Synergistic apoptotic effect of Doxil® and aminolevulinic acid-based photodynamic therapy on human breast adenocarcinoma cells

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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx

Available online at www.sciencedirect.com

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Synergistic apoptotic effect of Doxil® and aminolevulinic acid-based photodynamic therapy on human breast adenocarcinoma cells Soad Zakaria a, Amira M. Gamal-Eldeen PhD b,c,∗, Sherien M. El-Daly b,d, Samira Saleh e a

Department of Pharmacology and Toxicology, Faculty of Pharmacy, October 6 University, October 6 City, Giza, Egypt b Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt c Department of Biochemistry, National Research Center, Cairo, Egypt d Department of Medical Biochemistry, National Research Center, Cairo, Egypt e Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

KEYWORDS Breast cancer; 5-Aminolevulinic acid; Protoporphyrin IX; Doxil® ; Apoptosis; Death receptors

∗

Summary Background: 5-Aminolevulinic acid (ALA) is a natural heme precursor metabolized into protoporphyrin IX (PpIX). PpIX preferentially accumulates in tumor cells resulting in the formation of singlet oxygen upon exposure to visible light. Doxil® , an active agent against breast and ovarian cancer, is a nano-formulation of doxorubicin. This study aimed to investigate in vitro synergistic cytotoxic effect of low doses of combined chemotherapy and ALA/PDT to human breast adenocarcinoma cells (MCF-7) compared to high doses of each individual therapy. Methods: MCF-7 cells were pretreated with Doxil® (48 h) followed by ALA/PDT (4 h). The cell viability was evaluated by trypan blue assay and PpIX production was measured spectrofluorometrically. Alkaline phosphatase was determined as a marker for cellular differentiation. Apoptosis and necrosis were evaluated by fluorescence stains. The apoptosis cell death pathways were investigated: detection of mitochondrial membrane potential ( m ) and percent of DNA fragmentation, malondialdehyde, histone deacetylase (HDAC) activity, caspase-3 and death receptors (DR4 and DR5). Vascular endothelial growth factor (VEGF) was determined by ELISA, as an angiogenic mediator. Results: There was a higher reduction in cell viability in Doxil® + ALA/PDT-treated cells compared with their individual effect. The combined therapy showed enhanced apoptosis with a significant increase in the loss of  m , DNA fragmentation %, caspase-3, DR4, DR5 and lipid peroxides and inhibited HDAC. Pretreatment with Doxil® resulted in a twofold increase in the intracellular PpIX, by increasing the PDT killing of MCF-7 cells.

Corresponding author at: NRC, Dokki, 12622 Cairo, Egypt. Tel.: +20 1006053903; fax: +20 233370931. E-mail address: [email protected] (A.M. Gamal-Eldeen).

http://dx.doi.org/10.1016/j.pdpdt.2014.03.001 1572-1000/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Zakaria S, et al. Synergistic apoptotic effect of Doxil® and aminolevulinic acidbased photodynamic therapy on human breast adenocarcinoma cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.001

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S. Zakaria et al. Conclusion: The combined therapy using 50% of IC50 of ALA/PDT and Doxil® possessed a synergistic apoptotic effect on MCF-7 cells compared to 100% of IC50 of each therapy through enhancing both intrinsic and extrinsic apoptotic pathways, thus may minimize side effects of Doxil® and ALA. © 2014 Elsevier B.V. All rights reserved.

Introduction Breast cancer, the most common type of cancer among women in the world, affects approximately one out of every eight women over their lifetime. In recognition of the high invasiveness of surgical excision and severe side effects of chemical and radiation therapies, increasing efforts are made to seek minimally invasive modalities with fewer side effects [1]. Photodynamic therapy (PDT) has an advantage as a cancer therapy over surgery or ionizing radiation as PDT can eliminate tumors without causing fibrosis or scarring [2,3]. PDT involves the topical or systemic administration of a photosensitizer (PS), followed by activation of a PS with light of specific wavelength, which interacts with molecular oxygen to generate singlet oxygen and other reactive oxygen species (ROS) that eventually cause tumor cell death [4]. The cytotoxic effect of PDT is mainly through photodamage to subcellular organelles and biomolecules [5]. An alternative to the administration of exogenous photosensitizing compound is to stimulate the cellular synthesis of endogenous PSs [6]. Protoporphyrin is an important second generation PS endogenously generated by 5-aminolevulinic acid (5-ALA). ALA is a heme precursor that may have potential applications for photodynamic detection and photodynamic therapy-based treatment of solid tumors in a variety of malignancies [7]. Another important anticancer drug is doxorubicin. It is an effective chemotherapeutic drug, and therefore it became one of the main ‘‘first line’’ anticancer drugs almost from its discovery and it remains so till today. It is effective against more types of cancer (including leukemias, lymphomas, and breast, uterine, ovarian, and lung cancers) than any other class of chemotherapy agents [8—10]. However, their usefulness is limited by cumulative dose-dependent cardiotoxicity that may manifest as congestive heart failure, arrhythmias or conductivity dysfunction [11]. Nano-preparations of chemotherapeutic agents can significantly optimize drug delivery as well as its safety, and efficacy. Nanoparticles, up to 400 nm in size, have shown great promise for carrying, protecting and delivering potential therapeutic molecules with diverse physiological properties [12]. Pegylated liposomal doxorubicin (Doxil® ) is a unique formulation of doxorubicin hydrochloride in which the drug is encapsulated in the inner water phase of lipid vesicles of approximately 80—100 nm diameters. These vesicles are then surrounded by a dense layer of surface-bound methoxypolyethylene glycol (m-PEG). The PEG coating on the liposome creates a hydrophilic layer around the liposome that buffers the liposome wall from the surrounding milieu. This decreases proteins from binding to the lipid bilayer [13]. Doxil® is the first FDA-approved nano-drug formulations of doxorubicin that have been investigated clinically [14]. Due to the enhanced permeability and retention (EPR) effect, Doxil® is ‘‘passively targeted’’ to tumors and its

