Biometals (2014) 27:575–589 DOI 10.1007/s10534-014-9730-y

Water-soluble platinum phthalocyanines as potential antitumor agents Giuseppina Bologna • Paola Lanuti • Primiano D’Ambrosio • Lucia Tonucci • Laura Pierdomenico • Carlo D’Emilio • Nicola Celli • Marco Marchisio • Nicola d’Alessandro • Eugenio Santavenere • Mario Bressan • Sebastiano Miscia

Received: 11 December 2013 / Accepted: 17 March 2014 / Published online: 4 April 2014 Ó Springer Science+Business Media New York 2014

Abstract Breast cancer represents the second cause of death in the European female population. The lack of specific therapies together with its high invasive potential are the major problems associated to such a tumor. In the last three decades platinum-based drugs have been considered essential constituents of many therapeutic strategies, even though with side effects and frequent generation of drug resistance. These drugs have been the guide for the research, in last

Giuseppina Bologna, Paola Lanuti, Primiano D’Ambrosio, Lucia Tonucci have contributed equally in this work. Mario Bressan, Sebastiano Miscia, have equal senior authorship.

Electronic supplementary material The online version of this article (doi:10.1007/s10534-014-9730-y) contains supplementary material, which is available to authorized users. G. Bologna  P. Lanuti  L. Pierdomenico  C. D’Emilio  M. Marchisio (&)  E. Santavenere  S. Miscia Department of Medicine and Aging Science, School of Medicine and Health Sciences, University ‘‘G. d’Annunzio’’ of Chieti-Pescara, Via dei Vestini, 31, 66013 Chieti Scalo, Italy e-mail: [email protected] G. Bologna  P. Lanuti  L. Pierdomenico  C. D’Emilio  M. Marchisio  E. Santavenere  S. Miscia Center for Ageing Sciences (Ce.S.I.), University ‘‘G. d’Annunzio’’ Foundation, Via dei Vestini, 31, 66013 Chieti Scalo, Italy

years, of novel platinum and ruthenium based compounds, able to overcome these limitations. In this work, ruthenium and platinum based phthalocyanines were synthesized through conventional techniques and their antiproliferative and/or cytotoxic actions were tested. Normal mammary gland (MCF10A) and several models of mammarian carcinoma at different degrees of invasiveness (BT474, MCF-7 and MDAMB-231) were used. Cells were treated with different concentrations (5–100 lM) of the above reported compounds, to evaluate toxic concentration and to underline possible dose–response effects. The study included growth curves made by trypan blue exclusion test and scratch assay to study cellular motility and its possible negative modulation by phthalocyanine. Moreover, we investigated cell cycle and apoptosis through flow cytometry and AMNIS Image Stream

P. D’Ambrosio  N. d’Alessandro (&)  M. Bressan Department of Engineering and Geology (INGEO), University ‘‘G. d’Annunzio’’ of Chieti-Pescara, Viale Pindaro, 42, 65127 Pescara, Italy e-mail: [email protected] L. Tonucci Department of Philosophical, Educational and Economic Science, University ‘‘G. d’Annunzio’’ of Chieti-Pescara, Via dei Vestini, 31, 66013 Chieti Scalo, Italy N. Celli Environmental Sciences Center, Mario Negri Sud Foundation, Via Nazionale 8/A, 66030 Santa Maria Imbaro, CH, Italy

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cytometer. Among all the tested drugs, tetrasulfonated phthalocyanine of platinum resulted to be the molecule with the best cytostatic action on neoplastic cell lines at the concentration of 30 lM. Interestingly, platinum tetrasulfophtalocyanine, at low doses, had no antiproliferative effects on normal cells. Therefore, such platinum complex, appears to be a promising drug for mammarian carcinoma treatment. Keywords Breast cancer  Phthalocyanine  Anticancer drug  Platinum  Cell motility Abbreviations ICP MS Inductively-coupled-plasma massspectrometry DMEM Dulbecco’s modified Eagle medium PcS Tetrasulfophthalocyanina PtPcS Platinum (II) tetrasulfonated phthalocyanine PtPcC Platinum (II) tetracarboxylated phthalocyanine RuPcS Ruthenium (II) tetrasulfonated phthalocyanine RuPcC Ruthenium (II) tetracarboxylated phthalocyanine RuPcAlkC b-Tetrapentylcarboxylated ruthenium (II) phthalocyanine RPMI Roswell Park Memorial Institute DMEM/F12 Dulbecco’s modified Eagle medium: Nutrient mixture F-12 PI Propidium iodide

