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Review

Tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine for photodynamic cancer therapy Weronika Kuzyniak a , Eugeny A. Ermilov a,d , Devrim Atilla b , Ays¸e Gül Gürek b , Bianca Nitzsche a , Katja Derkow c , Björn Hoffmann a , Gustav Steinemann a , Vefa Ahsen b , Michael Höpfner a,∗ a

Institute of Physiology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany Department of Chemistry, Gebze Institute of Technology, PO Box 141, Gebze, 41400, Turkey c Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany d Institute of Physics, Humboldt-Universität zu Berlin, Newtonstr. 15, 12489 Berlin, Germany b

a r t i c l e

i n f o

Article history: Received 27 April 2015 Received in revised form 19 June 2015 Accepted 2 July 2015 Available online xxx Keywords: Photodynamic therapy Zinc (II) phthalocyanine Apoptosis Cell cycle CAM assay

a b s t r a c t Photodynamic therapy (PDT) has emerged as an effective and minimally invasive treatment option for several diseases, including some forms of cancer. However, several drawbacks of the approved photosensitizers (PS), such as insufficient light absorption at therapeutically relevant wavelengths hampered the clinical effectiveness of PDT. Phthalocyanines (Pc) are interesting PS-candidates with a strong light absorption in the favourable red spectral region and a high quantum yield of cancer cell destroying singlet oxygen generation. Here, we evaluated the suitability of tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine (ZnPc) as novel PS for PDT. ZnPc-induced phototoxicity, induction of apoptosis as well as cell cycle arresting effects was studied in the human gastrointestinal cancer cell lines of different origin. Photoactivation of ZnPc-pretreated (1–10 ␮M) cancer cells was achieved by illumination with a broad band white light source (400–700 nm) at a power density of 10 J/cm2 . Photoactivation of ZnPc-loaded cells revealed strong phototoxic effects, leading to a dose-dependent decrease of cancer cell proliferation of up to almost 100%, the induction of apoptosis and a G1-phase arrest of the cell cycle, which was associated with decrease in cyclin D1 expression. By contrast, ZnPctreatment without illumination did not induce any cytotoxicity, apoptosis, cell cycle arrest or decreased cell growth. Antiangiogenic effects of ZnPc-PDT were investigated in vivo by performing CAM assays, which revealed a marked degradation of blood vessels and the capillary plexus of the chorioallantoic membrane of fertilized chicken eggs. Based on our data we think that ZnPc may be a promising novel photosensitizer for innovative PDT. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Light source and irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. PDT treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.5. Determination of cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. Fax: +49 30 450 528 918. E-mail addresses: [email protected] (W. Kuzyniak), [email protected] (E.A. Ermilov), [email protected] (D. Atilla), [email protected] (A.G. Gürek), [email protected] (B. Nitzsche), [email protected] (K. Derkow), [email protected] (B. Hoffmann), [email protected] (G. Steinemann), [email protected] (V. Ahsen), [email protected] (M. Höpfner). http://dx.doi.org/10.1016/j.pdpdt.2015.07.001 1572-1000/© 2015 Elsevier B.V. All rights reserved.

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2.6. Measurement of growth inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.7. Determination of cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.8. Detection of apoptosis-specific caspase-3 activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.9. Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.10. Cell cycle analysis/apoptotic cell death by flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.11. Intravital microscopy of CAM vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.12. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Phototoxic effects of PDT with ZnPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Apoptotic effects of photoactivated ZnPc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Cell cycle regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Changes in CAM vasculature after ZnPc-PDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction The therapeutic effect of light has been known for thousands of years in China or ancient Egypt. Only in nineteenth century photodynamic therapy (PDT) has been introduced to modern medicine by Herman von Tappeiner and Albert Jesionek, who treated skin cancer by combining a photoactive agent and light [1]. In 1977, PDT was introduced in clinical studies and then approved for cancer treatment in several countries (e.g. United States, Canada, Russia and Germany) [2,3]. PDT is a cancer therapy modality that involves the use of light at specially selected wavelengths to selectively excite a photosensitizer of choice in the presence of molecular oxygen. The activation of the PS results in the generation of reactive oxygen species (ROS) which damage and kill the target cells [4]. The advantages of PDT treatment over conventional cancer therapies are repeatability, excellent cosmetic effects (e.g. skin lesion treatment) and minor pain during and after treatment [5–7]. Nonetheless, increase of survival ratio and probability of long-term local disease control make PDT extremely attractive itself or as complement to other therapies [8]. However, several side effects of the PSs that have been approved for PDT treatment so far, limit the clinical applicability. Still, the main problems include long term photosensitivity and poor light absorption of PS [9,10]. Hence, there is need for new, better photoactive compounds. Phthalocyanines (Pcs) belong to the advanced group of so called second generation photosensitizers, which gather most attention for PDT nowadays [11,12]. Pcs are organic macrocyclic compounds which are similar in structure to naturally occurring porphyrins. Pcs are photostable, have a strong absorption in the red spectral region (with maximum at ca. 680–700 nm), which is regarded as the “therapeutic window” for effective PDT, and show a high quantum yield of singlet oxygen generation. Upon illumination the Pcs cause cell death via necrosis and – even more important – also via induction of apoptosis [13,14]. The substitution of alkylated polyethyleneglycol moieties to Pc [15–17] increased the solubility of the phthalocyanines in highly polar solvents and water. Thus highly solubility properties of these compounds provide convenience for in vitro studies. Only recently, we reported on the synthesis, and the photochemical and photophysical properties of tetratriethyleneoxysulfonyl substituted zinc phthalocyanine [15]. Preliminary in vitro studies have shown that ZnPc applied to human breast cancer cells resulted in photoinduced cytotoxicity at low light doses of 5 mW/cm2 [15]. The major goal of the present study was to investigate the effectiveness of ZnPc PDT and to characterize the underlying mode of action of the treatment in terms of apoptosis induction, cell cycle effects, as well as

