sign in sign up
<<
Home , Photodynamic therapy

ANTICANCER RESEARCH 35: 3943-3952 (2015)

Phthalocyanine-mediated Photodynamic Treatment of Tumoural and Non-tumoural cell lines

BARBORA MANISOVA1,2, SVATOPLUK BINDER1,2, LUKAS MALINA1,2, JANA JIRAVOVA1,2, KATERINA LANGOVA1 and HANA KOLAROVA1,2

1Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic; 2Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic

Abstract. This study deals with the use of cationic far-red PDT, and all can be used in support of a patient who has absorbing (λmax ~740 nm) from the group received PDT (2). of the , in photodynamic . The The principle of PDT is based on the photochemical photosensitizers differed in their central atom, bearing either reactions which are initiated by activation of a hydrogen, zinc or magnesium. These photosensitizers were photosensitising drug, causing tumour . Light is tested in vitro on the tumour cell line HeLa (cervical ) mainly applied by superficial illumination of the tumour and and non-tumour cell line NIH3T3 (mouse fibroblast). The the surrounding tissue, which causes drug activation following tests were performed: measurement of reactive followed by tumour cell death (3). The excited species production, viability testing, Comet assay and may undergo two kinds of reactions. Firstly, cell type detection (apoptotic, necrotic and living cells). The in a type 1 reaction, it can react directly with a substrate, best results were achieved with zinc derivative at relatively such as a cell membrane or a molecule, and transfer a proton low half-maximum inhibitory concentration (0.04 μM) and a or an to form a anion or cation, respectively. total radiation dose of 15 J cm-2. These radicals may further react with oxygen to produce (ROS). Alternatively, in a type 2 Photodynamic therapy (PDT) is a promising method for reaction, the excited photosensitizer can transfer its energy cancer treatment (1). It is a simple non-invasive therapy directly to molecular oxygen to form excited-state singlet that shows great promise in treating malignant disease. It oxygen (4). Both reaction types can occur simultaneously, can be used before or after , ionizing and the ratio between these processes is highly influenced by radiation, or surgery, without compromising, or being the sensitizer, the substrate and the oxygen concentration, as compromised by any of these treatments. It has particular well as the binding of sensitizer to substrate (5). appeal in oncology because the use of chemotherapy, The molecular nature of the photo-oxidized target has ionizing radiation, or surgery does not preclude the use of profound influence on the signaling pathways and mode of cell death initiated following PDT. Generally, photoactive compounds localizing to the mitochondria or the endoplasmic reticulum promote apoptosis, within a certain threshold of Abbreviations: ROS, Reactive oxygen species; PDT, photodynamic oxidative stress, while PDT with photosensitizers targeting therapy; PS, photosensitizer; Pc, ; DNA, deoxyribonucleic acid; MTT, methylthiazol tetrazolium bromide; PBS, either the plasma membrane or lysosomes can either delay or block the apoptotic program predisposing the cells to necrosis phosphate-buffered saline; CM-H2DCFDA, 5-(and-6)-chloromethyl- 2’,7’-dichlorodihydrofluorescein diacetate; half-maximum inhibitory (6, 7). concentration, IC50. The phototoxic effect of PDT can be assessed by a comet assay. The comet assay is a simple, sensitive and Correspondence to: Manišová Barbora, Department of Medical quantitative technique for the detection of DNA damage Biophysics, Faculty of Medicine and Dentistry, Palacky University caused by double- and singlestrand breaks, alkali labile in Olomouc, Hněvotínská 3, 775 15Olomouc, Czech Republic. Tel: +420 585632200, e-mail: [email protected]. sites, oxidative base damage and cross linking with DNA or proteins. DNA damage is reflected by the extend to which Key Words: Photosensitizer, Phthalocyanine, photodynamic treatment, unwound DNA fragments form a comet-like image, which oxidative stress, cellular damage, phototoxicity, tumour cells. comprises a 'head' and a 'tail' (8). The head consists of intact

0250-7005/2015 $2.00+.40 3943 ANTICANCER RESEARCH 35: 3943-3952 (2015)

