Photodynamic effects of the cationic porphyrin, mesotetra(4N-methylpyridyl)porphine, on microtubules of HeLa cells

Journal of Photochemistry and Photobiology B: Biology 27 (1995) 47-53

ELSEVIER

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Photodynamic effects of the cationic porphyrin, mesotetra(4Nmethylpyridyl)porphine, on microtubules of HeLa cells A. Juarranz *, A. Villanueva, V. Diaz, M. Cafiete Departamento de Biologia, Facultad de Ciencias, Universidad Aut6noma de Madrid, E-28049 Madrid, Spain

Received 17 February 1994; accepted 20 July 1994

i~bstract The treatment of H e L a human carcinoma cells with mesotetra(4N-methylpyridyl)porphine (T4MPyP) and blue light led to damage of the microtubules (MTs). The morphologies of interphase MTs and the mitotic spindle apparatus were analysed by immunofluorescence staining of a-tubulin. The extent of MT damage depended on the light dose and the time after photodynamic treatment. After a period of 1 h after irradiation with doses of 0.3 or 1.5 J cm -2 (sublethal conditions, corresponding to survival rates of 90% and 60% respectively), the normal MT network arrangement of interphase cells and 1he mitotic spindle apparatus of many cells were dearly affected. However, these effects were found to be transient, and ~,everal hours after irradiation most MTs resumed control morphology. Higher irradiation doses (4.5 J cm - : , lethal conditions, less than 10% cell survival) resulted in the irreversible alteration of interphase and mitotic NITs. The change in MT organization appeared to be the reason for the observed increase in the mitotic index (MI) after sublethal doses. The largest increase in IvlI was detected 6 h after treatment (twofold increase over untreated cells) for both sublethal light doses. Most of the cells in mitosis corresponded to metaphase, the number of ana-telophase cells being greatly reduced for the first hours after irradiation with a dose of 1.5 J cm -2. The results, which resemble those observed with inhibitors of MT assembly, suggest 1hat MTs might represent an important target for T4MPyP action. ,'~,ywords: Mesotetra(4N-methylpyridyl)porphine; Microtubules; Photosensitization

I. Introduction Many photosensitizers are currently under investi,gation for application in the photodynamic therapy (PDT) of cancer. This treatment is based on the ability of certain compounds to be retained preferentially in tumours. Subsequent exposure to appropriate light produces a sequence of events which destroys the tumour :ells [1]. The photodynamic process of the sensitizers an neoplastic tissues is still not well understood, although it is generally accepted that singlet oxygen (IO2) , produced after the exposure of the sensitizer to light, is the main species responsible for cell inactivation [2]. /'he cellular targets at which 10 2 seems to act are numerous, including the cell membrane [3,4], lysosomes [5], mitochondria [6] and nucleus [7-9]. It has also been reported that cell killing may be caused by the perturbation of microtubules (MTs) which produces a blockage of the cell cycle at mitosis. In this sense, * Corresponding author.

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several compounds including porphyrins (Photofrin II and sulphophenyl porphines) and phthalocyanines (tetrasulphonated aluminium phthalocyanine) in combination with light have been shown to be mitotic inhibitors [10-12]. The cationic, meso-substituted porphyrin, mesotetra(4N-methylpyridyl)porphine (T4MPyP) has been revealed as a potent photosensitizer in vivo [13] and in vitro [9]. This porphyrin has been described as a DNA intercalator in vitro [14], and can photoinduce DNA-protein cross-linking in solution [15], as well as genotoxic effects in carcinoma cells in culture [9]. In addition, T4MPyP causes the inhibition of polymerization of tubulin in solution and in the dark [16], but, as far as we know, no reports are available on its effects on MTs in cells in culture. In this work, we studied the ability of T4MPyP in combination with blue light to destroy MTs of HeLa human carcinoma cells. This was done by immunofluorescence staining of a-tubulin cells and by analysis of the accumulation of the asynchronous cells in mitosis.

