Enhancing the Efficiency of 5-Aminolevulinic Acid-Mediated

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Photodiagnosis and Photodynamic Therapy 13 (2016) 297–302

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Photodiagnosis and Photodynamic Therapy

jou rnal homepage: www.elsevier.com/locate/pdpdt

Enhancing the efﬁciency of 5-aminolevulinic acid-mediated

photodynamic therapy using 5-ﬂuorouracil on human melanoma cells

a b,∗ c d

Hadis Tahmasebi , Karim Khoshgard , Ameneh Sazgarnia , Ali Mostafaie ,

Mohammad Taghi Eivazi

M.Sc. in Medical Physics, Student Research Committee, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran

Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran

Medical Physics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Department of Immunology, Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

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

Article history: Background: 5-Aminolevulinic acid-mediated photodynamic therapy (ALA–PDT) is an effective and non-

Received 8 May 2015

invasive modality for treatment of several types of non-melanoma skin cancers. This in-vitro study

Received in revised form 14 August 2015

attempted to know whether the killing effect of ALA–PDT on the human melanoma cells (Mel-Rm cell

Accepted 24 August 2015

line) could be increased by the presence of 5-ﬂuorouracil (5-FU).

Available online 28 August 2015

Methods: To evaluate the effect of ALA–PDT in combination with 5-FU on viability of human melanoma

Mel-Rm cells, the cells incubated with 5-ALA and 5-FU for 3 h in nontoxic concentrations, and sub-

Keywords:

sequently illuminated with a 630 nm light-emitting diode array. The cells viability and cytotoxicity

5-Aminolevulinic acid

5-Fluorouracil determined by mitochondrial activity and lactate dehydrogenase assays.

Results: Combination of ALA–PDT and 5-FU (FU–ALA–PDT) showed a considerable growth inhibition

Photodynamic therapy

Protoporphyrin IX (PpIX) according to the results of MTT assay compared to ALA–PDT. The results of LDH assay also showed

Mel-Rm cell line a cytotoxicity effect in ALA–PDT; however, the FU–ALA–PDT showed no signiﬁcantly enhancement in

cytotoxicity compared to ALA–PDT using LDH assay.

Conclusion: The Mel-Rm cells incubation with 5-FU before PDT enhances the efﬁciency of 5-

Aminolevulinic acid-mediated photodynamic therapy.

1. Introduction IX (PpIX), a potent Ps, during the heme synthesis cycle in cells. It

is known that PpIX can accumulate preferentially in tumor cells

Photodynamic therapy (PDT) is an effective treatment for sev- [4]. The most important limitations of PDT for providing effective

eral types of cancers especially for non-melanoma skin cancers treatment are the intracellular production and accumulation of the

[1]. PDT has several advantages such as noninvasive treatment, Ps, low depth of the prodrug penetration and visible light into the

repeatability without side effects and furthermore it causes little target. Previous studies have shown that topical application of 5-

scar [2]. PDT requires three factors, a photosensitizer (Ps), light with ALA and its methyl ester [5,6] and light sources at long wavelengths

speciﬁc wavelength and oxygen. After Ps administration and illu- [7] provide a sufﬁcient depth of penetration into the dermis. How-

mination, it will be excited and transfers the energy to molecular ever, inadequate production and accumulation of PpIX within the

oxygen, leading to generation of reactive oxygen species (ROS). ROS, tumoral cells have not been optimized yet.

1 *

mainly singlet oxygen O2 , is responsible for cellular oxidation and Malignant melanoma is an aggressive and fatal type of skin can-

subsequent necrosis [3]. 5-Aminolevulinic acid (5-ALA) is not a pho- cer and arises from the melanocytes, melanin-producing cells that

tosensitizer itself; however, it is converted to the protoporphyrin are located in the epidermis. Melanoma is largely resistant to radia-

tion therapy and chemotherapeutic drugs and too many alternative

therapies have been studied so far [8–13]. Photodynamic therapy

∗ has been used as a complementary treatment for melanoma; how-

Corresponding author at: Department of Medical Physics and Biomedical Engi-

neering, Kermanshah University of Medical Sciences, Sorkheh Lizheh, Kermanshah, ever, there are some promising results, melanoma also is resistant