Materials and Methods

Photosensitizers. Three different photosensitizers were used. These photosensitizers were synthetised by A. Cidlina (16). The photosensitizers used are chemically classified as magnesium(II) (MgPCs), zinc(II) (ZnPCs) and metal-free (HPCs) phthalocyanines (Figure 1). They do not aggregate in water due to the presence of eight substituents in non-peripheral positions. Their photophysical properties are highly advantageous for use in PDT. They absorb in the far-red region and produce at high to reasonable rate (ΦΔ=0.08, 0.25 and 0.91 in dimethylformamide for for metal- free, Mg and Zn derivatives) (16). Prior to their use, the photosensitizers were diluted in 1X phosphate-buffered saline (PBS). For methylthiazol tetrazolium bromide (MTT) test two different concentration scales were included. The first was identical for all cell lines (0.1, 1, 10 and 100 μM). The other scale was specific to the needs of each photosensitizer [0.01, 0.0215, 0.0464, and 0.1 μM for ZnPcs (HeLa); 0.1, 0.215, 0.464 and 1 μM for ZnPcs (NIH3T3), and MgPcs and HPcs] and the results from the Figure 1. The chemical structure of compounds used in the study. MgPcs: latter concentration scale were used in support of the half-maximum [1,4,8,11,15,18,22,25-Octakis[(2-(triethylammonio)-ethyl)sulfanyl]- inhibitory concentration (IC ) assessment. phthalocyaninato]magnesium(II) octaiodide; ZnPcs: [1,4,8,11,15,18,22,25- 50 octakis[(2-(triethylammonio)-ethyl)sulfanyl]-phthalocyaninato]zinc(II) octaiodide; 2HPcs: [1,4,8,11,15,18,22,25-octakis[(2-(triethylammonio)- Cell lines and culture conditions. Two cell lines were tested, HeLa ethyl)sulfanyl]- phthalocyanine octaiodide. (cervical cancer) and NIH3T3 (mouse embryonic fibroblast). Both cell lines were purchased from European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). These cell lines were grown in 96-well microplates 105 cells per well in Dulbecoo’s DNA, while the tail is created from broken fragments of modified Eagle’s medium (DMEM) containing photosensitizes for negatively-charged DNA or a relaxed chromatin. The a period of 24 hours in darkness at 37˚C and 5% CO2. Three wells were used as the negative controls (photodynamically treated cells amount of DNA damage is directly proportional to the without photosensitizer). amount of DNA liberated from the head (9). Phthalocyanines are a group of second-generation Light source and exposure. A homemade LED-based light source, photosensitizers which are structurally related to specifically designed for the irradiation of experimental microplates, but exhibit maximum absorption at longer was used. The samples were illuminated at a wavelength of 740 nm. wavelengths (10). Phthalocyanines are tetra pyrrolic The light source is protected by a National Patent CZ 302829 B6. macrocycles that have a number of unusual physical and In total 96-wells with a sample were exposed to a total irradiation -2 chemical properties arising from their characteristic 18 π- dose 15 J cm (total irradiation time was 10 min and intensity of radiation was 25 mW cm-2) at room temperature. electron de-localization, such as high chemical and thermal stability, which makes them valuable in different fields of ROS measurement. The cells were incubated with the photo - science and technology (11). sensitizers in darkness at 37˚C and 5% CO2. After a period of 24 h There exist numerous commercial uses associated with the DMEM was replaced by a solution of 10 μM probe phthalocyanines in textile, photography and electrical 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate industries, however, phthalocyanines have attracted attention in (CM-H2DCFDA, Life Technologies, Prague, Czech Republic) the field of PDT because of their strong absorption in the far- (Ex/Em: ~495/530 nm) in PBS. The cells were incubated for a further 30 min and irradiated at total irradiation dose 15 J cm-2 (total red region of the visible spectrum, good chemical stability, low irradiation time was 10 min and intensity of radiation was 25 mW photobleaching and sufficient quantum yields of fluorescence cm-2). After the irradiation the ROS production level was measured (ΦF) and singlet oxygen (ΦΔ). Production of ROS after by the microplate reader SYNERGYTM HT (BioTek Instruments, excitation of photosensitizer is essential for phototoxic effect United States of America). in PDT (12, 13). A disadvantage of these photosensitizers is their high tendency to form dimers and higher aggregates, MTT viability test. The phototoxic effect of the photosensitizers although this can be controlled by introduction of charged used on the HeLa and NIH3T3 cell lines was measured using a MTT assay. The DMEM was replaced by a solution containing substituents to the periphery of the molecule (14, 15). 50 μl of 0.5 mg ml-1 MTT dissolved in PBS. The cell lines were Aim of the present study is the utilization of three then incubated for a period of 4 hours at 5% CO2 and 37˚C. The phthalocyanine photosensitizers in photodynamic therapy. MTT solution was then replaced with 100 μl of dimethylsulfooxide Cellular impairment of the tumour and non-tumour cells after to help dissolve the formazan crystals. The absorbance level was PDT was also be evaluated. measured by a Synergy HT reader. The measured data were

3944 Manišová et al: Phthalocyanines in PTD

Figure 2. Dark toxicity measurement. The IC50 of each photosensitizer was higher than 100 μM. The control represents cells without photosensitizer and its value was set at 100%. Data are presented as the mean±SD of three independent measurements. *Statistically significant at p<0.05.

