Accepted Manuscript

The efficacy of Radachlorin-mediated photodynamic therapy in human hepa-

tocellular carcinoma cells

Hamidreza Mirzaei, Gholamreza Esmaili Javid, Mahnaz Hadizadeh, Maryam

Jahanshiri-Moghadam, Parastoo Hajian

PII: S1011-1344(14)00356-X
DOI: http://dx.doi.org/10.1016/j.jphotobiol.2014.11.007
Reference: JPB 9881

To appear in: Journal of Photochemistry and Photobiology B: Bi-

ology

Received Date: 20 June 2014

Revised Date: 22 November 2014
Accepted Date: 30 November 2014

Please cite this article as: H. Mirzaei, G. Esmaili Javid, M. Hadizadeh, M. Jahanshiri-Moghadam, P. Hajian, The
efficacy of Radachlorin-mediated photodynamic therapy in human hepatocellular carcinoma cells, Journal of
Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.11.007

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Title page

The efficacy of Radachlorin-mediated photodynamic therapy in human hepatocellular

carcinoma cells
Hamidreza Mirzaeia, Gholamreza Esmaili javidb, Mahnaz Hadizadehc *, Maryam Jahanshiri-
Moghadamb , Parastoo Hajiand
a
Laser application in medical sciences research center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
b
Medical Laser Research Center, Iranian Center for Medical lasers (ICML), Academic Center for
Education, Culture & Research (ACECR), Tehran, Iran
C
Biotechnology Center, Iranian Research Organization for Science and Technology (IROST),
Tehran, Iran
d
Radiotherapy and Oncology Department, Shohada Tajrish Hospital, Shahid Beheshti University
of Medical Sciences, Tehran, Iran

Corresponding author at: Biotechnology Center, Iranian Research Organization for Science and
Technology, Tehran, Iran. POBOX 33535111 Tel: +98-21-56276031; fax: +98-21-56276636
E-mail: Hadizadehmahnaz@gmail.com
Running head: Radachlorin-PDT of HepG2
Abstract

Background: Photodynamic therapy (PDT) is a relatively novel modality for the treatment of

cancer and some non-malignant lesions. PDT uses a photosensitive drug and light to destroy

malignant cells. The aim of this study was to determine in vitro efficacy of Radachlorin-based

PDT (Radachlorin-PDT) on human hepatocellular carcinoma (HCC).Methods: The study used

human liver cancer cells (HepG2) and normal liver cells (HFLF-PI4) to evaluate cell viability

using the standard 2-(4, 5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT)

assay. The mechanism of cell death following Radachlorin-PDT was determined by DNA

agarose gel electrophoresis and flow cytometry. Results: Radachlorin without light irradiation

had no toxic effect on HepG2 and HFLF-PI4 cells. Cell survival of HepG2 and HFLF-PI4 cells

were decreased following PDT in a concentration-dependent manner. However, HepG2 cells

were much more sensitive to Radachlorin-PDT than HFLF-PI4 cells. Radachlorin LD50 on

HepG2 cells was 30µg/ml and 20 µg/ml, 24 h after exposure to doses of 5 J/cm2 and 15, or 25

J/cm2 , respectively. Optimal Radachlorin and light dose to kill HepG2 cells with minimal

effects on normal HFLF-PI4 cells were 100µg/ml and 15 J/cm2, respectively. Our results also

showed that apoptosis is induced in HepG2 cells following Radachlorin-PDT. Conclusion: Our

in vitro data suggest that the use of PDT with Radachlorin can be effective in the treatment of

HCC.

Keywords: Photodynamic therapy- Radachlorin- Hepatocellular carcinoma- liver cancer

 1. Introduction

Worldwide, Hepatocellular carcinoma (HCC) is the most common form of liver cancer,

counting approximately for 5 to 10% of all cancer cases. The estimated annual incidence is more

than 500,000 cases with nearly 1 million deaths annually [1]. HCC is the fifth most common

malignancy in the world and the third leading cause of cancer related death, after lung and

stomach cancer [2]. The major risk factors for this cancer are chronic infection with hepatitis B

(HBV) and hepatitis C (HCV) viruses, alcoholism, diabetes and cirrhosis of the liver. Most cases

of HCC occur in East Asia and Africa, where hepatitis B infection is endemic [3]. Recently, the

prevalence of HCC is rising in developed countries such as the United States and Canada [4, 5].

