Molecular cytogenetic evaluation of the efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma MCF-7 cells

Photodiagnosis and Photodynamic Therapy (2013) 10, 194—202

Available online at www.sciencedirect.com

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

Molecular cytogenetic evaluation of the efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma MCF-7 cells Mona A.M. Abo-Zeid PhD a,b,c,∗, Thomas Liehr b, Sherien M. El-Daly c, Amira M. Gamal-Eldeen c, Michael Glei d, Ali Shabaka e, Samarth Bhatt b, Ahmed Hamid b a

Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, National Research Center, Dokki 12622, Cairo, Egypt b Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Kollegiengasse 10, D-07743 Jena, Germany c Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki 12622, Cairo, Egypt d Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University, Dornburger Strasse 25, D-07743 Jena, Germany e Department of Spectroscopy, Physics Division, National Research Center, Dokki 12622, Cairo, Egypt Available online 5 December 2012

KEYWORDS Photodynamic therapy (PDT); Indocyanine green; Laser irradiation; MCF-7; Interphase-FISH; TP53; HER-2; TOP2A

Summary Background: Photodynamic therapy (PDT) is used for the treatment of many types of predominantly epithelial cancers. Photosensitizer is taken up by fast growing tumor cells more actively than by other body cells and is activated by light, generating reactive oxygen species that cause cell death by necrosis or apoptosis. This study aimed to evaluate the efficacy of PDT with indocyanine green (ICG) through the investigation of TP53, HER-2 and TOP2A genes signals as breast cancer gene markers by interphase fluorescence in situ hybridization (nuc-FISH). Methods: The photosynthetizer ICG (200 ␮M) was applied to breast cancer cell line MCF-7 cells (adenocarcinoma) in combination with laser irradiation (807 nm) exposure for 20 min and then incubated for 12, 24 and 48 h. Cell viability was evaluated using trypan blue. The signals for nuc-FISH was investigated and counted for probes specific for the genes TP53 (17p13), HER-2 (17q11.2-q12), and TOP2A (17q21-q22), and BAC-probes RP11-746M1 in 17p11.2 and RP11-403E9 in 17q11.2. Results: The cell viability of MCF-7 did not reduced significantly when the cells were treated with ICG (200 ␮M) or exposed to laser irradiation for 20 min followed by incubation for 24 h.

∗ Corresponding author at: Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki 12622, Cairo, Egypt. Fax: +20 233370931. E-mail address: [email protected] (M.A.M. Abo-Zeid).

1572-1000/$ — see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.pdpdt.2012.11.006

Efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma

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ICG/PDT treatment with laser irradiation exposure for 20 min reduced the cell viability after incubating cells for 12, 24 and 48 h highly significantly in a time dependent manner. For nuc-FISH analysis, TP53, HER-2, TOP2A, RP11-746M1 and RP11-403E9 signals did not reduce or increase in a significant manner when the cells were treated with ICG or exposed to laser irradiation for 20 min then incubated for 24 h. PDT enhanced amplification of TP53 signals from nuc ish 17p13(TP53×2) to nuc ish 17p13(TP53×3) or nuc ish 17p13(TP53×4). However, the signals of HER-2 gene, TOP2A gene and BAC probes were reduced highly significantly when MCF-7 cells were treated with PDT with all time intervals. Conclusion: ICG/PDT and laser induced cytotoxic effect in MCF-7 cells. Also, PDT enhanced TP53 gene amplification, and reduced HER-2, TOP2A, and BAC probes RP11-746M1 and RP11-403E9 signals. Therefore ICG/PDT can be used for breast cancer treatment. It has the potential to induce apoptotic effect and reduce HER-2 and TOP2A genes propagation. Further in vivo studies are needed to evaluate ICG/PDT as a promising therapeutic approach for breast cancer. © 2012 Published by Elsevier B.V.

