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Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells
Journal of Photochemistry and Photobiology B: Biology 62 (2001) 146–152 www.elsevier.com / locate / jphotobiol
Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells a,b a, a a b b M.-H. Teiten , L. Bezdetnaya *, J.-L. Merlin , C. Bour-Dill , M.E. Pauly , M. Dicato , a F. Guillemin a
´ Unite´ de Recherche en Therapie Photodynamique, Centre Alexis Vautrin, Avenue de Bourgogne, Brabois, F-54511 Vandoeuvre-les-Nancy Cedex, France b Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Centre Universitaire de Luxembourg, L-1511 Luxembourg, Luxembourg Received 23 March 2001; accepted 26 July 2001
Abstract Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7 / DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light ( l5650 nm), was significantly greater in MCF-7 / DXR as compared to MCF-7 cells. A light dose of 2.5 J cm 22 inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7 / DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype. 2001 Elsevier Science B.V. All rights reserved. Keywords: Photodynamic therapy; mTHPC; Multidrug-resistance; Cytotoxicity
1. Introduction Photodynamic therapy (PDT) is a rapidly growing modality for the treatment of light-accessible tumors. PDT is based upon the administration of a photosensitizing compound (photosensitizer) with its following activation by visible light [1]. The resulting photodamages originate from the production of singlet oxygen and other reactive oxygen species [2]. Photodynamic therapy has been proposed in several studies as an alternative in overcoming multidrug resistance (MDR) phenotype. Triggered by the overexpression of P-glycoprotein (P-gp) efflux protein with the successive exclusion of hydrophobic and cationic drugs from cells, development of MDR phenotype results in a broad-spec*Corresponding author. Tel.: 133-3-8359-8306; fax: 133-3-83447635. E-mail address: [email protected] (L. Bezdetnaya).
trum of resistance, thus explaining a frequent failure of conventional chemotherapy. An effective outcome of photosensitization in MDR cells was reported for mesoporphyrin, phthalocyanines and Photofrin [3–5]. An equal or slightly better photocytotoxicity was observed with these photosensitizers in both sensitive and resistant cell lines under conditions of similar drug uptake. Meanwhile, there are several observations of cross-resistance between both treatment modalities. The failure of PDT could be translated either by an impaired accumulation of sensitizers due to their recognition by P-gp [6,7] or to other mechanisms that are not directly related to P-gp overexpression [8]. Experiences with the first generation photosensitizers (hematoporphyrin derivative (Hpd) and Photofrin) have led to the development of new photosensitizing molecules. Meta-tetra(hydroxyphenyl)chlorin (mTHPC), a second generation photosensitizer, has better characteristics for use in PDT over Photofrin, since it is a chemically pure compound with a high quantum yield of singlet oxygen
1011-1344 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 01 )00178-6
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generation, an enhanced absorption in far red region (650 nm) and reduced skin photosensitivity [9–12]. Our recent studies demonstrated favorable photochemical and immunological aspects of mTHPC-induced cell sensitization [13,14]. Presently, mTHPC is under investigation for potential clinical applications [15] as it has been presented as a molecule of interest for the PDT of ovary, skin and breast cancers [16,17]. The impact of mTHPC-induced sensitization on MDR cells has not been addressed so far. Therefore, the present study inspects the efficacy of mTHPC-based photosensitization in cells expressing the MDR phenotype. This goal was achieved by comparing mTHPC accumulation and photosensitization in MCF-7 human breast adenocarcinoma cell line and its doxorubicin resistant subline (MCF7 / DXR), which is characterized by the overexpression of P-gp. Our results indicate absence of cross-resistance to PDT in MDR cells. Moreover, considerably higher photocytotoxicity was demonstrated in chemoresistant cells compared to their normal counterparts and was attributed to a different mTHPC distribution pattern between both cell lines.
2. Experimental
2.1. Chemicals mTHPC was supplied by Scotia Pharmaceuticals (Stirling, UK). mTHPC stock solution was performed according to the manufacturer’s recommendations (20% ethanol, 30% polyethylene glycol 400 and 50% water). Further dilution was performed in phenol red free RPMI 1640 medium supplemented with 2% fetal calf serum to reach a final mTHPC concentration of 1.5 mM. mTHPC solution was prepared 1 h before use in experiments. Doxorubicin was purchased from Pharmacia (St. Quentin-Yvelines, France). SDZ-PSC-833 and cyclosporin A were obtained from Novartis (Basel, Switzerland). These two P-gp modulating agents were solubilized in sterile water and in ethanol, respectively. Further dilutions were realized in sterile water to reach a final concentration of 1.5 mM.
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MCF-7 / DXR, was maintained in 10 mM doxorubicincontaining medium. Our previous published study showed that such incubation conditions resulted in the development of 200-fold resistance to doxorubicin [18] and that MCF-7 / DXR cell line overexpresses P-glycoprotein independently of other efflux proteins such as the multidrug-resistanceassociated protein (MRP), the major vault lung resistance protein (LRP) and the anthracycline resistance associated protein (ARA) [19]. A wash-out period of 7 days in doxorubicin-free medium was allowed for MCF-7 / DXR cells before being used in experiments.
2.3. Intracellular accumulation and efflux of mTHPC Logarithmically growing MCF-7 and MCF-7 / DXR cells, inoculated in 25-cm 2 culture flasks, were washed twice and exposed for 3 h to different mTHPC concentrations or to 1.5 mM mTHPC for different incubation times. At different experimental points, cells were washed three times, trypsinized and re-suspended in RPMI 1640 supplemented with 2% FCS. mTHPC intracellular uptake was assessed by measuring mTHPC fluorescence from drug-loaded cells. Intracellular mTHPC was extracted by adding of 10% ScintiGest (70% cetyltriammonium bromide, Sigma; 20% methylalcohol, Prolabo; 9.9% potassium hydroxide, Sigma) to cell suspension with the successive overnight storage at 608C. Efflux of mTHPC from cells was studied in the following way. After 3 h incubation with mTHPC (1.5 mM), the medium was removed, cells were washed twice with phosphate-buffered saline (PBS) and re-incubated in fresh medium for different time with the following measurements of mTHPC fluorescence. Intracellular mTHPC fluorescence was assessed using a computer-controlled Perkin-Elmer LS50B luminescence spectrofluorimeter equipped with a front surface accessory (Perkin-Elmer L225 9051), providing small-angle (458C) excitation geometry. The excitation wavelength was 422 nm and spectra were collected for a wavelength range between 600 to 750 nm. Fluorescence intensity was monitored at an emission wavelength of 655 nm and normalized to 10 4 viable cells.