general cytotoxicity and antiproliferative potency. The effects were confirmed on the in vivo level by performing CAM assays to additionally monitor antiangiogenic effects of the ZnPcs. 2. Material and methods 2.1. Compound Tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine (ZnPc) was prepared by slightly modifying procedure described in literature [15]. The synthetic pathway of the preparation of ZnPc and its structure is shown in Scheme 1. Synthesis: 4(4,7,10-Trioxaundecan-1-sulfonyl) phthalonitrile [18] (0.30 g, 0.98 mmol) and anhydrous Zn(OAc) 2 (92 mg, 0.50 mmol) were refluxed in N,N -dimethylaminoethanol (2 ml) for 24 h under argon atmosphere. N,N -dimethylaminoethanolwas distillated off and the reaction mixture was diluted with dichloromethane. The product was precipitated from dichloromethane in hot hexane and filtered. The green waxy crude product was purified over a silica gel column using chloroform–ethanol mixture (15:1) as eluent (139 mg, 44%). C60 H72 N8 O12 S4 Zn, MW 1288.34; found C, 56.85; H, 5.26; N, 8.53; requires C, 56.12; H, 5.65; N, 8.72 1 H NMR: (DMSO-d6 ) ı 8.87 (d, 4H, aromatics), 8.82 (s, 4H, aromatics), 8.04 (d, 4H, aromatics), 4.04 (t, 8H, CH2 ), 3.78 (t, 8H, CH2 ), 3.70 (t, 8H, CH2 ), 3.67 (t, 8H, CH2 ) 3.57 (t, 8H, CH2 ), 3.48 (m, 8H, CH2) 3.42 (t, 8H, CH2 ), 3.22 (s, 12H, CH2 ). MS (ESI): an isotopic pattern peaking at m/z 1289.3 [98%, (M+H)+ ], 1291.3 [100%, (M+3H)+ ], 1293.3 [75%], 1294.3 [42%], 1295.3 [23%], 1296.3 [7%]. The compound was dissolved in DMSO of spectroscopic grade (Aldrich) which was used without further purification. Concentration of stock solutions was calculated by measuring their optical density at 694 nm with UV–vis spectrometer Ultraspec 2100 (Amersham Biosciences) and using Lambert–Beer relationship with 694nm = 2.04 × 105 M−1 cm−1 [15]. Fluorescence excitation and emission spectra, were recorded on a Varian Eclipse spectrofluoremeter using 1 cm pathlength cuvettes at room temperature in DMSO (Fig. 1). 2.2. Cell culture Human pancreatic carcinoid BON cells [19] were cultured in DMEM/Ham’s F-12 (1:1) medium (Biochrom AG) supplemented with 10% fetal calf serum (FCS, Biochrom), 100 U/ml penicillin and 100 ␮g/ml streptomycin (Biochrom AG). The human esophageal squamous carcinoma cell lines Kyse-70 and Kyse-140 [20] were cultured in RPMI 1640 medium (Biochrom AG) supplemented with 10% fetal bovine serum (FCS, Biochrom), 100 U/ml

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Scheme1. Synthetic pathway for the preparation of tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine (ZnPc) (i) zinc acetate, DMAE, reflux.