Iran, P.O. Box: 6714869914. Fax: +98 83 34276477. to PDT [14,15]. One of the melanoma resistance mechanisms to

E-mail addresses: [email protected] (H. Tahmasebi),

PDT is the efﬂux of photosensitizers from the cells and reduction

[email protected] (K. Khoshgard), [email protected] (A. Sazgarnia),

[email protected] (A. Mostafaie), [email protected] (M.T. Eivazi).

http://dx.doi.org/10.1016/j.pdpdt.2015.08.011

298 H. Tahmasebi et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 297–302

in accumulation of intracellular Ps that reduces efﬁcacy of PDT on Similarly, to determine the 5-ALA cytotoxicity, the cells were

melanoma cells [16]. incubated with 5-ALA at different concentrations of 0, 0.5, 1, 2 and

Some studies have tested common pharmacologic agents to 4 mM for 3 h. Then, the culture medium on the cells were removed

enhance PpIX production and accumulation in cancer cells [16]. and the cells washed twice with PBS and refed with fresh pre-

For example, it has been reported that vitamin D increase PpIX lev- warmed culture medium. The MTT assays were also performed

els signiﬁcantly in LNCaP prostate carcinoma cells [17]. Another after 48 h of incubation with 5-ALA.

study has shown that methotrexate (MTX) enhances selectively The probable synergetic effect of cytotoxicity of 5-FU and 5-ALA

PpIX production in skin carcinoma cells compared with normal ker- in the cells were also investigated; the cells were incubated with

atinocytes [18]. 5-Fluorouracil (5-FU) is known as an anticancer 5-FU (5, 10 and 25 ␮g/ml) and 5-ALA (1 mM) simultaneously for a

agent; it causes cell cycle arrest and apoptosis via inhibition of period of 3 h. The MTT assays were performed 48 h after incubation

thymidylate synthetase or through ﬂuoronucleotide misincorpo- of these agents.

ration into RNA and DNA [19]. Maytin et al. investigated the effect

of 5-FU in combination with ALA-PDT in squamous cell carcinoma; 5. Photodynamic therapy in combination with 5-FU

they have reported that 5-FU enhances PpIX accumulation signif-

icantly in the cells [20]. In the present study, we investigated the The Mel-Rm cells (10 cells /well) were cultured in 96-well

combination of 5-FU and ALA–PDT in enhancement of PDT efﬁ- plates. After 24 h of plating, when the cells reached to about 60%

ciency on human melanoma (Mel-Rm) cells. conﬂuency, they were incubated with 1 mM of ALA and also 5,

10, 25 ␮g/ml of 5-FU for 3 h. Then, the cells’ culture medium was

replaced by fresh culture medium. Subsequently, the cells were

2. Material and methods

illuminated by the 630 nm LED array for 5 and 10 min.

2.1. Chemicals and cell line

6. Cells illumination

5-Fluorouracil (50 mg/ml) and 5-aminolevulinic acid

A light-emitting diode array at the power density of

hydrochloride were purchased from Sigma–Aldrich (USA) and

± 2

44.8 2.7 mW/cm was used in this study as a light source. The

Santa Cruz Biotechnology Inc. (USA), respectively. The mate-

LED array had 8 lamps dedicated for 8 speciﬁc wells of 96-well

rials used for the cell cultures, including Dulbecco’s modiﬁed

plate. The peak wavelength of output light was 630 nm, and its full

Eagle’s medium (DMEM), fetal calf serum (FCS), trypan blue,

width at half maximum (FWHW) was 20 nm. Illuminations were

penicillin/streptomycin, and trypsin-EDTA 0.25% all purchased

performed for 5 and 10 min for delivering the light doses of 13 and

from Gibco Company (Invitrogen, Germany). The MTT (3-[4,5- 2

26 J/cm .

dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and

DMSO (dimethyl sulfoxide) used in the MTT assays were purchased

7. MTT assay

from Sigma–Aldrich (USA) and Merck (Germany), respectively.