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A. Juarranz et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 47-53

2. Materials and methods

2.1. Cell culture An HeLa human epithelial carcinoma cell line was used. The cells were routinely grown as a monolayer using Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum and antibiotics. The cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and the medium was changed daily. 2.2. Drug The porphyrin T4MPyP, as the tetraiodide (VentronAlfa Produkte, Germany), was prepared at a concentration of 10 -4 M in distilled water and sterilized by filtration through a Millipore filter. The stock solution was stored in the dark at 4 °C. For treatments, all dilutions were performed in DMEM with 1% foetal calf serum.

2.3. Treatments and light irradiation HeLa cells, grown on coverslips plated into 35 mm Petri dishes, were treated with 10 -5 M T4MPyP for 20 min. After incubation, the cells were washed three times with sensitizer-free DMEM with 10% serum and exposed to light doses of 0.3, 1.5 or 4.5 J cm -2, which corresponded to survival rates of approximately 90%, 60% and 10% respectively, estimated by the trypan blue method [9]. After irradiation, cells on coverslips were immediately (or further incubated) fixed and stained for scanning electron microscopy or immunofluorescence studies. Irradiation was carried out using a Reflecta slide projector equipped with a 150 W lamp. The light was filtered through a 3.0 cm water layer (to absorb heat) and a blue filter with a selected wavelength in the 360--460 nm range. The light intensity at the treatment site was 31 mW cm -2 (M8 spectrum power energy meter).

2.4. Scanning electron microscopy Cells for scanning electron microscopy were fixed by 2% glutaraldehyde in phosphate-buffered saline (PBS) solution (pH 7.3) and postfixed by 2% osmium tetroxide. The fixed cells were dehydrated in graded alcohol solutions, air dried, gold coated and examined in a Philips XL 30 scanning electron microscope.

2.5. lmmunofluorescence staining of a-tubulin At variable times after light irradiation (immediately, 1, 4, 6 and 24 h), cells were fixed in cold methanol ( - 2 0 °C) for 10 min and hydrated in graded ethanol-PBS solution (without CaClz and MgCI2). Cells

were later permeabilized with PBS containing 0.5% Triton X-100 for 30 min and labelled with a 1:1000 dilution of monoclonal anti-a-tubulin antibody (Amersham) for 1 h at 37 °C. After washing in PBS, cells were stained with a 1:50 dilution of anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody (Southern Biotechnology Associates) (1 h at 37 °C), rinsed again in PBS, and mounted in PBS-glycerol (1:9) containing 1,4-diazabicyclo(2.2.2)octane (25 mg ml-1, DABCO, Sigma). Photography and fluorescence observations were performed in an Olympus photomicroscope equipped with an HBO 100 W mercury lamp.

2.6. Mitotic index (MI) Coverslides treated for indirect immunofluorescence were used to determine MI. The mitosis was subdivided into prophase, metaphase and ana-telophase. Metaphases were further divided into typical, atypical and multipolar, depending on whether the spindle apparatus was disturbed or not, and on the number of spindles that could be seen in the same cell (see Fig. 1, Section 3). At least 5000 cells were counted, for each point, under the fluorescence microscope to estimate MI, and a minimum of 100 cells in division for the distribution within mitosis.

3. Results

The organization of the MTs of HeLa cells was studied after incubation with 10 -5 M T4MPyP (20 min) and subsequent exposure to light doses of 0.3, 1.5 or 4.5 J cm -2. These treatment conditions corresponded, as previously reported by Villanueva et al. [9], to survival rates of approximately 90%, 60% and 10% respectively. The polymerization state of the MTs of HeLa cells was examined by immunofluorescence staining of atubulin immediately and several hours after photodynamic treatment. Variable damage of MTs was found when cells incubated with T4MPyP were exposed to light. The extent of MT perturbation depended on both the irradiation dose and the time following treatment. Immediately after a light dose of 0.3 J cm -2, most of the interphase cells retained the flattened shape with apparently normal interphase MTs. Under these conditions, bundles of MTs irradiating from the perinuclear area to the cell edges, as in untreated cells, could be seen (Fig. I(A)). However, some of the cells were round in shape 1 h after light exposure, the number of long cytoplasmic MTs being greatly reduced and irregularly scattered over the cytoplasm (Fig. I(B)). MT damage was larger at doses of 1.5 J cm-2; the fraction of rounded cells immediately and 1 h after irradiation was greater than for a dose of 0.3 J cm-2 (Fig. I(B')). For both sublethal doses, the rounded