2.4. Photodynamic treatment 2.2. Cell culture conditions MCF-7 human breast adenocarcinoma cell line and its subline MCF-7 / DXR were cultivated in 75-cm 2 culture flasks (Costar, Dutscher, Brumath, France) in phenol red free RPMI 1640 medium (Life Technologies, Cergy-Pontoise, France) supplemented with 9% fetal calf serum (FCS) (Dutscher, Brumath, France), penicillin (10 000 IU) and streptomycin (10 000 IU). Cells were kept at 378C in a 5% CO 2 humidified atmosphere, trypsinized and re-seeded into fresh medium every 7 days. The resistant subline,
Logarithmically growing MCF-7 and MCF-7 / DXR in 96-well plates were washed twice with PBS and incubated with mTHPC alone or in the presence of SDZ-PSC-833 (1.5 mM) or cyclosporin A (1.5 mM) for 3 h. Nonincorporated reagents were removed by washing cells twice with PBS and cells were further re-incubated in fresh culture medium. Irradiation was carried out at different fluences at 650 nm with a dye laser (Spectra-Physics 375B), pumped with an argon dye laser (Spectra-Physics 2020, Les Ulis, France) with an output of 0.3 W. Light
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spots of 7 cm diameter provided a fluence rate of 8 mW cm 22 .
2.5. Measurement of cell viability Cell viability was assessed 24 h after illumination using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma] colorimetric assay, which measures the capacity of mitochondrial deshydrogenase in viable cells to reduce MTT to a purple formazan precipitate. The MTT method assesses the number of surviving cells and has shown a good correlation with other established measures of cytotoxicity as clonogenic assay [20]. Likewise, the MTT-test has been well characterized for use as a measure of PDT effect and it shows a good correlation with viability determined by a clonogenic assay for various types of cells photosensitized with HpD [21,22]. A 50-ml volume of 0.125 M MTT was added into each well for 3 h at 378C to allow metabolism of MTT. After incubation, the formazan crystals were solubilized by adding 50 ml of 25% sodium dodecyl sulfate solution into each well. The reaction product was quantified photometrically at 540 nm on a Multiskan plate reader (Flow Laboratories). Results are given as the percentage of the data obtained at similar fluence with control cultures exposed to drug-free medium.
2.6. Subcellular localization of mTHPC For the fluorescence microscopy observations, cells were plated into Slideflasks (Nunc, Polylabo, France), and allowed to attach to the dishes for 4 days at 378C before being tested. The use of the Slideflasks allowed the adhesive living cells to be maintained during observation. Cells were incubated with mTHPC solution (1.5 mM) for 3 h, rinsed three times with PBS and immediately observed under an AX 70 PROVIS upright epifluorescence microscope (Olympus, France) equipped with a 100 W mercury vapor lamp. The specific filter set for mTHPC fluorescence detection consisted of a 400–440 nm band-pass excitation filter associated with a 570 nm dichroic mirror and a 590 nm long pass filter. Neutral density filter was used in order to reduce photobleaching phenomenon. Fluorescence images were then recorded using a 403 oil immersion plan apochromatic objective (corrected for spherical and chromatic aberrations), a high numerical aperture and a strictly controlled integration time. No interfering autofluorescence signal was observed in our experimental conditions.
3. Results
3.1. Intracellular accumulation of mTHPC in MCF-7 and MCF-7 /DXR cells Cells were exposed to mTHPC at different concentrations and intracellular drug fluorescence was detected at the end of a 3-h incubation period. As follows from Fig. 1, the intracellular mTHPC uptake in both cell lines increased as a function of drug concentration. While in the concentration range from 0.75 to 3 mM, intracellular mTHPC accumulation was statistically not different in both sensitive and resistant cells, the highest applied concentration of 7.5 mM resulted in a significantly greater mTHPC accumulation in MDR cells. The mTHPC concentration of 1.5 mM was selected for the rest of experiments. Kinetic of intracellular uptake of the fluorescent forms of mTHPC (1.5 mM) is presented in Fig. 2. The photosensitizer accumulates in both cell lines as a function of incubation time and no plateau was registered during the whole observation period. The values of the relative mTHPC intracellular fluorescence after 3 h contact were not significantly different between both cell lines (0.6160.10 and 0.6860.06 for MCF-7 and MCF-7 / DXR, respectively; P50.109). The fluorescence measurements in living cells not always reflect the total photosensitizer content, therefore, the amount of intracellular mTHPC was estimated after the ScintiGest extraction procedure. No significant difference in mTHPC relative content was found between both cell lines after extraction (1.1460.15 and 1.1760.12 for MCF-7 and MCF-7 / DXR, respectively; P50.520). At the longer incubation time, mTHPC uptake became significantly greater in MCF-7 / DXR than in MCF-7 cells. To determine whether mTHPC could be a possible substrate for P-gp, MCF-7 / DXR cells were tested for
2.7. Statistical analyses Results were analyzed for statistical significant differences using non-parametric Mann–Whitney test. In all cases, the limit for significance was set to P,0.05.
Fig. 1. Intracellular uptake of mTHPC in MCF-7 (♦) and MCF-7 / DXR (j) cells as a function of sensitizer concentration. The results are expressed as normalized intracellular mTHPC fluorescence measured in both cell lines after 3 h incubation with mTHPC. Data are mean6S.E.M. of at least five experiments.