penicillin and 100 ␮g/ml streptomycin (Biochrom AG). The human esophageal adenocarcinoma cell line OE-33 was cultured in RPMI 1640 medium (Biochrom AG) supplemented with 10% fetal bovine serum (FCS, Biochrom), 100 U/ml penicillin and 100 ␮g/ml streptomycin (Biochrom AG) and 2 mML-glutamine (Biochrom AG) [20]. Human colorectal carcinoma HCT-116 cells were cultured in DMEM medium (Biochrom AG) supplemented with 10% fetal bovine serum (FCS, Biochrom), 100U/ml penicillin and 100 ␮g/ml streptomycin (Biochrom AG). Cells were maintained under standard conditions (37 ◦ C in a humidified atmosphere of 5% CO2 ). The culture medium was changed every second day and once a week the cells were passaged using 1% Trypsin/EDTA.

2.4. PDT treatment For PDT treatment cells were incubated in the dark at 37 ◦ C (5% CO2 , humidified atmosphere) with ZnPc (1–10 ␮M) for 24 h in the culture medium. Thereafter the medium was removed, and cells were irradiated in PS-free PBS for 21 min to reach 10 J/m2 of energy. During irradiation the temperature of the samples never exceeded 37 ◦ C. Temperature was measured with a digital thermometer placed inside of irradiation system and fans connected to the thermometer started to cool down the temperature inside the illumination unit before 37 ◦ C were reached. After irradiation PBS was removed and cells were maintained at 37 ◦ C (5% CO2 , humidified atmosphere) in PS-free culture medium. Dark control was treated in the same way except irradiation.

2.3. Light source and irradiation PDT was performed with a broad band light source equipped with a 100 W halogen lamp (EFR 12 V/100 W GZ—6.35 Lampe, OMNILUX, Germany). The spectral output of the lamp ranged from 400 to 800 nm. To prevent infrared irradiation, a heat-reflecting filter that cuts off transmission at 700 nm and above was inserted into the optical path. The illuminated area (5.5 × 4.5 cm) had an average power density of 80.2 W/m2 . The light energy dose was measured with a P-9710 radiometer controlled by a silicon photocell (OPTOMETER P 9710) from Gigahertz-Optik (Munich, Germany). The total light energy dose was calculated by integrating the energy signal over the entire period of irradiation.

2.5. Determination of cell viability Cell viability of irradiated BON and Kyse-70 cells was investigated by using a cell viability/cytotoxicity assay kit (live/dead assay) from Life Technologies (USA) as described earlier [21]. Cells grown on glass coverslips were incubated for 24 h with ZnPc. After washing off the incubation medium with PBS, cells were irradiated as described above. 24 h after PDT cells were incubated with calcein-AM (160 nM) and EthD-1 (2 ␮M) for 1 h in PBS at 37 ◦ C and examined by fluorescence microscope from Zeiss (Axioskop2; Jena, Germany). Live cells were identified by the presence of ubiquitous intracellular esterase activity, leading to the conversion of nonfluorescent cell-permeable calcein-AM to the greenfluorescent polyanionic dye calcein (excitation/emission approx. 495/510 nm), which is well retained within live cells. Dead cells were determined by EthD-1 (excitation/emission—495/635 nm), which becomes red-fluorescent upon binding to nucleic acids of cells with damaged membranes.

2.6. Measurement of growth inhibition

Fig. 1. Fluorescence excitation and emission spectra of ZnPc in DMSO. Excitation wavelength = 672 nm.

After PDT treatment, samples and controls were cultured for up to 3 days in PS-free medium. Evaluation of the changes in cell numbers induced by ZnPc-PDT was performed by crystal violet staining as described previously [22]. Briefly, the cells were fixed with 1% glutaraldehyde and stained with 0.1% crystal violet. The unbound dye was removed by washing with water. Bound crystal violet was solubilized with 0.2% Triton-X-100 in PBS. Light extinction, which

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Fig. 2. Time- and dose-dependent growth inhibition of BON and Kyse-70 cells by ZnPc-PDT. BON (A) and Kyse-70 (B) were treated for 24 h with increasing concentrations of ZnPc and illuminated with a light dose of 10 J/m2 , which resulted in a dose- and time-dependent decrease in tumor cell proliferation of up to >95% after 3 days post PDT. 24 h of incubation with non-photoactivated ZnPc did not affect proliferation of BON (C) and Kyse-70 (D) cells, which indicates that ZnPc does not possess any appreciable “dark toxicity”. By contrast, photoactivated ZnPc decreased the cell number of either cell line by up to almost 100%. Data are given as percentage of untreated but illuminated controls, whose growth was set 100% (mean ± SD of 3 independent experiments). *P < 0.05 non-photoactivated vs photoactivated cells.

increases linearly with the cell number, was analyzed at 570 nm using an ELISA-Reader.

wavelengths: excitation: 360/40 nm, emission: 460/10 nm) from Bio-Rad (Munich, Germany).