Figure 3. The dependence of reactive oxygen species (ROS) production in NIH3T3 and HeLa cells on the concentration of phthalocyanine photosensitizer. The dependence of the NIH3T3 cell ROS production on the concentration of phthalocyanine photosensitizer, using 5-(and-6)- chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate fluorescence probe, was measured immediately after irradiation with total irradiation dose 15 J cm-2 (total irradiation time was 10 min and intensity of radiation was 25 mW cm-2). The control represents cells irradiated without photosensitizer (the negative control). Data are presented as the mean±SD of three independent measurements. *Statistically significant at p<0.05.

3945 ANTICANCER RESEARCH 35: 3943-3952 (2015)

Figure 4. The dependence of HeLa and NIH3T3 cell viability on the concentration of phthalocyanine photosensitizer. The dependence of cell viability on the concentration of phthalocyanine photosensitizers was determined by measuring the enzyme activity of living cells using the methylthiazol tetrazolium bromide test. The total irradiation dose used was 15 J cm-2 (total irradiation time was 10 min and intensity of radiation was 25 mW cm- 2). The control represents cells irradiated without photosensitizer (the negative control) and its value was set at 100%. Data are presented as the mean±SD of three independent measurements. *Statistically significant at p<0.05.

Figure 5. The dependence of membrane potential in HeLa and NIH3T3 cells on the concentration of phthalocyanine photosensitizer. Mitochondrial membrane potential of HeLa and NIH3T3 was measured after they were incubated with different concentrations of photosensitizers. The higher the JC-1 fluorescence ratio, the greater the damage to cells. The total irradiation dose used was 15 Jcm-2 (total irradiation time was 10 min and intensity of radiation was 25 mW cm-2). The control represents the irradiated cells with an absence of a photosensitizer (the negative control). Data are presented as ±SD from three independent measurements.

3946 Manišová et al: Phthalocyanines in PTD

Table I. The IC50 values (μM) for each photosensitizer against HeLa and of 150 g and the cell pellet was dispersed in 20 μl of PBS and NIH3T3 cell lines with total irradiation dose 15 J cm-2 (total irradiation vortexed. Eighty-five microlitres of 1% low melting point (LMP) time was 10 min and intensity of radiation was 25 mW cm-2). agarose (Qbiogene, California, United States of America) was added to this solution and Eighty-five microlitres of this suspension was Cell line placed on the solidified agarose on the microscope slide and covered by coverslip. The microscope slides were then transferred to a Photosensitizer HeLa NIH3T3 refrigerator. After the agarose had solidified, the coverslips were immersed in lysis buffer [2.5 M NaCl (Sigma-Aldrich, St. Louis, 2HPc 0.37±0.04 0.67±0.07 United States of America), 100 mM EDTA ethylenediaminetetraacetic ZnPc 0.04±0.01 0.23±0.03 acid (EDTA; Sigma-Aldrich, St. Louis, United States of America), 10 MgPc 0.43±0.05 0.55±0.06 mM tris(hydroxymethyl)aminomethane (Tris; Sigma-Aldrich, St. Louis, United States of America), 1% Triton X-100 (SERVA Electrophoresis, GmbH, Heidelberg, Germany), pH 10] at 4˚C for a period of 60 min. After lysis the slides were placed in a electrophoretic tank and dipped for 40 min in a cool electrophoretic solution [300 mM NaOH, 1 mM EDTA (Sigma-Aldrich, St. Louis, calculated by Phototox Version 2.0 software (ZEBET, Berlin, United States of America)]. The electrophoresis was run at 350 mA Germany). Dark toxicity was measured in parallel under the same and 0.8 V cm-1for 20 min. Following completion of the conditions without irradiation. electrophoretic separation the slides were carefully rinsed twice for 10 min with a neutralisation buffer [0.4 M Tris (Sigma-Aldrich, St. Measurement of mitochondrial membrane potential. Mitochondrial Louis, United States of America), pH 7.5] at 4˚C. The samples were membrane potential was monitored using fluorescence probe stained by means of SYBR Green (Invitrogen, Thermofisher Scietific, 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolylcarbocyanine Massachusetts, United States of America) and manually scored using chloride (JC-1). JC-1 exists as a monomer at low concentrations and fluorescence microscope with CCD camera CometScore 1.5 software yields green fluorescence (emission at 530 nm), similar to fluorescein. (TriTek, Wilmington, United States of America). To help summarize At higher concentrations or higher mitochondrial potential, JC-1 the findings and prepare for our evaluation, we randomly chose 100 forms J-aggregates that exhibit a broad excitation spectrum and an cells from each given sample. Median values of Olive moment, the emission maximum at ~590 nm. Immediatelly after irradiation the amount of the DNA in the head and in tail, which is directly cells were incubated with PBS with JC-1 at a final assay proportional to the DNA damage, were evaluated. concentration of 2 μg/ml for 20 minutes at 37˚C, 5% CO2 and then washed with PBS. Total irradiation dose was 15 J cm-2 (total Statistics analysis. All assays were performed three times. The irradiation time was 10 min and intensity of radiation was 25 mW statistical analysis was performed using a Dunnett t-test for cm-2). Fluorescence was recorded by the fluorescence reader Tecan comparison of treated groups against the control (p<0.05). The data Infinite 200pro (Tecan Group Ltd., Männedorf, Switzerland). Results are presented as the mean±SD of three independent measurements. were expressed as the ratio of green to red fluorescence (530/590 nm).

