João Nuno Silva

Antoine Galmiche

João P C Tomé

Agnès Boullier

Maria G P M S Neves

Eduarda M P Silva

Jean-Claude Capiod

José A S Cavaleiro

René Santus

Jean-Claude Mazière

Paulo Filipe

Patrice Morlière
email: morliere.patrice@chu-amiens.fr

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes

Keywords: methyladenine, ALA, 5-aminolevulinic acid, BCC, Basal cell carcinoma , protoporphyrin IX, ROS, reactive oxygen species, SDS, sodium dodecyl sulfate, TBARS, thiobarbituric acid reactive substances, TX100, triton X100 PDT, photocytotoxicity, necrosis, autophagy, caspases, MAPK pathway

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tricationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene--D-galactopyranosyl (P-OGal) or N,N´dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC = P-Me >> P-H.

The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by -aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.

A c c e p t e d M a n u s c r i p t

Introduction

The treatment of malignant or benign skin diseases by photodynamic therapy (PDT) has developed rapidly [START_REF] Soler | A follow-up study of recurrence and cosmesis in completely responding superficial and nodular basal cell carcinomas treated with methyl 5-aminolaevulinate-based photodynamic therapy alone and with prior curettage[END_REF]. Current evidence indicates that topical PDT is effective in actinic keratosis, Bowen's disease and basal cell carcinoma (BCC) but it is a relatively poor treatment option for either squamous cell carcinomas or nodular BCC. In current skin PDT, topical application of aminolevulinic acid (ALA) or its methyl ester (MAL), precursors of protoporphyrin IX (PPIX), is followed by illumination of the diseased area with red light. The poor water solubility of these agents requires expensive preparation of formulations for systemic or topical administration.

Hence, development of new photosensitizers with better effectiveness is of current interest. Such new photoactive substances might demonstrate better clinical and histological efficacy in treating skin lesions for which topical MAL-PDT is ineffective (e.g. melanoma) or might be used as a palliative treatment for patients with plaque-type cutaneous T-cell lymphoma. Despite superior absorbance in the 700 nm region (molar absorptivity: ~2 10 5 M -1 cm -1 ) phthalocyanines are not currently used in the PDT of epidermal skin lesions (normally ~1-2 mm thickness). They tend to form aggregates of reduced uptake and photosensitizing properties and the phthalocyanine ring resists to biodegradation [START_REF] Conneely | Study of the white-rot fungal degradation of selected phthalocyanine dyes by capillary electrophoresis and liquid chromatography[END_REF]. However, their encapsulation in silica nanoparticles would appear to provide a promising approach to photosensitizer uptake and delivery as it has already been shown to improve the aqueous solubility and stability of the silicon phthalocynanine 4 [START_REF] Zhao | Enhanced photodynamic efficacy towards melanoma cells by encapsulation of Pc4 in silica nanoparticles[END_REF]. For these reasons, the synthesis of water-soluble derivatives of porphyrins and chlorins has been investigated. Conjugation of amino-acids, peptides and sugars to photosensitizers favours specific targeting since such groups play key roles in the recognition and metabolism in micro-organisms and cells [START_REF] Chen | Efficient synthesis and photodynamic activity of porphyrin-saccharide conjugates: targeting and incapacitating cancer cells[END_REF][START_REF] Tomé | Synthesis and anti-bacterial activity of new poly-S-lysine-porphyrin conjugates[END_REF].

In this study, new water-soluble derivatives of 5-(4-carboxyphenyl)-10,15,20-tris(4-A c c e p t e d M a n u s c r i p t 4 methylpyridinium-4-yl)porphyrin tri-iodide (P-R), whose absorbance maxima are shifted to the red by 15 to 20 nm as compared to PPIX, have been shown to be of potential interest in dermatological PDT since a poly-S-lysine conjugate is, in vitro, an effective photosensitizer towards rapidly proliferating human skin keratinocytes [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. Furthermore, tricationic porphyrins are promising molecules since they may bind to negatively charged skin cells after disruption of the Stratum Corneum barrier. In this study, the photocytotoxicity effectiveness of conjugates with a methyl (P-Me), a dicyclohexylureidooxy group (P-DDC), a di-O-isopropylidene--Dgalactopyranosyl group (P-OGal), a /-D-galactopyranosyl group (P-Gal) has been evaluated in cultured proliferating human skin keratinocytes for allowing the direct comparison of results obtained with the poly-S-lysine conjugate (P-(Lys) n ) [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. We have also determined the molecular mechanisms involved in the photo-induced cell death, the induction of a specific death mode being susceptible to result in a therapeutic advantage.

Materials and Methods

Chemicals, culture media and routine spectroscopic equipment

Reagents for cell culture, minimum essential medium with Earle's salts (EMEM), Hanks' balanced saline solution containing 20 mM Hepes (HBSS), Dulbecco's phosphate-buffered saline (DPBS), all without phenol red, fetal calf serum (FCS), trypsin and antibiotics were purchased from Sigma Chemical Co (St Louis, MO, USA). Chemicals and reagents were obtained from Sigma chemical Co, Merck (Darmstadt, Germany), Fluka (Saint-Quentin Fallavier, France) and Molecular Probes (Eugene, OR, USA) [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. Inhibitors of p38 (SB203580) and JNK (SP600125) were purchased from Calbiochem (Darmstadt, Germany). Specific antibodies against caspase-3, caspase-8, caspase-9 and p62/SQSTM1 were provided by Santa Cruz Biotechnology (Heidelberg, Germany) whereas those against total p38, phospho-p38, total JNK, phospho-JNK, total Akt, A c c e p t e d M a n u s c r i p t 5 phospho-Akt, total ERK, phospho-ERK were purchased from Cell Signaling (Beverley, MA, USA). The synthesis of the porphyrin conjugates (P-R) (Fig. 1) was performed as described by Tomé et al. [START_REF] Tomé | Synthesis and anti-bacterial activity of new poly-S-lysine-porphyrin conjugates[END_REF][START_REF] Tomé | Synthesis of neutral and cationic tripyridylporphyrin-D-galactose[END_REF]. The 500 µM stock solutions were prepared in water:DMSO (1:1 v/v) and stored at 0-4 °C. A UVIKON 943 spectrophotometer from Kontron Instruments (Montigny les Bretonneux, France) was used for optical absorption spectrophotometry. Fluorescence measurements were carried out with a SLM AMINCO-BOWMAN (series 2) fluorometer (Bioritech, Chamarande, France).