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Fig. 2. Intracellular uptake of mTHPC in MCF-7 (♦) and MCF-7 / DXR (j) cells as a function of incubation time. The results are expressed as normalized intracellular mTHPC fluorescence measured in both cell lines after different incubation times with mTHPC (1.5 mM). Data are mean6S.E.M. of three experiments.
possible changes in mTHPC intracellular fluorescence after 3 h exposure to 1.5 mM mTHPC. MCF-7 served as a positive control in that experiment. No significant variations in mTHPC intracellular fluorescence have been observed in both cell lines up to 8 h re-incubation in mTHPC-free culture medium (Fig. 3). Absence of mTHPC exodus from MDR cells rules out the possibility for mTHPC to be a P-gp substrate.
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function of light fluence after 3 h incubation with 1.5 mM mTHPC. These conditions provide a similar mTHPC uptake in sensitive and resistant cell lines. The percentage of cell survival was measured by normalizing the results of MTT assay with cells that were exposed to mTHPC only. It should be noted that cytotoxicity of mTHPC loaded cells (drug, no light) was not different from control cultures (no drug, no light). Cell viability decreased in a fluence-dependent manner for both cell lines. However, except of the lowest fluences applied (0.01 to 0.7 J cm 22 ), it was markedly enhanced in MCF-7 / DXR compared to its parental counterpart (Fig. 4). The light dose that induced 50% cell inactivation (LD 50 ) in MCF-7 cells was 2.4 J cm 22 and only 1.1 J cm 22 in MCF-7 / DXR. This trend was even more pronounced when cells were irradiated with greater fluences. Cell survival, following the highest applied fluence of 4.7 J cm 22 , was about 38.3 and 5% for MCF-7 and MCF-7 / DXR, respectively.
3.3. Role of P-gp in the photocytotoxic effect of mTHPC in MCF-7 /DXR mTHPC-sensitized photocytotoxicity was assessed after cells incubation in the presence or absence of MDRmodulating agents. Cyclosporin A and its derivative SDZPSC-833, the two P-gp inhibitors were used in the present study. The influence of both modulators on mTHPC intracellular fluorescence demonstrated that mTHPC up-
3.2. Effect of photodynamic treatment on MCF-7 and MCF-7 /DXR cell lines Photocytotoxicity in both cell lines was carried out as a
Fig. 3. mTHPC efflux kinetic from MCF-7 (♦) and MCF-7 / DXR (j) cells. After 3 h incubation with mTHPC (1.5 mM), the incubation medium was replaced by drug-free culture medium and normalized intracellular mTHPC fluorescence was measured. Data are the mean6S.E.M. of at least three experiments.
Fig. 4. Photocytotoxicity of MCF-7 (♦) and MCF-7 / DXR (j) cells exposed to mTHPC (1.5 mM) and red light irradiation (650 nm, 8.0 mW cm 22 ) at different light fluences. Survival was assessed by MTT colorimetric assay 24 h after light exposure. Data are mean6S.E.M. of at least three experiments.
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3.4. mTHPC intracellular distribution The subcellular distribution of mTHPC fluorescence detected after 3 h incubation with cells is shown in Fig. 6. Consistent with our previous work performed in the human colon adenocarcinoma HT 29 cells [23], mTHPC fluorescence images revealed diffuse pattern of dye localization throughout the cytoplasm with fluorescence intensification in the perinuclear area sparing the nucleus (Fig. 6). The only difference between the two cell lines was the presence of the punctate bright fluorescence pattern throughout the cytoplasm in MCF-7 / DXR.
4. Discussion
Fig. 5. Effect of SDZ-PSC-833 and cyclosporin A on mTHPC-sensitized photocytotoxicity on MCF-7 / DXR cells. (j) Irradiation of cells preloaded with mTHPC (1.5 mM); (♦) irradiation of cells co-incubated with mTHPC (1.5 mM) and SDZ-PSC-833 (1.5 mM); (d) irradiation of cells co-incubated with mTHPC (1.5 mM) and cyclosporin A (1.5 mM). Survival was assessed by MTT colorimetric assay 24 h after light exposure.
take was not modified by cell incubation in the presence of modulators (data not shown). As follows from Fig. 5, incubation of mTHPC-preloaded cells in the presence of cyclosporin A or SDZ-PSC-833 with the following irradiation did not affect the loss of cell viability compared to mTHPC alone.
Several studies proposed PDT as an alternative pathway in the treatment of chemoresistant cells expressing the MDR phenotype [3–5]. The present work focuses on the potency of mTHPC-based PDT in killing cells expressing the MDR phenotype. The MDR phenomenon involves a wide range of cytotoxic drugs, which do not share any clear functional or structural similarities. Impaired uptake was demonstrated for cationic photosensitizers, namely CDS1 or tetrabromorhodamine 123, and was attributed to the elevated P-gp expression in chemoresistant cell lines [6,7]. At the same time, the reduced accumulation of Photofrin, an anionic photosensitizer, was reported in RIF-8A cells and was suggested to be based on mitochondrial alterations [8,24,25] or altered subcellular targeting of sensitizer rather than classical MDR resistance mechanisms. Therefore, mTHPC was first tested for its intracellular accumulation in MCF-7 and MCF-7 / DXR cells. Photosensitizer uptake in the concentration range from 0.75 to 3 mM was
Fig. 6. Distribution of mTHPC fluorescence in MCF-7 (a) and MCF-7 / DXR (b) cells following 3 h of incubation with the drug (1.5 mM).