Background Photodynamic therapy (PDT) is increasingly recognized as an alternative treatment for superficial cancer. It involves the administration of a photosensitizer (PS) followed by a local illumination of the tumor with light of the appropriate wavelength to activate the specific PS, which absorbs the light then transforms from its ground state into an excited singlet state [1]. PDT is an approved treatment for several types of cancer as well as for age-related macular degeneration and is under investigation for different cancer, ocular, autoimmune and cardiovascular indications [2]. Indocyanine green (ICG) is a clinically approved, water soluble dye [3] which has low toxicity, rapid excretion and strong absorption at 800 nm in plasma [4]. Plasma disappearance rate of ICG (PDRICG) for clinical and experimental assessment of liver function ranges from 18% to 25%/min [5]. ICG is approved for use in humans by the Food and Drug Administration and in Europe in NIR fluorescence cancer-related surgery for three indications: sentinel lymph node (SLN) mapping, intraoperative identification of solid tumors, and angiography during reconstructive surgery [6,7]. ICG is a blood pooling agent that has different delivery behavior between normal and cancer vasculature. In normal tissue, ICG acts as a blood flow indicator in tight capillaries of normal vessels. However in tumors, ICG may act as a diffusible (extravascular) flow in leaky capillary of vessels. Therefore, pharmacokinetics of ICG has the potential to provide new tools for tumor detection, diagnosis and staging. Pharmacokinetic-rate images of ICG for breast tumors using near-infrared optical methods have been also investigated for breast cancer treatment with ICG-PDT [8]. Photothermal effects on murine mammary tumors using ICG and an 808-nm diode laser effectively caused tumor cell destruction and enhanced ablation and eradicatation of tumor tissue [9]. Fluorescence in situ hybridization (FISH) is a powerful technique for localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. The detection of nucleotidic sequences on examined DNA molecule consists in hybridizing a DNA probe to its complementary sequence on chromosomal preparations. Probes are labeled either directly, by incorporation of fluorescent nucleotides, or indirectly, by incorporation

of reporter molecules that are subsequently detected by fluorescent antibodies. Probes and targets are finally visualized in situ by microscopy analysis. FISH technique protocols and wide variety of current applications of FISH technology are presented [10—12]. In this study molecular cytogenetics was used to evaluate the efficacy of PDT in breast adenocarcinoma MCF-7 cell line. These malignant cells have functional p53, lack caspase-3 activity, maintain a functionally intact estrogen receptor, and have constitutively high levels of Bcl-2, a protein endowed with cytostatic and antiapoptotic activities [13,14]. The p53 tumor suppressor functions as a transcription factor which becomes activated upon a number of diverse stress stimuli including DNA damage, hypoxia, oncogene overexpression or metabolic limitations. Upon activation, p53 triggers growth arrest or apoptosis, which serves to eliminate heavily damaged cells [15]. Human epidermal growth factor receptor-2 (HER2) is the second member of the human epidermal growth factor receptor (HER) family [16]. It plays a key role in the HER family, cooperating with other HER receptors via a complex signaling network to regulate cell growth, differentiation and survival. The HER2 protooncogene encodes the transmembrane HER2 protein. HER2 is normally expressed in a variety of cell and tissue types excluding those of hematopoietic origin, and, importantly, is frequently overexpressed in a number of human cancers. In approximately 35—40% of patients with breast carcinoma that demonstrates HER2 gene amplification, topoisomerase II␣ (TOP2A) is co-amplified [17]. DNA topoisomerases are essential nuclear enzymes that play a crucial role in the control of DNA topology. They are involved in the resolution of conformational constraints that occur during fundamental nuclear metabolic processes such as replication, transcription, recombination or chromosome segregation [18]. These enzymes act by promoting transient DNA breakage in order to allow strand-passage events and DNA relaxation to occur before rejoining the broken DNA ends [19]. Two types of DNA topoisomerases have been described. Type I enzymes, encoded in humans by the TOP1, TOP3A and TOP3B genes, cleave only one strand of the DNA helix whereas type II enzymes, encoded by the human TOP2A and TOP2B genes [20,21], cleave both DNA strands. In mammalian cells, expression of the TOP2A isoform is closely linked to proliferation state, with maximal

196 protein levels and enzymatic activity in late S and G2/M phases, whereas TOP2B is constitutively expressed at high levels even in quiescent cells [22]. The present study was aiming to evaluate the molecular cytogenetic measurable effect of ICG with laser irradiation as PDT to reduce cell viability and induce apoptotic effect on breast cancer cell line to elucidate its efficacy as a novel breast cancer treatment.