Surgical resection, liver transplantation and local ablative therapies (radiofrequency ablation and

cryotherapy) are the most effective treatments for patients with an early-stage of HCC, with a 5-

year survival rate of 20-75% [6]. Unfortunately, most patients are diagnosed at late stages of

liver cancer with distant metastaseswhen surgical resection is not possible. There is also a high

possibility of local recurrence after surgical resection and liver transplantation. It is also reported

that other medical treatment modalities such as trans-arterial chemoembolization (TACE),

percutaneous ethanol injection, acetic acid injection and intra-arterial lipiodol chemotherapy are

not very effective [7, 8].

Photodynamic therapy (PDT) has been found to be a promising therapeutic modality for cancer

treatment and for some non malignancy diseases [9, 10]. This method uses a photosensitive

chemical compound called a photosensitizer and a specific wavelength of light to activate the

photosensitizer. The activated photosensitizer then interacts with molecular oxygen to produce

singlet oxygen and other reactive oxygen species which destroy the target cells and cause tumor
regression without affecting the surrounding normal tissues [11, 12]. The efficacy of PDT depends on

several factors, including the wavelength and dose of light, type of photosensitizer, tissue

concentration and intracellular localization of photosensitizer, incubation time of drug with cells,

oxygen availability and the type of treated cells [13]. Radachlorin is a second-generation

photosensitizer with favorable physicochemical properties [14]. Figure 1 presents the molecular

structure of Radachlorin's components. Radachlorin has an intensive absorption band at the

wavelength of 662 nm, with a better light penetration in the target tissue as compared to the 1st

generation PSs. Other significant advantages of Radachlorin are negligible dark toxicity and

rapid body excretion (only 48 h). These properties can help prevent hyper photosensitivity of the

skin [15]. The antitumor effects of PDT using Radachlorin as photosensitizer have been reported

in skin cancer [16, 17], cervical cancer [18] and early esophageal cancer [19] both in vitro and in

vivo. However, little is known about the potential of Radachlorin-mediated PDT for treatment of

hepatocellular carcinoma. In this study, we investigate the toxicity of Radachlorin-mediated PDT

in human hepatocellar carcinoma cells. The study also uses Radachlorin and a 660 nm diode

laser to investigate whether apoptotic cell death is induced by PDT.

2. Materials and methods

2.1. Cell lines and culture conditions

Human hepatocellular carcinoma cell line HepG2 (NCBI C158) and normal human fetal liver

fibroblast (HFLF-PI4) cell line (NCBI C168) were used in this study. Both cell lines were

obtained from the Pasteur institute of Iran. HepG2 cells have an epithelial-like cell morphology

with a doubling time of 48 h. HFLF-PI4 cells have a spindle-shaped morphology with a doubling

time of about 8 days (187 h). Cells were allowed to grow to confluence in 25 cm2 tissue culture

flasks (Jet Biofil) with 7 ml RPMI 1640 medium (Sigma, St. Louis, MO) and supplemented
with10% fetal bovine serum (PBS; Gibco BRL), 50 U.ml-1 penicillin and 50 mg.ml-
1
streptomycin (Gibco BRL). The cells were kept at 37 oC in a 5% CO2humidenvironment. For

the experiments, the cells were removed by trypsinizing (trypsin0.025%, EDTA 0.02%) and

washed with phosphate-buffered saline (PBS;Gibco/BRL) [20].

2.2. Radachlorin uptake

In order to evaluate the uptake of Radachlorin, the cellular Radachlorin content of HepG2 and

HFIF-IP4 cells was detected at different time points from 1 to 24 h. After incubation cells with

20 µg/ml of Radachlorin, cells were washed twice with PBS and lysed by addition of 200 µl

dimethyl sulfoxide (DMSO) at respective time points (1 h post incubation, up to 24 h).

