Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn(II)-phthalocyanine

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B:I~IOLOGY

ELSEVIER

Journal of Photochemistry and Photobiology B: Biology 33 (1996) 219-223

Apoptosis of mouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn (II)-phthalocyanine Chuannong Zhou a, Chi Shunji a, Deng Jinsheng a, Liang Junlin a, Giulio Jori b,., Carla Milanesi b a Laboratory of Electron Microscopy, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, People's Republic of China b Department of Biology, University of Padova, Via Trieste 75, Padova 35121, Italy Received 30 March 1995; accepted 6 October 1995

bstract

The destructive process of mouse MS-2 fibrosarcoma induced by photodynamic therapy (PDT) with liposome-administered Zn(II)pathalocyanine (ZnPc) was studied by electron microscopy. Pronounced ultrastructural changes characteristic of apoptosis were observed f~r several tumour cells, including early occurrence of condensation and margination of chromatin, disappearance of nuclear pores, karyopyknosis, karyorrhexis, protuberance formation at the cell surface and cell fragmentation. The findings indicate that apoptosis was involved ~,~the process of tumour cell death induced by ZnPc-PDT. The detailed mechanism and pathways controlling this phenomenon need to be ,.~ucidated further. ~,eywords: Zinc(II)-phthalocyanine; Photodynamic therapy; Tumour cells; Apoptosis; Ultrastructure

1. Introduction Zn (II)-phthalocyanine (ZnPc) is a promising second gen~'ration photosensitizer for use in photodynamic therapy PDT) of tumours, because of its high selectivity of tumour t ~trgeting [ 1,2] and the high efficiency of photogeneration of ~ytotoxic singlet oxygen [3]. ZnPc gave excellent photot aerapeutic results when tested as a PDT agent in a variety of t amour models in mice [4]. Owing to its very low solubility i n water, ZnPc needs to be administered in vivo by means of Iiposome delivery systems [ 5 ], which favours its quantitative ~acorporation into serum lipoproteins [2,5]. This transport modality is likely to enhance the accumulation of the photo:,ensitizer in malignant ceils of a tumour tissue [ 6], although .tn additional contribution to tumour targeting could occur hrough liposome uptake by host macrophages and lympho:ytes which have infiltrated the cancer [7]. Previous ultra~.tructural studies from our laboratories [ 8] on the mechanism hy which ZnPc photosensitizes the necrosis of an intramusularly transplanted fibrosarcoma, demonstrated early and fighly preferential damage of malignant cells, especially at he level of several membranous systems. From this point of Aew, ZnPc differs significantly from both Photofrin (the * Corresponding author. Tel: + 39 49 8276333; Fax: + 39 49 8276344. !011 - 1344/96 / $15.00 © 1996 Elsevier Science S.A. All rights reserved ;;SDIIO1 1 - 1 3 4 4 ( 9 5 ) 0 7 2 5 0 - 0

PDT agent which is most frequently used for clinical treatments) and other second generation photosensitizers which act largely via vascular impairment [ 6]. In order to gain further information on these peculiar features of ZnPc photosensitization, we carried out more detailed electron microscope observations on tumour specimens taken at different times after PDT; the indications emerging from our studies suggest that apoptosis is involved in ZnPcphotoinduced death of malignant cells.

2. Materials and methods 2.1. Animals and tumour

Healthy female Balb/c mice ( 18-20 g body weight) were obtained from Charles River (Como, Italy). The MS-2 fibrosarcoma was supplied by Istituto Nazionale dei Tumori, Milan, Italy. The tumour was implanted intramuscularly in the right hind leg by injection of 0.25 ml of a cell suspension containing at least 106 cells ml-1. The experiments were started at 7 days after tumour implantation, when the tumours were 0.7-0.8 cm in diameter. Mice were housed in standard cages with free access to normal dietary chow and were taken care of according to the guidelines established by the Italian

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Committee for Experiments on Animals. Mice were anaesthetized by intraperitoneal injection of Ketalar during irradiation. At 3, 6, 12, 24 and 48 h after the end of the phototreatment, groups of three mice were killed by prolonged exposure to ether vapours. Small pieces of tumour tissue were taken and fixed immediately in 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 for 2 h at 4 °C, post-fixed in 1% osmium tetroxide for 1 h, dehydrated and embedded in Epon. The thin sections were double stained with uranyl acetate and lead citrate and examined with a Philips EM-410 transmission electron microscope. Control samples were taken from tumours of three untreated mice, as well as three mice that received light irradiation alone and three mice that received ZnPc alone. 2.2. Photosensitizer formulations