2.7. Determination of cytotoxicity

2.9. Western blotting

Cells were seeded at a density of 4000 cell/well into a 96well microtiter plate and incubated with rising concentrations of ZnPc for 6 and 24 h without illumination. Thereafter, release of the cytoplasmic enzyme lactate dehydrogenase (LDH), indicating unspecific cytotoxicity, was measured in the supernatant of the samples by using colorimetric kit from Roche Diagnostics as described elsewhere [23]. Maximum release of LDH was measured after adding 2% Triton X-100 to untreated cells. The absorbance of the samples was measured at 490 nm, using an ELISA-Reader.

Western blotting was performed as described in [25]. In brief, whole-cell extracts were prepared by lysing cells with RIPA buffer. Lysates containing 25 ␮g proteins was subjected to gel electrophoresis. Proteins were transferred to PVDF membranes by electroblotting for 1.5 h. Blots were blocked in 5% skim milk powder solution (Merck, Darmstadt, Germany) for 1 h, and then incubated at 4 ◦ C overnight with antibodies directed against BAX (1:1000), ERK ½ (1:1000), GAPDH (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2 (1:1000), Cyclin D1 (1:200), ␤-actin (1:2000) (Sigma-Aldrich, USA) or AKT (1:1000) (Cell Signaling Technology, MA, USA). After incubation with horseradish peroxidase-coupled anti-IgG antibodies (1:10000, Amersham, Uppsala, Sweden) at room temperature for at least 1 h, the blot was developed using enhanced chemiluminescent detection (Amersham, Uppsala, Sweden) and exposed to Hyperfilm ECL film (Amersham, Uppsala, Sweden) for 0.5–1.5 min.

2.8. Detection of apoptosis-specific caspase-3 activity Changes in caspase-3 activity were calculated from cleavage of the fluorogenic substrate AC-DEVD-AMC (CalbiochemNovabiochem, Bad Soden, Germany), as described previously [24]. Irradiated cells (BON and Kyse-70) were maintained in PS-free culture medium in an incubator (37 ◦ C, 5% CO2 , humidified atmosphere) for 24 h. Thereafter, cells were lysed by treatment with lysis buffer and the lysates were incubated for 1 h at 37◦ C with a substrate solution containing 20 ␮g/ml AC-DEVD-AMC, 20 mM HEPES, 10% glycerol, 2 mM DTT and pH 7.5. Substrate cleavage was measured fluorometrically using a VersaFluor fluorometer (filter

2.10. Cell cycle analysis/apoptotic cell death by flow cytometry Cell cycle analysis was performed according to the previously described procedure [26]. 24 h after PDT cells were washed with PBS and fixed in PBS/2% formaldehyde (vol/vol) on ice for 30 min.

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Fig. 3. Phototoxic effects of ZnPc-PDT. 24 h after PDT, BON (A) and Kyse-70 (B) cells were incubated with calcein-AM and EthD-1 to determine the toxicity of photoactivated ZnPc. ZnPc-PDT treatment led to a dose-dependent decrease in viable (green) cells while the number of dead (red) cells increased in both cells lines. Phase-contrast and corresponding fluorescence images of representative preparations out of n = 4 independent preparations for each cell line are depicted. Scale bar 20 ␮m.

Afterwards cells were incubated in ethanol/PBS (2:1 vol/vol) for 30 min and pelleted. Resuspension in PBS containing 40 ␮g/ml RNase A followed. After incubation for 30 min at 37 ◦ C, cells were pelleted again and resuspended in PBS containing 50 ␮g/ml propidium iodide. Cells were then analyzed by using FACSCanto II (BD Biosciences, Heidelberg, Germany) and FCS Express Software (De Novo, Los Angeles, CA).

2.11. Intravital microscopy of CAM vasculature In vivo studies were performed by using chicken chorioallantoic membrane assay (CAM) as described previously [27,28]. Briefly, two silicon rings (5 mm in diameter) were placed on the CAM of fertilized chicken eggs at day 11 of the chicken embryo development.