Thereafter, the quantity of Radachlorin in the lysate was indicated as fluorescence signal

measured by a multiplate reader system (Synergy H4). The optimal incubation time for

Radachlorin mediated photodynamic therapy was selected according the time point

corresponding to the highest fluorescence signal in HepG2 cells.

2.3. Photosensitizer and light source

The Radachlorin (RADA-PHARMA Co, Ltd., Moscow, Russia) was dissolved in distilled water

to obtain 1 mM stock solution, which was stable in solutions at 0 ± 4 oC in the dark. The

working drug solutions (2.5-100 µg/ml) were prepared by diluting the stock solution with serum-

free RPMI 1640 medium. Light Source: A diode laser device (Lasotronic MED 131 Switzerland,

power output 35 mW) was the source of monochromatic red light (660 nm) used in this study .

2.4. Photodynamic treatment

HepG2 and HFLF-PI4 cells were seeded at a density of 5 x 104 cells/well in 96-well flat-

bottomed micro titer plates (Jet Biofil Cat. No. TCP011096). After 24 h, cells were incubated

with 0-100 µg/ml of Radachlorin for 4 h in the dark. After removing the drug and washing with

PBS, the treated cells were irradiated using different irradiation times to deliver light doses of 5

J/cm2, 15 J/cm2 or 25 J/cm2. All irradiations were performed at room temperature (25 o
C). Post-

irradiated cells were incubated for 24 h before cell viability was measured by the MTT assay.

Controls for each experiment were cells exposed to light (at each light dose) without

Radachlorin. Phototoxicity results were expressed as percentage of treated cells viability

compared to control. To determine the dark toxicity of Radachlorin, additional groups of cells

were incubated with different concentrations of Radachlorin (0-100 µg/ml) and cell viability was

measured without exposing cells to the laser treatment. All experiments were repeated three

times [14].

2.5. Assessment of cell viability

Cell survival was evaluated by MTT assay. In brief, culture medium was removed, and cells

were incubated in medium with 0.5 mg/ml 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium

bromide (MTT; Sigma, St. Louis, MO) for 3 h at 37° C to allow MTT metabolization. The

resulting formazan crystals were dissolved with 100 mol/L dimethyl sulphoxide (DMSO; Gibco

BRL) and the absorbance was measured at 540 nm using an ELISA reader (Hyperion, Inc., FL,

U.S.A.) [21].

2. 6. Analysis of DNA fragmentation by gel electrophoresis

Human HepG2 cells at a density of 2 x 106 were cultured in 50 mm dishes and incubated for 24
h. The 90% confluent dishes were washed with phosphate buffered saline before cells were

treated with 30 µg/ml Radachlorin. A dish of untreated cells was also prepared simultaneously. After

4 h, the Radachlorin treated dish was irradiated with diode laser (660 nm, 15 J/cm2). After

photodynamic treatment, the cells were incubated at 37° C in a 5% CO2 incubator for another 24

h. Both untreated and treated cells were then harvested by scraping and centrifugation. The cells

were lysed in 400 µl lysis buffer (200 mM Tris, pH 8.3, 100 mM EDTA, 1% SDS), 20 µl

proteinase K (10 mg/ml) at 37 °C and incubated for 10 min at 60 °C. Then, 200 µl isopropanol

was added at room temperature. After vigorous shaking, the sample was centrifuged at 8000 rpm

for 1 min. Supernatant was collected and the DNA was precipitated with 500µl washing buffer.

After centrifugation (8000 rpm for 1min), the precipitate obtained was mixed with cold 75%

ethanol and centrifuged again at 8000 rpm at 4 °C for 1 min. The final precipitate was allowed to

dry. One microgram of each DNA preparation was loaded into each lane of a 1.5% agarose gel.

Electrophoresis was carried out at 80 V for 3 h. DNA in gel was visualized under UV light after

staining with ethidium bromide [22].