ZnPc was a gift from Ciba-Geigy (Basel, Switzerland) and was used as received. The liposome-ZnPc system (final ratio [phospholipid] / [phthalocyanine] = 7 0 ) was prepared by incorporation of ZnPc into small unilamellar vesicles of L-a-dipalmitoylphosphatidylcholine (DPPC) following the procedure described by Valduga et al. [9]. The liposomes were prepared in 0.9% aqueous NaC1 solution and had a diameter of ca. 50 nm. The ZnPc concentration in the liposome vesicles was measured by absorbance at 673 nm using ~= 2.41 × 105 M -~ cm -~ in pyridine [9]. 2.3. Phototherapeutic studies

The mice bearing an MS-2 fibrosarcoma were depilated in the area of the neoplastic mass and injected in the femoral vein with 0.12 m g / k g ZnPc incorporated into DPPC liposomes. At 24 h post-injection the tumour area was irradiated by 600-700 nm light isolated from the emission of a quartz/ halogen lamp (Teclas, Lugano, Switzerland) by means of optical filters. The light beam was focused into a bundle of optical fibers whose tip was kept at a distance of 1 cm from the irradiated tumour area. The lamp was operated at a fluence rate of 150 m W c m - 2 for a total light dose of 300 J c m - 2; a circular light spot of 1.5 cm diameter was focused on the tumour area. Under these conditions, extensive tumour necrosis is obtained, eventually leading to tumour eradication as shown in previous PDT studies with ZnPc in the same animal model [ 2,8]. However, in the absence of photosensitizer, no effect of irradiation on the tumour is noticed either at the ultrastructural level or by comparison of the rate of tumour growth with that typical of untreated control mice.

3. Results The main ultrastructural features of untreated MS-2 fibrosarcoma in Balb/c mice were described previously [8,10]. The images obtained during the present investigations were identical with those reported previously; in particular, no

Fig. 1. A tumour cell from a Z,nrc-treated mouse, 3 h after PDT. The cell has shrunk and cap-like condensed chromatio is present at one pole of the nucleus (N), × 14 000. Fig. 2. At 3 h after PDT, a tumour cell shows crescent chromatin condensation at the periphery of the nucleus (N). The arrows indicate a roughly spherical apoptotic body, containing strongly condensed chromatin, × 14 ooo. typical apoptosis was observed in nine thin sections from three control mice. The overall mechanism of ZnPc-photosensitized tumour destruction, as deduced by ultrastructural analysis, was also essentially identical with that described elsewhere [ 10]. The peculiar micrographs shown in this paper refer to malignant cells and do not involve endothelial cells. In ZnPc-treated mice, at 3 h after the end of PDT, few tumour cells show cap-like or crescent chromatin condensation in their nuclei, as well as dilatation of perinuclear space and RER profiles (Figs. 1, 2). Furthermore, one can observe the presence of roughly spherical apoptotic bodies, which contain strongly condensed chromatin material (Fig. 2). Some tumour cells have shrunk and a number of blunt protuberances are present at the cell surface (Fig. 3). At 6 h after PDT, a few cells show characteristic cap-like condensed chromatin at one nuclear pole (Fig. 4) and occa-

Fig. 3. At 3 h after PDT, several cells have shrunk and show an elongated shape, while blunt protuberancesare evident at the cell surface, as indicated by the arrows, × 10 000. Fig. 4. At 6 h after PDT, a tumour cell shows condensationof the cytoplasm and cap-like condensedchromatinmass adherent on the nuclear membrane, × 15 000.

C. Zhou et al. / Journal of Photochemistry and Photobiology B: Biology 33 (1996) 219-223

Fig. 5. At 6 h after PDT one can observe the characteristic cap-like condensed aromatin in the nucleus (N) and the oedematous protuberance (arrows) the cell surface, X 14 000. F ~g. 6. Micrograph taken at 12 h after PDT: disintegration of a dead tumour ; ~11 with a highly condensed nuclear chromatin and numerous large cyto~qasmic vacuoles, x 13 000.

'igs. 7, 8. Tumour cells from a ZnPc treated mouse, 24 h (Fig. 7) and 48 h Fig 8) after PDT. The tumour tissue is completely disintegrated, with 4 vidence of karyorrhexis and disruption of the plasma membrane, Fig. 7, .,' 10 000;Fig. 8, ×5000.

:~ionally cell fragments still surrounded by a membrane can !~e detected. Furthermore, some neoplastic cells show oedenatous protuberances at their cell surface (Fig. 5). A severe degeneration of most tumour cells takes place by 2 h after irradiation. As shown in Fig. 6, there is karyopyk~osis with prominent condensed chromatin aggregates and ~aumerous cytoplasmic vacuoles of varying size. Some cells ;tre even disintegrated. At this time the features of apoptotic :ell degeneration cannot be differentiated clearly from those ypical of cell necrosis. Lastly, at 24 and 48 h after PDT, the tumour tissue is ,eriously damaged and a large number of dead tumour cells ~re present (Figs. 7,8). The general organization of the tissue s lost.