A. Juarranz et al. / J. Photochem Photobiol. B: BioL 27 (1995) 47-53

Fig. 1. Immunofluorescence staining of a-tubulin in HeLa cells treated with 10 -~ M T4MPyP (20 min), exposed to variable light doses and fixed at different times after photodynarnic treatment. (A, A') Untreated cells in interphase and in different stages of mitosis; typical metaphase (lower), ana-telophase (right) and end telophase-early G~ (left). (B, B') Interphase ceils exposed to doses of 0.3 and 1.5 J cm -2 respectively, and fixed 1 h after irradiation for immunostaining. (C, C') Cells treated with 4.5 J cm -2 and fixed immediately or 1 h after light exposure. (D, D') Main morphologies of atypical metaphases with perturbed mitotic spindle apparatus and a multipolar metaphase.

state was found to be transient, and 4 h after light irradiation the MTs of interphase cells returned to the control morphology. When lethal conditions (4.5 J cm -2) were used, most cells showed an irregular plasma membrane with bleb projections under the scanning electron microscope immediately after irradiation (Fig. 2). Whereas untreated cells showed a typical surface morphology, with numerous randomly distributed microvilli (Fig. 2(A)), treated cells showed numerous blebs on the plasma membrane (Fig. 2(B)). These membrane deformations seemed to be filled with a-tubulin (Fig. I(C)). After a period of 1 h after light exposure, most treated cells lacked blebs and showed an irregular rounded mor-

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Fig. 2. Scanning electron micrographs of HeLa cells. (A) Morphology of an untreated cell in interphase. (B) Cells treated with 10 -5 M of T4MPyP (20 min) and subsequent exposure to light (4.5 J cm-2); notice the blebs on the plasma membrane.

phology that made the observation of MTs difficult (Fig. I(C')); 4 h after treatment, most cells exhibited a very fiat shape reacting negatively with a-tubulin antibody. The structure of the mitotic spindle apparatus of the cells in division was clearly affected during the first hours after treatment with both sublethal doses when compared with controls (Fig. I(A')). Many metaphase cells exhibited a disorganized mitotic spindle, and cells with tubulin in grains dispersed over the cytoplasm could also be seen (Fig. I(D)). Multipolar mitosis was also observed, in particular, 6 h after treatment with 1.5 J cm -2 (Fig. I(D')). With lethal doses, the photosensitization of the mitotic cells was greater, the spindle of most mitotic cells being highly perturbed. Fig. 3 shows the MI after all the photodynamic treatments. Immediately after any of the light doses used, the MI was significantly lower (twofold lower) than untreated cells. Afterwards, MI progressively

50

A. Juarranz et al. / Z Photochem. Photobiol. B: Biol. 27 (1995) 47-53

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Fig. 3. MI of HeLa cells pretreated with T+MPyP and exposed to light doses of 0.3 J cm -2 ( e ) , 1.5 J cm -2 (A) or 4.5 J em -2 (11); control (neither T+MPyP nor light) ( - - - ) . The MI values of cells treated only with light or T+MPyP are not included; 5000 cells were counted for each point.