Introduction Breast cancer represents the second cause of death in the European female population (Camerlingo et al. 2011). It includes many histological subtypes, characterized by different clinical signs and molecular mechanisms (Lorico and Rappa 2011). The lack of specific therapies, together with its high invasive potential are the major problems associated to such a tumor (Camerlingo et al. 2011). Many transition metal complexes have attracted a growing interest for their biological activities against cancer diseases (Allardyce and Dyson 2006; Meggers 2007). In particular, the discovery of the anticancer potential of cis-platinum dates to the mid-sixties (Rosenberg et al. 1965) and its following clinical

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success has opened large interest in the use of metal compounds (Biersack and Schobert 2013). Starting from 1971, when for the first time, cis-platinum was administrated to a patient affected by testicular cancer, other platinum derivatives, like carboplatin and oxaliplatin, were diffused and, consequently, largely employed as chemotherapy drugs (Lebwohl and Canetta 1998). Despite their widespread use, platinum complexes could have heavy side effects such as nephro- and neurotoxicity and resulted ineffective against some resistant tumors (Merrin 1976; Ward et al. 1977; Godwin et al. 1992). Recently, some Pt(IV) compounds, like ormaplatin, iproplatin and satraplatin, have been proposed for clinical trials in humans (Hall et al. 2007), with the aim to test their improved chemical stability in biological fluids (they are often orally administrable: e.g. see satraplatin) which may reduce the related side effects (Choy et al. 2008). Unfortunately, it has been demonstrated that iproplatin therapeutic effects are not better than the ones demonstrated by old Pt-compounds, and, at the same time, ormaplatin showed big and unpredictable neurotoxicity problems, while satraplatin is still currently undergoing clinical trials (Merrin 1976; Wheate et al. 2010). Therefore, the studies addressed to the researches of novel anticancer therapies, having the aim to overcome the disadvantages of platinum administration, have been also focused on some other transition metals (e.g. ruthenium, gold, gallium, palladium, iron, cobalt, copper) (Graf and Lippard 2012; Komeda and Casini 2012; Lessa et al. 2013; Aliwaini et al. 2013; Selvamurugan et al. 2013), because of their large range of potential oxidation states and their variety of molecular geometries (tetrahedral, planar, octahedral, etc.). In Dwyer et al. (1952) published a pioneering study demonstrating the anticancer activity of ruthenium polypyridyl complexes. In the mid-eighties, novel ruthenium compounds, active against cancer cells, have been designed, synthesized and tested (Alessio et al. 1988). Today, two anionic Ru(III) derivatives, namely NAMI-A (Sava et al. 1997) and KP109 (Lipponer et al. 1996), are in phase II clinical trials as antimetastatic and antitumor agents, respectively and other interesting organoruthenium complexes have been recently proposed as promising new drugs (Gianferrara et al. 2009; Ang et al. 2011). The action mechanism of cis-platinum and its analogues has been established. In fact, it has been