2.7. Apoptosis measurement by flow cytometry

Annexin V- FITC/PI flow cytometry was performed as previously described [23]. HepG2 cells

(1 x 106) were treated with 30 µg/ml Radachlorin for 4 h and were then exposed to red light of 15

J/cm2. The treated and untreated cells were harvested and washed with PBS. The collected

pellets were resuspended in binding buffer. The cell suspension was incubated with Annexin V

and PI in the dark for 20 and 10 min. After incubation, cells were analyzed by flow cytometry

(Partec CyFlow Space).

3. Results

3.1. Radachlorin uptake

The cellular uptake of Radachlorin as a function of photosensitizer incubation time is shown in

Figure 2, Radachlorin started to enter HepG2 cells 1 h after incubation, and a fluorescence

decrease started after 4 h of incubation. For HFIF-IP4 cells, the rate of Radachlorin uptake was

lower than HepG2 cells and increased slowly during 24 h. As shown in Figure 2, the cellular

accumulation of Radachlorin was higher in cancer HepG2 cells than normal HFLF-PI4 cells, with

the ratio approximately 2.5:1.

3.2. Dark cytotoxicity of Radachlorin

The dark cytotoxicity of Radachlorin was initially evaluated by MTT reduction assay. For this

purpose, HepG2 and HFLF-PI4 cells were incubated for 4 h with different concentrations of

Radachlorin (0-100 µg/ml), without irradiation. As shown in Figure3, after 4 h incubation, no

significant dark cytotoxicity of Radachlorin was observed in the HFLF-PI4 cells at all tested

concentrations of Radachlorin. For HepG2 liver cancer cells, cell viability values were more than

90% in the presence of concentrations of Radachlorin of 10 µg/ml and less. Radachlorin

concentrations of 20 to 100 µg/ml, was found to lower cell viability in HepG2 cancer cells by

only 20 to 25 percent.

3.3. Effect of photodynamic therapy with Radachlorin on HepG2 cells

Figure 4 shows the percentage cell viability of HepG2 cells incubated with different Radachlorin

concentrations (0-100 µg/ml) and exposed to 660 nm red light delivered at three different light

doses (5 J/cm2, 15 J/cm2 and 25 J/cm2). Twenty-four hours after PDT with each light dose, the

viability of irradiated HepG2 cells gradually decreased with increasing Radachlorin

concentration. Radachlorin at photosensitizing concentrations of 2.5 µg/ml, 5 µg/ml, 10 µg/ml,

 g/ml and 100 µg/ml and a light dose of 5 J/cm2 lowered the cell
20 µg/ml, 30 µg/ml, 50 µg/ml

viability of HepG2 cancer cells to 87%, 85%, 75%, 54%, 53%, 42% and 40%, respectively.

While red light dose of 15 J/cm2 with each of the tested concentrations of Radachlorin showed a

higher cell-destruction effect than that induced by 5 J/cm2. The viability percent of irradiated

HepG2 cells with 15 J/cm2 of light decreased to 72%, 67%, 57%, 50%, 44%, 34% and 30% in

the presence of 2.5 µg/ml,

g/ml, 5 µ
µg/ml, 10 µg/ml, 20 µg/ml, 30 µg/ml, 50 µg/ml and 100 µg/ml

Radachlorin, respectively. As shown in Figure 4, the viability percent of irradiated HepG2 cells

with 25 J/cm2 of light decreased to 60% and 54% in the presence of 2.5 µg/ml and 5 µg/ml

Radachlorin, respectively. However, when HepG2 cells incubated with higher concentrations of

g/ml),we found no significant difference between viability of HepG2 cells

Radachlorin (>5 µg/ml),we

irradiated with 15 J/cm2 or 25 J/cm2. Radachlorin LD50 on HepG2 cells was 30 µg/ml and 20

µg/ml 24 h after exposure to doses of 5 J/cm2 and (15 or 25 J/cm2), respectively. By contrast,

laser irradiation alone (5 J/cm2, 15 J/cm2 or 25 J/cm2) in the absence of Radachlorin was found to

have no effect on the cell viability of HepG2 cancer cells.

