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In vitro cytotoxicity assessment of phthalocyanines on virus-transformed animal cells Radostina Alexandrovaa, Elena Stoykovab, Rodica-Mariana Ionc, Kristina Nedkovab, Elena Ivanovab, Kaloian Zdravkovb, Georgi Minchevb, a Institute of Experimental Pathology and Parasitology, Acad. G. Bonchev Str., Bl. 25, 1113 Sofia, Bulgaria, b Central Laboratory for Optical Storage and Processing of Information, Acad. G. Bonchev Str., Bl.101, P.O. Box 95, 1113 Sofia, Bulgaria c Institute for Chemical Research - ICECHIM, Chemical Analysis Dept., Splaiul Independentei 202, Bucharest-77208, Romania ABSTRACT In this study we evaluate in vitro the cytotoxic activity of two newly synthesized phthalocyanines (metallo-complexes) with absorption bands in the red part of the spectrum: zinc-tri-sulphonated phthalocyanine (ZnS3Pc) and zinc- tetrasulphonated phthalocyanine (ZnS4Pc). Two permanent animal tumor cell lines are used in the experiments: LSR- SF(SR) - transplantable sarcoma in rat induced by Rous sarcoma virus strain Schmidt-Ruppin and LSCC-SF(Mc29) - transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29. Irradiation has been performed with a laser diode emitting at 672 nm in a wide range of radiant exposures (2 – 100 J/cm2) and irradiance of 120 mW/cm2. The neutral red uptake cytotoxicity assay has been used to evaluate the effect of the photosensitizers on cell viability. The light dose-response curves and light exposures that ensure viability drop to 50 % and 10 % have beenobtained for each cell line. The ability of the compounds to induce apoptosis has been determined by acridine orange dye staining. Keywords: photodynamic therapy, phthalocyanines, metallo-complexes, cytotoxicity, apoptosis . I. INTRODUCTION The outcome of the photodynamic therapy of cancer1,2 depends on too many parameters that characterize photochemistry, photobiology and photophysics involved in the photodynamic effect. One of the main problems in this type of treatment is to ensure an over-threshold fluence within the bulk of the treated tumors. This task stimulates development of second-generation photosensitizers with absorption in the “therapeutic window” (600 – 1000 nm) - a region that is far from the absorption peaks of the main tissue chromophores. Phthalocyanines (free-base, metallo- complexes) are promising photosensitizers (PS) for the photodynamic therapy. One of their strong features is the intense absorption in the red and near-infrared region. Their use permits deeper penetration of the laser light into biological tissues. In the present work we report on the cytotoxicity of two newly synthesized metallo-complexes of phthalocyanines - zinc-tri-sulphonated phthalocyanine (ZnS3Pc) and zinc-tetrasulphonated phthalocyanine (ZnS4Pc) - using animal permanent cell lines established from virus-induced transplantable tumors and irradiation at wavelength of 672 nm. A strong additional motive to use these cells is the still existing need to clarify the photodynamic effect on virus-induced cancers. The neutral red uptake assay and fluorescence assay after staining with acridine orange were used to study cytotoxicity and apoptotic/necrotic potential of the photosensitizers. II. MATERIALS AND METHODS Cell lines. The following cell lines were used: LSR-SF(SR) - transplantable sarcoma in rat induced by Rous sarcoma virus strain Schmidt-Ruppin; LSCC-SF(Mc29) - transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29. Cells were routinely grown as monolayer cultures in a combination of E-199 and Minimum Essential medium AppliChem, Germany) supplemented with 5 to 10% fetal calf serum (BioWhittaker, Belgium), penicillin (100 U/ml) and streptomycin (100g/ml). Cytotoxicity assays. The effect of both photosensitisers on viability of tumor cells was evaluated using a neutral red uptake cytotoxicity test. The cells were seeded in 96-well (8 rows, 12 columns) plates (Nunclon) at 2 x 10 4 cells/well. At the 24th h the cells from the monolayers were washed and covered with media modified with one of the PSs. 24 h after the irradiation, the cells were washed with phosphate-saline buffer (PBS) and 100 µl culture medium containing 0.0075% neutral red was added. The cells were incubated for 3 hours at 37 oC in a humidified atmosphere of 5% CO2, washed with PBS and 150 µl ethanol/water/acetic acid (50:49:1) solution was added. The plates were shaken on a micro-titer plate-shaker for 10 min. The absorption of the resulting colored solution was measured at 540 nm in a micro- plate reader. The readings of the reader itr ,c , where “i” is a well number, serve as a measure of the number of living cells, i.e. of cells’ viability. To avoid the unwanted activation