Cell culture and treatment

The immortalized NCTC 2544 human skin keratinocyte cell line was purchased from ICN Flow (Fontenay sous Bois, France). Cells were propagated, grown and seeded as earlier described [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF].

For fluorescence microscopy, cells were seeded on 30 mm diameter and 0.15 mm thick coverglass at a density of about 1,700 cells/cm 2 (2.5 mL at about 5,000 cells/mL) and were grown for 4 days. The incubations were carried out with 1 mL of P-R at various concentrations and for various times in either HBSS or 10% FCS-supplemented EMEM. In the case of endogenous PPIX synthesis, incubations were performed with 0.2 to 0.8 mM ALA in 10% FCS-supplemented EMEM. After incubation, cells were washed twice with 4 mL of HBSS before measurements.

Irradiation

Washed cell monolayers, covered with 1mL of HBSS without photosensitizer, were irradiated with broad band red light provided by a custom-built table consisting of two 300 watt tungstenhalogen lamps whose light was filtered with Balzers Y54 and calflex 3000 optical filters [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF][START_REF] Morlière | Tolyporphin: A natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo[END_REF].

Petri dishes were placed on a 30 x 25 cm thermostated (37 °C) glass table above the lamps. Under these conditions, most of the light originates from wavelengths in the range 500 -750 nm as determined with a CS 1000A Minolta spectroradiometer. The lamp actinometry is described in the supplementary data.

A c c e p t e d M a n u s c r i p t 6

Determination of intracellular photosensitizer concentration

Immediately after P-R or ALA incubation and washings, cells were mechanically scrapped in 1 mL of water. After collection, 100 µL of an 11% SDS solution in 10 mM phosphate buffer (pH 7.0) were added to the disrupted cell suspension. Fifty µL of this solution were saved for protein determination using Lowry's method with Folin reagent. The remainder was utilized for fluorometric measurement of photosensitizer concentration, using standard P-R solutions for calibration (λ exc = 427 nm and λ em = 659 nm) or standard PPIX solutions (λ exc = 407 nm and λ em = 636 nm) [START_REF] Reyftmann | Interaction of human serum low density lipoproteins with porphyrins: a spectroscopic and photochemical study[END_REF]. Data are the mean ± SD of at least three independent experiments, each performed in triplicate.

Neutral Red uptake assay

The assessment of photocytotoxicity by the NR uptake assay has been validated by the European Union for the testing of phototoxic chemicals and for the classification and labelling of hazardous chemical (EU Commission Directive 2000/33/EC). This assay has been fully described by Filipe et al. [START_REF] Filipe | Contrasting action of flavonoids on phototoxic effects induced in human skin fibroblasts by UVA alone or UVA plus cyamemazine, a phototoxic neuroleptic[END_REF].

Thiobarbituric acid reactive substances (TBARS) assay

Immediately after irradiation or sham-irradiation, 0.9 mL of supernatants was collected. After addition of 90 µL of butylated hydroxytoluene (2% w/w in ethanol) samples were kept frozen until TBARS assay. Cells were washed twice with DPBS, scrapped and collected for protein determination. TBARS were fluorometrically determined in terms of malondialdehyde equivalent as earlier described [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. Background fluorescence obtained from cell-free areas was subtracted from all the acquired images.

Fluorescence microscopy equipment for photosensitizer localization

Flow cytometry

At various times after photosensitization, adherent cells were washed twice in ice-cold DPBS and detached with Accutase™ (Sigma-Aldrich Co). Cells were transferred to 15 mL conical tubes and gently washed with serum supplemented medium by low speed centrifugation at 1500 rpm for 5 minutes. Cell pellets were washed once with DPBS and then re-suspended in assay buffer (0.1M HEPES/NaOH, pH7.4; 140mM NaCl; 25mM CaCl 2 ) at a concentration of 1x10 6 cells/mL. Annexin V-FITC binding was assessed using the Santa Cruz Biotechnology Apoptosis Kit (Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer's protocol. After 15 min of incubation in the dark with annexin V-FITC (5 µg/mL) and propidium iodide (PI) (5 µg/mL), cell fluorescence was measured in the spectral regions 505-545nm (FL1) for A and 610-650nm (FL3) for PI with a Cytomics FC 500 flow cytometer (Beckam Coulter, Villepinte, France). Data analysis was performed using Cytomics Analysis software.

DNA fragmentation assay

DNA fragmentation was assessed through determining cytoplasmic accumulation of histoneassociated DNA fragments by an enzyme-linked immunosorbent assay (Roche Diagnostics, Mannheim, Germany). At different times after irradiation, the cell supernatant was removed, cells were washed twice with ice-cold DPBS and lysed with the kit buffer. Equal amounts of protein from the cytoplasmic fraction (100 µg, Bradford assay from Bio-Rad Laboratories, Marne-la-

Caspase assays

After irradiation cells were lysed in buffer (10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride, supplemented with 10 μg/mL pepstatin, aprotinin and leupeptin), sonicated and centrifuged at 14 000×g for 5 min at 4 °C. Supernatants were saved as representative cytosol extracts. Aminomethylcoumarin (AMC)conjugated peptide substrates at a final concentration of 20 μM were added to 40 μg of total cytosol protein and incubated for 30 min at 37 °C. The specific substrates used were: acetyl-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC) for caspase-3, acetyl-Ile-Glu-Thr-Asp-AMC (Ac-IETD-AMC)

for caspase-8 and acetyl-Leu-Glu-His-Asp-AMC (Ac-LEHD-AMC) for caspase-9 (AnaSpec, Inc, San Jose, CA, USA). The fluorescence of coumarin dyes was measured with a 96-microwell plate reader under excitation at 360 nm and emission at 460 nm.

Immunoblot analysis

Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. The protein concentration of cell extracts was determined using the Pierce BCA assay (Thermo Fisher Scientific, Cergy Pontoise, France) and equal amounts of proteins (40 µg) were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were immunoblotted using conventional procedures and revealed using an enhanced chemiluminescence detection kit from Amersham (Saclay, France). Western blots were scanned and quantified using the software ImageJ (National Institutes of Health).