doxorubicin is released and becomes available to tumor cells [14]. Higher drug levels in tumor tissue have been observed with Doxil® than free doxorubicin in multiple cancer models [15,16]. It has shown the ability to clinically reduce cardiotoxicity, a hallmark side effect of free doxorubicin treatment [17,18]. However it is associated with hand—foot syndrome (HFS) a potentially dose-limiting effect causing a painful erythema and swelling on the palms of the hand and the soles of the feet [19]. Combined therapy is a common practice in many medical disciplines. PDT could be used as an approach to enhance the efficacy of anticancer drugs, facilitating the delivery of macromolecular agents [20]. The use of PDT in addition to one of the nano-formulations of doxorubicin in the treatment of breast cancer could be useful through reaching a desirable death proportion of malignant cells with lower doses of chemotherapeutic drugs. Since in many studies, doxorubicin and ALA each have previously shown to independently enhance apoptosis-dependant cell death in tumor cells, therefore, we investigated the hypothesis that the simultaneous administration of Doxil® and ALA could have a synergistic apoptotic effect in vitro against breast adenocarcinoma MCF-7 cells.

Materials and methods Doxil® was purchased from Schering-Plough, Utrecht, Netherlands, while ALA and all chemicals were purchased from (Sigma/Aldrich, VA, USA) except mentioned. ALA stock solution (100 mM) was prepared in PBS and kept in dark at −20 ◦ C.

Cell culture Human breast adenocarcinoma cells (MCF-7) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-7 cells were routinely cultured in RPMI 1640 Media (Gibco® /Invitrogen) with 2 mM Lglutamine, and was supplemented with 10% fetal bovine serum (Gibco® /Invitrogen), penicillin (100 units/ml), and streptomycin (100 ␮g/ml). Cells were maintained in humidified air containing 5% CO2 at 37 ◦ C. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37 ◦ C. All experiments were repeated four times independently, and the data were represented as the mean ± SD.

Cytotoxicity of Doxil® The cytotoxic effect of different concentration of Doxil® on MCF-7 cells was tested using the trypan blue exclusion test of cell viability. MCF-7 cells (5 × 104 cells/well) were treated with different concentrations (5, 10, 20, and 40 ␮M)

Please cite this article in press as: Zakaria S, et al. Synergistic apoptotic effect of Doxil® and aminolevulinic acidbased photodynamic therapy on human breast adenocarcinoma cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.001

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Synergistic apoptotic effect of Doxil® and aminolevulinic acid of Doxil® and incubated for 48 h. After cell collection, 20 ␮l of cell suspension was added to 20 ␮l of 0.4% trypan blue and then viable and dead cells were counted by a hemocytometer using a light microscope. The percentage of viability was determined by the following equation: % of cell viability = (number of viable cells/total cell number) × 100.

Cytotoxicity of ALA/PDT To evaluate the effect of different laser irradiation intervals on ALA-induced phototoxicity, MCF-7 cells were cultured in cell culture inserts (SPL, Life science, Korea) and treated with ALA 1 mM in serum free RPMI 1640 medium. After 4 h, attached cells were washed with the media and irradiated for different intervals (0, 1, 2, 4 and 8 min) using a helium—neon laser (He—Ne ion laser, Melles Griot, Singapore) at a wavelength of 633 nm and power of 0.25 W, and then incubated for further 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. The longest safe irradiation interval on cell viability was 4 min, which we select to evaluate the influence of different concentrations of ALA (0.5—2 mM) on MCF-7 cells. Cells were cultured with ALA in serum-free RPMI 1640 medium for 4 h, and then irradiated for 4 min. The cytotoxicity was then evaluated using Trypan blue exclusion test.