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found to be similar in both sensitive and resistant cells (Fig. 1). In addition, no drug efflux was observed in our experimental conditions up to 8 h re-incubation period (Fig. 3). These results suggest that cells expressing the MDR phenotype exhibited none of the anthracyclinesmediated transport mechanisms when mTHPC was the transport substrate. Our data could be compared with studies of Dellinger et al. [3] and Kessel and Erickson [4] which showed no difference in mesoporphyrin and Photofrin accumulation between sensitive and MDR cells. mTHPC accumulation becomes significantly greater in MCF-7 / DXR cells when high drug concentrations or long incubation periods were employed. Possible explanation could be afforded by the study of Bohmer and Morstyn [26], who demonstrated that environmental factors such as differences in intracellular pH, and / or size of targets cells influence the uptake of HpD. Consistent with this study, a better 5-aminolaevulinic acid (ALA)-induced Protoporphyrin IX uptake was observed in MDR resistant bladder cell line compared to their parental counterpart and was attributed to the increased cell surface area [27]. Our fluorescence microscopy studies demonstrated that cell surface area in MCF-7 / DXR cells was 1.2-fold greater compared to MCF-7 and could explain an enhanced mTHPC uptake in MCF-7 / DXR under certain experimental conditions. An end-point of the present study was the testing of photocytotoxicity in both cell lines. In spite of similar mTHPC intracellular content, the photocytotoxicity was considerably greater in MCF-7 / DXR compared to their parental counterparts. Indeed, the LD 50 was reduced by a factor of 2.2 in MCF-7 / DXR cells (Fig. 4). This observation contrasts other studies performed in MDR murine cell lines with porphyrin related photosensitizers [6,8,25]. This contradiction could probably be explained by the specific targeting of mTHPC in the human cell line. To determine whether an enhanced cell photoinactivation in MDR cells was related to the overexpression of P-gp, the photocytotoxicity was assessed after cells coincubation with mTHPC in the presence of MDR-modulating agents. Cyclosporin A and SDZ-PSC-833, P-gp modulators used in the present work, have been reported to be powerful MDR inhibitors [28]. Both inhibitors act predominantly through their binding to P-gp with subsequent inhibition of efflux [29]. We did not observe influence of any applied modulators on mTHPC-based photocytotoxic effect (Fig. 5). Therefore, an enhanced photokilling of MCF-7 / DXR could not be attributed to the over-expression of P-gp. The plausible explanation could be the difference in the sites of intracellular mTHPC localization between two cell lines. Specific photosensitizer localization contributes largely to the targeted photochemical reactions with a highly effective outcome [30]. The only difference in mTHPC distribution pattern between MCF-7 and MCF-7 / DXR was the observed mTHPC sequestration in in-
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tracytoplasmic vesicles in MCF-7 / DXR (Fig. 5b) that resemble lysosomes both in location and size. Several studies indicate that the targeted photosensitizer localization into lysosomes could contribute to markedly greater cell photoinactivation [31–33]. Considering an elevated pH gradient in MCF-7 / DXR cells due to their more acidified vesicles and more alkalinized cytosol [34], the directed mTHPC transport into the lysosomes of MCF-7 / DXR was anticipated. However, the results of double staining (1.5 mM mTHPC / 10 mM Lysotracker blue) in MCF-7 / DXR did not provide a direct evidence of colocalization of mTHPC with putative vacuoles. A distinct red fluorescence from mTHPC-loaded vesicles and blue fluorescence from Lysotracker stained lysosomes was observed in the combined fluorescence images (data not shown). Microspectrofluorometric studies that allow the real time monitoring of biological response would be useful in further identification of mTHPC-based PDT subcellular damage and are the subject of our ongoing experiments. PDT might be of clinical value for the patients with recurrent breast cancer, especially when classical treatments are considered ineffective [35]. Frequently, conventional modes of breast cancer therapy fail to control local disease because of the appearance of resistant cells expressing the MDR phenotype. Testing of the second generation photosensitizers in favor to the first generation is promising since a treatment at greater depth from skin surface could be achieved [36,37]. The data reported in this study clearly show that mTHPC-mediated PDT could be considered as an efficient way for destruction of neoplastic MDR cells.
Acknowledgements This work was supported by Alexis Vautrin Cancer Center Research Funds, French Ligue Nationale contre le Cancer. We are grateful to the National Research and Education Ministry of Luxemburg for awarding Scholarship to M.H.T. We are thankful to Dr. V. Melnikova for her contribution to this study. Finally, we gratefully acknowledge Scotia Pharmaceuticals Ltd. (Stirling, UK) for providing the mTHPC.
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[22] J.L. Merlin, S. Azzi, D. Lignon, C. Ramacci, N. Zeghari, F. Guillemin, MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy, Eur. J. Cancer 28A (1992) 1452–1458. [23] V.O. Melnikova, L.N. Bezdetnaya, C. Bour, E. Festor, M.P. Gramain, J.L. Merlin, A. Potapenko, F. Guillemin, Subcellular localization of meta-tetra(hydroxyphenyl) chlorin in human tumor cells subjected to photodynamic treatment, J. Photochem. Photobiol. B: Biol. 49 (1999) 96–103. [24] S.M. Sharkey, B.C. Wilson, R. Moorehead, G. Singh, Mitochondrial alterations in photodynamic therapy-resistant cells, Cancer Res. 53 (1993) 4994–4999. [25] G. Singh, B.C. Wilson, S.M. Sharkey, G.P. Browman, P. Deschamps, Resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and Chinese hamster ovary-multi-drug resistant cells in vitro, Photochem. Photobiol. 