20 ␮l of ZnPc (10 ␮M) was applied topically to one of the silicon rings while the other ring was treated with 20 ␮l of NaCl (0.9%) as a negative control. The eggs were sealed with clear tape and incubated in the dark at 37 ◦ C in a humidified incubator. After 24 h the treatment was repeated followed by illumination of the CAM with light dose of 10 J/m2 . Then the eggs were kept in the incubator for additional 24 h. PDT-induced changes in CAM microvasculature were recorded after 24 h by intravital microscopy using a Zeiss Axiotech microscope, equipped with a 4× objective. Video images were acquired by a CCD camera (CF8/5 MX, Kappa Optronics, Gleichen, Germany) and recorded digitally (DVCAM, DSR-25, Sony, Berlin, Germany). Image analysis was performed offline by using an in-house software [29]. Briefly, the video images were stacked to create a time series of 200–500 frame sequences.

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Table 1 Inhibitory concentrations IC50 of photoactivated ZnPc when applied to the human gastrointestinal cancer cell lines of different origin. Cell line

Origin

IC50 value

BON Kyse-70 Kyse-140 OE-33 HCT-116

Pancreatic carcinoid Esophageal squamous carcinoma Esophageal squamous carcinoma Esophageal adenocarcinoma Colorectal carcinoma

1.3 ␮M (±0.5) 3.1 ␮M (±0.3) 1.1 ␮M (±0.2) 3.75 ␮M (±0.3) 2.3 ␮M (±0.1)

Values calculated based on concentration-response curves obtained by measuring the percentage of vital cells relative to control cells (48 h after ZnPc-PDT) using crystal violet staining.

After movement correction the intensity values for each pixel location throughout the time series were calculated and correlated. The resultant image produced a time series reconstruction of blood flow during the time interval of the image stack and allowed visualization the small capillaries in the vascular bed of the chicken CAM. 2.12. Statistical analysis If not stated otherwise, means of three independent experiments ± SD were shown. Caspase-3 measurements were evaluated by using the Mann–Whitney U-test. Significance between controls and treated samples was analyzed by the Holm-Sidak method. P < 0.05 was considered statistically significant. 3. Results 3.1. Phototoxic effects of PDT with ZnPc The growth inhibitory potency of ZnPc PDT was determined in a panel of human cancer cell models of different origin. PDT of ZnPc-pretreated (1–10 ␮M) cancer cells led to a pronounced doseand time-dependent growth inhibition of pancreatic carcinoid BON cells, colorectal HCT-116 cancer cells, squamous esophageal carcinoma cells Kyse-70 and Kyse-140 as well as adenocarcinomatous esophageal OE-33 cancer cells with IC50 values in the low micromolar range (Table 1, Fig. 2A and B). By contrast irradiated but ZnPc untreated cells did not show any appreciable cell number reduction, indicating the non-toxic properties of the compound in its non-irradiated/non-activated form (Fig. 2C and D). At least after PDT with higher ZnPc doses (5–10 ␮M) those cells that survived the initial PDT treatment did not seem to be able to (re-) proliferate in the following days (Fig. 2A and B). The dose-dependent phototoxic effect of ZnPc-PDT was visualized by live/dead fluorescence microscopy. PDT with ZnPc led to a decrease in viable (green) cells while the percentage of dead (red) cells increased. Moreover, morphological changes of BON (Fig. 3A) and Kyse-70 (Fig. 3B) cells became apparent, as the cells appeared flat and shrunken after PDT treatment. By contrast, control cells, which were irradiated but not incubated with the PS, remained healthy and morphologically unaffected. Cytotoxicity of non-photoactivated ZnPc was determined by measurement LDH release. Incubating BON and Kyse-70 cells with 1–10 ␮M of non-photoactivated ZnPc for up to 24 h did not result in an increase in LDH release, indicating that in the absence of light ZnPc does not directly affect cell membrane integrity and does not exhibit cytotoxic effects even at concentrations as high as 10 ␮M (data not shown). 3.2. Apoptotic effects of photoactivated ZnPc Both BON and Kyse-70 cells showed a dose-dependent and significant increase in caspase-3 activity after ZnPc-PDT (Fig. 4).

Fig. 4. Photoactivated ZnPc causes a dose-dependent increase in apoptosis specific caspase-3 activity of Kyse-70 and BON cells. Changes in caspase-3 activity were calculated 24 h after ZnPc-PDT. Data are given as percentage of caspase-3 activity of untreated but illuminated control cells, which was set 100% (mean ± SD of 3 independent experiments). *P < 0.05 vs untreated control.