~. Discussion In spite of active research spanning over more than 20 years, the mechanisms by which PDT induces tumour

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destruction are not yet completely understood. At least in part, conflicting experimental results may reflect differences in the subtissular and intracellular distribution of the photosensitizing agent. Thus, while hydrophilic or moderately hydrophobic porphyrinoids (such as Photofrin, or tetrasulphonated phthalocyanines) act largely via early vascular damage, more hydrophobic derivatives, which are transported almost exclusively by serum lipoproteins [6], cause the direct preferential inactivation of malignant cells [ 8,10]. Macrophages also appear to play an important role in the photosensitizer uptake by tumours and the subsequent photodynamic effect [7]. Previous studies from our laboratories [ 10] pointed out that liposome-delivered ZnPc photosensitizes an early and irreversible damage of neoplastic cells, at least in the present tumour model; other tissue compartments are affected at a significantly lower rate. In general, cell death may occur through either necrosis, namely a degenerative phenomenon of uncontrolled and random nature, or apoptosis, which is a process triggered and regulated by specific steps of gene expression and metabolic pathways, and requiring active cell participation [ 11,t2]. Cells undergoing apoptosis show typical morphological changes including deformation of nuclei, chromatin condensation, karyopyknosis and subsequent karyorrhexis, floating of chromatin aggregates in the cytoplasm, formation of blebs on the plasma membrane, and DNA fragmentation consequent to the activation of endogenous endonucleases [ 13 ]. Several of these morphological features are observed repeatedly in the micrographs obtained with ZnPcphotosensitized tumour specimens at different post-irradiation times; hence, the overall pattern is consistent with the hypothesis that ZnPc-PDT of the MS-2 fibrosarcoma induces cell apoptosis. Apoptosis would be expected at a low frequency even in untreated tumours [ 12]. However, we did not find typical apoptosis in nine examined thin sections cut from three control tumours, while we observed at least one tumour cell (one to four cells) with typical features of apoptosis in each section (three sections from each mouse) from tumours taken at 3 and 6 h after PDT. Therefore, it would be highly unlikely that the apoptosis detected in the PDT-treated tumour tissues were occurring spontaneously. This conclusion is not unexpected, since different photosensitizers, including Photofrin, Pc IV (a specially developed phthalocyanine) and chloroaluminium-phthalocyaninetetrasulphonate were shown to photoinduce apoptosis of cells in vitro and tumour cells in vivo, including murine lymphoma, hepatoma and a radiation-induced fibrosarcoma (RIF-1) grown in C3H/HeN mice [ 14-18]. Apoptotic changes, such as chromatin condensation around the periphery of nuclei and the formation of nucleosome-size DNA fragments, were evident in the tumour tissue by 1 h after the end of phototreatment, suggesting that apoptosis represents an early event in tumour shrinkage following PDT treatment [ 14,15]. Analogously, we observed a number of apoptotic bodies in spec-

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imens taken at 3 h, i.e. the shortest post-irradiation time analysed by us. PDT-induced apoptosis has been detected in a variety of photosensitized cells [ 14,16-19]. In some cellular systems, however, PDT does not induce evident apoptosis [ 17,18 ], suggesting that PDT-induced apoptosis is not a universal event for all cancer cell lines [ 19,20]. Cytoplasmic free calcium plays an important role in the regulation of many intracellular systems, for example as a second messenger in signal transduction. The process of apoptosis involves an important participation of Ca 2+, through activation of a Ca 2 +-dependent endonuclease [20]. PDT was shown to cause a transiently increased intracellular concentration o f Ca 2+ ions [21,22]. However, it has been reported [ 21 ] that an increased Ca 2 + concentration after PDT contributed to survival o f the treated Chinese hamster ovary ( C H O ) cells and T24 human bladder transitional carcinoma cells by triggering a cellular rescue response. The proper explanation o f such a unique phenomenon and the exact relationship between increased intracellular free calcium concentration and photodynamic induction of apoptosis are not yet completely elucidated. Apoptosis is antagonized by Bcl-2 protein, which is located in the outer mitrochondrial membrane, endoplasmic reticulum and nuclear membrane [ 23,24] ; if one or more membranous systems are photosensitized by porphyrin-type compounds, it appears reasonable to correlate this event with the known localization of this class of photosensitizer in the cellular membranes. This is also in agreement with the PDTinduced rapid activation of phospholipase c and breakdown of membrane phosphoinositides [ 16]. Thus, photodynamic damage of cell membranes could mimic natural stimuli of phospholipases and initiate apoptosis of tumour cells [ 16]. Although we demonstrated that the highly hydrophobic photosensitizer Zn(II)-phthalocyanine incorporated in liposomes is able to induce photodynamic death of tumour cells via apoptosis, the detailed mechanism and regulating pathways of this process remain to be explored further.

Acknowledgement

This research received financial support from EU under the project " H u m a n Capital and M o b i l i t y " ( P D T network), contract No. E R B C H R X C T 930178.

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