Cell transfection with GFP-LC3

The construct encoding microtubule-associated-protein-light-chain-3 (LC3) protein tagged at its N-terminus with green fluorescent protein (GFP), GFP-LC3, has been used to monitor autophagy Science, Japan) [START_REF] Kabeya | LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[END_REF]. NCTC 2544 cells grown on glass coverslips were transfected with GFP-LC3 using Lipofectamine 2000 according to the supplier's protocol (Invitrogen). After transfection, cells were incubated for 3 hours with 5 µM P-OGal or for 18 h with 0.8 mM ALA and irradiated for 15 min. At various times after irradiation, cells were fixed with paraformaldehyde, rinsed and mounted on glass slides using Mowiol (Calbiochem). Cells were observed under a Nikon Eclipe TE2000-U fluorescence microscope equipped with a plan APO VC 60X / 1.40 objective under oil immersion. Cells with active autophagy were defined as those displaying 3 or more puncta of GFP-LC3. Results are given under each experimental condition as percentage of cells with more than 3 puncta of GFP-LC3, and are based on the counting of over 500 cells expressing GFP-LC3.

Cytochrome c immunofluorescent staining

Cells grown on glass coverslips and treated as indicated were washed with PBS, fixed with 4% paraformaldehyde , and permeabilized with 0.1% TX100. Immunolabelling was performed using a mouse antibody raised against cytochrome c (BD Bioscience, Le Pont de Claix, France) and a secondary antibody labeled with Cy3 (Jackson Immunoresearch). Nuclei were counterstained with DAPI. The stained samples were rinsed and mounted in Mowiol (Calbiochem) and the slides were examined and photographed using a Nikon Eclipe TE2000-U fluorescence microscope equipped with a plan APO VC 60X / 1.40 objective under oil immersion.

Statistical analyses

Statistical analyses were performed with the Student's t-test. Statistical significances are provided in the figures (* p < 0.05, ** p < 0.01).

A c c e p t e d M a n u s c r i p t 10

Results

Uptake of P-R derivatives by cells

NCTC 2544 keratinocytes have been incubated at various durations with increasing concentrations of porphyrins in FCS-supplemented EMEM. As shown in Fig. 2A, the uptake of all derivatives is extremely fast and reaches a plateau within less than an hour. No further uptake is observed with incubation of up to 18 h. P-H and, to a lesser extent, P-Gal are poorly incorporated when compared to P-Me, P-DDC and P-OGal. After a 3 h incubation in FCS-supplemented EMEM, the uptake increases almost linearly as a function of the incubation concentration for all derivatives (Fig. 2B). The relative order of magnitude of the uptake is the same as that observed in Fig. 2A.

Though incubation in FCS-supplemented culture medium is more relevant to the in vivo situation, incubation in serum-free HBSS has also been performed to determine whether serum proteins play any peculiar role in the uptake process. The P-R uptake profiles as a function of incubation time or of incubation concentration are found to be similar in the presence and absence of serum (data not shown). This suggests that, in contrast to the behavior of P-(Lys) n , there is no strong interaction between the other P-R shown in Fig. 1 and serum proteins [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF].

Photocytotoxicity of P-R derivatives

To reach maximum uptake, cells were incubated for 3 h with porphyrins in FCS-supplemented EMEM prior to irradiation. No cytotoxicity was observed with untreated sham-irradiated cells or with untreated irradiated (up to 60 min) cells in the presence of 0.5% or less DMSO in the incubation medium (data not shown). Moreover, none of these porphyrins exhibited dark cytotoxicity at incubation concentrations up to 5 µM. Figure 2C (irradiation with increasing light doses) demonstrates that P-Me, P-DDC and P-OGal all exhibit a much larger photocytotoxicity than P-H

A c c e p t e d M a n u s c r i p t 11 or P-Gal at an incubation concentration of 5 µM. Conversely, lower concentrations of P-Me, P-DDC and P-OGal are sufficient to achieve high photocytotoxicity (Fig. 2D). The incubation concentrations necessary to obtain a 50% decrease in NR uptake after a 30 min irradiation can be estimated from Fig. 2D. These values, hereafter referred to as "photocytotoxic concentrations" (PC 50 ), are reported in Table 1, which also includes earlier data obtained with the P-(Lys) n derivative. Table 1 provides another photocytotoxicity index, the "intracellular phototoxic concentration" (IPC 50 ) which represents the intracellular concentration which produces a 50% decrease in NR uptake after a 30 min irradiation. Low IPC 50 values of ~50 pmol per mg of protein are obtained with P-Me, P-DDC or P-OGal and a relative efficiency --P-OGal > P-DDC = P-Me >> P-Gal > P-H --is deduced from Table I. Experiments performed after incubation in the absence of serum proteins lead to similar data for these conjugates, futher illustrating that little if any role is played by serum proteins in uptake of these porphyrins (data not shown).

Oxidative stress photo-induced by P-R derivatives

Singlet oxygen is the main cytotoxin involved in PDT with porphyrins [12]. The photophysical studies here revealed no major difference in the efficiency of P-H, P-Me, P-DDC, P-OGal and P-Gal to produce singlet oxygen in a high yield whereas P-(Lys) n was found earlier to be about 4 times less effective [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF][START_REF] Silva | Tricationic porphyrin conjugates: evidence for chain-structure-dependent relaxation of excited singlet and triplet states[END_REF]. Inhibition of cell photosensitization by specific 1 O 2 quenchers such as N 3 -ions (Table 1) demonstrates the role of singlet oxygen in photo-oxidative stress induced by the tricationic porphyrins. Lipid peroxidation has been used as an overall index of oxidative stress. It has been quantified by measuring TBARS formation in cell supernatants since previous studies demonstrate practically no intracellular TBARS formation in photosensitized cells themselves. The TBARS production is shown to increase nearly linearly with the irradiation time (Fig. 3A). The ability of P-R to trigger lipid peroxidation parallels their photocytotoxic potential, and the strongly photocytotoxic P-Me, P-DDC and P-OGal as well as P-(Lys) n (see Fig. 4 in [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]), may

A c c e p t e d M a n u s c r i p t 12 be seen to be the most effective TBARS producers. Again, similar data were obtained in the absence of serum proteins (data not shown).