Materials and methods ICG (Sigma/Aldrich, USA) was dissolved in deionizer distilled water before application to cancer cells. The laser source was a diode array laser from Quanta System (Milan, Italy) emitting at 807 nm. The nominal output energy (continuous wave) was 0.4 W. All exposures were performed under the following conditions: wavelength: 807 nm, average power: 400 mW, beam diameter: 3.0 cm, and power density: 51 mW/cm2 . The time period of exposure was 20 min. The laser irradiation was coupled into a monocore fiber, and the use of a biconvex lens allowed the homogeneous exposure of the 12-well plate. In all of the cellular experiments, photothermal effect was investigated and no thermal effect was detected, data not shown.

Cell culture Human breast adenocarcinoma cell line (MCF-7, ATCC number HTB-22, USA) was used to carry out the in vitro study. Cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-7 was routinely cultured in RPMI 1640 Media with 2 mM L-glutamine, and was supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 ␮g/ml). Cells were maintained in humidified air containing 5% CO2 at 37 ◦ C. Cells were maintained in humidified air containing 5% CO2 at 37 ◦ C. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37 ◦ C.

Treatments Four different experiments were performed. In the first experiment, MCF-7 cells were treated with ICG (200 ␮M) then incubated for 24 h. In the second experiment, MCF-7 cells were exposed to laser irradiation for 20 min then incubated for 24 h. In the third experiment, cells were treated with ICG for 2 h, and then washed by medium, followed by laser irradiation exposure for 20 min as PDT, then incubated for 12, 24 and 48 h. In the fourth experiment, cells were not treated and incubated for 24 h. The experiments were repeated three times independently.

Cell viability with trypan blue Cell viability was carried out using the trypan blue exclusion method to detect the safety of ICG treatment and/or laser irradiation exposure on MCF-7 cells (5 × 104 cells/well). Trypan blue exclusion method is based on the fact that viable cells possess intact cell membranes that exclude trypan blue dye which is known to be negatively charged

M.A.M. Abo-Zeid et al. and does not react with cells unless there membranes are damaged (non-viable cells). After collecting cells, 20 ␮l of cell suspension was added to 20 ␮l of 0.4% trypan blue and then viable and dead cells were counted by a hemocytometer using a light microscope. The percentage of viability was determined by the following equation; and data were represented as the mean ± the standard deviation (SD).