Combined therapy of Doxil® and ALA/PDT MCF-7 cells, cultured in cell culture inserts at a density of 5 × 104 cells/well, were treated with RPMI 1640 medium containing Doxil® (half of the IC50 concentration; 9.925 ␮M) and incubated for 48 h, then the medium was aspirated and replaced with serum free medium containing ALA (half of the IC50 concentration; 0.55 mM) and incubated for additional 4 h. Attached cells were washed by medium and irradiated for 4 min at a wavelength of 633 nm and power of 0.25 W and incubated for further 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. In order to investigate the effect each of laser alone or with ALA treatment on Doxil® on MCF-7 viability, other inserts of Doxil® treated cells were exposed to laser only or treated with ALA in dark conditions. Trypan blue exclusion assay was used to detect the synergistic cytotoxic effect of both Doxil® and ALA on MCF-7 cells.

Quantification of protoporphyrin IX production For quantification of protoporphyrin IX (PpIX) production in MCF-7 cells, cells were cultured in six well plates at a density of (5 × 105 cells/well) and treated with RPMI 1640 medium containing Doxil® 9.925 ␮M as final concentration. The cells were incubated for 48 h, and then media were replaced with ALA (0.55 mM) in serum-free media for final 4 h incubation. In a parallel experiment, MCF-7 cells were directly treated with ALA (1.12 mM) and incubated for 4 h. All manipulations of ALA-treated cells were performed under reduced light conditions. After 4 h, quantification of PpIX was performed as previously described by Ortel et al. [21]. In brief, cell pellets were suspended in 1% SDS in 0.1 N NaOH and submitted to quantitative spectrofluorometry (excitation 400 nm,

3 emission 580—720 nm, peak 630 nm). The level of PpIX was expressed as a fluorescence intensity value.

Determination of alkaline phosphatase activity Alkaline phosphatase (AP) activity was evaluated in MCF-7 cells as a marker for cellular differentiation. After treatment of MCF-7 cells either with individual or combined therapy, cell pellets were washed three times with phosphate buffer saline (PBS) and lysed by three repetitive freezing/thawing cycles (thawing at 37 ◦ C for 2 min and freezing at −80 ◦ C for 15 min), followed by passing the cells through a 20 G needle. ALP was assessed according to the method of Tietz et al. [22] using a commercial kit (Biosystems S.A., Spain) and was expressed as U/mg protein.

Apoptosis and necrosis staining To screen the effect of the Doxil® and ALA alone or in combined on the mode of cell death, apoptosis and necrosis ratios were investigated using acridine orange/ethidium bromide staining (AO/EB) [23,24]. MCF-7 cells were treated Doxil® and/or ALA as described above, collected cells were stained using the nucleic acid-binding dye mixture of 100 ␮g/ml AO and 100 ␮g/ml EB in PBS, and were examined by fluorescence microscopy. For each sample, at least 500 cells/well in the six wells were counted, and the percentage of apoptotic or necrotic cells was determined as: % of apoptotic or necrotic cells = (total number of apoptotic or necrotic cells/total number of cells counted) × 100.

Determination of mitochondrial transmembrane potential Determination of mitochondrial transmembrane potential ( m ) was carried out using cell staining with MitoTracker® Red CMX-Ros (Life Technologies, Carlsbad, USA). MitoTracker® Red CMX-Ros was dissolved in dimethylsulfoxide (DMSO) for a final concentration of 1 mM stock solution prior to use. MCF-7 cells (1 × 106 cells) treated with Doxil® and/or ALA/PDT were incubated with 100 nM MitoTracker® Red CMX-Ros in RPMI 1640 medium for 15 min at 37 ◦ C in the dark. After staining is complete, the cell pellets were collected by centrifugation and re-suspended in fresh pre-warmed PBS. The cells were visualized at 40× magnification using a Carl Zeiss automated fluorescence microscope equipped with Zen 2011 software. A total of 1000 cells were examined for the evaluation of MitoTracker® Red CMX-Ros staining intensity as a measure of mitochondrial function.

DNA fragmentation The DNA fragmentation assay allows determining the amount of DNA that is degraded upon treatment of cells, and was performed as reported previously [25]. Briefly after treating MCF-7 cells with Doxil® and/or ALA, cell pellet was re-suspended in 250 ␮l 10 mM Tris, 1 mM EDTA, pH 8.0 (TEbuffer), and incubated with an additional volume of lysis buffer (5 mM Tris, 20 mM EDTA, pH 8.0, 0.5% Triton X-100) for 30 min at 48 ◦ C. After lysis, the intact chromatin (pellet)

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was separated from DNA fragments (supernatant) by centrifugation for 15 min at 13,000 × g. Pellets were resuspended in 500 ␮l TE buffer and samples were precipitated by adding 500 ␮l of 10% trichloroacetic acid at 48 ◦ C. Samples were pelleted at 4000 rpm for 10 min and the supernatant was removed. After addition of 300 ␮l of 5% trichloroacetic acid, samples were boiled for 15 min. DNA contents were quantified using diphenylamine reagent [26]. The percentage of DNA fragmented was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.