Cytotoxicity and oxidative stress photo-induced by PPIX

To compare the effects of P-R photosensitization to those of ALA, the pro-drug used in the PDT of skin lesions, cells have been incubated with various concentrations of ALA prior to irradiation in order to induce endogenous PPIX. Subsequently, the intracellular PPIX content was determined by fluorescence measurement. The photocytotoxicity or the lipid peroxidation, or both have also been determined. Data in Fig. 3B show that the IPC 50 is approximately 50 pmol per mg of protein. About 1.8 mol of TBARS per mol of intracellular PPIX are released after a 30 min irradiation (Fig. 3C).

Fluorescence microscopy of P-R derivatives

P-R derivatives fluoresce near 660 nm and exhibit comparable fluorescence quantum yields under similar solvent conditions [START_REF] Silva | Tricationic porphyrin conjugates: evidence for chain-structure-dependent relaxation of excited singlet and triplet states[END_REF]. ,C) the localization patterns of the green and red fluorescences are very similar, although a total superimposition cannot be achieved. This suggests a localization in other non-lysosomal structures, most probably, endocytotic or pinocytotic vesicles beside a minor lysosomal incorporation. These vesicles have a pH higher than that of lysosomes (pH 5-6) and are therefore inefficiently labeled by LysoTracker Green ® . On the other hand, lysosomal staining by the probe may be partially hidden by FRET from the probe (the donor) to P-R (the acceptor). Such behavior is observed in the case of another PDT model where tolyporphin is the photosensitizer [START_REF] Morlière | Tolyporphin: A natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo[END_REF]. Since P-R are positively charged, and since the inner mitochondrial membrane exhibits a negative potential, mitochondria are expected to be a preferential site for P-R localization. However, micrographs obtained with the specific mitochondrial probes, rhodamine 123 and MitoTracker ® show a perinuclear network extending all over the NCTC 2544 keratinocytes (see also Gaullier et al. [START_REF] Gaullier | Subcellular localization of and photosensitization by protoporphyrin IX in human keratinocytes and fibroblasts cultivated with 5-aminolevulinic acid[END_REF]) incompatible with the pattern of P-Me, P-DDC and P-OGal localization (Figs. 5D,E,F). This observation confirms previous results obtained with P-(Lys) n [START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. Although tetracationic porphyrins readily accumulate in the cell nucleus [START_REF] Georgiou | Measurement of the rate of uptake and subcellular localization of porphyrins in cells using fluorescence digital imaging microscopy[END_REF][START_REF] Kuimova | Singlet oxygen in a cell: spatially dependent lifetimes and quenching rate constants[END_REF], the fluorescence depression on the nuclear area observed for all P-R strongly suggests very little, if any, localization in the nucleus of NCTC 2544 keratinocytes.

Mechanisms of the PDT-induced death in NCTC 2544 cells

Necrosis induced in vitro by PDT with porphyrins has long been established, and is essentially characterized by increased cell volume, organelle swelling and plasma membrane alterations the most effective photosensitizer --was chosen as representative P-R for this study.

Is apoptosis involved in P-R photocytotoxicity?

Translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane is observed in the initial stages of apoptosis. To detect this translocation, we used annexin V, a protein that binds to cells with exposed phosphatidylserine. A simultaneous cytometric analysis of annexin V binding and propidium iodide uptake by dead cells was carried out. According to Fig. 3A, incubation with 5 µM P-OGal for 3 h, followed by a 15 min irradiation, leads to about 25% cell killing. However, no significant change in the percentage of annexin V positive cells (Fig. 6A) could be observed up to 9 h following irradiation. Similar results were observed after a 3 h incubation of cells with 1 µM P-OGal followed by 15 min of irradiation or with 5 µM P-OGal followed by 30 min of irradiation (data not shown).

Another hallmark of apoptosis is nuclear DNA breakdown into oligonucleosomal units of about 200 bp. The enzyme-linked immunosorbent assay used to quantify the cytoplasmic accumulation of histone-associated DNA fragments shows no significant increase in cytoplasmic histone-associated DNA fragments 0, 1, 3, 6, 9 12 h (Fig. 6B) or 24 h (data not shown) after PDT with 5 µM P-OGal.

Activation of intracellular proteases called caspases, is a key event during apoptosis. The caspase cascade is regulated through their partial cleavage. To further examine whether apoptosis is involved in the P-R-induced photokilling, caspase-3, caspase-8 and caspase-9 activities have been assayed. Cleavage of proteins by caspases leads to chromatin condensation and DNA degrada- and Annexin V staining readily show apoptosis induction only 9 h after treatment. These data support the conclusion that apoptosis induction after photodynamic injury induced by P-R is insignificant.

Autophagy of NCTC 2544 keratinocytes after PDT with tricationic porphyrins

Autophagy is a normal process that helps the cell get rid of damaged organelles or macromolecules. This highly regulated process is morphologically evidenced by the formation of autophagosomes and autolysosomes [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF][START_REF] Kondo | The role of autophagy in cancer development and response to therapy[END_REF]. Recently, a new form of programmed cell death characterized by autophagic hallmarks has been discussed [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF][START_REF] Galluzzi | Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes[END_REF]. To determine if programmed cell death associated with PDT might exhibit such characteristics, we monitored the autophagic activ- tagged with a green fluorescent protein (GFP). Upon autophagy induction, LC3 is proteolytically processed and recruited to autophagosomal membranes [START_REF] Kabeya | LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[END_REF]. After P-OGal PDT, we have observed the subcellular redistribution of the fluorescent marker GFP-LC3 fusion protein from a uniform, cytosolic, to a punctated, vacuolar pattern [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF][START_REF] Galluzzi | Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes[END_REF] (Fig. 7A). After a 3 h incubation of cells with 5 µM P-OGal, followed by 15 min of irradiation, a time-dependent increase in the number of cells showing the characteristic vacuolar pattern of GFP-LC3 is observed (Fig. 7B).