 % of viability =

Number of viable cells Total cell number

 × 100

Interphase-FISH analysis (nuc-FISH) Different experiments of MCF-7 cells were trypsinized by (trypsin/EDTA) and centrifuged and then the pellet was re-suspended in 5 ml 0.075 M KCl and incubated at 37 ◦ C for 20 min. After addition of 2 ml Carnoy fixative [methanol:acetic acid (3:1)], the mixture centrifuged, resuspended, and incubated at −20 ◦ C for 5 min in 3 ml Carnoy fixative, then the preparation was finalized with a last centrifugation. The supernatant is discarded, and the cells are diluted in 200 ␮l of the remaining supernatant and placed on a slide [23]. For interphase FISH purposes, the air dried slides with the target DNA were denatured in 70% formamide/2× SSC at 70—75 ◦ C for 2—3 min transferred to chilled 70% ethanol and then dehydrated through ethanol series. The slides were air dried and used for commencing FISH. Two types of probes were used: Vysis LSI TP53 SpectrumOrange/CEP 17 SpectrumGreen Probe and Vysis LSI TOP2A SpectrumOrange/HER-2 SpectrumGreen/CEP 17 SpectrumAqua Probe (Abbott Molecular Inc., USA). We found an unknown defect in chromosome 17 centromere (17CEP) region. Therefore, we replaced it with Bacterial Artificial Chromosomes (BACs) RP11-746M1 (17p11.2) directly labeled nucleotides with diethylaminocoumarin (DEAC, blue) and RP11-403E9 (17q11.2) directly labeled nucleotides with fluorescein isothiocyanate (FITC, green). The fluorescently labeled DNA probes were hybridized to the samples after divided into two groups. The first group included Vysis 17p13.1 LSI TP53, RP11-746M1 and RP11403E9. The second group included Vysis 17q11.2-q12 LSI HER-2, Vysis 17q21-q22 LSI TOP2A and RP11-746M1. The denatured DNA probes were applied on denatured target DNA on slides, covered by a 22 mm × 22 mm cover slip and sealed with rubber cement. The probes and target DNA were hybridized by incubation of slides at 37 ◦ C for 16—24 h. The slides then were washed and stained with nuclear counterstain 4,6-diamidino-2-phenylindole (DAPI). Fluorescence microscopy (Axio Imager Z2, Carl Zeiss, Jena, Germany) with MetaSystems Isis software was used to analyze and record all observed signal patterns. At least 100 interphase cells were analyzed for each sample with filters specific for each of the probe fluorophores. ISCN-FISH nomenclature [24] was used to identify interphase cells (nuc ish).

Statistical analysis The statistical analysis of cell viability data was carried out by t-test, while data were analyzed by 2 test for nuc-FISH.

Efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma

Figure 1 The mean percentage of viable cells in MCF-7 cells treated with ICG, laser and PDT for different time intervals as evaluated by trypan blue (Mean ± SD).

Results Cell viability In the MCF-7 cells treated with ICG (200 ␮M) for 24 h, the percentage of viable cells reached 84.36% which was a nonsignificant difference in comparison to non-treated cells (91.28%). Also, the cell viability did not decrease in a significant manner and it reached to 87.49% when MCF-7 cells were exposed to laser irradiation for 20 min and then were incubated for 24 h before collecting samples (Fig. 1). In PDT experiment, the MCF-7 cells were treated with ICG for 2 h then exposed to laser irradiation for 20 min followed by incubation for different time intervals. The ICG/PDT treatment of MCF-7 cells reduced the cell viability in highly significant manner (p < 0.001) at all time intervals (12, 24 and 48 h). The maximum reduction of cell viability went down to 14.23% after 48 h from starting point of treatments (Fig. 1).

nuc-FISH analysis The fluorescently labeled DNA probes were divided into two groups before hybridized to the DNA of experimental study, SpectrumGreen and SpectrumOrange were repeated with Vysis probes. The first group included TP53 (SpectrumOrange), RP11-746M1 (DEAC, blue), and RP11-403E9 (FITC, green). The nuc-FISH analysis for nontreated MCF-7 cells demonstrated that the standard signals for TP53, RP11-746M1 and RP11-403E9 in this group were nuc ish 17p13(TP53×2), 17p11.2(RP11-746M1×2), and 17q11.2(RP11-403E9×3) (Fig. 2). It was demonstrated that MCF-7 cells were not affected by reducing or increasing number of signals of TP53 gene or RP11-403E9 when treated with ICG for 24 h. Also, ICG did not amplify