3,4. Effect of photodynamic therapy with Radachlorin on HFLF-PI4 cells

Figure 5 shows the percentage cell viability of normal hepatic cells (HFLF-PI4) incubated with

different Radachlorin concentrations (0-100µg/ml) and exposed to 660 nm red light delivered at

three different light doses (5 J/cm2, 15 J/cm2 and 25J/cm2). Cell viability was 100% for HFLF-

PI4 cells irradiated with each light dose in the absence of Radachlorin. Twenty-four hours after

laser exposure of 5 J/cm2 and incubation with different concentrations of Radachlorin, the

viability percent of irradiated normal hepatic cells (HFLF-PI4) slightly decreased from 93% to

85% in proportion to the Radachlorin concentration increment from 2.5 µg/ml to 100 µg/ml.
After increasing the light dose of 5 to 15 J/cm2, no significant reduction in the survival of HFLF-

PI4 cells was observed. The viability percent of irradiated HFLF-PI4 cells with 25 J/cm2 of light

decreased to 78%, 73%, 71%, 67%, 65%, 62% and 60% in the presence of 2.5 µg/ml, 5 µg/ml,

10µg/ml, 20 µg/ml, 30 µg/ml, 50 µg/ml and 100µg/ml Radachlorin, respectively (Figure 5).

Indeed, the results indicate that normal hepatic cells (HFLF-PI4) are more resistant to

Radachlorin-mediated PDT than HepG2 cancer cells.

3.5. Induction of apoptosis by Radachlorin-mediated photodynamic therapy

In order to evaluate whether the cell death induced by Radachlorin-mediated photodynamic

therapy involved apoptotic pathways, DNA gel electrophoresis was performed to detect the

presence of intranucleosomal DNA fragmentation 24 h after treatment. Electrophoretic analysis

showed a DNA ladder pattern, a hallmark of apoptosis, after HepG2 cells had been treated with

30 µg/ml Radachlorin for4 h and exposed to laser light (660 nm, 15 J/cm2). In contrast to HepG2

cells investigated after PDT, no ladder pattern was observed in control cells and cells treated

with either Radachlorin or the light irradiation alone (Figure 6A).

To confirm that Radachlorin-PDT induces cell apoptosis, HepG2 cells were stained with

Annexin V-FITC and PI for apoptosis and necrosis analysis, respectively. As shown in Figure

6B, 46.22% of the treated cells were at early apoptosis phase (Annexin V-FITC positive and PI

negative) compared with 3.18% of the untreated control. A small percentage of cells were

distinguished at later stage of apoptosis or necrosis (Annexin V-FITC positive and PI positive) in

both treated and untreated cells. Our results showed that Radachlorin-PDT is capable of inducing

cell death via apoptosis in the HepG2 cells.

4. Discussion

PDT is a relatively new modality for the treatment of cancer, microbial infections, and some

non-malignant lesions. This therapeutic modality uses a selective uptake of a photosensitizer in

tumor tissues and irradiation of light of an appropriate wavelength, producing singlet oxygen and

other cytotoxic species that destroy malignant cells [9-12]. The antitumor effects of PDT using

Radachlorin as photosensitizer have been reported in some types of cancer both in vitro and in

vivo [16-19]. However, there is little known about the potential of Radachlorin-PDT for the

treatment of HCC, the most common form of liver cancer. The primary objective of this study is

to fill this gap and evaluate the efficacy of Radachlorin-mediated PDT on human HCC.

The first criterion to study the feasibility of Radachlorin mediated photodynamic therapy for the

treatment of liver cancer is the capacity of HepG2 cancer cells to absorb Radachlorin. First, we

determined the incubation time when Radachlorin accumulation in HepG2 cancer cells was

maximum. It was found that the cellular uptake of Radachlorin reaches its maximum value 4 h

after treatment as evidenced by the fluorescence intensity detected using a spectrophotometer.