54 (1991) 307–312. [26] R.M. Bohmer, G. Morstyn, Uptake of hematoporphyrin derivative by normal and malignant cells: effect of serum, pH, temperature, and cell size, Cancer Res. 45 (1985) 5328–5334. [27] S.N. Datta, R. Allman, C. Loh, M. Mason, P.N. Matthews, Effect of photodynamic therapy in combination with mitomycin C on a mitomycin-resistant bladder cancer cell line, Br. J. Cancer 76 (1997) 312–317. [28] J. Petriz, J. Sanchez, J. Bertran, J. Garcia-Lopez, Comparative effect of verapamil, cyclosporin A and SDZ PSC 833 on rhodamine 123 transport and cell cycle in vinblastine-resistant Chinese hamster ovary cells overexpressing P-glycoprotein, Anticancer Drugs 8 (1997) 869–875. [29] M.C. Cabot, A.E. Giuliano, T.Y. Han, Y. Y Liu, SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells, Cancer Res. 59 (1999) 880–885. [30] N.L. Oleinick, H.H. Evans, The photobiology of photodynamic therapy: cellular targets and mechanisms, Radiat. Res. 150 (1998) S146–S156. [31] K. Berg, K. Prydz, J. Moan, Photochemical treatment with the lysosomally localized dye tetra(4-sulfonatophenyl)porphine results in lysosomal release of the dye but not of b-N-acetyl-D-glucosmaminidase activity, Biochim. Biophys. Acta 1158 (1993) 300–306. [32] C.W. Lin, J.R. Shulok, S.D. Kirley, L. Cincotta, J.W. Foley, Lysosomal localization and mechanism of uptake of Nile Blue photosensitizers in tumors cells, Cancer Res. 51 (1991) 2710–2719. [33] R. Bachor, C.R. Shea, R. Gillies, T. Hasan, Photosensitized destruction of human bladder carcinoma cells treated with chlorin e6conjugated microspheres, Proc. Natl. Acad. Sci. USA 88 (1991) 1580–1584. [34] R. Belhoussine, H. Morjani, S. Sharonov, D. Ploton, M. Manfait, Characterization of intracellular pH gradients in human multidrugresistant tumor cells by means of scanning microspectrofluorometry and dual-emission-ratio probes, Int. J. Cancer 81 (1999) 81–89. [35] H. Koren, G. Alth, G.M. Schenk, R.H. Jindra, Photodynamic therapy – an alternative pathway in the treatment of recurrent breast cancer, Int. J. Radiat. Oncol. Biol. Phys. 28 (1994) 463–466. [36] P.W. Sperduto, T.F. DeLaney, G. Thomas, P. Smith, L.J. Dachowski, A. Russo, R. Bonner, E. Glatstein, Photodynamic therapy for chest wall recurrence in breast cancer, Int. J. Radiat. Oncol. Biol. Phys. 21 (1991) 441–446. [37] S.W. Taber, V.H. Fingar, C.T. Coots, T.J. Wieman, Photodynamic therapy using mono-L-aspartyl chlorin e6 (Npe6) for the treatment of cutaneous disease: a Phase I clinical study, Clin. Cancer Res. 4 (1998) 2741–2746.
Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells a,b a, a a b b M.-H. Teiten , L. Bezdetnaya *, J.-L. Merlin , C. Bour-Dill , M.E. Pauly , M. Dicato , a F. Guillemin a
´ Unite´ de Recherche en Therapie Photodynamique, Centre Alexis Vautrin, Avenue de Bourgogne, Brabois, F-54511 Vandoeuvre-les-Nancy Cedex, France b Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Centre Universitaire de Luxembourg, L-1511 Luxembourg, Luxembourg Received 23 March 2001; accepted 26 July 2001
Abstract Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7 / DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light ( l5650 nm), was significantly greater in MCF-7 / DXR as compared to MCF-7 cells. A light dose of 2.5 J cm 22 inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7 / DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype. 2001 Elsevier Science B.V. All rights reserved. Keywords: Photodynamic therapy; mTHPC; Multidrug-resistance; Cytotoxicity
1. Introduction Photodynamic therapy (PDT) is a rapidly growing modality for the treatment of light-accessible tumors. PDT is based upon the administration of a photosensitizing compound (photosensitizer) with its following activation by visible light [1]. The resulting photodamages originate from the production of singlet oxygen and other reactive oxygen species [2]. Photodynamic therapy has been proposed in several studies as an alternative in overcoming multidrug resistance (MDR) phenotype. Triggered by the overexpression of P-glycoprotein (P-gp) efflux protein with the successive exclusion of hydrophobic and cationic drugs from cells, development of MDR phenotype results in a broad-spec*Corresponding author. Tel.: 133-3-8359-8306; fax: 133-3-83447635. E-mail address: [email protected] (L. Bezdetnaya).
trum of resistance, thus explaining a frequent failure of conventional chemotherapy. An effective outcome of photosensitization in MDR cells was reported for mesoporphyrin, phthalocyanines and Photofrin [3–5]. An equal or slightly better photocytotoxicity was observed with these photosensitizers in both sensitive and resistant cell lines under conditions of similar drug uptake. Meanwhile, there are several observations of cross-resistance between both treatment modalities. The failure of PDT could be translated either by an impaired accumulation of sensitizers due to their recognition by P-gp [6,7] or to other mechanisms that are not directly related to P-gp overexpression [8]. Experiences with the first generation photosensitizers (hematoporphyrin derivative (Hpd) and Photofrin) have led to the development of new photosensitizing molecules. Meta-tetra(hydroxyphenyl)chlorin (mTHPC), a second generation photosensitizer, has better characteristics for use in PDT over Photofrin, since it is a chemically pure compound with a high quantum yield of singlet oxygen
1011-1344 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 01 )00178-6
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generation, an enhanced absorption in far red region (650 nm) and reduced skin photosensitivity [9–12]. Our recent studies demonstrated favorable photochemical and immunological aspects of mTHPC-induced cell sensitization [13,14]. Presently, mTHPC is under investigation for potential clinical applications [15] as it has been presented as a molecule of interest for the PDT of ovary, skin and breast cancers [16,17]. The impact of mTHPC-induced sensitization on MDR cells has not been addressed so far. Therefore, the present study inspects the efficacy of mTHPC-based photosensitization in cells expressing the MDR phenotype. This goal was achieved by comparing mTHPC accumulation and photosensitization in MCF-7 human breast adenocarcinoma cell line and its doxorubicin resistant subline (MCF7 / DXR), which is characterized by the overexpression of P-gp. Our results indicate absence of cross-resistance to PDT in MDR cells. Moreover, considerably higher photocytotoxicity was demonstrated in chemoresistant cells compared to their normal counterparts and was attributed to a different mTHPC distribution pattern between both cell lines.