197

RP11-746M1 signals/nucleus after 24 h (Table 1). When MCF7 cells were exposed to laser irradiation for 20 min then were incubated for 24 h, laser irradiation enhanced TP53 signals amplification highly significantly (p < 0.01) in comparing to non-treated cells. The RP11-746M1 and RP11-403E9 were not affected by deletion or amplification of signals after 24 h (Table 1). MCF-7 cells were treated with ICG (200 ␮M) for 2 h and then exposed to laser irradiation for 20 min before incubation for 12, 24 and 48 h as PDT. This treatment did not affect in TP53 gene negatively. The TP53 gene signals were highly significantly amplified at all time intervals and that significance reached to p < 0.001 after 48 h from incubating cells. The number of signals per nucleus was reduced with RP11-746M1 and RP11-403E9. This signal reduction was highly significant at p < 0.001 with RP11-746M1 after 12 h but reduction became slightly significant at p < 0.05 after 48 h. RP11-746M1 and RP11-403E9 signals were highly significantly reduced at all time intervals (p < 0.001) (Table 1). The second group of fluorescently labeled DNA probes included HER-2 (SpectrumGreen), TOP2A (SpectrumOrange) and RP11-746M1 (DEAC, blue). The nuc-FISH investigation for non-treated MCF-7 cells demonstrated that the standard signals for HER2, TOP2A and RP11-746M1 in this group were nuc ish 17q11.2-q12(HER2×3), 17q21-q22(TOP2A×3), and 17p11.2(RP11-746M1×2) (Fig. 3). Neither ICG nor laser irradiation induced imbalance in HER2, TOP2A nor RP11-746M1 signals significantly when applied to MCF-7 cells separately for 24 h (Table 2). When PDT was applied to MCF-7 cells, it was observed that there is a great reduction in HER2 gene and TOP2A gene demonstrated by the deletion of signals number of the two genes per nucleus. Deletion of HER2 gene signals was highly significant (p < 0.001) at all of incubation times in cells after PDT treatment (Table 2). Deletion of TOP2A gene signals were highly significant (p < 0.001) after 12 h but the proportion of reduction was significant at p < 0.01 after 48 h. RP11-746M1 signals were reduced highly significantly at p < 0.01 after 12 h from treating MCF-7 cells with PDT but the ratio of reduction was non-significant after 24 and 48 h from PDT treatment (Table 2).

Discussion The efficacy of PDT in the treatment of cancer depends on PS type, drug concentration and intracellular localization, light dose (fluency) and dose rate (fluency rate), oxygen availability, and the treatment regimen given [1]. When MCF-7 cell line was treated with ICG alone then incubated for 24 h, ICG did not induce cytotoxic effect on the cells. The ICG concentration of 200 ␮M was observed to be a safe dose. Also, laser irradiation did not induce cytotoxic effect on MCF-7 cells after exposure for 20 min then incubation for 24 h. These results are in agreement with our previous studies [25] which declared that neither MCF-7 nor HepG2 cell lines indicated significant cytotoxic effects when treated with ICG, ICG-ormosil PEBBLE without laser exposure in comparing to non-treated cells at concentrations ranging from 25 to 200 ␮M. Also, the studies of Mamoon et al. [26] evaluated ICG treatment with different concentrations (0—500 ␮M) on Human skin melanoma cells (Sk-Mel-28) with

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Figure 2 A representing photos panel showing examples of variable numbers of signals per nucleus demonstrated with different DNA probes such as TP53 (orange)/RP11-746M1 (blue)/RP11-403E9 (green), nuclei with inverted DAPI in MCF-7 cells treated with ICG/PDT.

Figure 3 A representing photos panel showing examples of variable numbers of signals per nucleus demonstrated with different DNA probes such as HER2 (green)/TOP2A (orange)/RP11-746M1 (blue), nuclei with inverted DAPI in MCF-7 cell line treated with ICG/PDT.

Efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma

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Table 1 The number and percentage of deleted or amplified signals of different DNA probes TP53, RP11-746M1 and RP11-403E9 in MCF-7 cells after treatment with ICG, laser and PDT for different time intervals. MCF-7 cells treatments

Time (h)

Type of probea

No. of signals/nucleusb 1

Untreated cells

2

3

4

5

6

Nuclei with deleted signals

Nuclei with amplified signals

No.

No.