Cell lysate preparation Cell lysate was prepared to be used in ELISA and lipid peroxides analysis. The treated and untreated cells were trypsinized, washed and centrifuged for 10 min at 1000 × g. Cell pellet was lysed in 0.5 ml of ice-cold lysis buffer [50 mM Tris—HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3 VO4 , 0.5% NP40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)]. Cell lysates were passed through a 21-gauge needle to break up cell aggregates, and then centrifuged at 14,000 × g for 15 min at 4 ◦ C. The supernatants (total cell lysate) were submitted to ELISA and lipid peroxides estimation. The total protein content of the lysates was measured according to Smith et al. [27], by bicinchoninic acid (BCA) and using bovine serum albumin (BSA) as a standard (data not mentioned).

Evaluation of death receptors and caspase 3 levels Death receptors (DR4 and DR5) and caspase 3 levels were estimated in cell lysates by indirect ELISA. All steps of Indirect ELISA were followed according to the method described by Gamal-Eldeen et al. [28] with some modifications, in which 96-well flat bottom polystyrene plates were coated with cell lysates samples (100 ␮l/well). After overnight incubation at 4 ◦ C the plates were washed and then 200 ␮l of blocking buffer were delivered into each well. The plates were covered and incubated for 1.5 h at 37 ◦ C. After washing step, rabbit polyclonal antibody to DR4, DR5 or caspase 3 (Abcam Inc., Cambridge, MA, USA) each was adjusted to 0.2 ␮g/ml and was dispensed as (100 ␮l/well). After 1 h of incubation at 37 ◦ C, the plates were washed and 100 ␮l/well of polyclonal Goat anti-rabbit peroxidase-conjugate (diluted 1:1000) were added and the plates incubated for another 1 h at 37 ◦ C. At the end of the incubation period, the plates were washed, and the substrate solution was prepared (equal volumes of 3 , 5, 5 -tetramethyl benzidine (TMB) and H2 O2 ; Kirkegaard and Perry Labs, Gaithersburg, MD). Color development was stopped by the addition of (100 ␮l/well) of stopping buffer (1 M HCl). The color intensity was measured at 450 nm using the microplate reader (FLUOstar OPTIMA; BMG Labtech GmbH, Offenburg, Germany). The levels of caspase 3, DR4 and DR5 in the cell lysates were expressed as milliabsorbance.

Estimation of histone deacetylase activity The activity of histone deacetylase (HDAC) was measured using a colorimetric assay kit (BioVision, Mountain View, kit no. K331-100). The procedure involves the use of the HDAC colorimetric substrate (Boc-Lys(Ac)-pNA), which comprises an acetylated lysine side chain and is incubated with a sample containing nuclear extract. Deactivation sensitizes the substrate, and treatment with the lysine developer produces a chromophore, which can be analyzed using a colorimetric plate reader. HeLa cell nuclear extract was used as a positive control. A standard curve was prepared using the known amount of the deacetylated standard (Boc-Lys-pNA) included in the kit. A similar volume of control sample was added to 100 ng/ml trichostatin A (TSA), as a known inhibitor of HDAC.

Estimation of vascular endothelial growth factor The level of vascular endothelial growth factor (VEGF) in cell lysate was determined using Sandwich ELISA, as described by Talaat et al. [29] with some modifications. Monoclonal antibody against the N-terminal region of VEGF (4 ␮g/ml) is bound to a flat bottom 96-well polystyrene plate (50 ␮l/well). After overnight incubation at 4 ◦ C, the plates were washed with washing buffer (PBS/0.05% polyoxyethylene-20; Tween-20) then 200 ␮l of blocking buffer (PBS/0.05% Tween-20/5% fetal bovine serum) was delivered into each well, and incubated for 1.5 h at 37 ◦ C. After a washing, samples or standards (0.195—100 ng/ml) were added (50 ␮l/well) and incubated for 2 h at 37 ◦ C. Wells were washed, then diluted goat antibody (200 ng/ml) of high specificity for the C-terminus of VEGF (biotin-labeled antiVEGF polyclonal antibody) was added as (50 ␮l/well) for 1 h at 37 ◦ C. After a washing, an incubation for 1 h at 37 ◦ C with 50 ␮l/well of peroxidase conjugated streptavidin of a rabbit anti-Goat IgG (1:1000). The plates were washed, and 50 ␮l/well of TMB/H2 O2 were added, then 50 ␮l/well of 1 M HCl. The color intensity was measured at 450 nm using the microplate reader. Standards of the highly purified recombinant VEGF were used to generate a standard curve.

Determination of lipid peroxidation The level of malondialdehyde (MDA), which is the end product of lipid peroxidation was evaluated using a commercially supplied kit (Biodiagnostic). In which thiobarbituric acid reactive substances detected spectrophotometrically at 535 nm [30].