Dark controls with nonphotoactivated ZnPc did not exhibit any measurable increase in capase-3 activity (not shown). To confirm apoptosis-inducing effects of ZnPc-PDT the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Akt was evaluated by Western blot analysis, showing a dose-dependent decrease in antiapoptotic Bcl-2 (Fig. 5A), while the level of proapototic Bax protein remained unchanged. Accordingly, mitogenic and prosurvival pathways, such as ERK-MAPK pathway or the mTOR/AKT pathway were dose-dependently down-regulated, as determined by measuring ZnPc-PDT induced decrease in the expression of ERK ½ and Akt (Fig. 5B). 3.3. Cell cycle regulation Effect of ZnPc-PDT on the cell cycle of BON and Kyse-70 cells was measured by flow cytometry and analyzed using FCS Express Software (De Novo). 24 h after PDT, a pronounced and dose-dependent accumulation of BON cells in G0 /G1 -phase was observed. Correspondingly, the amount of cells in S- or G2 /M-phase decreased (Fig. 6). Comparable, although less pronounced results were found for Kyse-70 cells (data not shown). To confirm the G1 -phase arrest and the underlying change in cell cycle regulating proteins, we additionally evaluated changes in the expression of cyclin D1. Cyclin D1 is well-known cell cycle promoter, acting at the transition from the G1 —to the S-phase of the cell cycle. Corresponding to the observed G1 -arrest, we found a dose-dependent decrease in cyclin D1 expression after ZnPc-PDT in BON and Kyse-70 cells (Fig. 5B). 3.4. Changes in CAM vasculature after ZnPc-PDT Changes in the vascular network monitoring vascular adaptation and potential antiangiogenic effects of ZnPc-PDT were studied by performing CAM assays. Visualization of the blood flow allowed examination of effects on the level of the small capillaries. In the control treated area, shown in Fig. 7A, the blood vessel of the CAM branches out into a network of finest capillaries. 24 h after ZnPc-PDT (10 ␮M) a marked degeneration of the blood vessels and capillary plexus was observed, leading to a decreased blood perfusion of the ZnPc-PDT treated area, compared to the control area of PDT-treatment in the absence ZnPc. Either the morphological changes as well as the reduced number of vessels in the ZnPc-PDT

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Fig. 5. Effects of ZnPc-PDT on the expression apoptosis and cell cycle related proteins. 24 h after ZnPc-PDT antiapoptotic Bcl-2 was markedly downregulated in BON and Kyse-70 cells, while the expression of proapototic Bax remained unchanged (A). The cell cycle promoter cyclin D1, Akt and mitogenic ERK ½ were also dose-dependently down-regulated (B). ␤-actin and GAPDH were used as housekeeping genes. Control cells were neither incubated with ZnPc nor illuminated.

treated area suggest that the novel ZnPc photosensitizer possesses avascular and antiangiogenic potency (Fig. 7B). 4. Discussion PDT is a promising treatment modality for several diseases, including some types of cancer [8]. The advantages of PDT are its minimal invasiveness, the almost painless and patient friendly

Fig. 6. Induction of cell cycle arrest measured by flow cytometry. 24 h after ZnPcPDT, a dose-dependent accumulation of BON cells in G0 /G1 -phase was observed. Correspondingly, the amount of cells in S- or G2 /M-phase decreased. Means of >3 independent experiments are shown. *P < 0.05 vs untreated control.

application and the possibility to apply it repeatedly—if needed. The development and improvement of specific light delivery systems via laser fibres or endoscopic light sources increased the field of PDT applications in the last years. Organs previously impossible to reach with a lamp, today are reachable with laser fibres [30]. Although the PDT technique has been further developed, the basic principle remained the same—a photosensitive drug destroys target cells or tissues, after its activation by light which leads to the production of highly reactive oxygen-species (ROS). ROS destroy the target cell by damaging cell- and organelle membranes, cellular proteins and enzymes. The goal of our study was to evaluate the potential of tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine as a potential, new PS for PDT. There are several properties, an ideal PS should exhibit. Among those are (a) high light absorption in the infrared spectrum (600–800 nm), which allows deep tissue penetration, (b) the absence of cytotoxicity in the nonphotoactivated state, (c) the induction of apoptosis for an inflammation-free cancer cell dying and elimination and (d) high cost effectiveness due to low manufacturing costs because of an easy to produce and well-known chemical structure [31,32]. ZnPcs meet all of those aforementioned criteria, making them especially promising as new photosensitizers for innovative PDT treatment. Depth of tissue penetration can extensively cut back the efficiency of PDT. Short wavelengths (in UV spectral range) do not penetrate the tissue enough and do not sufficiently activate PS in deeper (cancer) tissue areas, while long wavelengths (IR spectral region) often do not provide enough energy to drive a sufficient singlet oxygen production of the PS that are so far available. Moreover,