(%)

(%)

24

TP53

5

358

5

32

0

0

5

1.25

37

9.25

24 24

RP11-746M1 RP11-403E9

7 4

358 9

5 349

30 3

0 4

0 31

7 13

1.75 3.25

35 38

8.75 9.50

ICG

24 24 24

TP53 RP11-746M1 RP11-403E9

10 11 5

365 368 5

4 2 368

21 19 1

0 0 0

0 0 21

10 11 10

2.50 2.75 2.50

25 21 22

6.25 5.25 5.50

Laser

24 24 24

TP53 RP11-746M1 RP11-403E9

4 4 3

329 352 13

11 3 336

42 39 7

0 0 5

0 2 36

4 4 16

1.00 1.00 4.00

53 44 48

13.25 11.00 12.00

PDT

12 24 48 12 24 48 12 24 48

TP53

10 6 14 27 23 20 8 10 12

308 329 297 341 350 340 45 57 43

43 42 44 11 17 13 310 299 302

39 23 41 21 10 25 11 19 13

0 0 1 0 0 1 7 5 5

0 0 3 0 0 1 19 10 25

10 6 14* 17*** 23** 20* 53*** 67*** 55***

82*** 65** 89*** 32 27 40 37 34 3

20.50 16.25 22.25 8.00 6.75 10.00 9.25 8.50 10.75

a b * ** ***

RP11-746M1

RP11-403E9

2.50 1.50 3.50 6.75 5.75 5.00 13.25 16.75 13.75

Standard signals with each probe for MCF-7 cells (TP53: 2 signals; RP11-746M1: 2 signals; RP11-403E9: 3 signals). Total No. of nuclei (400/experiment). p < 0.05 in comparing to non-treated cells (using 2 test). p < 0.01 in comparing to non-treated cells (using 2 test). p < 0.001 in comparing to non-treated cells (using 2 test).

trypan blue. They demonstrated that the percentage of dead cells revealed the safety of ICG in the tested concentrations, where it led to 25.7% death in the cells at 500 ␮M, in comparison with untreated cells, and the theoretical calculated half maximum inhibitory concentration (IC50 ) was 949.4 ␮M. In this study, the ICG/PDT treatment of MCF-7 cells reduced the cell viability highly significantly at all time intervals (12, 24 and 48 h). The maximum reduction of cell viability went down to 14.23% after 48 h from starting point of treatments. This result is in agreement with our previous study which demonstrated that cell viability of MCF-7 and HepG2 cell lines (comparing to control cells) reduced highly significantly when the cells treated with ICG/PDT or ICG-ormosil PEBBLE/PDT [25]. Also, Bozkulak et al. [27] measured cell viability by MTT assay at 0, 3, 6, 9, 12, 24 and 48 h after Laser irradiation (809 nm). They demonstrated that ICG/PDT application exerted its photo-oxidative effect on MDA-MB231 breast cancer cells immediately. Radzi et al. [28] found that ICG at a concentration of 150 ␮M induced inhibition in cell proliferation from 0 to 24 h post-photodynamic hyperthermal therapy (PHT) treatment of B16F10 murine melanoma cells with a threefold decrease in cell viability compared to the control group. In the present work, MCF-7 cells had unknown defect in the centromeric position of chromosome 17; D17Z1, the alpha satellite probe gave no signal in MCF-7, while it worked perfectly in controls. Therefore, we replaced centromeric

probes by centromere-near positioned BAC probes for RP11746M1 and RP11-403E9. The centrosome amplification might be useful in monitoring chromosomal instability and in turn phenotypic diversity in breast cancer [29]. Therefore, it is useful in predicting outcomes and survival of MCF-7 cells before and after treatment with PDT. It was observed that both RP11-746M1 and RP11-403E9 signals did not reduce or increase in a significant manner when the cells were treated with ICG or exposed to laser irradiation. However, these centromere-near regions were affected significantly after treating MCF-7 cells with PDT. There was a moderate significant deletion in the RP11-746M1 signals especially after 12 h. When MCF-7 cells treated with PDT, RP11-403E9 signals were deleted highly significantly with all time intervals, indicating that PDT have the potential to induce chromosome instability and in turn reduce amplification of breast cancer cells. Hirano et al. [30] first demonstrated that ICG could induce singlet oxygen [1 O2 ] formation in response to light with a wavelength between 600 and 800 nm, which means that ICG has properties that could make it useful as a photosensitizer during photodynamic therapy. The mode of cell death induced by ICG or ICG-ormosil PEBBLE alone or followed by laser exposure on MCF-7 and HepG2 cell lines was demonstrated in our previous studies [25]. The ratio of apoptotic and necrotic cells were determined comparing to control cells that both MCF-7 and HepG2 cell lines responded