By contrast, no significant change in the accumulation of GFP-LC3 is observed (Fig. 7B) after incubation of NCTC 2544 cells with 0.8 mM ALA for 18 h followed by a 15 min irradiation, which induces similar photocytotoxic effects as those observed with P-R (see Fig. 3C).

In another series of measurements, the immunoblotting analysis for the modulation of the p62 protein, also called sequestosome 1 (SQSTM1), by P-R has been performed. Since p62/SQSTM1 accumulates when autophagy is inhibited and decreases when autophagy is induced, it may be used as a marker of autophagic activity [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF]. Western blotting analysis reveals that 6 h after irradiation the amount of p62/SQSTM1 is decreased by more than 60% in the P-OGal-treated cells but not in cells treated with P-OGal but not irradiated (Fig. 7D).

To further assess a possible contribution of autophagy to P-OGal-induced cell death, we have studied the effect of 3-methyladenine (3-MA), a well-characterized autophagy inhibitor [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[END_REF]. After 15 min of irradiation of cells incubated for 3 hours with 5 µM P-OGal and 5mM 3-MA, the percentage of cells showing the characteristic vacuolar pattern of GFP-LC3 in the P-OGal+3-MA-treated group is decreased to about 70% of the value obtained with the group treated with P-OGal alone (Fig. 7C). By contrast P-OGal-induced cell death is inhibited by about 40% after incubation with 5mM 3-MA (Fig. 6C). It is well established that the inhibition of autophagy by 3- PDT (Fig. 6C). Furthermore, no leakage of mitochondrial cytochrome c in the cytoplasm has been observed with 3-MA-treated cells up to 6 h after irradiation (data not shown), but the p62 degradation was decreased by about 80% (Fig. 8D).

These data suggest that cells undergoing P-OGal-PDT show increased autophagy levels and that autophagy may be associated with cell death induced by P-OGal photosensitization. The MAPK phosphorylation has been assessed in NCTC 2544 cells treated by P-OGal-PDT and lysed 0, 1, 3, 6 or 9 h after irradiation. The p38 and JNK phosphorylation was significantly increased. This increase is stronger with p38 and maximal 3 h after irradiation (Fig. 8A). After this interval, p38 and JNK phosphorylation steadily declines, becoming barely detectable 9 h after irradiation. On the other hand, no significant phosphorylation of p38 and JNK is observed before irradiation in untreated (control) cells or after sham-irradiation of P-OGal-treated cells.

MAPK participate in death of

Regarding Akt and ERK, the level of total Akt and ERK is unchanged whereas the p-Akt and p-ERK levels are markedly decreased after P-OGal-PDT in a time dependent manner (Fig. 8A).

In contrast to results obtained with p38 and JNK, substantial amounts of phosphorylated ERK1/2 To examine the role of MAPKs activation in cell death, P-OGal-treated cells were preincubated with the p38 (SB203580) and JNK (SP600125) inhibitors. As a matter of fact, SB203580 and SP600125 inhibit p38 and JNK phosphorylation in the NCTC 2544 cells. Thus, after an osmotic stress induced by 0.4 M sorbitol for 10 min, immunoblot analysis demonstrates that 25 µM SB203580 and 25 µM SP600125 reduced p-p38 and p-JNK over 75% of the control value (data not shown). Addition of SP600125 3 h before irradiation, increases the photo-induced cell death by approximately 50%, whereas SB203580 has no significant effect (Fig. 8B). Incidentally, it must be noted that the viability of control cells treated with P-OGal and pre-incubated with SB203580 and SP600125 but left in the dark was unchanged. Figure 8B also suggests that an insignificant effect on the percentage of annexin V positive cells is observed 6 h after P-OGal-PDT of cells pre-incubated with 25 µM of p38 and JNK inhibitors. By contrast, 6 h after irradiation, the number of cells showing the vacuolar pattern of GFP-LC3 is increased by 31% (p < 0.001) in cells treated with P-OGal and 25 µM SP600125 (Fig. 8C) although SP600125 has no effect on the basal level of GFP-LC3-puncta. On the contrary, no significant change has been observed in the P-OGal PDT-induced accumulation of GFP-LC3 after pre-incubation of cells with SB203580. Under the same experimental conditions, SP600125 but not SB203580 increases the p62 degradation after PDT (Fig. 8D). Taken together, these results suggest that JNK exerts a negative regulator effect on P-OGal PDT-induced autophagy and PDT-dependent programmed cell death. The comparison of the photocytotoxic potential of P-R is straightforward since they absorb identical light under the same experimental conditions (see supplementary data). The greater photocytotoxic efficiency of P-OGal, P-DDC and P-Me compared to that of P-H and P-Gal (Figs. 2C,D) parallels the magnitude of the lipid peroxidation (Fig. 3A) suggesting that their phototoxicity and their overall ability to produce an oxidative stress correlate, irrespective of the targets in the cell lines studied.

Interestingly, P-OGal, P-DDC and P-Me favorably compare with endogenous PPIX produced by ALA treatment. Figure 3B demonstrates that a 50% loss in Neutral Red uptake is obtained with an intracellular PPIX concentration (~50 pmol/mg of cellular protein) similar to that found The intracellular localization may explain different photocytotoxic responses. Microfluorometry confirms that P-H and P-Gal are taken up less by NCTC 2544 keratinocytes than is P-Me, P-DDC or P-OGal, whose fluorescence is more specifically localized in spots that may correspond to endo-or pinocytotic vesicles, but not to mitochondria and nucleus. The observations reported here demonstrate that the nature of the conjugated side chain is more critical than the electric charge of the porphyrin ring in controlling the uptake and site of localization of the P-R and, consequently, their greater photosensitizing efficiency. It may be thought that major direct damage to the mitochondria and the nucleus by 1 O 2 produced by P-R is rather unlikely since 1 O 2 does not diffuse a great distance from its site of production [START_REF] Kuimova | Singlet oxygen in a cell: spatially dependent lifetimes and quenching rate constants[END_REF].

The photo-induced cell death shows hallmarks of autophagy

Beside demonstrating for the first time the chain structure dependence of the photocytotoxic potential of tri-cationic porphyrins, this study provides new information about mechanisms of cell death and MAPK-mediated stress signalling in our PDT model. It has been demonstrated that many PDT photosensitizers including those used to treat skin lesions in clinical practice, e.g.