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Fig. 7. ZnPc-PDT treatment induced changes in the vascular bed of the chicken chorioallantoic membrane (CAM). 24 h after PDT (10 J/cm2 ) pictures were taken of the control CAM area (preincubated with NaCl 0.9% instead of ZnPc) (A). In the control CAM the vascular network consisted of symmetrically arranged blood vessels with a fine structured capillary bed. ZnPc-PDT altered the architecture of the CAM and dramatically reduced the number of capillaries leading to large nonperfused areas (B). Arrows show the direction of the blood flow in veins (v) and arteries (a). Scale bar 100 ␮M.

multiple scattering caused by naturally occurring chromophores, such as hemoglobin, myoglobin and cytochromes can hamper the efficacy of a PDT. Referring to multiple studies high quality PS should have high absorption peak between 600 and 800 nm. ZnPc absorb light at 694 nm and to our knowledge, there is no photosensitizer recently approved or undergoing clinical trials which can be robustly excited at this particular wavelengths, making ZnPc particularly interesting for a powerful and deeply tissue penetrating PDT treatment [8,33]. Our in vitro studies clearly demonstrated the effectiveness of ZnPc-PDT to kill human cancer cells of different origin. The underlying mode of action of ZnPc-PDT was exemplarily investigated in squamous esophageal carcinoma and pancreatic carcinoid cells. While PDT is already a well-established treatment modality for esophageal cancer, but is becoming also a promising new possibility to treat pancreatic cancer [34,35]. Thus it was particularly interesting to check out the anticancer effects of ZnPc PDT in esophageal Kyse-70 and pancreatic BON cancer .We could show that even at low micromolar concentrations (1–10 ␮M) photoactivated ZnPc produced marked cytotoxicity leading to the dose-dependent killing of BON and Kyse-70 cells of up to almost 100% at 24 h post PDT. Interestingly, no significant reproliferation of cells that escaped from immediate, cytotoxic cell death was observed and Kyse-70 or BON cells that were pretreated with 5–10 ␮M ZnPc did not show signs of reproliferation for up to 3 days post PDT. Accordingly, the expression of Akt and ERK ½, both being associated with cell survival and proliferation [36,37] was dose-dependently downregulated in both cell lines. By contrast, clinically available second generation photosensitizers, such as ␦-aminolaevulenic acid (5ALA), have been shown to solely exhibit immediate cytotoxicity but no long lasting growth inhibitory effects on 5-ALA-PDT treated cancer cells [21]. Thus, the long lasting growth inhibitory effects assessed for ZnPc in this study confirm the extraordinary potential of ZnPc as a new photosensitizer for PDT. Dark toxicity – cytotoxicity in absence of light – is an unwanted feature of some PS [38], because it leads to PDT-independent and unspecific systemic effects. Therefore, we examined if non-illuminated ZnPcs may exhibit any appreciable unspecific cytotoxicity. However, by performing respective LDH release assays as well as proliferation tests we could not observe any indication of unspecific “dark toxicity” of ZnPc. Accordingly, Western Blots

with non-illuminated ZnPc-treated cancer cells did not bring forward any changes in the expression of mitogenic, cell cycle- and apoptosis-related proteins, such as Akt, ERK ½, Cyclin D1, Bax or Bcl-2, and as compared to the ZnPc-untreated controls. Our findings support the notion, that the novel ZnPc-PS does not exhibit unwanted dark toxic effects, which further qualifies it as an interesting new compound for PDT. The latest new generation of PS should preferably induce apoptotic cell death, which is a tightly controlled cell suicide and unlike necrosis does not cause e.g. inflammatory events in the treated area. Most of the PSs currently undergoing clinical or pre-clinical studies have been shown to induce apoptosis due accumulation and subsequent light-induced damage of the mitochondria, which induces mitochondria-dependent apoptosis [39]. Phthalocyanines have also been shown to bind strongly to mitochondria and the Golgi apparatus, which suggests that our new phthalocyanine-based ZnPc may also exert its apoptotic effects by induction of mitochondria-dependent apoptosis [40–44]. By performing caspase-3-assays, we could show that photoactivated ZnPc caused a pronounced increase in caspase-3 activity in both cell lines, indicating the execution of apoptosis [44]. Further characterizing the apoptotic effects of ZnPc-PDT we determined the regulation of the antiapoptotic Bcl-2 and proapoptotic Bax. As expected from the caspase-3 data, a downregulation of Bcl-2 was observed in BON and Kyse-70 cells. We suggest that ZnPc-PDT triggers the mitochondrial pathway of apoptosis. However, the exact underlying signaling events need further investigations, which will be performed in a forthcoming study. Induction of cell cycle arrest is a valuable attribute of any drug used in anticancer therapy. Some cytostatic drugs inhibit the cancer cell division (e.g. taxanes, such as Paclitaxel), while others preferably work in a particular phase of cell cycle (e.g. S-phase dependent antimetabolites (5-FU)), or G1 -phase dependent working alkylating drugs (cyclophosphamide). Thus, knowledge of cell cycle arresting effects of ZnPc-PDT may be of particular interest for combination therapies with clinically relevant cytostatics. Thus, we determined the cell cycle arresting effects of ZnPc-PDT and could observe a marked downregulation of the cell cycle promoter cyclin D1, leading to a G1 cell cycle arrest in both cancer cell models. To confirm the G1 -phase arrest, we additionally performed flow cytometry analyses of ZnPc-PDT