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Table 2 The number and percentage of deleted or amplified signals of different DNA probes HER2, TOP2A, and RP11-746M1 in MCF-7 cells after treatment with ICG, laser and PDT for different time intervals. MCF-7 cells treatments

Time (h)

Type of probea

No. of signals/nucleusb 1

2

3

4

5

Nuclei with deleted signals

Nuclei with amplified signals

6

No.

No.

(%)

(%)

Untreated cells

24 24 24

RP11-746M1 HER-2 TOP2A

12 4 4

360 16 17

2 348 346

26 1 3

0 5 4

0 26 26

12 20 21

3.00 5.00 5.25

28 32 33

7.00 8.00 8.25

ICG

24 24 24

RP11-746M1 HER-2 TOP2A

4 0 0

377 16 19

3 353 353

16 7 4

0 6 6

0 18 18

4 16 19

1.00 4.00 4.75

19 31 28

4.75 7.75 7.00

Laser

24 24 24

RP11-746M1 HER-2 TOP2A

10 0 0

354 16 12

6 344 346

30 4 5

0 7 7

0 29 30

10 16 12

2.50 4.00 3.00

36 40 42

9.00 10.00 10.50

PDT

12 24 48 12 24 48 12 24 48

RP11-746M1

28 23 20 2 1 2 1 2 2

352 360 343 46 51 48 46 49 41

6 3 10 323 325 304 326 324 309

14 14 27 10 8 12 9 10 12

0 0 0 1 1 8 4 2 10

0 0 0 18 14 26 14 13 26

28* 23 20 48** 52** 50** 47** 51* 43*

7.00 5.75 5.00 12.00 13.00 12.50 11.75 12.75 10.75

20 17 37 29 23 46 27 25 48

5.00 4.25 9.25 7.25 5.75 11.5 6.75 6.25 12.00

a b * **

HER-2

TOP2A

. Standard signals with each probe for MCF-7 cells (RP11-746M1: 2 signals; HER-2: 3 signals; TOP2A: 3 signals). . Total No. of nuclei (400/experiment). p < 0.01 in comparing to non-treated cells (using 2 test). p < 0.001 in comparing to non-treated cells (using 2 test).

to PDT with ICG predominantly by necrosis. While in case of PDT with ICG-ormosil PEBBLE, the apoptotic mode of cell death preferentially occurred in both of cell lines. Mamoon et al. [26] demonstrated that ICG/PDT can efficiently and rapidly induce apoptosis in Human skin melanoma cells (Sk-Mel-28). They demonstrated that the cell death pathway, 1 O2 generation evoked rapid multiple consequences that trigger apoptosis after laser exposure for only 15 min including the release of cytochrome c, the activation of total caspases, caspase-3, and caspase-9, the inhibition of NF- B P65, and the enhancement of DNA fragmentation, and histone acetylation. These 1 O2 generated by PDT can directly kill tumor cells by the induction of apoptosis and/or necrosis [1,25,26,28,30]. Radzi et al. [28] demonstrated that the morphological observation revealed apoptotic and some degree of necrotic features in the PHT-treated B16F10 murine melanoma cells. Therefore, the released singlet oxygen from ICG after exposure to laser irradiation could be activate TP53 gene and enhance the apoptotic pathway to start programmed cell death. These results were clear when the signals of TP53 gene remained constant (nuc ish TP53×2) or amplified to (nuc ish TP53×3) or (nuc ish TP53×4) after treating MCF-7 cells with PDT. In the latent form, p53 has low affinity to its specific DNA sequences but once activated, p53 regulates the expression of many target genes whose products are the important players in cell cycle regulation, apoptosis, senescence, metabolism and many others [31—33]. In addition, the transcription-independent function of p53 can further

