that 0.1 mM of
Ppa facilitated the enhanced accumulation in TC-1 cells,
which is consistent with the phototoxicity of TC-1 cells at
0
.125 mM. This increased accumulation and penetration
may be attributed to the efficient internalization of Ppa
by the endocytic cell uptake mechanisms of TC-1 cells.
Tumors treated with Ppa (1.25 or 2.5 mg/kg) showed
rapid shrinkage after irradiation, while tumors treated
with the same amount of Ppa-only (no irradiation)
showed a slow growth (Fig. 6). In case of mice treated
with PBS and PBS-irradiation, the tumor growth
continuously increased up to 19 days. After 3 days, tumor
growth in mice treated with Ppa (2.5 mg/kg) significantly
decreased and was comparable to that treated with Ppa
Fig. 6. PDT effect of Ppa on tumor growth in C57BL/6 mice
bearing TC-1 cells. Groups of five mice were given intravenous
injection of Ppa (1.25 or 2.5 mg/kg) or PBS as a control. At 6 h
after administration, the tumors were irradiated by laser with a
6
1
70 ± 10 nm wavelengths and interstitial total impact energy of
2
50 J/cm
difference from each other, they exhibited greater effects
relatively on the tumor recurrence after their application.
These data were consistent with the results of in vitro
experiments.
(
1.25 mg/kg). Until 10 days we observed shrinkage in
tumor size by as much as half the initial tumor size. After
3 days, tumor recurrence was observed in mice treated
1
with Ppa (2.5 mg/kg) compared to 5 days in mice treated
with Ppa (1.25 mg/kg). Although in vivo the PDT effect
of Ppa (1.25 or 2.5 mg/kg) on the tumor shrinkage in
the mice bearing TC-1 cells did not show a significant
CONCLUSION
We tested the phototoxicity and dark toxicity of Ppa on
two cell lines; TC-1 and CaSki. It was evident that Ppa at
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PHOTOTOXICITY EFFECTS OF PYROPHEOPHORBIDE-A FROM CHLOROPHYLL-A ON CERVICAL CANCER CELLS
187
concentration of 0.06 and 0.125 mM exhibit much higher
70–90% higher) growth inhibition effect on TC-1 cells
compared to CaSki cells. However, Ppa at concentrations
0.25 mM displayed similar effects on both cell lines.
These results were consistent with the cellular uptake
assay. Taken together, Ppa at lower concentration is more
toxic to TC-1 cells than CaSki cells. Our future work
will be focused on determining whether it is cell line-
dependent phototoxicity or cell-specific localization of
Ppa [26].
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Cellular uptake of Ppa in TC-1 and CaSki cells was
observed by fluorescence microscope (Figures S1–
S6) after Ppa treatment (0.0, 0.01, 0.1 and 1.0 mM).
Fluorescence was recorded at 1, 3, 6, 12, 24 and 48 h.
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