Breast cancer band

Received 21 December 2002; accepted 14 March 2003

ABSTRACT

To determine the initial photodamage sites of Foscan(R)-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan(R)-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan(R) photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan(R)-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan(R)-mediated MCF-7 cell death by late and indirect mechanism.

Abbreviations: CSA, cyclosporin A; cyt c, cylochrome c; cyt c^sub red^, reduced form of cyt c; ER, endoplasmic reticulum; FCS, fetal calf serum; MPT, membrane permeability transition; mTHPC, meta-tetra(hydroxyphenyl)-chlorin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NADH, nicotinamide adenine dinucleotide reduced; PDT, photodynamic therapy; PE, phycoerythrin; PT, permeability transition; Rh123, rhodamine 123; TPZ, trifluoroperazine; UDP, uridine 5'-diphosphate.

INTRODUCTION

Photodynamic therapy (PDT) consists in the activation with visible or near-infrared light of photosensitizing compound accumulated in tumor or other abnormal tissues (1). Most of the photosensitizers studied thus far induce cell or tissue photodamage through the production of reactive cytotoxic species, mainly singlet oxygen, which has a short lifetime in biological systems ([asymptotically =]=40 ns) and, consequently, a radius of action shorter than 0.02 [mu]m (2). This implies that the initial cell damage sites are very close to those of singlet oxygen formation and strictly related to the distribution of the photosensitizer in the cell. However, the probability always exists that a low level of incorporation into a given compartment may give rise to high impact of photochemical reaction. Identification of primary photodamage sites can be done by measuring the activities of proteins typical of subcellular organelles immediately after irradiation. The targeted organelles in cell photoinactivation with hematoporphyrin derivative, tetraphenylporphyrins and zinc phthalocyanine have been thus approached (3-6).

Photosensitizer localization has also been shown to affect the mode of photodamage. In a series of studies, Kessel et al. (7,8) have shown that photosensitizers that localize in mitochondria are very efficient inducers of apoptosis. For nonmitochondrially localizing photosensitizers, mitochondrial damage may still be the effector pathway leading to cell death. Production of different messengers photoinitiated by different affector pathways may ultimately converge on certain mitochondrial events that lead to downstream effector pathways, culminating in cell death (9,10). These mitochondrial events include the induction of permeability transition (PT), release of cytochrome c (cyt c) and mitochondrial membrane depolarization (11-13).

Meta-tetra(hydroxyphenyl)chlorin (mTHPC, Foscan(R)) is a second-generation photosensitizer (14) approved in the European community (EC) countries for the treatment of advanced head and neck cancers (15) and has been studied in basal cell carcinoma (16) and locoregional breast cancer recurrences (17). Foscan(R)-induced cell photokilling is predominantly mediated by singlet oxygen (18,19). Our recent study, based on the microspectrofluorometric analysis of MCF-7 cells costained with Foscan(R) and organelle-specific probes, unambiguously demonstrated that the endoplasmic reticulum (ER) and the Golgi apparatus are the preferential sites of Foscan(R) accumulation. This sensitizer exhibited a weak accumulation in mitochondria and was barely delectable in lysosomes (20). The critical subcellular site(s) of Foscan(R)-mediated photodamage remains to be determined.

The present investigation extends our studies to evaluate the primary therapy-induced damage sites in MCF-7 cells. With this aim, we measured the post-PDT activities of enzymes typical of mitochondria, Golgi apparatus and ER and compared them with the loss of clonogenicity. We further addressed the milochondrial response after Foscan(R) cell photosensitization and its contribution to the overall cell death. We report a dose- and time-dependent mitochondrial membrane depolarization and cyt c release.

MATERIALS AND METHODS

Foscan(R) preparation

Foscan(R) was kindly supplied by Biolitec Pharma Ltd. (Edinburgh, UK). Foscan(R) stock solution (2 mg/mL, methanol) was further diluted in phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, France) supplemented with 2% fetal calf serum (FCS, Costar, Brumath, France) to reach a final Foscan(R) concentration of 1 [mu]g/mL.

Cell culture conditions

MCF-7 human breast adenocarcinoma cell line was cultivated in phenol red-free RPMI 1640 medium (Life Technologies) supplemented with 9% FCS, penicillin (10 000 IU) and streptomycin (10 000 IU). Cells were kept at 37[degrees]C in a 5% CO^sub 2^-humidified atmosphere, trypsinized and reseeded into fresh medium every 7 days. Unless otherwise indicated, four days before treatment, 42 x 10^sup 2^ cells/cm^sup 2^ were seeded in Nunc petri flasks.

Photodynamic treatment

Logarithmically growing MCF-7 cells were incubated with 1 [mu]g/mL Foscan(R) for 3 h. Nonincorporated reagents were removed by washing cells three times with phosphate-buffered saline. Cells were then reincubated in fresh culture medium and irradiated with a 650 nm diode laser light (Coherent, France) at a fluence rate of 2.12 mW/cm^sup 2^ for different light doses. When needed, 5 [mu]M cyclosporin A (CSA) and 5 [mu]M trifluoroperazine (TPZ) were added 30 min before irradiation and maintained until cell harvest, 4 h after PDT.

Assay of loss of clonogenicity

Immediately after treatment, cells were harvested by trypsinization and seeded into six-well plates. Clonogenicity was assessed by using double-agar layer technique as described previously (21). After 14 days, colonies composed of more than 50 cells per dish were counted using an automatic image analysis program (AnalySiS 3.1). The plating efficiency of untreated (no light, no drug) cells was 20-30%.

NADPH cyt c reductase and UDP galactosyl transferase measurements

Cells were scraped in ice-cold 0.25 M sucrose solution and then sonicated. The homogenate was cleared of cell debris, nuclei and mitochondria by centrifugation for 10 min at 10 000 g. The resulting supernatant was collected and centrifuged for l h at 100 000 g to give microsomal pellets, which were resuspended in 0.25 M sucrose.

The activity of the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) cyt c reductase, a specific enzyme of the ER (22,23), was measured as described by Beaufay et al. (24). This method is based on the formation of the reduced form of cyt c, cyt c^sub red^, induced by [beta]-NADPH (Sigma, France), which can be recorded spectrophotochemically at 550 nm.

The activity of uridinc 5'-diphosphate (UDP) galactosyl transferase (uridine diphospho-D-[6-^sup 3^H]galactose, ammonium salt; Amersham Pharmacia Biotech, France), a specific enzyme of the Golgi apparatus (25), was measured by a method based on the ability of the enzyme to bind radiolabeled uridine diphospho-D-[6-^sup 3^H]galaclose to ovalbumin (Sigma). The resulting activity was monitored by scintillation counting of the radioactivity with a Beckman automatic liquid scintillation system as described by Brandli et al. (25).

Cyt c oxidase measurements

Cells were scraped, swollen in an ice-cold hypotonic buffer (10 mM NaCl, 1.5 mM MgCl^sub 2^, 10 mM Tris-HCl, ph 7.5) and ruptured with a few strokes in a Dounce homogenizer using a tight-fitting pestle. The homogenate was centrifuged at 1300 g for 5 min to remove nuclei, unbroken cells and large membrane fragments. The supernatant was then centrifuged at 17 000 g for 15 min to pellet mitochondria. The mitochondrial cyt c oxidase enzymatic activity was measured as described by Gibson and Hilf (26) by a spectropholometric technique recording the ;disappearance of cyt c^sub red^ (reduced form of the substrate cytochrome c) at 550 nm.

MTT assay