The kit of lactate dehydrogenase (LDH) assay was purchased from

In all experiments, the cells viability was determined using MTT

German company of Roche (Germany). The Mel-Rm cell line used

method after 48 h. In this method, the yellow tetrazolium MTT

in this study was separated from patient who had suffered from

is reduced by metabolically active cells. The resulting purple for-

melanoma in the melanoma division of Newcastle and Sydney in

Australia. mazan can be solubilized and quantiﬁed by the spectrophotometric

technique. After unloading the wells from the cell-culture medium,

200 ␮l of fresh culture medium and 20 ␮l of MTT were added to each

3. Cell culture well. The plates were kept in an incubator for 4 h. Then, the wells

medium were refreshed by 150 ␮l dimethyl sulphoxide (DMSO).

The Mel-Rm cells were grown as a monolayer in DMEM culture Finally, optical densities (ODs) of the samples were measured at

medium supplemented with 5% FCS in 25 cm ﬂasks (Orange Sci- 570 nm against 630 nm using an ELISA reader (Stat Fax-2100, USA).

entiﬁc, Belgium). The cell growth was carried out in a humidiﬁed

◦

atmosphere containing 5% CO2 at 37 C and the culture medium

8. LDH assay

was changed periodically when the cells reached to about 70–80%

conﬂuency, they were washed with PBS and incubated with the

LDH as a cytoplasmic enzyme normally is not discharged out-

trypsin-EDTA to detach them from the ﬂask. The cells were then

side the cells, but upon damage to the cell membrane’s integrity

re-suspended in the culture medium and manually counted using

leaks out. LDH test is a colorimetric assay for quantiﬁcation of cell

trypan blue staining using a light microscope.

death. This method measures the LDH activity released from the

cytosol of damaged cells into the supernatant. All the test sam-

ples in this assay were similar to that in the MTT assays; also, the

4. Cytotoxicity of 5-FU and 5-ALA

LDH assays were performed after 24 h of illumination. Fifty ␮l of

4 culture medium from each well was transferred to a new 96-well

A number of 10 cells per well were seeded in 96-well plates

plate and 50 ␮l of substrate mix solution was added to these wells.

and were cultured for 24 h to attach on the wells’ ﬂoor in different

LDH that has been released from the cells catalyzes the reaction in

experimental groups. Then, the cells at about 60% conﬂuency were

substrate mix solution and converts the INT-tetrazolium salt into

incubated with several different concentrations (0, 5, 10, 15, 20, 25,

a red formazan salt, which can be quantiﬁed. The wells were incu-

30, 35 ␮g/ml) of 5-FU for 48 h. The cells were subsequently washed

bated with the substrate mix solution for 30 min. Optical density of

using PBS and refed with fresh pre-warmed culture media. The

the samples (Test) were measured at 490 nm by the ELISA reader.

MTT assay was performed after 24 h to evaluate the cells viability.

The percentage of cytotoxicity was calculated using the following

Thereafter, experiments were repeated using nontoxic concentra-

equation:

tions of 5-FU (5, 10 and 25 ␮g/ml); when the cells reached to about

60% conﬂuency after plating, they were incubated with 5, 10 and testvalue − lowcontrol

Cytotoxicity(%) = × 100

25 ␮g/ml of 5-FU for 3 h. Then they washed and the MTT assays

highcontrol − lowcontrol

were performed after 48 h of incubation with 5-FU.

H. Tahmasebi et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 297–302 299

Fig. 1. Viability percentage of Mel-Rm cells incubated at different concentrations of Fig. 2. Viability percentage of Mel-Rm cells incubated at different concentrations of

5-FU for 48 h using MTT assay. Statistically signiﬁcant differences observed between 5-ALA for 3 hours using MTT assay. Statistically signiﬁcant differences were found

treated groups (with different concentration of 5-FU) and control group (p < 0.005). between some treatment groups (which are signed with star) and control group.

(*p < 0.05; **p < 0.01; ***p < 0.005).

Where the high control corresponds to OD of maximum released

LDH; it is obtained by incubation of Tritons X-100 (10%) with the

cells. The Low control value is OD of spontaneously released LDH

from the cells, which had any treatment.