2. Experimental
2.1. Chemicals mTHPC was supplied by Scotia Pharmaceuticals (Stirling, UK). mTHPC stock solution was performed according to the manufacturer’s recommendations (20% ethanol, 30% polyethylene glycol 400 and 50% water). Further dilution was performed in phenol red free RPMI 1640 medium supplemented with 2% fetal calf serum to reach a final mTHPC concentration of 1.5 mM. mTHPC solution was prepared 1 h before use in experiments. Doxorubicin was purchased from Pharmacia (St. Quentin-Yvelines, France). SDZ-PSC-833 and cyclosporin A were obtained from Novartis (Basel, Switzerland). These two P-gp modulating agents were solubilized in sterile water and in ethanol, respectively. Further dilutions were realized in sterile water to reach a final concentration of 1.5 mM.
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MCF-7 / DXR, was maintained in 10 mM doxorubicincontaining medium. Our previous published study showed that such incubation conditions resulted in the development of 200-fold resistance to doxorubicin [18] and that MCF-7 / DXR cell line overexpresses P-glycoprotein independently of other efflux proteins such as the multidrug-resistanceassociated protein (MRP), the major vault lung resistance protein (LRP) and the anthracycline resistance associated protein (ARA) [19]. A wash-out period of 7 days in doxorubicin-free medium was allowed for MCF-7 / DXR cells before being used in experiments.
2.3. Intracellular accumulation and efflux of mTHPC Logarithmically growing MCF-7 and MCF-7 / DXR cells, inoculated in 25-cm 2 culture flasks, were washed twice and exposed for 3 h to different mTHPC concentrations or to 1.5 mM mTHPC for different incubation times. At different experimental points, cells were washed three times, trypsinized and re-suspended in RPMI 1640 supplemented with 2% FCS. mTHPC intracellular uptake was assessed by measuring mTHPC fluorescence from drug-loaded cells. Intracellular mTHPC was extracted by adding of 10% ScintiGest (70% cetyltriammonium bromide, Sigma; 20% methylalcohol, Prolabo; 9.9% potassium hydroxide, Sigma) to cell suspension with the successive overnight storage at 608C. Efflux of mTHPC from cells was studied in the following way. After 3 h incubation with mTHPC (1.5 mM), the medium was removed, cells were washed twice with phosphate-buffered saline (PBS) and re-incubated in fresh medium for different time with the following measurements of mTHPC fluorescence. Intracellular mTHPC fluorescence was assessed using a computer-controlled Perkin-Elmer LS50B luminescence spectrofluorimeter equipped with a front surface accessory (Perkin-Elmer L225 9051), providing small-angle (458C) excitation geometry. The excitation wavelength was 422 nm and spectra were collected for a wavelength range between 600 to 750 nm. Fluorescence intensity was monitored at an emission wavelength of 655 nm and normalized to 10 4 viable cells.
2.4. Photodynamic treatment 2.2. Cell culture conditions MCF-7 human breast adenocarcinoma cell line and its subline MCF-7 / DXR were cultivated in 75-cm 2 culture flasks (Costar, Dutscher, Brumath, France) in phenol red free RPMI 1640 medium (Life Technologies, Cergy-Pontoise, France) supplemented with 9% fetal calf serum (FCS) (Dutscher, Brumath, France), penicillin (10 000 IU) and streptomycin (10 000 IU). Cells were kept at 378C in a 5% CO 2 humidified atmosphere, trypsinized and re-seeded into fresh medium every 7 days. The resistant subline,
Logarithmically growing MCF-7 and MCF-7 / DXR in 96-well plates were washed twice with PBS and incubated with mTHPC alone or in the presence of SDZ-PSC-833 (1.5 mM) or cyclosporin A (1.5 mM) for 3 h. Nonincorporated reagents were removed by washing cells twice with PBS and cells were further re-incubated in fresh culture medium. Irradiation was carried out at different fluences at 650 nm with a dye laser (Spectra-Physics 375B), pumped with an argon dye laser (Spectra-Physics 2020, Les Ulis, France) with an output of 0.3 W. Light
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spots of 7 cm diameter provided a fluence rate of 8 mW cm 22 .
2.5. Measurement of cell viability Cell viability was assessed 24 h after illumination using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma] colorimetric assay, which measures the capacity of mitochondrial deshydrogenase in viable cells to reduce MTT to a purple formazan precipitate. The MTT method assesses the number of surviving cells and has shown a good correlation with other established measures of cytotoxicity as clonogenic assay [20]. Likewise, the MTT-test has been well characterized for use as a measure of PDT effect and it shows a good correlation with viability determined by a clonogenic assay for various types of cells photosensitized with HpD [21,22]. A 50-ml volume of 0.125 M MTT was added into each well for 3 h at 378C to allow metabolism of MTT. After incubation, the formazan crystals were solubilized by adding 50 ml of 25% sodium dodecyl sulfate solution into each well. The reaction product was quantified photometrically at 540 nm on a Multiskan plate reader (Flow Laboratories). Results are given as the percentage of the data obtained at similar fluence with control cultures exposed to drug-free medium.
2.6. Subcellular localization of mTHPC For the fluorescence microscopy observations, cells were plated into Slideflasks (Nunc, Polylabo, France), and allowed to attach to the dishes for 4 days at 378C before being tested. The use of the Slideflasks allowed the adhesive living cells to be maintained during observation. Cells were incubated with mTHPC solution (1.5 mM) for 3 h, rinsed three times with PBS and immediately observed under an AX 70 PROVIS upright epifluorescence microscope (Olympus, France) equipped with a 100 W mercury vapor lamp. The specific filter set for mTHPC fluorescence detection consisted of a 400–440 nm band-pass excitation filter associated with a 570 nm dichroic mirror and a 590 nm long pass filter. Neutral density filter was used in order to reduce photobleaching phenomenon. Fluorescence images were then recorded using a 403 oil immersion plan apochromatic objective (corrected for spherical and chromatic aberrations), a high numerical aperture and a strictly controlled integration time. No interfering autofluorescence signal was observed in our experimental conditions.