ALA and photofrin  , activate several pathways leading to apoptosis [12 and references therein].

Our results are in sharp contrast with these reports. Consistent with the absence of apoptosis, no annexin V cell staining (Fig. 6A), no oligonucleosome fragmentation (Fig. 6B) and no caspase activation (see supplementary data) are observed after P-OGal-PDT. Among other factors, these results may be related to the non-mitochondrial localization of the most photocytotoxic P-R (Fig. 4). The absence of major mitochondrial damage is also suggested by the lack of cytochrome c leakage (Fig. 7A). On the other hand, we show the appearance of a punctate pattern of GFP-LC3 and a decrease in the amount of p62 following PDT with P-OGal (Figs. 7A,B,C). Consistently, 3- ). The incorporation of P-R in lysosomes or pre-lysosomes in the vicinity of the ER and the Golgi apparatus might be responsible for this conjunction of pro-autophagic and anti-apoptotic events.

There are relatively few recent reports on the induction of autophagy by photodynamic therapy and most deal with apoptosis-deficient cells or cells whose caspase activity has been inhibited [12,[START_REF] Kessel | Initiation of apoptosis and autophagy by photodynamic therapy[END_REF]. We used apoptosis-competent cells in which porphyrin conjugates induced autophagic cell death in the absence of apoptosis inhibitor. Treatment with the autophagy inhibitor 3-MA is not associated with a significant change in the percentage of annexin V positive cells and thus with apoptosis activation (Fig. 6C). Possibly, autophagy may constitute an attempt by cells to remove photo-oxidized organelles or large cross-linked aggregates formed after photodynamic damage to protein complexes.

Consequences of P-OGal-PDT on MAPKs in NCTC 2544 cells

Several studies performed with different cell lines and photosensitizers have shown that MAPKs participate in the oxidative signalling events induced by PDT [12]. Previous research presented conflicting data concerning the roles of p38 and JNK in cell viability after PDT. Results suggest that the degree of p38 and JNK phosphorylation as well as their effect on cell death regulation after PDT may depend on cell type, photosensitizer and light dose [START_REF] Xue | Promotion of photodynamic therapy-induced apoptosis by stress kinases[END_REF][START_REF] Assefa | The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin[END_REF]. In contrast to apoptosis, relatively little is known about PDT-induced autophagy signalling in terms of MAPK activation and possible cross-talk among different pathways.

The role of JNK in cell death has long been controversial as it exhibits a pro-survival function or it promotes lethality depending on the physiological or pathological condition studied [START_REF] Davis | Signal transduction by the JNK group of MAP kinases[END_REF].

The paradoxical nature of this effect could be partially due to different modes of JNK activation A c c e p t e d M a n u s c r i p t 22 [START_REF] Ventura | Chemical genetic analysis of the time course of signal transduction by JNK[END_REF], different kinetics [START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF] or might depend on the nature of the chemical inducers used including reactive oxygen species [START_REF] Ventura | Chemical genetic analysis of the time course of signal transduction by JNK[END_REF][START_REF] Shimizu | Involvement of JNK in the regulation of autophagic cell death[END_REF]. An early transient JNK activation determines cell survival whereas sustained JNK activation can induce cell death signals [START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF]. Consistent with these data, we observed a rapid and transient JNK activation after P-OGal-PDT associated with a negative regulatory effect on cell mortality and autophagy (Fig. 8A). We may speculate that this effect might be partially explained by JNK1 mediated Bcl-2 phosphorylation known to inhibit the binding of Bcl-2 to Beclin 1 which activates autophagy [START_REF] Wei | JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy[END_REF]. Of note, it has recently been reported that JNK activation, induced by 2-methoxyestradiol in Ewing sarcoma cells, modulates autophagy either through Bcl2 phosphorylation or up regulation of the damaged regulated autophagy modulator [START_REF] Lorin | c-Jun NH2-terminal kinase activation is essential for DRAM-dependent induction of autophagy A c c e p t e d M a n u s c r i p t 28 and apoptosis in 2-methoxyestradiol-treated Ewing sarcoma cells[END_REF][START_REF] Lorin | Evidence for the interplay between JNK and p53-DRAM signalling pathways in the regulation of autophagy[END_REF].

While several reports support the role of p38 as inhibitor of autophagy [START_REF] Webber | New insights into the function of Atg9[END_REF], others imply the opposite [START_REF] Zhang | Involvement of PKC signal pathways in oridonin-induced autophagy in HeLa cells: a protective mechanism against apoptosis[END_REF]. Although we observed a p38 activation after P-OGal PDT, our data suggest that p38 is not directly implicated in induction of cell death. A downstream event in the mitogenic pathway is ERK activation through binding of ligands to extracellular growth factor receptors involved in regulation of growth and cell cycle progression. The Ras/Raf/ERK activation pathway can promote opposite pro-survival or anti-proliferative cellular responses, such as apoptosis and autophagy. This wide variety of processes triggered by the activation of a single pathway depends on the timing, duration and strength of activation, on subcellular localization and on the presence of reactive oxygen species (ROS) [START_REF] Cagnol | ERK and cell death: mechanisms of ERK-induced cell death-apoptosis, autophagy and senescence[END_REF]. Available evidence suggests that the photooxidative stress induced by PDT may modulate ERK activity as does other ROS such as H 2 O 2 , which is produced in a variety of tumor cell lines by 1,3-dibutyl-2-thiooxo-imidazolidine-4,5dione [START_REF] Wong | Simultaneous induction of non-canonical autophagy and apoptosis in cancer cells by ROS-dependent ERK and JNK activation[END_REF]. Depending on the photosensitizer and cell culture, ERK phosphorylation variably responds to the PDT-induced oxidative stress by irreversible inhibition [START_REF] Assefa | The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin[END_REF], moderate attenuation [START_REF] Schieke | Singlet oxygen-induced attenuation of growth factor signaling: possible role of ceramides[END_REF], insignificant modulation [START_REF] Xue | Promotion of photodynamic therapy-induced apoptosis by stress kinases[END_REF] or transient activation [START_REF] Tong | Sustained activation of the extracellular signal-regulated kinase pathway protects cells from photofrin-mediated photodynamic therapy[END_REF]. It is noteworthy that ALA-PDT A c c e p t e d M a n u s c r i p t 23 has no effect on ERK expression in HaCat cells [START_REF] Klotz | Activation of JNK and p38 but not ERK MAP kinases in human skin cells by 5-aminolevulinate-photodynamic therapy[END_REF]. We found a decrease in the p-ERK content to almost undetectable levels 3 h after after P-OGal-PDT despite high pre-existing expression (Fig. 8A).