9. Data analysis

All experiments were performed in triplicate. The results

are expressed as means ± standard deviations and are plotted

using OriginPro (v8.0724) software. The values of cell viability

and cytotoxicity among different groups were compared using

Kruskal–Wallis and Mann–Whitney tests with a 95% conﬁdence

interval by SPSS16 software.

To evaluate the additive or synergistic cytotoxicity effect of

combining the 5-FU and ALA–PDT fractional product values (Bliss

synergy), which described by Duska et al. [21], were calculated.

The fractional product value is deﬁned as c.f.[combination]/{c.f.[5-

FU] + c.f. [ALA–PDT] − (c.f. [5-FU] × c.f. [ALA–PDT])} in which c.f.

Fig. 3. Viability percentage of Mel-Rm cells incubated at different concentrations of

means cytotoxicity fractions. C.f. values greater than 1 indicate Bliss

5-FU and 5-ALA (1 mM) for 3 and then the MTT assay was done after 48 h. Statisti-

synergism, values approximately equal to 1 indicate additivity, and cally signiﬁcant differences observed between treated groups with 5-ALA and their

values less than 1 indicate Bliss antagonism [21]. control groups (p < 0.05).

In addition, to quantify the 5-FU–ALA–PDT efﬁcacy which can

be deﬁned as a reduction in needed light dose to have an effective

11. PDT efﬁciency

PDT, the median effective dose (ED50) values (the required light

dosage to cause 50% inhibition of cell growth) were estimated.

The results of the MTT assays showed that illumination of the

groups receiving 5-FU causes no substantial cytotoxicity effect on

10. Results Mel-Rm cells (Fig. 4). The mean values of cells viability were 85.6%

and 77.3% after 5 and 10 min illumination time, respectively.

10.1. Cytotoxicity of 5-FU and 5-ALA ALA-PDT caused the cytotoxicity levels of 35.4 ± 2.9 and

67.5 ± 2.5 percentage after 5 and 10 min illuminating, respectively;

The MTT results of cytotoxicity assessment of 5-FU after 48 h however, combination of ALA–PDT and 5-FU (5, 10 and 25 ␮g/ml)

incubation time is shown in Fig. 1. The results of cytotoxicity assess- (FU–ALA–PDT) caused the signiﬁcant growth inhibition (Fig. 5).

ment of ALA after 3 h incubation time is shown in Fig. 2. In addition, Increasing the 5-FU concentration signiﬁcantly increased the cell

cytotoxicity effect of 5-FU and 5-ALA in the cells after 3 h incubation growth inhibition (p = 0.004–0.016 < 0.05).

time is shown in Fig. 3. The cells viability decrease with increasing The mean of fractional product obtained 1.62 and 1.16 at light

concentration of 5-FU. The results showed a signiﬁcant difference doses of 13 and 26 J/cm , respectively.

(p < 0.004) between the control group and the groups in which the The ED50 values were estimated for the treatment groups of

cells incubated with 5-FU for 48 h. ALA–PDT and FU–ALA–PDT by the data interpolation from the cells

␮

Concentrations of 5, 10 and 25 g/ml of 5-FU and 1 mM of 5-ALA viability graphs. The ED50 for the group receiving ALA–PDT was

were chosen for the main experiments, because they have shown about 19.5 J/cm , however the ED50 values for the groups receiving

less than 50% inhibitory effect in viability and proliferation after 3 h FU–ALA–PDT with 5-FU at concentrations of 5, 10 and 25 ␮g/ml,

incubation time in the cells. were 8.0, 5.0, and 4.3 J/cm , respectively.

300 H. Tahmasebi et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 297–302

Fig. 4. Viability percentage of Mel-Rm cells obtained by MTT assay after incuba-

tion with the different concentrations of 5-FU for 3 h and then illumination with

light doses of 13 and 26 J/cm . A signiﬁcant difference (p < 0.05) was noted only in

treatment group, in which the cells incubated with 25 ␮g/ml and illuminated with

26 J/cm of light dose, compared to its control group.

Fig. 6. Results of PDT obtained using LDH assay. ALA–PDT causes a signiﬁcant

increase in released LDH compared to that of the control group, but combination of

5-FU and ALA–PDT (FU–ALA–PDT) causes no increase in LDH compared to ALA–PDT.