enhance the apoptotic response [34,35]. The main function of p53 is to defend the organism against genome alterations resulting from widely understood DNA damage. Even though the precise p53 pathway is still not understood, on the whole it has now been well documented that p53 confers a crucial barrier for cancer progression. This is why p53 inactivation during tumorigenesis occurs with a high frequency and via multiple mechanisms. p53 was found to be inactivated in a very broad variety of human tumors via two distinct mechanisms: point mutations in the TP53 gene which render p53 inactive or de-regulated murine double minute 2 (MDM2) pathway [35]. The direct evidence supporting the notion that p53 is indeed involved in PDT response came recently from the work by Mitsunaga et al. [36]. The authors reported that induction of early apoptosis and cell death in colon cancer cells HCT 116 by ATX-S10Na(II)® (Photochemical Co.)mediated photodynamic therapy is Bax- and p53-dependent. The chlorine PS ATX-S10Na(II) localizes in lysosomes [37] and after their photodamage induces mitochondrial-dependent apoptosis. The 1 O2 released from PDT [1,25,26,28,30] may be responsible for the reduction of the signals of HER-2 gene highly significantly. However it does not affect on TP53 gene in the same manner. The data in which p53, but not HER-2/neu, sequences are preferentially repaired following DNA damage in breast cancer cell lines were consistent with previous data using a bladder cancer cell line [38] and may relate to preferential repair of domains normally required for cell cycle checkpoint control [38,39]. In

Efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma multiple studies, HER-2/neu amplification/overexpression has been shown to be an independent prognostic and predictive marker of response to therapy with Herceptin. However, the mechanism(s) by which HER-2/neu is selectively amplified/overexpressed in some cancers remains poorly understood. In addition, the chromosomal machinery through which the oncogene amplification is generated is largely unknown [40—42]. TOP2A gene is important to complete processes of cell division and any defect on this gene will lead to chromosomes accumulation and will stimulate cell death via apoptosis [43]. ICG/PDT induced DNA damage due to the induction of 1 O2 from ICG after exposure to laser irradiation [25,26], the generation of 1 O2 evoked rapid multiple consequences that trigger apoptosis after laser exposure. The 1 O2 interact with TOP II causing the accumulation of enzyme—DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis. These results are in agreement with Cowell et al. [43], who demonstrated that exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with TOP II. The most promising result is that ICG/PDT has the potential to delete HER2 and TOP2A genes as they are correlated with overexpression in MCF-7 cells. This is in agreement with other reports which demonstrated that anthracycline therapy can affect in amplification and deletion of TOP2A [44—46]. Thus, the simultaneous amplification of HER2 and TOP2A has been proposed as a molecular predictor of response to anthracycline-based regimens [47]. In conclusion, ICG/PDT was successful in initiating cell death in breast cancer cells and reducing cancer cell propagation significantly by enhancing the programmed cell death, which is correlated to TP53 gene. Also, ICG/PDT reduced the aggressiveness of MCF-7 cells line by reducing HER-2 gene marker significantly, since over-expression of this gene is correlated with higher aggressiveness in breast cancer. ICG/PDT negatively affected TOP2A gene, which is involved in processes such as chromosome condensation, chromatid separation, and the relief of torsional stress during DNA transcription and replication. Further in vivo and in vitro experimental studies are needed to evaluate ICG/PDT as a promising therapeutic approach for breast cancer treatments.

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Conflict of interest statement The authors declare no conflict of interest. [18]

Acknowledgements The experimental studies were financially supported by NRC, Egypt. The molecular cytogenetic studies were funded by DFG Program Bonn Award (LI 820/39-1).

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