Activation of PI3-K/Akt is another important signaling pathway. The Akt acts on a variety of ways including mTOR activation, Bad phosphorylation, IκB kinase phosphorylation and caspase-9 inhibition, thereby preventing cell death [START_REF] Kondo | The role of autophagy in cancer development and response to therapy[END_REF]. The PDT effect on Akt regulation varies from dephosphorylation and subsequent activation of caspase-assisted death [START_REF] Matsubara | Photodynamic therapy induces caspase-dependent apoptosis in rat CNV model[END_REF] to phosphorylation stimulation [START_REF] Bozkulak | Multiple components of photodynamic therapy can phosphorylate Akt[END_REF]. Our results show a time-dependent decrease in the content of p-Akt after PDT treatment (Fig. 8A), consistent with increased photo-induced autophagic death.

Conclusions

Beside necrosis, autophagy may contribute to the death of NCTC 2544 keratinocytes by P-OGal-PDT with no evidence for significant apoptosis. JNK activation exerts a negative regulatory effect on photocytotoxicity and autophagy while p38 activation is not involved in cell death. Several studies show that ERK and PI3-K/Akt pathways are aberrant in human cancers [START_REF] Sebolt-Leopold | Targeting the mitogen-activated protein kinase cascade to treat cancer[END_REF] suggesting them as targets for the development of novel cancer treatments [START_REF] Cheng | The Akt/PKB pathway: molecular target for cancer drug discovery[END_REF]. The inhibition of ERK1/2 and Akt activities suggests that water-soluble tri-cationic porphyrins may thus play a beneficial role in cancer treatment by PDT given their potentially easy formulation. However, the role of MAPKs modulation on photocytotoxicity and autophagic cell death induced by P-OGal-PDT still needs clarification. In this regard, the use of the siRNA technology would of definite interest to understand the role played by the JUNK pathway in the photo-induced death mechanism. Finally, the present study has allowed the characterization of P-OGal as the most potent photoactive conjugate in the NCTC 2544 cell model. In view of its potential interest as a PDT photosensitizer in dermatology, it would also be valuable to assess its photobiological activity towards SCC or BCC

A c c e p t e d M a n u s c r i p t 7 view/

 7 An inverted fluorescence microscope (Eclipse TE 2000 DV Nikon) equipped with a CoolS-NAP HQ ™ detector cooled to -30 °C (Princeton Instruments; Division of Roper Scientific, Evry, France) was used for cell microfluorometry. This system is controlled by the Meta-Metamorph software, which is also used for image analyses. The fluorescence recording conditions (objective, camera binning and exposure time, dichroic mirrors, filters) are fully described in ref[START_REF] Silva | Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine[END_REF]. No porphyrin fluorescence was detected with the green emission filter while negligible fluorescence of the LysoTracker Green was recorded with the red emission filter.

A c c e p t e d M a n u s c r i p t 8 Coquette

 8 , France) were transferred into an anti-DNA precoated microtiter plate and analyzed using the ELISA procedure recommended by the manufacturer.

A c c e p t e d M a n u s c r i p t 9 through

 9 direct fluorescence microscopy and was provided by Drs. T. Yoshimori and N. Mizshima (Department of Bioregulation and Metabolism, The Tokyo Metropolitan Institute of Medical

Figure 4

 4 illustrates the phase contrast and fluorescence images obtained for control cells (sham-treated cells) and for cells treated for 3 h with P-R in FCSsupplemented culture medium. The fluorescence images have been obtained with an excitation in the near UV (330-380 nm) and an emission in the red-visible range (610-700 nm). Figures 4G to R are matching fluorescence images of the phase contrast images (Figs.4A-F). For ready comparison, fluorescence images are displayed on the same scale in panels G to L, while they are displayed between the minimum and maximum fluorescence levels in M to R. As expected, untreated cells exhibit a very low fluorescence arising from the cytoplasm with larger intensities in the perinuclear area and the mitochondrial network. Cells treated with P-Me, P-DDC and P-OGal fluoresce more intensely, in agreement with greater uptake. A rather diffuse fluorescence with some spots in the perinuclear area is observed from cells treated with P-H and P-Gal, suggesting a main cytoplasmic and plasma membrane localization and little accumulation at specific sites.

  A c c e p t e d M a n u s c r i p t 13 By contrast, cells treated with P-Me, P-DDC and P-OGal exhibit stronger fluorescence, mostly localized in perinuclear spots which suggests more specific localization in lysosomes or lysosome-like structures. A double labeling of the cells (Figs. 5A,B,C) was performed with P-R and LysoTracker Green, a lysosome specific probe emitting a green fluorescence centered toward 510 nm. As shown for P-DDC (Figs. 5B

[ 17 ]

 17 . Apoptosis and autophagy are induced by a variety of stress factors, notably those encoun-A c c e p t e d M a n u s c r i p t 14 tered in cancer therapies. Two reviews have been recently published on guidelines concerning the use and interpretation of assays for determing mechanisms of cell death [18, 19]. It is of interest to understand the death mechanisms underlying the efficient photocytotoxicity of P-Me, P-OGal and P-DDC. As P-Me, P-OGal and P-DDC exhibit similar intracellular localization, P-OGal --