However, the results showed a decrease in the released LDH with increasing the con-

2 2

centration of 5-FU; light doses were 13 J/cm (a) and 26 J/cm (b) in PDT. (*p < 0.05;

**p < 0.01; ***p < 0.005).

The results of LDH assay showed that ALA–PDT causes the

cytotoxicity effect upto 47.6 ± 3.7 and 55.8 ± 5.5 percent after

5 and 10 min illuminating, respectively. The groups receiving

FU–ALA–PDT showed no signiﬁcant enhancement in cytotoxicity

compared to ALA–PDT. The results of FU–ALA–PDT even showed

a reduction in cytotoxicity level, especially with 25 ␮g/ml of 5-FU

(p < 0.004) (Fig. 6).

12. Discussion

Most studies in the ﬁeld of PDT were focused on effects of PDT

induced cell death. PDT causes phototoxic reactions via production

1 *

of reactive oxygen species (ROS), mainly singlet oxygen O2 , which

can only be distributed to a range of 20 nm in cells [22]. Therefore,

intracellular localization of the PS is important to damage subcel-

lular organelles and to have therapeutic success in PDT. One of the

most common reasons for resistance to PDT is efﬂux of photosen-

sitizers by ATP-binding cassette (ABC) transporters from cell that

are located at the cell membrane and many intracellular organelles

[23]. There is a group of ABC transporters in melanoma cells, which

reduce intracellular accumulation of PS and limit efﬁcacy of PDT on

these cancer cells [16]. Some approaches including pharmaceuti-

Fig. 5. Results of PDT obtained by MTT assay. 5-Fluorouracil (5-FU) increased the cell

2 2 cal agents have been examined to overcome the ABC transporter

killing effect of ALA-PDT of Mel-Rm cells; light doses were 13J/cm (a) and 26J/cm

(b) in PDT. (*p < 0.05; **p < 0.01; ***p < 0.005). function and the resistance to PDT, which can inhibit drug-efﬂux of

photosensitizers [16]. For example, preconditioning by low-dose

H. Tahmasebi et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 297–302 301

methotrexate is enhanced the efﬁcacy and cancer selectivity of 13. Conclusion

ALA-PDT in skin carcinoma cells [24]; that such enhancement can

be related to the MTX function in impairment of ABC transporter This in vitro study showed using 5-FU in combination with 5-

[16]. Another study investigated the special inhibition of ABCG2 Aminolevulinic acid-mediated photodynamic therapy can increase

transporter by Ko-134, a non-toxic fumitremorgin C analog; the the efﬁciency of photodynamic therapy of the human melanoma

result indicated that pretreatment with Ko-134 increased the sen- cells.

sitivity of HaCaT keratinocytes to a red light dose of 1.5 J/cm [25].

In addition, a comparison between MTX and 5-FU, suggests 5-

Acknowledgments

FU for combination with ALA-PDT for enhancing the PpIX level

in squamous carcinoma cells [19]. MTX and 5-FU induce apo-

The authors gratefully acknowledge the Research Council of Ker-

ptosis in a similar pathway; however, potential toxicity of MTX

manshah University of Medical Sciences for the ﬁnancial support

is more than 5-FU [19]. Therefore, for overcoming to resistance

(Grant Number: 93009). This work was performed in partial ful-

of melanoma cells and consequently increasing the efﬁciency of

ﬁllment of the requirements for the Master of Science of Hadis

ALA–PDT, we used 5-FU at nontoxic concentrations and low incu-

Tahmasebi, in Faculty of Medicine, Kermanshah University of Med-

bation time with ALA–PDT simultaneously. We followed treatment

ical Sciences, Kermanshah, Iran.

effects using MTT assay as well as LDH assay. The results of pre-

treatment with 5-FU and illuminating after 3 h showed that, 5-FU

does not cause the signiﬁcant photosensitizing effect in the absence

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