increased with time, when light doses of 0.3 and 1"5 J cm-2 were used" F°r b°th sublethal d°ses' the largest MI increase (twofold increase over untreated cells) was observed 6 h after treatment; 24 h following light irradiation, no significant differences in MI could be detected between the treated cells and controls. Cells in mitosis following the lethal light dose of 4.5 J cm -2 were negligible from 1 h after treatment. Fig. 4 shows the distribution of cells in the different phases of mitosis as a function of the time following photodynamic treatment with sublethal doses. It can be seen that the increase in MI observed in Fig. 3 is mainly due to an increase in the number of metaphase cells (typical, atypical and multipolar) under both irradiation conditions. The highest accumulation of metaphase cells was found 6 h after treatment with both doses (three times the control values). In the first 2 h after treatment, the number of ana-telophase cells was reduced with respect to the control, especially with doses of 1.5 J era-2 (Fig. 4(B)). The cell distribution in the different stages of mitosis 24 h after treatment was within the control values (prophase, 0.86% + 0.04%; metaphase, 2.45% + 0.06%; anatelophase, 0.89% +0.03%). Fig. 5 shows the percentage of typical and atypical metaphases with the mitotic spindle organized or not

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Fig. 4. The percentage of mitotic phases after doses of 0.3 J cm -2 (A) or 1.5 J cm-2 (B) of cells treated with 10 -5 M T, MPyP: prophases ( e ) ; total metaphases (A, including typical, atypical and multipolar); ana-telophases (R). The indices of the different phases of mitosis of untreated cells were estimated to be: prophase, 0.86% +0.04%; metaphase, 2.45% + 0.06%; ana-telophase, 0.89%---0.03%.A minimum of 100 mitoses were counted for each point.

(see Fig. 1), obtained from the total number of cells in division (prophase, metaphase and ana-telophase), following irradiation with sublethal doses. As depicted, for the first hours after irradiation, the percentage of atypical metaphases for a dose of 0.3 J c m - 2 (Fig. 5(A)) was lower than for a dose of 1.5 J era -2 (Fig. 5(B)). The highest accumulation of atypical metaphases was found, in both cases, 4-6 h after treatment, but 24 h after treatment the percentage of atypical meta-

A. Juarranz et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 47-53

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Fig. 5. Percentage of typical and atypical (including multipolar cells) metaphases, obtained from the total number of cells in division, as a function of the time after light irradiation doses of 0.3 J cm -2 (A) or 1.5 J era-2 (B). The greatest standard deviation (SD) ( + 10.14) w a s found for atypical metaphases 1 h after treatment with d o s e s of 0.3 J c m -2.

phases was within the control range. The number of multipolar mitoses increased significantly with time at doses of 1.5 J cm -2, reaching a maximum 24 h after irradiation (0.67% + 0.14% (treated) vs. 0.11% 5- 0.06% (untreated)). No significant increase in the number of multipolar mitoses was found at any time after doses of 0.3 J c m -2. The MTs of HeLa cells treated only with light were unaffected; however, the MTs of unirradiated cells immediately after incubation with T4MPyP were slightly disturbed, although they resumed control organization 1 h after drug removal. In both cases, the MI and cell