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treated BON cells, which showed a pronounced accumulation of in G1 -phase with a concomitant decrease of cells in the S- and G2 /Mphase. The findings give a first and valuable hint for respective combination therapies that will be evaluated in the future. It has been already shown, that PDT combined with chemotherapy increase remission ratio over 20% and prolonged survival time over 6 months in treatment of advanced esophageal carcinoma [45,46]. A very promising approach for combination therapies with enhanced efficacy was recently introduced by Weiss et al., who combined PDT with antiangiogenic chemotherapy for treating A2780 human ovarian tumor by using known photosensitizer and angiostatic drugs [47]. The use of anti-angiogenic drugs may significantly improve efficiency of PDT. Depending of the treatment schedule, angiostatic drugs can be used before or after PDT. It is well-known that angiostatic drugs may normalize tumor vascularity, which is important for drug delivery (chemotherapy) or oxygen-dependent radiotherapy. On the other hand if used after PDT where the blood vessels may be already damaged, a supportive antiangiogenic therapy may help to prevent vascular regrow constantly. In our preliminary in vivo study, we observed a degeneration of blood vessels and capillary plexus of the CAM after ZnPc-PDT treatment, which suggests avascular and/or antiangiogenic potency of the new PS. It is tempting to speculate on possible additive or even synergistic antineoplastic effects of a combination of ZnPc-PDT and subsequent antiangiogenic treatment with small molecules, such as sorafenib or sutent. In order to ensure good tolerance of the treatment for the patients the time of treatment should be as short as possible. In addition, prolongation of illumination time can cause pain in treated area [48]. Thus, efficacy of light excitation and utilization (i.e. strong light absorption and high intersystem crossing S1 → T1 quantum yield) of a photosensitizer is very important. Using a simple halogen lamp, we here demonstrated that at least in vitro ZnPcs can be effectively activated at rather low light doses of 10 J/cm2 which already caused cell death of almost 100% of the treated tumor cells. Based on these encouraging data it is thus, conceivable that the efficiency may even be enhanced when using more powerful light sources (e.g. laser), which could further improve the in vivo applicability of a ZnPc-based PDT. In conclusion, our findings show the extraordinary photoactive potential of the novel ZnPc photosensitizer, which may become an interesting candidate for innovative PDT treatment. Further investigations are required to explore the suitability and effectiveness of ZnPc-PS in different tumor entities in vitro and in vivo, alone or in combination with already established chemotherapeutics or antiangiogenic treatment. Conflicts of interest Authors declare no conflict of interest. Acknowledgements Weronika Kuzyniak was funded by a scholarship of the Studienstiftung des deutschen Volkes. Gustav Steinemann was supported by a scholarship of the FAZIT-Stiftung, Frankfurt a. M., Germany. References [1] B.R. Prasad, N. Nikolskaya, D. Connolly, et al., Long-term exposure of CdTe quantum dots on PC12 cellular activity and the determination of optimum non-toxic concentrations for biological use, J. Nanobiotechnol. 8 (2010) 7. [2] T.J. Dougherty, J.E. Kaufman, A. Goldfarb, et al., Photoradiation therapy for the treatment of malignant tumors photoradiation therapy for the treatment of malignant tumors, Cancer Res. 38 (1978) 2628–2635. [3] S. Anand, B.J. Ortel, S.P. Pereira, et al., Biomodulatory approaches to photodynamic therapy for solid tumors, Cancer Lett. 326 (2012) 8–16.

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Tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine for photodynamic cancer therapy.

Photodynamic therapy (PDT) has emerged as an effective and minimally invasive treatment option for several diseases, including some forms of cancer. H...
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