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demonstrated that, in the biological fluids, the chloride leaving groups, surrounding the central platinum atom, are replaced by one or two aqua-ligands; such hydrolyzed species can bind to DNA (Mello et al. 2009), mainly to softer ligands, like thiols and thioethers which than quickly form strong bonds with the metal aquo-species (Wexselblatt et al. 2012). Unfortunately, such simple reaction scheme must be optimized and revised, since often cancer cells recognize the damage induced by Pt drugs on the DNA, providing to its repair and inducing therapeutic resistance (Micouskova et al. 2012). On the other hand, the action mechanism carried out by ruthenium compounds is not completely clear and many hypothesis have been formulated. However, it seems that before it becomes active, Ru(III) is in vivo reduced to the more reactive Ru(II) species which then evolves in many different mechanistic pathways (Clarke et al. 1999; Van Rijt and Sadler 2009). Among metal-derivatives, metal-phthalocyanines have recently attracted an increasing and renewed interest in several fields (Sorokin and Kudrik 2011; Ryan et al. 2012) and also in medical applications, because of their behavior under irradiation (generators of singlet oxygen) (D’Ambrosio et al. 2011; Ishii 2012). In particular, specific zinc, silicon and aluminum (PhotosensÒ) phthalocyanines, with various degrees of sulfonated substituents or incorporated into liposomes have been widely employed as drugs in the photodynamic therapy against cancer (Ali and van Lier 1999; Allison and Sibata 2010). The relative low toxicity demonstrated by the previous administrations of metal-phthalocyanines for photodynamic therapy, together with the well-documented anticancer properties of Pt and Ru metals, prompted us to in vitro investigate their effects. In the present study, the attention has been focused on newly-synthesized Pt and Ru compounds, used as phthalocyanine ligands, being phthalocyanine peripherally functionalized by polar groups, such as sulfonate or carboxylate (Fig. 1), to allow the solubility of the aforementioned compounds in water media, with the aim to reduce their potential side effects. In particular, the above reported compounds, ones synthesized, were tested for their ability, in non-photodynamic conditions, to induce cell death and/or cell cycle and cell motility arrest on several breast cancer cell lines (MDA-MB-231, BT474 and MCF-7 cell

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lines), characterized by different ability in terms of proliferation, invasiveness and carcinogenicity. Results were compared to data obtained by treating, in the same conditions, MCF10A cells, stemming from normal breast tissue and used as control cell line (Bertagnolo et al. 2007).

Materials and methods Syntheses of metal phthalocyanines Phthalocyanine tetrasulfonate hydrate was purchased from Frontier Scientific (Logan, UT, USA). Tetrasulfonated phthalocyanines of platinum II (PtPcS) and ruthenium II (RuPcS) were prepared by template synthesis, following previously reported general procedures (Weber and Bush 1965). Briefly, K2PtCl4 (or RuCl3), 4-sulfopthalic acid, urea and ammonium molybdate, used as catalyst, were added in a flask containing a solution of nitrobenzene and the mixture was refluxed for 6 h. Characterization and purity check of both compounds were performed by ESI MS and UV Vis spectroscopy. The diagnostic Q bands appeared at 590 nm (PtPcS) and 630 nm (RuPcS) with e = 26,000 M-1cm-1 and 29,500 M-1cm-1, respectively. Clear parent ion peaks centred at m/z 1,028 u (PtPcS) and m/z 935 u (RuPcS) were observed, in both cases with the well known isotopic pattern of the used metal (Nicastro et al. 2007; d’Alessandro et al. 2005). Likewise, the tetracarboxylated phthalocyanines of Ru and Pt (RuPcC and PtPcC) were prepared by an analogue procedure, by substituting the 4-sulfopthalic acid with benzene1,2,4-tricarboxylic acid 1,2-anhydride. Again, visible analyses and ESI MS were carried out for the compound characterization; data were perfectly in agreement with those reported in the literature. For PtPcC the parent ion appeared centered at m/z 883 u while the parent ion of RuPcC appeared centered at m/z 790 u, in both cases the isotopic pattern of the metal was respected. The vis spectra, recorded in alkaline conditions, showed the Q bands were around 600 nm: in detail, kmax PtPcS = 598 nm with e = 26,400 M-1cm-1 and kmax RuPcC = 588 nm with e = 22,800 M-1cm-1 (Carchesio et al. 2010). To obtain the b-tetrapentylcarboxylated ruthenium (II)

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Integration times were 0.3 s/mass for holmium (m/z = 165), and 1.5 s/mass for Pt (m/z = 195) and spectra were acquired in quintuplicate for each analysis.