of the PS, all procedures during the experiments were performed in a darken room. Samples of cells grown in non-modified medium served as a control. The induction of apoptosis was determined by acridine orange dye staining. Briefly, cells cultered on cover slips in 24- well plates were cultivated for 18-22h in the presence of different concentrations of the photosentitisers and exposed to irradiation. Twenty four hours after the light treatment the cells were fixed in methanol and incubated in 0.1% solution of acridine orange in 0.1M citrate buffer (pH 3.0) for 30 min at 37 oC. The cell observation was performed by fluorescent microscope . Irradiation. The normalized absorption spectra of both photosensitizers measured with a two-beam UV-Vis-NIR- spectrophotometer Cary-5E (Varian) are shown in Fig.1. The phthalocyanines have been prepared in the laboratory, and purified by thin layer chromatography. Their purity has been checked by X-ray diffractometry, FTIR spectrometry and UV-Vis spectrophotometry. The cells were exposed to irradiation from a laser diode emitting at 672 nm. The laser spot had a rectangular shape that covered uniformly four adjacent wells. The spectral width of the emitted light was ~ 10 nm. As it can be seen, the emission wavelength practically coincides with the main absorption peak of the ZnS 3Pc. In the case of the ZnS4Pc emission at 672 nm corresponds to 70 % of the maximal absorption. Radiant exposures from 2 to 100 J/cm2 were delivered at irradiance of 120 mW/cm2. This irradiance had been chosen after test measurements at 12, 24, 60 and 120 J/cm2, which showed a rather weak influence of this parameter on the photodynamic effect. No influence of the irradiance and radiant exposure on the viability of the control cells had been established. Fig.2 gives the histograms of itr ,c fluctuations around the mean value for the control cells at different irradiances after delivery of 8 J/cm2. The histograms obtained at higher radiant exposures are characterized with the same spread. III. RESULTS AND DISCUSSION. ˆ ˆ ˆ The output of the neutral red uptake cytotoxicity test is the estimate, Vtr Vc 100% , of the mean relative cell N ˆ viability, , where Vtr ,c 1 N v i i tr ,c are the estimates of the mean values of viability of the treated and control cells respectively, and N is the number of the used wells. As a rule, N = 8. The plots in Figs. 3-6 present the so-called light- dose response curves, which give as a function of the radiant exposure, in J/cm2. To decrease the possible sources of errors, the data comprising a given curve were obtained in the same ambient conditions. For the purpose we divided the 96-wells micro-plate in 5 or 6 sections, each containing two columns with control and treated cells respectively, and delivered increasing radiant exposures to them. As it has been discussed earlier3, survival curves consist of a part of moderate decrease and a rapid fall that starts after a certain exposure has been exceeded. The lower the PS’s concentration, the longer is the plateau section of the survival curve. For this reason, the delivered 5 or 6 radiant exposures on a single micro-plate did not ensure 90 %-mortality in all the experiments. To improve the accuracy of viability evaluation, we subtracted the background from the itr ,c data. To estimate the background level, we left an empty row in each microplate and added the neutral red to the cells of this row. The vertical bars in Figs.3-6 give the standard deviation, 2 2 2 N 1 tr Vtr c Vc2 , of ˆ 2 2 2 which depends on the standard errors of both ˆ mean estimates Vtr ,c , where tr ,c are the standard deviations of fluctuations for the treated and the control cells. The ˆ dashed lines in Figs.3-6 indicate 50 and 90 %-mortality of the treated cells. The values of the light dose P50%, which causes 50% mortality, are given for each figure. Fig.1. Normalized absorption spectra of the tested phthalocyanines. 1.0 1.0 Normalized absorption coefficient Normalized absorption coefficient zinc-tetra- 0.8 sulphonated zinc-tri-sulphonated 0.8 phthalocyanine phthalocyanine ZnS4Pc 0.6 ZnS3Pc 0.6 0.4 0.4 0.2 0.2 0.0 0.0 450 500 550 600 650 700 750 450 500 550 600 650 700 750 Wavelength, nm Wavelength, nm 70 Counts Counts 70 Control cells LSR-SF-SR 60 LSR-SF-SR 60 50 50 40 40 2 Control cells 24 mW/cm 30 2 30 12 mW/cm 20 20 10 10 0 0 0.7 0.8 0.9 1.0 1.1 1.2 0.7 0.8 0.9 1.0 1.1 1.2 Viability/mean value Viability/mean value LSR-SF-SR Counts Counts 70 Control cells LSR-SF-SR 70 2 60 2 60 120 mW/cm 60 mW/cm 50 50 40 40 Control cells 30 30 20 20 10 10 0 0 0.7 0.8 0.9 1.0 1.1 1.2 0.7 0.8 0.9 1.0 1.1 1.2 Viability/mean value Viability/mean value Fig.2. Fluctuations of the viability of the control cells at different irradiances. The delivered light dose is 8 J/cm2. 