Statistical analysis Statistical analysis for the data obtained was performed using the Statistical Package for the Social Sciences (SPSS) program version 11. Data were expressed as mean ± standard division. Data were analyzed using one way ANOVA test followed by Tukey’s Post Hoc Test. The results were considered to be significant when P value is less than 0.05, (a) is significant comparing with the untreated group, (b) is significant comparing with the ALA/PDT group, and (c) is significant comparing with Doxil® -treated group.

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Fig. 1 The cytotoxicity using trypan blue exclusion assay. (a) Cytotoxicity of Doxil® on MCF-7 cells after 48 h incubation. (b) The effect laser irradiation intervals (1—8 min) on MCF-7 cells viability using in absence (black bars) and in presence of ALA/PDT (1 mM, 4 h, gray bars). (c) Cytotoxicity of ALA on MCF-7 cells after 4 h, in dark (white squares) or after laser irradiation (black squares) for 4 min (at wavelength of 633 nm and power of 0.25 W). (d) The effect of individual treatment with IC50 of Doxil® or ALA/PDT compared with combined therapy using 50% of each drug IC50 on MCF-7 cells viability. Data expressed as a percentage of control untreated cells. Results represent the mean ± SD of three independent experiments.

Results Cytotoxicity of Doxil® , ALA/PDT and combined therapy The effect of different concentrations of Doxil® on the viability of MCF-7 cells was assessed by trypan blue test. Cell viability was reduced by increasing the dose Doxil® compared to untreated cells. The calculated IC50 was 19.85 ␮M

(Fig. 1a). The optimum laser irradiation time at a wavelength of 633 nm and power of 0.25 W was 4 min, which were safe for irradiated cells, but it enhanced ALA-induced phototoxicity; (Fig. 1b). As shown in Fig. 1c, ALA/PDT induced a significant cytotoxicity on MCF-7 cells in a concentration dependant manner comparing with both untreated cells and ALA-treated cells in dark conditions. The calculated IC50 of ALA/PDT was 1.12 ␮M. The effect of combined therapy on the viability of MCF-7 was evaluated using 50% of each IC50

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PpIX Flluorescence intensity(IFU)

6000 5000 4000 3000 2000 1000 0 Control

ALA

Doxil+ALA

Fig. 2 Effect of Doxil® (50% of IC50 , 48 h) pretreatment on the amount of ALA-induced PpIX production in MCF-7 cells using ALA (50% of IC50 , 4 h). Results represent the mean ± SD of three independent experiments.

of both Doxil® and ALA/PDT and compared with the cytotoxicity of each drug individually using their IC50 . As shown in Fig. 1d, a significant cell viability reduction was observed when both therapies (Doxil® + ALA/PDT) were used in low doses compared with the high doses of each individual therapy.

Quantification of Protoporphyrin IX production When MCF-7 cells were pretreated with Doxil® IC50 for 48 h followed by treatment with ALA IC50 , it remarkable enhanced the PpIX accumulation. Doxil® -pretreated cells produced at least twofold the amount of PpIX as compared to MCF-7 cells treated with ALA only (Fig. 2).

Evaluation of apoptosis percentage and its mediators To screen the mode of cell death induced by either Doxil® alone or when followed by ALA/PDT, on MCF-7 cell line. As shown in Fig. 3a, AO stained both live and dead cells, while EB stained only cells that have lost membrane integrity. Vital cells will appear uniformly green, while early apoptotic cells are stained green and contain bright green to yellow dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells also incorporated EB (orange), but, in contrast to necrotic cells, the late apoptotic cells showed condensed and often fragmented nuclei. Necrotic cells stain deep orange to red, but have a nuclear morphology resembling that of viable cells, with no condensed chromatin. The percentages of vital, apoptotic and necrotic cells were determined comparing to untreated cells. As shown in Fig. 3b, MCF-7 cells treated with combined therapy (Doxil® + ALA/PDT), using half IC50 concentration of each, showed a significant (P < 0.05) increase in the total percentage of cell death into 77%, where there was a significant increase in both of apoptotic and necrotic

Fig. 3 Representative images for the mode of cell death in MCF-7 cells for control, Doxil® (IC50 ), ALA/PDT (IC50 ) and combined drugs Doxil® + ALA/PDT (50% of IC50 ) treated cells, as stained by AO/EB and captured under fluorescence microscope (x200). Vital cells (green), early apoptotic (bright green to yellow), late apoptotic (orange) and necrotic cells (red). (b) The cell population percentage of vital (black segments), apoptotic (gray segment) and necrotic (white segments) cells were counted. Results represent the mean ± SD of three independent experiments.

cell populations, compared to cells treated with IC50 concentration of each individual therapy. For further investigation of cell death pathways initiated by ALA/PDT alone or in combined with Doxil® , death receptors and caspase-3 were estimated in cell lysates by indirect ELISA. As shown in Fig. 4a and b, the levels of DR4, DR5 and caspase-3 were significantly increased (P < 0.01) in MCF-7 cells treated with the combined therapy Doxil® + ALA/PDT compared to untreated cells and also compared to each individual therapy (P < 0.05).