Apoptosis/necrosis assay. Prior to the start of measurement DMEM Results was replaced by binding buffer containing annexin and propidium iodide and the samples were then incubated at 37˚C and 5% CO2 Dark toxicity measurement. The dark toxicity of the each for 10 min. The fluorescence signals of annexin and propidium photosensitizer on both cell lines was evaluated. The iodide were recorded 4 h after irradiation by a fluorescence concetrations used were 0.1, 1, 10 and 100 μM. According to microscope with a CCD camera. Fluorescent cells were manually scored and counted as a percentage of the total number of cells. the results, the IC50 for each photosensitizer under dark conditions was higher than 100 μM. The results are shown in Comet assay. DNA damage caused to the cells was tested by a Figure 2. The values represent average values from the three comet assay. DMEM was replaced by PBS and the cells were independent measurements. irradiated by means of LEDs with a maximum emission of 740 nm -2 at total irradiation dose 15 J cm (total irradiation time was 10 min ROS measurement. The ROS production of each and intensity of radiation was 25 mW cm-2). Following the photosensitizer was tested using CM-H DCFDA fluorescence irradiation PBS was replaced by the DMEM and the cells were 2 incubated for a period of 24 hours. probe and measured immediately after irradiation . The ROS Microscope slides were first pre-coated with 1% high melting production of NIH3T3 and HeLa cell lines induced by each point (HMP) agarose (SERVA Electrophoresis GmbH, Heidelberg, photosensitizer is shown in Figure 3. The highest increase in Germany) dissolved in distilled water and placed in a drying oven for ROS production was demonstrated by use of the zinc 30 minutes at a temperature of 60˚C. Eighty-five microlitres of 1% derivative for both cell lines. HMP agarose in PBS was applied onto the precoated slides and covered with a coverslip and placed in a refrigerator in order to MTT assay. All of the photosensitizers were tested on the enhance gelling of the agarose. The cells were trypsinized for 10 min in order to detach the cells from the well bottom. The trypsinisation two cell lines. From the previous results, more specific was stopped using fetal bovine serum (FBS). The isolated cells were concentrations (from which the half maximum inhibitory centrifuged for a period of 4 minutes, at a relative centrifugal force concentration was assessed) was determined for each

3947 ANTICANCER RESEARCH 35: 3943-3952 (2015)

Table II. Comet assay parameters for NIH3T3 cells treated at two Table III. Comet assay parameters for HeLa cells treated at two different concentrations of photosensitizer with a total irradiation dose different concentrations of photosensitizer with an irradiation dose of of 15 J cm-2 (total irradiation time was 10 min and intensity of radiation 15 J cm-2 (total irradiation time was 10 min and intensity of radiation was 25 mW cm-2). Data are presented as the mean±SD of three was 25 mW cm-2). Data are presented as the mean±SD of three independent measurements. independent measurements