0.5 g/ml 100 LSCC-SF-Mc29 0.5 g/ml 100 1 g/ml 90 1 g/ml 90 5 g/ml 80 5 g/ml 80 10 g/ml 10 g/ml Relative viability, % Relative viability, % 70 70 60 60 50 50 40 40 ZnS3Pc 30 30 20 LSR-SF-SR 20 ZnS3Pc 10 10 0 0 10 100 10 100 2 2 Radiant exposure, J/cm Radiant exposure, J/cm Fig.3. Cell viability of the tumor cell line LSR-SF-SR treated Fig.4. Cell viability of the tumor cell line LSCC-SF- with the ZnS3Pc as a function of the radiant exposure at Mc29 treated with the ZnS3Pc as a function of the different PS’s concentrations. The values of P 50% are as radiant exposure at different PS’s concentrations. The follows: - > 100 J/cm2, - 35-40 J/cm2, - 18–20 J/cm2, values of P50% are as follows: - 70-80 J/cm2, - 45- - 5 - 6 J/cm2. 50 J/cm2, - 18–20 J/cm2, - < 4 J/cm2 . 100 LSR-SF-SR 100 1 g/ml 0.5 g/ml 90 90 5 g/ml 1 g/ml 80 80 10 g/ml 5 g/ml Relative viability, % Relative viability, % 70 70 10 g/ml 60 60 50 50 40 ZnS4Pc 40 LSCC-SF-Mc29 30 30 20 ZnS4Pc 20 10 10 0 0 10 2 100 10 100 Radiant exposure, J/cm Radiant exposure, J/cm 2 Fig.5. Cell viability of the tumor cell line LSR-SF-SR Fig.6. Cell viability of the tumor cell line LSCC-SF- treated with the ZnS4Pc as a function of the radiant Mc29 treated with the ZnS4Pc as a function of the exposure at different PS’s concentrations. The values radiant exposure at different PS’s concentrations. The of P50% are as follows: - ~ 30 J/cm2, - 15-20 J/cm2, values of P50% are as follows: - 65-70 J/cm2, - > - 15–20 J/cm2, - 3 J/cm2. 60 J/cm2, - 30-40 J/cm2 . We observed the photodynamic action of both PSs at the tested small concentrations from 0.5 up to 10 g/ml. The obtained results confirm the general observation that the photodynamic effect produced by a certain PS can strongly vary with the cell line. Comparing the P 50% doses in the case of 10 g/ml we see that they are rather low (3-5 J/cm2). It should be noted the large difference between P50% (10 g/ml) and P50% (5 g/ml) which is about 16-20 J/cm2. The only exception is the line LSCC-SF-Mc29 when being treated with the ZnS4Pc. The obtained low values of P50% (10 g/ml) are comparable with P50% (10 g/ml) doses for the LSCC-SF-Mc29 line when being treated with the TS4PP [5,10,15,20- tetra (4-sulfophenyl) porphyrin] at 511 nm3. In the case of the cell line LSR-SF-SR both ZnS3Pc and ZnS4Pc show much better results than the TS4PP, which requires P50% (10 g/ml) about 16-20 J/cm2. It is interesting to compare the obtained values of P50% with the results of treatment of human tumor cell lines 4. Ref.4 gives P50% (10 g/ml) between 5 and 10 J/cm2 and P50% (5 g/ml) about 14-15 J/cm2 for both ZnS3Pc and ZnS4Pc when being applied to 8 MG BA (glioblastoma multiforme) and MCF-7 (breast adenocarcinoma). The fluorescent microscopic observation of the PS/light treated tumor cells after acridine orange staining revealed some typical for the apoptosis changes such as cell shrinkage, cytoplasmic condensation, pyknotic nuclear chromatine and membrane blebbing. (Fig.7 and 8 ). Fig.7. Acridine orange staining of LSCC-SF(Mc29) Fig. 8. Acridine orange stainiing of LSCC-SF(Mc29) cells cells treated with 1 ug/ml ZnS3Pc, 20 J/cm2 (x 250) treated with 5 ug/ml ZnJ4Pc, 20 J/cm2 (x 250) CONCLUSION Both photosensitisers tested exhibit pronounced cytotoxic activity on the virus-transformed cells used in the experiments. A series of experiments are underway to study the potential mechanism of action of these metallo- complexes of phthalocyanines, especially their apoptotic/necrotic potential. Further in vitro investigations are planned to reveal the difference between cytotoxic and cytostatic effects of these compounds as well as to compare the influence of the photosensitisers on tumor and non tumor cells. ACKNOWLEDGEMENTS The authors would like to thank the National Science Fund to the Ministry of Education and Science of Bulgaria, Project Ph1313, and COST D20, WG 0012/02. REFERENCES 1. McCaughan Jr., J.S.. “Photodynamic therapy”, Drugs&Aging, 15, 49-68, 1999 2. Sibata C.H., V.C. Colussi, N.L. Oleinick, T.J. Kinsella, “Photodynamic therapy in oncology”, Expert. Opin.Pharmacother., 2, 1-11, 2001 3. Alexandrova R., Sabotinov O., Stoykova E., Ion R.-M. , Shurulinkov S., Minchev G., “In vitro cytotoxicity assessment of [5,10,15,20-tetra (4-sulfophenyl) porphyrin] on tumor and non-tumor cell lines”, Proc. SPIE, vol. 5449, pp.227-234, 2004. 4. E. Stoykova, R. Alexandrova, K.Nedkova, K. Zdravkov, O. Sabotinov, G. Minchev, R. Ion, “In vitro cytotoxicity assessment of phthalocyanines on human tumour cells”, CD-Proc. of the 4th European Symp. on Biomedical Engineering, Patras, Greece, 25-27 June 2004 (4 pages).
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