Therefore, it can be suggested that HepG2 cells may need 4 h to achieve optimal uptake of

Radachlorin.

Low dark toxicity of a photosensitizer would minimize the undesirable side effects on the normal

tissues during PDT treatment of tumors. To this end, we studied the effect of Radachlorin

without light illumination on HepG2 and HFLF-PI4 cells. Our dark toxicity studies indicated that

Radachlorin at concentrations 2.5 µg/ml and 5 µg/ml

g/ml had no effect on cell viability of both

HFLF-PI4 cells and HepG2 cells. At higher concentrations of Radachlorin (10-100µg/ml), a

small reduction in cell viability was observed in HepG2 cancer cells but not in normal HFLF-PI4

cells, indicating a low level of dark toxicity for Radachlorin alone. Low dark toxicity of
Radachlorin has also been reported by other researchers. Bae et al [18] reported that Radachlorin

(2.5-20 µg/ml)
g/ml) without laser irradiation had no toxicity side effect on TC-1 cells.

Our phototoxicity studies showed that the viability of cells exposed to red laser light (660 nm)

depends on the photosensitizer concentration. For each light dose tested, the percentage of viable

cells (HepG2 and HFLF-PI4 cells) decreased with increasing Radachlorin concentration.

However HepG2 cells were more sensitive to the lethal effects of Radachlorin photodynamic

therapy than normal HFLF-PI4 cells. For HepG2 cells, the cell viability was reduced by up to

60% using a light dose of 5 J/cm2 with 100 µg/ml Radachlorin. By contrast, the viability of

HFLF-PI4 cells was only reduced by up to 15%. This could be due to faster cell division of
15%.This

HepG2 cells, resulting in a greater accumulation of Radachlorin in cancer cells than normal

HFLF-PI4 cells. HepG2 cell line had a doubling time of about 48 h, whereas that of HFLF-PI4

cell line was 8 days.

In addition, our results showed that Radachlorin LD50 on HepG2 cells was 30 µg/ml and 20

g/ml, 24 h after exposure to doses of 5 J/cm2 and (15 or 25 J/cm2), respectively. However,
µg/ml,

Douillard et al have reported a LD50 0.59 µg/ml for Radachlorin on HT29 and A549 cells treated

with photosensitizer for 3 h incubation followed by 500 mW, 20 J/cm2 [14, 24]. Also, Privalov et

al have reported a LD50 1 µg/ml

g/ml for Radachlorin on PC12 cells, 39 h after exposure to dose of

50 J/cm2 [25]. In fact, by an unknown reason our studies have demonstrated a 7-40 times lower

LD50 than found in the literature.

For the use of PDT as a safe and effective treatment, suitable concentrations of the

photosensitizer should be used with an appropriate light dose. The influence of different light

doses (5 J/cm2, 15 J/cm2 and 25 J/cm2) on the cell viability of liver cancer HepG2 cells and

normal HFLF-PI4 cells photosensitized with the different Radachlorin concentrations was
evaluated during the light dose study. Our results showed that concentration of 100 µg/ml

Radachlorin with the light dose of 15 J/cm2 or 25 J/cm2 were the most lethal for the HepG2 cells

in comparison with other concentrations and light doses tested. While the cell viability of healthy

normal HFLF-P I4 cells photosensitized with Radachlorin (100

(100µg/ml) and the light dose of 5

J/cm2 or 15 J/cm2 was more than 80%. The cell viability of HFLF-PI4 cells decreased with the

increase in light dose from 15 J/cm2 to 25 J/cm2. This indicates that the use of a laser light dose

of 15 J/cm2 at a wavelength of 660 nm would be a better combination with the optimum

Radachlorin concentration (100 µg/ml) than 5 J/cm2 and 25 J/cm2 for killing of cancer HepG2

cells.