A c c e p t e d M a n u s c r i p t 15 tion. 3 ( 6 -

 1536 Several families of PDT photosensitizers (porphyrins, chlorins, phthalocyanines...) are strong inducers of apoptosis in various cell lines[12]. Because apoptosis is a dynamic process, samples have been collected up to 24 h following PDT. No evidence for cleavage of the caspase-3, caspase-8 and caspase-9 substrates was found after P-OGal-PDT under the conditions outlined above (see Fig.S1in the supplementary data). The absence of procaspase-3, procaspase-8 and procaspase-9 cleavage in Western blots, also shown in the supplementary data, confirms the lack of caspase activities. In addition, no cytochrome c leakage has been observed with P-OGal photosensitized cells (Fig.7A). To verify that under our experimental conditions, NCTC 2544 cells may readily undergo apoptosis, such cells were incubated 30 min with 10 µM 5-methoxypsoralen in DPBS and then were subjected to UVA irradiation (2.5 J/cm 2 ). Twenty four hours later, NCTC 2544 treatment with 5-methoxypsoralen+UVA had resulted in extensive activation of caspasefold increase in activity) and caspase-9 (2-fold increase in activity) in cell lysates. No change in activity was observed with caspase-8 (data not shown). These results are fully consistent with those previously presented by Viola et al.[START_REF] Viola | Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544[END_REF]. Another positive control was performed by treating cells with 5 µM campothecin. Measurement of caspase-3 activity and flow cytometry after PI

A c c e p t e d M a n u s c r i p t 16 ity

 16 in NCTC cells exposed to P-OGal. Autophagic activity was followed using a fluorescent marker (GFP-LC3) consisting in a fusion microtubule-associated protein light-chain 3 (LC3)

A c c e p t e d M a n u s c r i p t 17 MA

 17 may potentiate apoptosis induced by a variety of chemicals[START_REF] Kondo | The role of autophagy in cancer development and response to therapy[END_REF]. Autophagy inhibition does not significantly change the percentage of annexin V positive cells measured 6 h after P-OGal-

  NCTC 2544 keratinocytes after PDT with tricationic porphyins Several lines of evidence indicate that MAPK participate in closely related signalling cascades that contribute to the regulation of gene expression in response to photodynamic treatment. Activation of ERK 1/2 is generally associated with cell proliferation and survival, while p38 and JNK are linked to induction of apoptosis [12, 22]. Recent reports show, however, that the role of MAPKs in cell death induced by photosensitization is largely cell type and photosensitizerdependent [12].

A c c e p t e d M a n u s c r i p t 18 and

 18 Akt are detected prior to irradiation. Although the p-ERK and p-Akt levels remain unchanged in sham-irradiated cells and untreated controls, p-Akt decreased progressively after the photodynamic challenge while p-ERK has a significant abrupt drop at 3 hours and becomes barely detectable. These results suggest that MAPKs activity modulation by P-OGal-PDT may be implicated in the cytotoxicity.

A c c e p t e d M a n u s c r i p t 19 4. Discussion 4 . 1 .

 1941 The nature of the conjugated chain determines the intracellular localization of tricationic conjugates and their cytotoxicity.

  for P-OGal, P-DDC and P-Me. Since light doses absorbed by equal concentrations of P-R and PPIX are essentially identical (see supplementary data), P-OGal, P-DDC and P-Me are photosensitizers as powerful as PPIX in our cell model. Comparison of data in Figs. 2B, 3A, 3C demonstrates that at equivalent light doses, oxidative membrane damage is more pronounced with PPIX than with P-Me, P-DDC or P-OGal. This behavior is consistent with the higher hydrophobicity of PPIX favouring its intracellular localisation in lipophilic microenvironments.With the exception of P-(Lys) n , P-R derivatives have comparable singlet oxygen formation quantum yield. Consequently, they have similar intrinsic photosensitizing potential. From Fig.2B, it may be estimated that incubation with P-Me, P-DDC or P-OGal concentrations in the range 0.7-0.8 µM leads to the same P-R intracellular contents as incubations with 5 µM P-H or 1.4-1.6 µM P-Gal. It may be deduced from Figs. 2C,D that these differences cannot account for the ob-A c c e p t e d M a n u s c r i p t 20 served differences in cytotoxicity.

A c c e p t e d M a n u s c r i p t 21 methyladenine

 21 , a well-known inhibitor of autophagy, inhibits the photocytotoxicity, lowers the percentage of cells showing punctate GFP-LC3 and decreases p62 degradation (Figs. 6C, 7C and 8D

Fig. 1 -Fig. 2 -

 12 Fig. 1 -Chemical structure of P-R derivatives.

Fig. 3 -

 3 Fig.3-A: Lipid peroxidation photosensitized by P-H, P-Me, P-DDC, P-OGal and P-Gal

Fig. 4 -

 4 Fig. 4 -A-R: Phase contrast images (A-F) and fluorescence micrographs (G-R) obtained with

Fig. 5 -

 5 Fig. 5 -Double-labeling experiments: A-C: Phase contrast images (A) and fluorescence micro-

Fig. 6 -

 6 Fig.6 -A: Flow cytometric analysis of NCTC 2544 keratinocytes after P-OGal photosensitiza-

Fig. 7 -

 7 Fig. 7 -P-OGal induced autophagic cell death in NCTC 2544 keratinocytes. A: Cells transfected

Fig. 8 -

 8 Fig. 8 -MAPKs protein expressions were analyzed in lysates of NCTC 2544 cells. A. Represent-

Figure2A

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Figure4 A c c e p t e d M a n u s c r i p

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Figure5 A c c e p t e d M a n u s c r i p t

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Figure6 A c c e p t e d M a n u s c r i p t

  Figure6

Figure7

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Acknowledgements

This work was supported by an INSERM/GRICES exchange agreement, a Franco-Portuguese "PESSOA" exchange program (07958NF), a co-tutelage "PAUILF" Franco-Portuguese program (J.N.S., P.F., J-C.M., R.S., P.M.) and grants from the Gulbenkian Foundation (J.N.S., P.F.) and the "Fundação para a Ciência e a Tecnologia" (J.P.C.T., M.G.P.M.S.N., E.M.P.S., J.A.S.C.). A portion of the epifluorescence microscopy experiments was performed with the equipment of the "Centre de Microscopie du Muséum". The authors wish to thank Drs. M. Gèze and M. Dellinger for their advices. The authors wish to thank Pr. L.K. Patterson (Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana) for his careful reading of the manuscript and improvement of the English language. We thank Drs. Yoshimori and Mizshima (Tokyo Metropolitan Institute of Medical Science, Japan) for the kind gift of the GFP-LC3 construct.

A c c e p t e d M a n u s c r i p t