Photosensitizer Concentration DNA in DNA in Olive Photosensitizer Concentration DNA in DNA in Olive (μM) head (%) tail (%) moment (μM) head (%) tail (%) moment

Control 0 (0 J cm-2) 99.72±0.19 0.28±0.19 0.12±0.05 Control 0 (0 J cm-2) 98.79±0.13 1.21±0.13 0.22±0.04 0 (15 J cm-2) 96.90±0.17 3.10±0.17 0.66±0.06 0 (15 Jcm-2) 95.04±0.05 4.96±0.05 0.93±0.02

2HPc 0.1 87.13±0.09 12.87±0.09 2.65±0.02 2HPc 0.1 79.27±3.45 20.73±3.45 4.51±1.21 1 31.51±0.79 68.49±0.79 32.42±1.26 1 25.25±2.55 74.75±2.55 40.41±3.23

ZnPc 0.1 75.01±1.01 24.99±1.01 6.06±0.21 ZnPc 0.01 77.65±7.25 22.35±7.25 4.52±1.88 1 23.46±7.68 76.54±7.68 46.59±6.21 0.1 20.85±0.70 79.15±0.70 58.43±0.69

MgPc 0.1 82.56±7.37 17.44±7.37 2.66±0.82 MgPc 0.1 79.96±0.23 23.04±0.23 5.72±0.05 1 33.98±8.57 66.02±8.57 31.35±2.34 1 28.68±4.52 71.32±4.52 39.38±1.34

photosensitizer as 0.01, 0.0215, 0.0464 and 0.1μM for that the main cause of cell death was by necrosis. The ZnPcs against HeLa cells; and 0.1, 0.215, 0.464 and 1 μM proportion of apoptotic cells was less than 10% of total cells. for HPcs, MgPcs against both cell lines and ZnPcs against The results are shown in Figure 7. NIH3T3). The efficacy of cell death induced by each photosensitizer against HeLa and on the NIH3T3 cells is Comet assay of DNA damage to HeLa and NIH3T3 cells. shown in Figure 4. The most efficient photosensitizer was From the results of the MTT test two concentrations were the zinc derivative of phthalocyanine. chosen for each photosensitizer for which the differences in Based on the results from the MTT viability assay the tested parameters were observed. Olive moment and the IC50 values were calculated using Phototox Version 2.0 amount of DNA in the head and tail, which are directly software. The IC50 values are shown in Table I. The most proportional to the DNA damage, were evaluated. The Olive effective was the zinc derivative. Its IC50 value was very low moment was computed as the summation of each tail especially in the tumour cell line compared to the non- intensity integral value, multiplied by its relative distance tumour cell line. The IC50 values for photosensitizer with a from the center of the head (the point at which the head central hydrogen or magnesium atom were comparable for integral was mirrored) and divided by the total comet both cell lines. intensity. The median values for the each of these parameters are shown in the Tables II and III. The highest levels of DNA Mitochondrial membrane potential. Mitochondrial membrane damage were caused by the zinc derivative of phthalocyanine. potential was measured using JC-1 fluorescence probe. From the results of the MTT assay, two concentrations were chosen Discussion for each photosensitizer. Results are expressed as the ratio of green to red fluorescence (530/590 nm). The higher the JC-1 In this study we analyzed the effect of three phthalocyanine fluorescence ratio, the higher the damage to cells. The final photosensitizers in vitro on the cervical line HeLa assay concentration was 2 μg/ml of JC-1. Results showed the and mouse fibroblast cell line NIH3T3. decrease of mitochondrial membrane potential dependent on It is generally accepted that the higher the photosensitizer the increased photosensitizer concentration. The values concentration, the higher and faster ROS production is and represent average values from three independent the lower the cell viability is (10, 17, 18). Our results are in measurements. The results are shown in the Figure 5. agreement with this. We confirmed that the higher concentration of photosensitizer reduced cell viability and Apoptosis/necrosis assay. The cell death type induced at the increased ROS production. The results demonstrated that the IC50 was tested using the ANNEXIN V kit. Apoptotic cells zinc derivative was the most effective against both tested cell were coloured green, necrotic cells were coloured orange and lines. We observed the highest rise of ROS in both cell lines living cells remained colourless (Figure 6). It was discovered when the zinc derivative was applied (see Figure 3).