The proliferation of cancer cells after PDT can be suppressed by different mechanisms such as

autophagy, necrosis and apoptosis [26, 27]. Chlorine e6, an important component of

Radachlorin, is shown to have the potential to induce apoptosis in cancer cells [28]. In this study,

we investigated whether PDT using Radachlorin can induce apoptosis of HepG2 cells. The

degradation of DNA down into oligonucleosomal fragments and DNA ladder formation is a late

event of apoptosis, which can be visualized by agarose gel electrophoresis [29, 30].

Therefore, we performed agarose gel electrophoresis to determine the presence of DNA ladder in

HepG2 cells treated to Radachlorin-PDT. DNA ladder formation was only observed in the

Radachlorin-PDT treated HepG2 cells. In order to confirm cell death process, flow cytometric

analysis of apoptotic cells following PDT was performed. About 46% HepG2 cells treated with

30 µg/ml Radachlorin and 15 J/cm2 light dose appeared at early apoptosis phase. These data were

consistent with the findings in cytotoxicity assay where the cell survival level was about 44%

when treated with the same dose.

The Induction of apoptosis in HepG2 cells after Radachlorin-PDT is an advantage for this
treatment because the escape of cancer cells from apoptosis is one of the major causes of their

uncontrolled proliferation [31].

In summary, this in vitro study demonstrates that Radachlorin-mediated photodynamic therapy is

effective in reducing liver cancer cells (HepG2 cells) by inducing apoptosis with low phototoxic

effects on normal liver cells (HFLF-PI4 cells). Although further investigations are needed both in

vitro as well as in vivo, our data suggest that the use of PDT with Radachlorin can be effective in

the treatment of HCC.

5. Acknowledgments

This work was supported by the laser application in medical sciences research center of Shahid

Beheshti University of Medical Sciences and Academic Center for Education, Culture and

Research (ACECR).

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Fig. 1. The molecular structure of Radachlorin's components: Chlorin e6 (A), Purpurin ) B) and
Chlorine p6 (C).

Fig. 2. Pharmacokinetics of Radachlorin on HepG2 cells (■) and HFLF-IP4 cells (O) assessed by
a spectrophotometer. The detected fluorescence intensity indicates the cellular uptake of
Radachlorin after incubation with 20 µg/ml Radachlorin for different times (1, 2, 3, 4, 5, 6, 12
and 24 h). All results are presented as mean ± SD of five independent experiments, each
measured in triplicate.

Fig. 3. The cell viability (%) of HepG2 and HFLF-PI4 cells after photosensitization with
different concentrations of Radachlorin in the absence of laser irradiation. Data points represent
the mean± standard deviation, n = 3.

Fig. 4.The graph comparing the effect of different light doses of 5 J/cm2,15 J/cm2, 25 J/cm2 on
the cell viability (%) of HepG2 cancer cells incubated with different Radachlorin concentrations
(0-100 µ g/ml).

Fig. 5. The graph comparing the effect of different light doses of 5 J/cm2, 15 J/cm2 and 52 J/cm2
on the cell viability (%) of HFLF-PI4 cells incubated with different Radachlorin concentrations
(0-100 µg/ml).

Fig. 6. Radachlorin-PDT induces apoptotic cell death. (A) Analysis of DNA fragmentation in
human HepG2 cells. Lane 1: control (untreated cells); lane 2: cells treated with Radachlorin (30
µg/ml) without light illumination; lane 3: cells treated with Radachlorin (30 µg/ml) with light
illumination (15 J/cm2); and lane 4: DNA markers. (B) Flow cytometry analysis of apoptosis in
HepG2 cells. a) Control (untreated cells), b) HepG2 cells treated with Radachlorin (30 µg/ml)
with light illumination (15 J/cm2). Apoptotic cells were indicated in the lower right quadrant and
necrotic cells in the upper right quadrant.
Highlights

• Radachlorin-photodynamic therapy effects were compared between HepG2 and HFLF-PI4 cells.
• HepG2 cells are more sensitive to Radachlorin-photodynamic therapy than HFLF-PI4 cells.
• Radachlorin-mediated photodynamic therapy induces apoptosis in the HepG2 cells.