G

G N N

N

N

M

N

Cell cultures

N

N N

G

G

G = -CH2CH2CH2CH2COOH

M = Pt,

Ru, 2H

-COOH -SO3 Na

+

Fig. 1 Tetrasubstituted phthalocyanines used

phthalocyanine (RuPcAlkC) we developed an appropriated synthetic procedure (see the Supplementary Section and Supplementary Figs. 1, 2, 3 and 4). ICP MS measurements Cell samples (pellets) and cell media (0.5 ml) were digested with 1 ml 67–69 % nitric acid (Superpure, Carlo Erba Reagenti, Rodano, Milan, Italy) at 80 °C for 30 min, then adding 0.1 ml of 30–32 % hydrogen peroxide (Ultrapure, Carlo Erba Reagenti) and incubating for further 30 min at 80 °C. After cooling to room temperature, samples were diluted to 10 ml with ultrapure water (Milli-Q System, Millipore Corp., Billerica, MA, USA) and then analysed for Pt quantitation by ICP-MS using an Agilent 7500cx ICP-MS system (Agilent Technologies, Santa Clara, CA, USA) equipped with a ASX-520 autosampler (Cetac Technologies, Omaha, NE, USA). ICP-MS Chemstation software B.03.07 (Agilent Technologies) was used for instrument control and data handling. Instrument parameters were optimized before each analytical session using a tuning solution containing 1 lg/l Li, Co, Y, Ce and Tl in 2 % HNO3 (w/w) (Agilent Technologies). Holmium standard solution (10 mg/l, ICP-MS grade, Carlo Erba Reagenti) was diluted to 100 lg/l in 2 % HNO3 (v/v) and used as on-line internal standard (final concentration 10 lg/l) in order to correct possible instrumental drift and variations due to the matrix. Calibration curves in the 0.1–100 lg/l range were prepared from Pt standard solution (10 mg/l, ICP-MS grade, Carlo Erba Reagenti) in 2 % HNO3 (v/v).

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MDA-MB-231, BT474, MCF-7 and MCF10A breast cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown at 37 °C in an atmosphere of 5 % CO2, as already reported (Lanuti et al. 2009a). Briefly, MDAMB-231 and MCF-7 cells were grown in DMEM-HG medium (Gibco, Carlsbad, CA, USA) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) and 10 % fetal bovine serum (Gibco); BT474 cells were grown in RPMI medium (Gibco, Carlsbad, CA, USA) added by 0.01 mg/ml Insulin, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) and 10 % fetal bovine serum (Gibco). MCF10A cells were grown in DMEM/F12 medium (Gibco, Carlsbad, CA, USA) supplemented by 10 lg/ml Insulin, 0.5 lg/ml Hydrocortisone, 20 ng/ml EGF, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) and 10 % fetal bovine serum (Gibco). Experiments were carried out on exponentially growing cells. Trypan blue assay Both the cytotoxic and the anti-proliferative effects of ruthenium and platinum metal complexes were determined by the trypan blue exclusion test, as already reported (Lanuti et al. 2006). Briefly, approximately 1.5 9 105 cells were seeded into each well of a 6-well plate (BD Falcon) in 3 ml of medium. After 12 h, solutions of each metal complex at different concentrations (5, 30 or 100 lM) were added into the wells. Metal deprived phthalocyanine and injectable cisplatinum (5, 30 or 100 lM) were used to treat negative and positive control samples, respectively. After 24, 48, and 72 h of incubation with the aforementioned compounds, cells were detached by adding to each well 500 ll Trypsin-EDTA; cells were than washed and re-suspended in 50 ll of medium; 10 ll of cell suspension was diluted with the same amount of trypan blue and cells were counted by a Neubauer cell chamber. Metal complexes and cis-platinum were

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Fig. 2 RuPcAlkC effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested RuPcAlkC concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). RuPcAlkC effects were evaluated on MDA-MB-231,

MCF-7 and BT474 breast cancer cell lines and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

used at the concentration ranges already reported in the literature (Prabhakaran et al. 2013). Drug effects on cell numbers were expressed as fold increases. Fold increases represent the increase of cell numbers, calculated for all tested drugs, the respective concentrations and time points of analyses, normalized with respect to plated cell numbers (0 h).

iodide (PI, Sigma) and 200 lg/ml of RNAse (Sigma). Cells were incubated overnight at 4 °C in the dark and then acquired by flow cytometry.