3948 Manišová et al: Phthalocyanines in PTD

Structurally similar azaphthalocyanines were tested by Zimcik et al. (19). They state that zinc as central metal appears to be the most suitable because it increases triplet lifetimes, resulting in a very high singlet oxygen quantum field. The zinc derivatives of phthalocyanines are considered to be a very promising group of substances used in PDT (20, 21, 22). We found that the IC50 value for the zinc photosensitizer was very low especially in the tumour cell line compared to the non-tumour cell line. A low effective concentration of the photosensitive is important for prevention of non-specific uptake of the photosensitizer by the non-tumour cells, which results in skin sensitivity as the main side-effect of administered drugs nowadays (1, 3). Our results showed that the IC50 values for photosensitizer with a central hydrogen or magnesium atom were comparable for Figure 6. NIH3T3 cells irradiated with total irradiation dose 15 J cm-2 both cell lines, which could lead to the unselective uptake of (total irradiation time was 10 min and intensity of radiation was 25 mW the photosensitizer by non-tumour cells and thus to skin -2 cm ) with sensitizer ZnPc at half maximum inhibitory concentration. Cells sensitivity. For both cell lines, the efficacy of these were stained with annexin and propidium iodide and observed in the fluorescence microscope with a CCD camera. Apoptotic cells are coloured photosensitizers was lower compared to that of the zinc green, necrotic cells are coloured orange and living cells remain colourless. derivative. The IC50 of each photosensitizer without

Figure 7. Type of HeLa and NIH3T3 cells present depending on the photosensitizer used. Cells were treated with photosensitizer at the half maximum inhibitory concentration and irradiated with the total irradiation dose 15 J cm-2 (total irradiation time was 10 minutes and intensity of radiation was 25 mW cm-2) The control represents cells irradiated without photosensitizer (the negative control). Data are presented as the mean±SD of three independent measurements.