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Fig. 4 The effect of treatment with Doxil® (IC50 ), ALA/PDT (IC50 ) and combined drugs Doxil® + ALA/PDT (50% of IC50 ) on apoptotic cell death pathway in MCF-7 cells. (a) DR4 (black bars) and DR5 (white bars) and (b). caspase-3 levels in treated cells comparing to control cells. The data represented as number of folds in relation to control absorbance readings, which were 7.2, 5.6 and 6.5 mA, respectively.

Mitochondrial transmembrane potential Mitochondrial function was determined by fluorescent microscopic examination of the  m -dependant uptake and retention of CMX-Ros in mitochondria, as represented in Fig. 5a. Fig. 5b demonstrates the effect of combined therapy and each individual therapy on  m in MCF-7 cells. Treatments with Doxil® followed by ALA/PDT enhanced the reduction in  m compared to untreated cells and to cells treated with individual therapy, as shown in Fig. 5b.

Oxidative stress and DNA damage To evaluate the role of oxidative stress, lipid peroxidation was studied by estimation of MDA level. As shown in Fig. 6a, lipid peroxidation was increased significantly (P < 0.05) in

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Fig. 5 Representative images for MCF-7 cells stained with MitoTracker® Red fluorescent dye, showing cells with normal transmembrane potential (control) and with reduced transmembrane potential (Doxil® + ALA/PDT). (b) Effect of treatment with Doxil® (IC50 ), ALA/PDT (IC50 ) and combined drugs Doxil® + ALA/PDT (50% of IC50 ) on MitoTracker® Red staining intensity in MCF-7 cells. Data represent the percent of fluorescent staining intensity (mean ± SD) as compared to untreated cells.

both Doxil® and ALA/PDT treated cells, further increase was obtained with combined therapy. We investigated the effect of Doxil® and ALA/PDT as individual or combined therapy on the degree of DNA fragmentation in MCF-7 cells, which was assayed by diphenylamine. As shown in Fig. 6b, the results indicated that the percentage of DNA fragmentation was increased in ALA/PDT and Doxil® -treated cells by 1.8 ± 0.07 and 2 ± 0.05-folds, respectively, compared to untreated cells (P < 0.05). However, DNA fragmentation in cells treated with the combined therapy Doxil® + ALA/PDT increased by 3.75 ± 0.2-folds compared to untreated cells (P < 0.05). HDAC activity was evaluated in MCF-7 cell lysate and showed that HDAC activity was markedly inhibited (P < 0.05) in cells treated either Doxil® or ALA/PDT as well as combined therapy Doxil® + ALA/PDT with a percentage of inhibition of 67%, 55% and 65%, respectively, compared to untreated cells (Fig. 6c), however there was a non

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Fig. 7 The effect of treatment of MCF-7 cells with Doxil® (IC50 ), ALA/PDT (IC50 ) and combined drugs Doxil® + ALA/PDT (50% of IC50 ) on: a. ALP activity data is expressed as U/mg protein. (b) VEGF levels; data is expressed as ng/ml.

significant inhibition in the combined therapy compared with the individual treatment by each therapy (P > 0.05).

Effect of Doxil® and ALA/PDT on cell differentiation and angiogenesis

Fig. 6 The effect of treatment of MCF-7 cells with Doxil® (IC50 ), ALA/PDT (IC50 ) and combined drugs Doxil® + ALA/PDT (50% of IC50 ) on: (a) MDA levels; data is expressed as nM/mg protein. (b) The percentage of DNA fragmentation, data is expressed as mean ± SE. (c) HDAC; data expressed as ␮M deacetylated substrate/mg protein.

Alkaline phosphatase activity was assessed and we found that after 48 h incubation with Doxil® in all Doxil® -treated groups, the ALP activity was enhanced by to threefold of the of control, while the treatment with ALA in absence or presence of laser irradiation resulted in a non significant change in ALP (P > 0.05) and it also did not further enhance the Doxil® -induced ALP in the combined therapy, as shown in Fig. 7a. A sandwich ELISA was established to evaluate VEGF level in MCF-7 cell lysate. The results showed that VEGF concentration in cells treated with the combined therapy Doxil® and ALA/PDT was significantly reduced compared to

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Synergistic apoptotic effect of Doxil® and aminolevulinic acid untreated cells or cells treated with each treatment individually (Fig. 7b).