Flow cytometry cell cycle assay Cell cycle profiles were characterized as already reported (Lanuti et al. 2009b; Erba et al. 2001); briefly 5 9 105 cells/sample for each drug concentration and incubation time were fixed by adding 500 ll of 70 % cold ethanol and then stored at 4 °C. After at least 24 h, samples were washed and stained by adding 500 ll of a solution containing 50 lg/ml of propidium

Flow cytometry measurement PI fluorescence data were collected using linear amplification. Debris was excluded from the analysis by gating on morphological parameters and at least 10,000 events were recorded for each sample. Samples were acquired on a FACSCalibur flow cytometer (twolasers, four-color configuration) equipped with CellQuest 3.2.1.f1 (BD) software or on the ImageStream IS100 (AMNIS Seattle, USA) imaging flow cytometer, using a 488 nm solid state laser (40 mW) and equipped with Inspire software (v 4.1.434.0). Data were analyzed using FlowJoTM software v 8.8.6 (TreeStar,

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Fig. 3 RuPcC effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested RuPcC concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). RuPcC effects were evaluated on MDA-MB-231, MCF-7 and BT474

breast cancer cell lines and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

Ashland, OR, USA) and IDEAS software v 5.0 (AMNIS).

length and related percentages of scratch reductions were calculated by Image J software v1.41.

In vitro wound healing scratch assay

Statistical analysis

PtPcS was evaluated for its ability to inhibit MDAMB-231 and MCF10A cell motility by scratch test assay, performed as already reported (Anderson et al. 2012; Liang et al. 2007). Briefly, approximately 4 9 104 cells were seeded into each well of a 6-well plate (BD Falcon) in 3 ml of medium. A single vertical scratch was made in each well of exponentially growing cells by a pipet tip of approximately 1 mm width; therefore, 30 lM PtPcS was added. Cell migration across the wound was observed 24 h after drug incubation using a phase-contrast microscopy with a 49 objective (Nikon, Eclipse TS 100). Scratch

Data were compared by the Student’s t test and showed as average ± SD. Statistical significance was defined as p \ 0.05.

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Results PtPcS induces an effective growth arrest on breast cancer cells Different concentrations (5, 30 and 100 lM) of several Ru- and Pt-complexes, recently synthesized

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Fig. 4 PtPcC effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested PtPcC concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). PtPcC effects were evaluated on MDA-MB-231, MCF-7 and BT474

breast cancer cell lines and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

in our laboratories (RuPcAlkC, RuPcC, PtPcC and PtPcS), were tested for their anticancer properties on different breast cancer cell lines (MDA-MB-231, MCF-7 and BT474), as well as on MCF10A normal cell line. Exponentially growing cells were treated by the above reported concentrations of the aforementioned compounds and enumerated by trypan blue exclusion test after 24 and 48 h of drug incubations. As shown, RuPcAlkC (Fig. 2), RuPcC (Fig. 3) and PtPcC (Fig. 4) did not induce any significant inhibition of cell proliferation neither in breast cancer cell lines, nor in normal cells. RuPcS induced a significant cell growth slowdown on both MCF-7 and BT474 breast cancer cell lines but no effect on MDA-MB-231 cells (Fig. 5). Unfortunately, the RuPcS strongest effect was detected on normal cells (MCF10A). On the

other hand, PtPcS treatment induced a marked timeand concentration-dependent reduction of MDA-MB231 cell proliferation (Fig. 6), whereas a consistent slowdown of MCF10A normal cell growth was observed only when cells were treated by the highest concentration of PtPcS (100 lM). To validate aforementioned PtPcS abilities, metal deprived phthalocyanine (negative control) and cis-platinum (positive control) were tested on MCF10A and MDA-MB-231 cell lines. As evidenced in Fig. 7, metal deprived phthalocyanine treatment did not induce any significant alteration in terms of cell growth in both cell lines. The cis-platinum treatment showed a dose- and timedependent effect on both cell lines, by being more effective than PtPcS in inducing MCF10A cell growth arrest (Fig. 8). Trypan blue exclusion tests performed

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Fig. 5 RuPcS effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested RuPcS concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). RuPcS effects were evaluated on MDA-MB-231, MCF-7 and BT474

breast cancer cell lines and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

on PtPcS-, phthalocyanine- and cis-platinum-treated MCF-7 and BT474 breast cancer cell lines, confirmed above reported data (not shown). Taken together, these data demonstrated that among all tested compounds, only PtPcS resulted potentially effective as chemotherapy agent.