3949 ANTICANCER RESEARCH 35: 3943-3952 (2015) irradiation were higher than 100 μM, so we considered these more severe DNA damage in cancer than in normal cells. photosensitizers as non-toxic under dark conditions. Unrepaired or incorrectly repaired double-strand breaks can lead However, the results from the MTT assay did not correlate to chromosomal damage and cell death (33). In our research, with the ROS measurement results, in which the production we studied the DNA damage to Hela and NIH3T3 cells. The of ROS was higher on the NIH3T3 non-tumour cell line DNA damage was higher in the tumour cell line compared to compared to the HeLa tumour cell line. This might be caused the non-tumour cell line. We determined that the highest level of by a reduced amount of glutathione in the HeLa cell line and DNA damage was caused by the zinc derivative in both cell simultaneously by non-changing level of gluthatione during lines. This correlates with the results of ROS measurement hyperoxia, thereby causing increased sensitivity in HeLa because ROS can cause oxidative damage to DNA and are cells to destruction by ROS (23). believed to the major endogenous toxic agents (34). PDT causes irreversible photodamage to vital sub-cellular We conclude that the induction of high ROS production, low targets which subsequently leads to cell death. We used the effective concentration (mainly against the tumour cell line) and Annexin V kit for the detection of cell death type. All low dark toxicity make the zinc derivative of phthalocyanine a photosensitizers used led to cell death mainly through potentially promising substance for use in PDT. necrosis. This is probably related to sub-cellular localization, when photosensitizers targeting the plasma membrane or Acknowledgements lysosomes can either delay or block the apoptotic programme predisposing the cells to necrosis (6, 7). Structurally similar This study was supported by LO1304, IGA_LF_2015_008 and phthalocyanine derivative ClAlPcS2 was tested and it was CZ.1.07/2.3.00/30.0004 grants. The Authors would like to thank the shown that this substance also causes cell death through Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University in Prag, Hradec Králové, Czech necrosis (24). Although apoptosis is considered to be the Republic for providing photosensitizers used in this study. more advantageous cell death type, necrosis can also be beneficial because primary necrotic cell death, without References apoptosis, inherently elicits many of the immune alarm signals-such as heat shock protein induction, designed to trigger immediate unequivocal immune responses (25, 26). 1 Zimcik P and Miletin M 2004: Fotodynamická terapie jako nová perspektivní metoda léčby nádorových onemocnění I. Přehled Development of resistance to apoptosis is an important step fotosenzitizérů. Ceska Slov Farm 53: 271-279, 2004. in carcinogenesis, thus the photosensitizers used in this study 2 Hopper C: Photodynamic therapy: a clinical reality in the may be effective even in cancer cells resistant to apoptosis. . Lancet Oncol 1: 212-219, 2000. High amount of ROS can cause changes of mitochondrial 3 Kübler AC: Photodynamic therapy. Med Laser Appl 20: 37-45, 2005. membrane potential and cell death because excessive ROS 4 Robertson CA, Hawkins Evans D and Abrahamse H: Photodynamic production may increase the permeability of lysosomal therapy (PDT): A short review on cellular mechanism and cancer membranes, leading to release of lysosomal proteases, which research applications for PDT. J Photochem Photobiol 96: 1-8, 2009. further contribute to mitochondrial membrane impairment and 5 Henderson BW and Dougherty TJ: How does photodynamic therapy work? J Photochem Photobiol 55: 145-157, 1992. cell death (27, 28). In this study, JC-1 was used for 6 Kessel D, Luo Y, Deng Y and Chang CK: The role of subcellular mitochondrial membrane potential measurement. Our results localization in initiation of apoptosis by photodynamic therapy. showed the decrease of mitochondrial membrane potential Photochem Photobiol 65: 422-426, 1997. dependent on the photosensitizer concentration. Mitochondrial 7 Buytaert E, Dewaelw M and Agostinis P: Molecular effectors of damage generally leads to cell death by apoptosis. Our finding multiple cell death pathways initiated by photodynamic therapy. that the photosensitizers used cause cell death through necrosis Biochim Biophys Acta 1776: 86-107, 2007. is in contrast with this. This can be explained by the 8 Choucrouna P, Gilleta D, Dorangeb G, Sawickia B and Dewitte JD: Comet assay and early apoptosis, Mutat Res Fundam Mol accumulation of phthalocyanine photosensitizers in different Mech Mutagen 478: 89-96, 2001. cell organelles such as mitochondria, lysosomes, nucleus and 9 Collins AR: The comet assay for DNA damage. Mol Biotechnol plasma membrane, which may result in organelle impairment 26: 249-261, 2004. leading to necrotic cell death (29, 30). 10 Pizova K, Bajgar R, Fillerova R, Kriegova E, Cenklova V, Induction of DNA damage in cancer cells is a well- Langova K, Konecny P, and Kolarova H: C-MYC and C-FOS recognized therapeutic strategy for killing cancer cells (31), expression changes and cellular aspects of the photodynamic inasmuch as cancer cells have lost or partially lost response to reaction with photosensitizers TMPyP and ClAlPcS2. DNA damage, and in turn loss of checkpoint controls, due to Photochem Photobiol 142: 186-196, 2015. 11 Gürel E, Pişkin M, Altun S, Odabaş Z and Durmuş M: Synthesis, etc mutations such as in p53, ataxia telangiectasia mutated . (32). characterization and investigation of the photophysical and Moreover, cancer cells often also have an impaired DNA repair photochemical properties of highly soluble novel metal-free, mechanism. Thus damaged DNA will not be repaired as zinc(ii), and indium(iii) phthalocyanines substituted with 2,3,6- efficiently as in normal cells, and genotoxic treatments cause trimethylphenoxy moieties. Dalton trans 44: 6202-6211, 2015.