Discussion PDT is a clinically approved, minimally invasive therapeutic procedure involving the administration and irradiation of a PS in presence of oxygen, resulting in the production of ROS, mainly singlet oxygen that lead to direct tumor cell death [31]. In our study ALA/PDT induced cell death in MCF7 cells in a dose dependent manner with IC50 1.12 mM. This is in accordance with the study of Tsai et al. [32], which reported that ALA/PDT decreased the survival rate of MCF-7 cells by increasing ALA concentration as well as treatment of cells with 1 mM ALA and irradiation with different light doses showed enhanced phototoxicity in a light dose dependent manner. PpIX, induced by the exogenous addition of ALA, reaches different levels in different tumor cells [33]. PpIX is one of the effective PSs with great potential to enhance light induced tumor cell damage exerting anti-tumor effects [34]. ALA-induced PpIX has shown to have preferential accumulation in cancerous breast cells [35], making it a promising agent in breast cancer diagnosis and treatment. Doxil® is a pegylated nano-liposomal carrier for doxorubicin characterized by improved pharmacokinetics and reduced cardiotoxicity [17]. In this study, Doxil® exerted a cytotoxic effect on MCF-7 cells in a dose dependent manner with IC50 of 19.85 ␮M. Previous in vitro study, showed that Doxil® has less cytotoxic effects when compared to free doxorubicin, which diffuses more rapidly to the cells than the encapsulated form, to be accumulated in the nucleus where it interchelates with DNA and inhibits topoisomerase II [36]. However, the in vitro study of Wang et al. showed that the IC50 of liposomal doxorubicin was less than that of the free form in multidrug resistant rat prostate cancer cells especially when using stealth liposomes coencapsulating verapamil with doxorubicin [37]. Moreover, other in vivo studies reported that Doxil® is characterized by long circulation time with significant increase in intratumoral doxorubicin delivered by Doxil® compared to free doxorubicin administration [16,38]. Its accumulation in tumor tissue is mainly via passive targeting, which is known as the enhanced permeability and retention effect [39]. Numerous studies have documented the use of PDT strategies along with conventional cancer treatments to enhance antitumor response [40]. In this study, we tried to evaluate whether using low doses of both Doxil® and ALA/PDT will show a synergistic anti-tumor effect more efficient than using high doses of each individually, in order to achieve maximal therapeutic effect with minimal side effects. According to the cytotoxicity experiments, we found that 50% of the IC50 value of both Doxil® (9.925 ␮M) followed by ALA/PDT (0.55 ␮M) has significantly enhanced the cytotoxic effect on MCF-7 cells rather than using 100% of the IC50 of Doxil® or ALA/PDT individually, indicating that combined therapy has the benefit of increasing treatment efficacy using lower doses of both drugs, thus may minimize their side effects. In the present study, the DNA changes of MCF-7 cells after treatment were examined by AO/EB staining to evaluate the apoptotic and/or necrotic features. It was obvious that after treating MCF-7 cells with

9 low doses of Doxil® and ALA/PDT a significant percentage of both apoptotic and necrotic cells were detected however; the percentage of apoptotic cells was higher than that of necrotic cells indicating that the apoptotic mode of cell death could be the predominant mode of cell death. Apoptosis can be initiated through two distinct pathways, the death receptor (extrinsic) pathway or the mitochondria (intrinsic) mediated pathway [41]. Both events eventually lead to activation of caspase cascades known as ‘‘executioner caspases’’ such as caspase-3, -6 and 7 [42]. The active executioner caspases cleave cellular substrates, leading to characteristic biochemical and morphological changes observed in dying cells [43]. This is followed by chromatin condensation, nuclear shrinkage and DNA fragmentation. Cleavage of cytoskeletal proteins leads to cell fragmentation and formation of apoptotic bodies [44,45]. Previously, ALA/PDT decreased the levels of mitochondrial membrane potential, and induced cell death through both apoptosis and necrosis [46], indicating that ALA/PDT mediated death of cancer cells is caused via mitochondrial-dependant pathway due to PpIX accumulation and localization in the mitochondria and cytoplasm of cancer cells [47]. This is in concordance with the results of our study, which showed that the combined Doxil® ALA/PDT treatment enhanced the reduction in mitochondrial transmembrane potential compared to untreated cells and to each individual therapy, which may be due to the elevated ROS production evidenced by increased MDA levels. Moreover the study of Diez et al. [48] reported that the use of ALA/PDT in addition to conventional chemotherapy as doxorubicin or vincristine significantly increased superoxide anion production and subsequently marked reduction in mitochondrial membrane potential compared to each individual treatment in murine leukemic cells. Doxorubicin was shown to induce apoptosis via caspase3 activation and DNA fragmentation, as the study of Wang et al. reported that small doses of doxorubicin 0.5 ␮M for 16 h in MCF-7 and PA-1 cells induces caspase-3 activity which is P53 dependent in tumor cells compared to normal cells [49]. Moreover, we reported that the synergistic effect of Doxil® that provides slow release of free doxorubicin- on ALA/PDT-mediated mitochondrial dysfunction resulted in a caspase-dependent cell death in MCF-7 cells. According to the results of the present study, caspase3 activity and the percent of DNA fragmentation were markedly increased in MCF-7 cells treated with the combined therapy (Doxil® + ALA/PDT) compared to that treated with each individual therapy. DRs are members of the TNF receptor (TNFR) superfamily, which is characterized by a cytoplasmic region known as the death domain that enables the receptors to initiate survival and/or apoptotic signals upon engagement of their cognate DR ligands [50]. Doxorubicin has been shown to induce the expression of death receptors, DR4 and DR5, in breast cancer cells [51]. The study of Turner et al. [52] showed that treatment of MCF-7 and MDA-MB-231 cells with doxorubicin 10 ng/ml resulted in enhanced cell surface expression of death receptors starting at 48 h after treatment which reached significantly higher levels after 72 h. There is also strong evidence that PDT-induced apoptosis usually proceeds through the extrinsic or cytoplasmic pathway via DRs, as well as the intrinsic or mitochondrial pathway [53]. Our