In this work, the effect of treatment with T,MPyP and light on the MTs of HeLa cells was studied. Indirect immunofluorescence staining of ~-tubulin showed damage to interphase MTs as well as perturbation of the mitotic spindle apparatus. The extent of damage was clearly dependent on the light dose. For sublethal light doses (more than 60% survival), MT disruption seemed to be transient, and control morphology was resumed several hours after exposure to light. However, lethal dose conditions (around 10% cell survival) resulted in an irreversible alteration of MTs. MTs are dynamic structures involved in many cellular functions, such as chromosome segregation, determination of cell shape and intracytoplasmic organization. All these processes are based on the capability of MTs to polymerize and depolymerize, and thus they are labile structures which are very sensitive to drugs [17,18]. The mitotic spindle is an example of MT dynamics; it is formed after cytoplasmic MT disassembly at the onset of mitosis, and thus is in a state of rapid assembly and disassembly, which explains the extreme sensitivity of this structure to MT inhibitors [18,19]. Therefore the effect of MT inhibitors on spindle MTs can be inferred from alterations in mitotic cells, such as metaphase arrest and the formation of multinucleated cells [20]. In this sense, we have experienced that treatment with T4MPyP and sublethal light doses leads to a temporary increase in the MI of HeLa cells. At lethal doses, no accumulation of cells in mitosis is observed as a result of irreversible cell damage. The increase in the MI detected after sublethal conditions is mainly due to an accumulation of metaphase cells for the first hours after treatment. Many of these metaphases are atypical, with a perturbed mitotic spindle, and the number of ana-telophase cells is greatly reduced. Similar results on cell accumulation in metaphase for human cell lines in vitro have been reported previously for several anionic porphyrins, such as Photofrin II, haematoporphyrin derivative (HpD), mono-, di- and tetrasulphophenyl porphines and tetrasulphonated aluminium phthalocyanine [10-12,21]. Metaphase arrest observed with these compounds is also due to perturbation of MTs in the spindle as in the case of T4MPyP. In addition, we have observed the photoinduction of blebs on the plasma membrane of HeLa cells immediately after incubation with TaMPyP and exposure to lethal doses. Such membrane deformations have been reported previously for several anionic porphyrins when they are linked to this structure [21,22]. Since T4MPyP

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A. Juarranz et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 47-53

has been described to be located preferentially at the nuclear level in cultured cells [9], the photoinduction of blebs could be explained by the location of sensitizer at the membrane level during irradiation (passing through the membrane on the way to the nucleus) (irradiation is carried out immediately after drug incubation). This could explain the sensitivity of treated mitotic cells to light, reflected by the reduced MI of treated cells compared with controls (see Fig. 3) immediately after irradiation; the possibility of detachment of treated mitotic cells should also be considered. However, the blebs of the interphase cells are filled with a-tubulin; thus it is possible to speculate that the photosensitizing effects on the plasma membrane may also be due to direct effects on the MTs linked to the membrane [23]. Indirect evidence indicates that blebs induced by disulphophenyl porphines are filled with unpolymerized tubulin [21]. It has been reported previously that T4MPyP, as well as other porphyrins, inhibits the assembly of MTs in solution and in the dark [16]. We have observed slight damage to MTs of HeLa cells immediately after incubation with T4MPyP; the damage to MTs and the increase in MI are much greater after light irradiation. Since the radius of action of singlet oxygen, the main photoproduct involved in cellular damage, is much shorter than 100 nm [24], T4MPyP should be located close to MTs to induce photodamage. In addition, specific binding of the sensitizer to tubulin, as has been suggested for some s~ )hophenyl porphines [11,12], may better explain the cytologic effects observed in HeLa cells, in particular the general reduction of the polymerization level of MTs and the mitotic arrest. Finally, the results shown indicate that MTs are important subcellular targets for the photodynamic action of T4MPyP in HeLa human carcinoma cells. Whether the sensitizer binds or not to tubulin cannot be concluded from these results,. The damage to the cytoskeleton induced by the PDT of cancer has not been studied in detail compared with other subcellular structures. However, the data presented here, as well as previous observations reported by other workers, strongly suggest that photodamage to the cytoskeleton could also contribute to cellular inactivation. Further studies of the effect of PDT on cytoskeletal elements, not only MTs, should be performed.

Acknowledgements We wish to thank Mrs E. Ilundain for valuable collaboration. This work was supported by a grant (PB870129) from the Direcci6n General de Investigaci6n Cientffica y Trcnica (DGICyT), Spain.

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[20] W.N. Dahl, R. Oftebro, E.O. Petersen and T. Brustad, Inhibitory and cytotoxic effects of oncovin (vincristine sulfate) on cells of human line NHIK 3025, Cancer Res., 36 (1976) 31013105. [21] K. Berg, J. Moan, J.C. Bommer and J.W. Winkelman, Cellular inhibition of microtubule assembly by photoactivated sulphonated meso-tetraphenylporphines, Int. J. Radiat. Biol., 58 (1990) 475-487.

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