3. Results
3.1. Intracellular accumulation of mTHPC in MCF-7 and MCF-7 /DXR cells Cells were exposed to mTHPC at different concentrations and intracellular drug fluorescence was detected at the end of a 3-h incubation period. As follows from Fig. 1, the intracellular mTHPC uptake in both cell lines increased as a function of drug concentration. While in the concentration range from 0.75 to 3 mM, intracellular mTHPC accumulation was statistically not different in both sensitive and resistant cells, the highest applied concentration of 7.5 mM resulted in a significantly greater mTHPC accumulation in MDR cells. The mTHPC concentration of 1.5 mM was selected for the rest of experiments. Kinetic of intracellular uptake of the fluorescent forms of mTHPC (1.5 mM) is presented in Fig. 2. The photosensitizer accumulates in both cell lines as a function of incubation time and no plateau was registered during the whole observation period. The values of the relative mTHPC intracellular fluorescence after 3 h contact were not significantly different between both cell lines (0.6160.10 and 0.6860.06 for MCF-7 and MCF-7 / DXR, respectively; P50.109). The fluorescence measurements in living cells not always reflect the total photosensitizer content, therefore, the amount of intracellular mTHPC was estimated after the ScintiGest extraction procedure. No significant difference in mTHPC relative content was found between both cell lines after extraction (1.1460.15 and 1.1760.12 for MCF-7 and MCF-7 / DXR, respectively; P50.520). At the longer incubation time, mTHPC uptake became significantly greater in MCF-7 / DXR than in MCF-7 cells. To determine whether mTHPC could be a possible substrate for P-gp, MCF-7 / DXR cells were tested for
2.7. Statistical analyses Results were analyzed for statistical significant differences using non-parametric Mann–Whitney test. In all cases, the limit for significance was set to P,0.05.
Fig. 1. Intracellular uptake of mTHPC in MCF-7 (♦) and MCF-7 / DXR (j) cells as a function of sensitizer concentration. The results are expressed as normalized intracellular mTHPC fluorescence measured in both cell lines after 3 h incubation with mTHPC. Data are mean6S.E.M. of at least five experiments.
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Fig. 2. Intracellular uptake of mTHPC in MCF-7 (♦) and MCF-7 / DXR (j) cells as a function of incubation time. The results are expressed as normalized intracellular mTHPC fluorescence measured in both cell lines after different incubation times with mTHPC (1.5 mM). Data are mean6S.E.M. of three experiments.
possible changes in mTHPC intracellular fluorescence after 3 h exposure to 1.5 mM mTHPC. MCF-7 served as a positive control in that experiment. No significant variations in mTHPC intracellular fluorescence have been observed in both cell lines up to 8 h re-incubation in mTHPC-free culture medium (Fig. 3). Absence of mTHPC exodus from MDR cells rules out the possibility for mTHPC to be a P-gp substrate.
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function of light fluence after 3 h incubation with 1.5 mM mTHPC. These conditions provide a similar mTHPC uptake in sensitive and resistant cell lines. The percentage of cell survival was measured by normalizing the results of MTT assay with cells that were exposed to mTHPC only. It should be noted that cytotoxicity of mTHPC loaded cells (drug, no light) was not different from control cultures (no drug, no light). Cell viability decreased in a fluence-dependent manner for both cell lines. However, except of the lowest fluences applied (0.01 to 0.7 J cm 22 ), it was markedly enhanced in MCF-7 / DXR compared to its parental counterpart (Fig. 4). The light dose that induced 50% cell inactivation (LD 50 ) in MCF-7 cells was 2.4 J cm 22 and only 1.1 J cm 22 in MCF-7 / DXR. This trend was even more pronounced when cells were irradiated with greater fluences. Cell survival, following the highest applied fluence of 4.7 J cm 22 , was about 38.3 and 5% for MCF-7 and MCF-7 / DXR, respectively.
3.3. Role of P-gp in the photocytotoxic effect of mTHPC in MCF-7 /DXR mTHPC-sensitized photocytotoxicity was assessed after cells incubation in the presence or absence of MDRmodulating agents. Cyclosporin A and its derivative SDZPSC-833, the two P-gp inhibitors were used in the present study. The influence of both modulators on mTHPC intracellular fluorescence demonstrated that mTHPC up-
3.2. Effect of photodynamic treatment on MCF-7 and MCF-7 /DXR cell lines Photocytotoxicity in both cell lines was carried out as a
Fig. 3. mTHPC efflux kinetic from MCF-7 (♦) and MCF-7 / DXR (j) cells. After 3 h incubation with mTHPC (1.5 mM), the incubation medium was replaced by drug-free culture medium and normalized intracellular mTHPC fluorescence was measured. Data are the mean6S.E.M. of at least three experiments.
Fig. 4. Photocytotoxicity of MCF-7 (♦) and MCF-7 / DXR (j) cells exposed to mTHPC (1.5 mM) and red light irradiation (650 nm, 8.0 mW cm 22 ) at different light fluences. Survival was assessed by MTT colorimetric assay 24 h after light exposure. Data are mean6S.E.M. of at least three experiments.
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3.4. mTHPC intracellular distribution The subcellular distribution of mTHPC fluorescence detected after 3 h incubation with cells is shown in Fig. 6. Consistent with our previous work performed in the human colon adenocarcinoma HT 29 cells [23], mTHPC fluorescence images revealed diffuse pattern of dye localization throughout the cytoplasm with fluorescence intensification in the perinuclear area sparing the nucleus (Fig. 6). The only difference between the two cell lines was the presence of the punctate bright fluorescence pattern throughout the cytoplasm in MCF-7 / DXR.
4. Discussion
Fig. 5. Effect of SDZ-PSC-833 and cyclosporin A on mTHPC-sensitized photocytotoxicity on MCF-7 / DXR cells. (j) Irradiation of cells preloaded with mTHPC (1.5 mM); (♦) irradiation of cells co-incubated with mTHPC (1.5 mM) and SDZ-PSC-833 (1.5 mM); (d) irradiation of cells co-incubated with mTHPC (1.5 mM) and cyclosporin A (1.5 mM). Survival was assessed by MTT colorimetric assay 24 h after light exposure.
take was not modified by cell incubation in the presence of modulators (data not shown). As follows from Fig. 5, incubation of mTHPC-preloaded cells in the presence of cyclosporin A or SDZ-PSC-833 with the following irradiation did not affect the loss of cell viability compared to mTHPC alone.