Please cite this article in press as: Zakaria S, et al. Synergistic apoptotic effect of Doxil® and aminolevulinic acidbased photodynamic therapy on human breast adenocarcinoma cells. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.001

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10 results showed that Doxil® and ALA/PDT both synergize the effect of each other by enhancing apoptotic cell death through the extrinsic pathway, which was indicated by the significant increase in the levels of DR4 and DR5 in addition to the intrinsic pathway. HDAC activity is another important marker for the detection of the apoptotic pathway either intrinsic or extrinsic pathway [54]. Acetylation of histones represents one of the key posttranslational modifications that contribute to the regulation of chromatin remodeling. Histone acetylation is governed by the balance between enzymes that put acetyl groups on histone tails or, alternatively, remove them [55]. Inhibition of HDAC leads to transcriptional changes of a very large number of genes, which affect signaling pathways, inhibit cell cycle progression and angiogenesis, and induce apoptosis in cancer cells [56]. According to the results of the present study, a significant inhibition in the activity of HDAC activity was observed in MCF-7 cells treated with Doxil® or ALA/PDT individually or in combined therapy comparing to the untreated cells. This is an indication that the observed apoptotic cell death is mediated by regulating histone function and subsequently gene transcription. Ma et al. [57] showed that doxorubicin treatment significantly inhibited HDAC activity in both neonatal cardiomyocytes and H9c2 cells. PDT could induce histone acetylation as shown by the study of Gamal-Eldeen et al. [28], which stated that PDT using indocyanine green entrapped in polymeric nanoparticles and their anti-EGFR-conjugate significantly decreased HDAC activity in skin cancer in CD1 mice. According to our results, it was shown that Doxil® increased cellular differentiation in MCF-7 cells and this was demonstrated by increased ALP activity. This could be explained by slow intracellular doxorubicin release from its encapsulated form [36], since it was previously reported that doxorubicin in low concentrations induces differentiation of breast cancer cells [58]. According to Maytin et al. [59] induction of cellular differentiation, in solid tumors, could increase PpIX level within diseased tissue as a result of increased ALA uptake. Other studies used other differentiation-inducing therapies combined with ALA to increase ALA-induced PpIX production, such as using of low doses of methotrexate [60], vitamin D [59] or synthetic androgen to induce differentiation in prostate cancer cells [61], since differentiation dependent-increased PpIX may be due to increased ALA uptake that enhances PpIX production and decreases PpIX export into culture media [21]. In the present study, ALA-induced PpIX production was markedly increased after treatment with Doxil® in MCF-7 cells, indicating that Doxil® could induce PpIX production, resulting in enhanced phototoxicity due to differentiation-dependent increase in cellular PpIX levels leading to significant reduction in cell viability. However, this induced-PpIX did not affect ALP after the treatment with ALA in absence or presence of laser irradiation and it also did not further enhance the Doxil® -induced ALP in the combined therapy. VEGF has a potent angiogenesis functions and play an important role in progression of breast carcinoma [62]. Inoue et al. [63] reported that the application of ALA/PDT resulted in lowering the rate of metastatic spreading and decreased VEGF level in blood serum of tumor-bearing mice. In addition, ALA/PDT potentiates the antiangiogenic action on neovacular endothelial cells [64]. It was obvious in this study

S. Zakaria et al. that MCF-7 cells submitted to ALA/PDT showed reduced VEGF level comparing to untreated cells, this reduction was significantly enhanced by Doxil® pretreatment, since chemotherapeutics have been shown to be able to suppress angiogenesis. It was previously reported that PDT using benzoporphyrin derivative monoacid ring A as PS plus doxorubicin exerts enhanced antitumor effect on breast cancer, through extrinsic apoptotic pathways and the inhibition of tumor angiogenesis [65]. In conclusion, the combined therapy using low doses (50% of IC50 ) of ALA/PDT and Doxil® possessed a synergistic cytotoxic effect on MCF-7 cells compared to the treatment with high doses (100% of IC50 ) of each individual therapy mainly through enhancing both intrinsic and extrinsic apoptotic mode of cell death. Thus the adverse effects of Doxil® or ALA could be minimized with ensuring a maximal therapeutic benefit in breast cancer treatment.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We are grateful to laser Research Unit, NRC, Egypt, in particular Prof. Dr. Ali Shabaka for their efforts. This work was financially supported by October 6 University and National Research Center, Cairo, Egypt.

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