MDA-MB-231 breast cancer cells, being MDA-MB231 cells characterized by the highest proliferation potential, among the tested tumor cell lines, therefore allowing better clarification of PtPcS specific cell growth effects. The effects of PtPcS on cell cycle profiles were then investigated by flow cytometry. Figure 9 shows representative histograms of cell cycle distributions in untreated cells (left histograms) and in samples treated for 48 h with 30 lM PtPcS (right histograms) of MDA-MB-231 (lower histograms) and MCF10A (upper histograms) cell lines: in MDA-MB231, 48 h of 30 lM PtPcS induced an accumulation of cells in S phase, whereas MCF10A cells did not undergo in the same conditions any perturbation in terms of cell cycle distribution. The absence of apoptotic sub-G1 populations both in MDA-MB-231 and in MCF10A,

PtPcS induces cell cycle perturbations With the aim to better define the role of PtPcS in inducing breast cancer cell growth alterations, further experiments were performed, using PtPcS 30 lM for 48 h, in order to avoid the impairment of its specific effects on breast cancer cell growth, without affecting cell proliferation of normal cells. For further experiments, MCF10A normal cells were paralleled only with

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Fig. 6 PtPcS effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested PtPcS concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). PtPcs effects were evaluated on both MDA-MB-231 breast cancer cell

line and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

Fig. 7 Metal-deprived phthalocyanine effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested metal-deprived phatlocyanine concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). Metal-deprived

phatlocyanine effects were evaluated on both MDA-MB-231 breast cancer cell line and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

after 48 h of 30 lM PtPcS also demonstrated that PtPcS did not induce apoptosis (Fig. 9). Such data were confirmed by analysing the same samples by image flow cytometry (ImageStrem): as shown in Fig. 9 (brightfield—Ch01—and PI fluorescence—Ch04—images) no typical apoptotic features were detected after specific PtPcS treatments. Of note, as reported in the literature (Ting Chang et al. 2011), we confirmed that both tested

cell lines evidenced an accumulation of cells in G2/M phase, when treated for 48 h by 30 lM cis-platinum (Supplementary fig. 5). PtPcS inhibits breast cancer cell migration PtPcS was administered to MDA-MB-231 and MCF10A cell lines and tested for its ability to improve

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Fig. 8 Cis-platinum effects on cell growth. Bars show fold increases, in terms of cell numbers, calculated for all tested cisplatinum concentrations (5, 30, 100 lM) and time points (0, 24 and 48 h), normalized with respect to plated numbers of cells (0 h). Cis-platinum effects were evaluated on both MDA-MB-

231 breast cancer cell line and MCF10A normal cell line. Data are representative of four separate experiments, performed in triplicate. *p \ 0.005 indicates statistically significant values with respect to control cells

cell motility and proliferation (Fig. 10 and Table 1), by being the effect of 30 lM PtPcS on cell motility less severe than the one registered for MDA-MB-231.

polyanionic (i.e. tetra-anionic) PtPcS and RuPcS molecules induced cell growth perturbations in breast cancer and/or normal cell lines, thus indirectly demonstrating the ability of the aforementioned drugs to cross the cell membrane. However, if the demetallated tetrasulfonated phthalocyanine (PcS) was used, any biological effect was detected. The chemical surrounding comparable to that of the PtPcS and RuPcS allows us to hypothesize that PcS may equally cross the cellular barrier, but the absence of any effect induces us to conclude that the central metal atom plays a crucial role in each type of antiproliferative effect. Moreover, since the tested carboxylated phthalocyanines (PtPcC, RuPcAlkC and RuPcC) do not interfere with cell growth, they are probably unable to pass the cell membrane. It is published that only the neutral form of Pt-drugs is able to cross the cell membrane. In particular, for example, cis-platinum undergoes, in the extracellular environment, an hydrolysis step, replacing a chloride anion with water and finally leading to the formation of the neutral hydroxochlorodiammino platinum derivative, which results capable to cross the cell membrane barrier trough passive diffusion, endocytosis or carrier mediated import (i.e. organic cation transporters or copper transporter 1) (Arnesano et al. 2013). In our case, the ligand-exchange processes must be ruled out, since involving unlikely decoordination of