3950 Manišová et al: Phthalocyanines in PTD

12 O’Connor AE, Gallagher WM and Byrne AT: and 24 Tomankova K, Kolarova H, Vachutka J, Zapletalova J, Hanakova nonporphyrin photosensitizers in oncology: preclinical and A and Kaplova E: Study of photodynamic, sonodynamic and clinical advances in photodynamic therapy. Photochem Photobiol antioxidative influence on HeLa cell line. Indian J Biochem 85: 1053-1074, 2009. Biophys 51: 19-28, 2014. 13 Novakova V, Miletin M, Kopecky K and Zimcik P: Red-Emitting 25 Melcher A, Gough M, Todryk S and Vile R.: Apoptosis or with Photophysical and Photochemical Properties necrosis for tumor immunotherapy: what’s in a name? J Mol Controlled by pH. Chemistry 17: 14273-14282, 2011. Med 77: 824-833, 1999. 14 Zimcik P, Miletin M, Kopecky K, Musil Z, Berka P, Horakova V, 26 Zong WX and Thompson CB: Necrotic death as a cell fate. Kucerova H, zbytovska J and Brault D: Influence of aggregation Genes Dev 20: 1-15, 2006. on interaction of lipophilic, water-insoluble azaphtalocyanines 27 Chang MC, Chan CP, Wang YJ, Lee PH, Chen LI, Tsai YL, Lin with DOPC vesicles. Photochem Photobiol 83: 1497-1504, 2007. BR, Wang YL and Jeng JH: Induction of necrosis and apoptosis 15 Zimcik P, Novakova V, Kopecky K, Miletin M, Uslu Kobak RZ to KB cancer cells by sanguinarine is associated with reactive Svandrlikova E, Vachova L and Lang K: Magnesium oxygen species production and mitochondrial membrane azaphtalocyanines: an emerging family of excellent red-emitting depolarization. Toxicol Appl Pharmacol 218: 143-151, 2007. . Inorg Chem 51: 4215-4223, 2012. 28 Kim R, Emi M, Tanabe K, Murakami S, Uchida Y and Arihiro 16 Cidlina A: Syntéza kationických ftalocyaninů. Diploma thesis K: Regulation and interplay of apoptotic and non-apoptotic cell Charles University in Prague, Faculty of Pharmacy in Hradec death. J Pathol 208: 319-326, 2006. Králové, 2011. 29 Ndhundhuma I, Hauser C, Scalfi-Happ C, Rück A and Steiner 17 Hanakova A, Bogdanova K, Tomankova K, Binder S, Bajgar R, R: Subcellular co-localization of aluminum (III) phthalocyanine Langova K, Kolar M, Mosinger J and Kolarova H: Study of chloride tetrasulphonate with fluorescent markers in the human photodynamic effects on NIH 3T3 cell line and bacteria. Biomed melanoma cell-line HT-144. Med Las Appl 26: 93-100, 2011. Pap Med Fac Univ Palacky Olomouc Czech Repub 158: 201- 30 Castano AP, Demidova TN and Hamblin MR: Mechanisms in 207, 2014. photodynamic therapy: part one-photosensitizers, 18 Kolarova H, Tomankova K, Bajgar R, Kolar P and Kubinek R: and cellular localization. Photodiagnosis Photodyn Ther 1: 279- Photodynamic and sonodynamic treatment by phthalocyanine on 93, 2004. cancer cell lines. Ultrasound Med Biol 35: 1397-1404, 2009. 31 Liao W, McNutt MA and Zhu WG: The comet assay: A sensitive 19 Zimcik P, Miletin M, Radilova H, Novakova V, Kopecky K, Svec method for detecting DNA damage in individual cells. Methods J and Rudolf E: Synthesis, properties and in vitro photodynamic 48: 46-53, 2009. activity of water-soluble azaphthalocyanines and azanaphthalo - 32 Zhang J, Lou X, Jin L, Zhou R, Liu S, Xu N and Liao DJ: cyanines. Photochem Photobiol 86: 168-175, 2010. Necrosis, and then stress induced necrosis-like cell death, but 20 Makhseed S, Machacek M, Alfadly W, Tuhl A, Vinodh M, not apoptosis, should be the preferred cell death mode for Simunek T, Novakova V, Kubat P, Rudolf E and Zimcik P: chemotherapy: clearance of a few misconceptions. Oncoscience Water-soluble non-aggregating zinc phthalocyanine and in vitro 1: 407-422, 2014. studies for photodynamic therapy. Chemi Commun (Camb) 49: 33 El-Hussein A, Harith M and Abrahamse H: Assessment of DNA 11149-11151, 2013. damage after photodynamic therapy using a metallophthalocyanine 21 Vachova L, Novakova V, Kopecky K, Miletin M and Zimcik P: photosensitizer. Int J Photoenergy 2012: 1-10, 2012. Effect of intramolecular charge transfer on fluorescence and 34 Bouwman P and Jonkers J: The effects of deregulated DNA singlet oxygen production of phthalocyanine analogues. Dalton damage signalling on cancer chemotherapy response and Trans 41: 11651-11656, 2012. resistance. Nat Rev Cancer 12: 587-598, 2012. 22 Moeno S, Krause RW, Ermilov EA, Kuzyniak W and Höpfner M: Synthesis and characterization of novel zinc phthalocyanines as potential photosensitizers for photodynamic therapy of . Photochem Photobiol Sci 13: 963-970, 2014. 23 Gille JJ, Wortelboer HM and Joenje H: Effect of normobaric Received April 16, 2015 hyperoxia on antioxidant defences of HeLa and CHO cells. Free Revised May 13, 2015 Radic Biol Med 4: 85-91, 1988. Accepted May 15, 2015

3951