Several studies proposed PDT as an alternative pathway in the treatment of chemoresistant cells expressing the MDR phenotype [3–5]. The present work focuses on the potency of mTHPC-based PDT in killing cells expressing the MDR phenotype. The MDR phenomenon involves a wide range of cytotoxic drugs, which do not share any clear functional or structural similarities. Impaired uptake was demonstrated for cationic photosensitizers, namely CDS1 or tetrabromorhodamine 123, and was attributed to the elevated P-gp expression in chemoresistant cell lines [6,7]. At the same time, the reduced accumulation of Photofrin, an anionic photosensitizer, was reported in RIF-8A cells and was suggested to be based on mitochondrial alterations [8,24,25] or altered subcellular targeting of sensitizer rather than classical MDR resistance mechanisms. Therefore, mTHPC was first tested for its intracellular accumulation in MCF-7 and MCF-7 / DXR cells. Photosensitizer uptake in the concentration range from 0.75 to 3 mM was
Fig. 6. Distribution of mTHPC fluorescence in MCF-7 (a) and MCF-7 / DXR (b) cells following 3 h of incubation with the drug (1.5 mM).
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found to be similar in both sensitive and resistant cells (Fig. 1). In addition, no drug efflux was observed in our experimental conditions up to 8 h re-incubation period (Fig. 3). These results suggest that cells expressing the MDR phenotype exhibited none of the anthracyclinesmediated transport mechanisms when mTHPC was the transport substrate. Our data could be compared with studies of Dellinger et al. [3] and Kessel and Erickson [4] which showed no difference in mesoporphyrin and Photofrin accumulation between sensitive and MDR cells. mTHPC accumulation becomes significantly greater in MCF-7 / DXR cells when high drug concentrations or long incubation periods were employed. Possible explanation could be afforded by the study of Bohmer and Morstyn [26], who demonstrated that environmental factors such as differences in intracellular pH, and / or size of targets cells influence the uptake of HpD. Consistent with this study, a better 5-aminolaevulinic acid (ALA)-induced Protoporphyrin IX uptake was observed in MDR resistant bladder cell line compared to their parental counterpart and was attributed to the increased cell surface area [27]. Our fluorescence microscopy studies demonstrated that cell surface area in MCF-7 / DXR cells was 1.2-fold greater compared to MCF-7 and could explain an enhanced mTHPC uptake in MCF-7 / DXR under certain experimental conditions. An end-point of the present study was the testing of photocytotoxicity in both cell lines. In spite of similar mTHPC intracellular content, the photocytotoxicity was considerably greater in MCF-7 / DXR compared to their parental counterparts. Indeed, the LD 50 was reduced by a factor of 2.2 in MCF-7 / DXR cells (Fig. 4). This observation contrasts other studies performed in MDR murine cell lines with porphyrin related photosensitizers [6,8,25]. This contradiction could probably be explained by the specific targeting of mTHPC in the human cell line. To determine whether an enhanced cell photoinactivation in MDR cells was related to the overexpression of P-gp, the photocytotoxicity was assessed after cells coincubation with mTHPC in the presence of MDR-modulating agents. Cyclosporin A and SDZ-PSC-833, P-gp modulators used in the present work, have been reported to be powerful MDR inhibitors [28]. Both inhibitors act predominantly through their binding to P-gp with subsequent inhibition of efflux [29]. We did not observe influence of any applied modulators on mTHPC-based photocytotoxic effect (Fig. 5). Therefore, an enhanced photokilling of MCF-7 / DXR could not be attributed to the over-expression of P-gp. The plausible explanation could be the difference in the sites of intracellular mTHPC localization between two cell lines. Specific photosensitizer localization contributes largely to the targeted photochemical reactions with a highly effective outcome [30]. The only difference in mTHPC distribution pattern between MCF-7 and MCF-7 / DXR was the observed mTHPC sequestration in in-
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tracytoplasmic vesicles in MCF-7 / DXR (Fig. 5b) that resemble lysosomes both in location and size. Several studies indicate that the targeted photosensitizer localization into lysosomes could contribute to markedly greater cell photoinactivation [31–33]. Considering an elevated pH gradient in MCF-7 / DXR cells due to their more acidified vesicles and more alkalinized cytosol [34], the directed mTHPC transport into the lysosomes of MCF-7 / DXR was anticipated. However, the results of double staining (1.5 mM mTHPC / 10 mM Lysotracker blue) in MCF-7 / DXR did not provide a direct evidence of colocalization of mTHPC with putative vacuoles. A distinct red fluorescence from mTHPC-loaded vesicles and blue fluorescence from Lysotracker stained lysosomes was observed in the combined fluorescence images (data not shown). Microspectrofluorometric studies that allow the real time monitoring of biological response would be useful in further identification of mTHPC-based PDT subcellular damage and are the subject of our ongoing experiments. PDT might be of clinical value for the patients with recurrent breast cancer, especially when classical treatments are considered ineffective [35]. Frequently, conventional modes of breast cancer therapy fail to control local disease because of the appearance of resistant cells expressing the MDR phenotype. Testing of the second generation photosensitizers in favor to the first generation is promising since a treatment at greater depth from skin surface could be achieved [36,37]. The data reported in this study clearly show that mTHPC-mediated PDT could be considered as an efficient way for destruction of neoplastic MDR cells.
Acknowledgements This work was supported by Alexis Vautrin Cancer Center Research Funds, French Ligue Nationale contre le Cancer. We are grateful to the National Research and Education Ministry of Luxemburg for awarding Scholarship to M.H.T. We are thankful to Dr. V. Melnikova for her contribution to this study. Finally, we gratefully acknowledge Scotia Pharmaceuticals Ltd. (Stirling, UK) for providing the mTHPC.
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