PtPcS concentration inside MDA-MB-231 and MCF10A cell lines Another set of experiments were performed measuring, by ICP MS, the PtPcS content per cell in both MDA-MB-231 and MCF10A cell lines (Fig. 11). We found a clear increasing trend with time and with the concentration of PtPcS used in each single experiment. Furthermore, the platinum content inside the MCF10A cells is always lower than in the content inside the MDA-MB-231 cells.

Discussion Metal phthalocyanines are molecules of planar geometry with strong bonds between metal and nitrogen. When in solution, especially in water, they present stacking (molecular aggregation), particularly pronounced when square planar coordinating metals are involved. Furthermore, their peripheral substituents significantly contribute to their physical behavior in water (i.e. solubility) (Leznoff and Lever 1996). All together, the data reported in the present study showed that, among all tested drugs, only the

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Fig. 9 Impairment of cell cycle profiles induced by PtPcS. Flow cytometry analysis of cell cycle profiles were performed on MDAMB-231 and MCF10A cell lines, following the DNA staining by propidium iodide (PI). Representative DNA distributions (histograms) and representative brightfield (Ch1) and PI (Ch4) 409 images, obtained by image flow cytometry (ImageStream) are shown. Data are representative of four separate experiments performed in duplicate

the macrocyclic ligands, but also the fact that the negative charge of the anionic species is in fact extensively delocalized over the entire aromatic phthalocyanine moiety, cannot explain the clearlycut differences between the ionic PcS and neutral Pt and Ru carboxylated complexes. The only thing we can say at present is that PtPcS can across more efficiently the cell barrier when in presence of MDA-

MB-231 cell line (see Fig. 11) and this finding probably can explain the major activity observed. The fact that tetraanionic phthalocyanine, although presenting four polar sulfonic groups, can equally cross the cellular membrane, is not new since other researcher, in studies related to photodynamic therapy, proved the presence of zinc tetrasulfophthalocyanine inside some tumor cells. Also, in the above cited case,

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Biometals (2014) 27:575–589 Table 1 PtPcS treatment modulates cell migration with in vitro scratch assay; cell lines were cultured with/without 30 lM PtPcS for 0 and 24 h and subjected to the in vitro scratch assay Cells lines

With/ without PtPcS

MDAMB231

Ctrl

MCF10A

Ctrl

0

1

0

PtPcS

0

1

0

Ctrl

24

0

100

PtPcS

24

0

100

PtPcS

Time (hr)

0 0

Ctrl

24

PtPcS

24

Length scratch ± SD (mm) 1 1

% Reduction

0 0

0.36 ± 0.06

77

0.71 ± 0.07

37

SD standard deviation; data are representative of four separate experiments

Fig. 10 PtPcS effects on cell migration, evaluated by the in vitro scratch test. MDA-MB-231 and MCF10A cell lines were treated by PtPcs 30 lM for 24 h. Cells of both cell lines, were then subjected to the in vitro scratch assay. Images (49) were captured by a phase-contrast microscope at 0 and 24 h after the scratch. Data are representative of two separate experiments

they observed that the presence of drug inside the more metastatic cells is higher than inside the less aggressive cells (Valduga et al. 1996). Uptake in cells was demonstrated to correlate well with the hydrophobicity of the drug and inversely with its aggregation outside the cellular environment. Although the hydrophobicity of PtPcS is far to be marked, the fact that we did not used a single positional isomer should facilitate the penetration inside the cells, since the degree of

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Fig. 11 Pt content per single cell after 0, 24 and 48 h for MDAMB-231 (rhombs-30 lM and squares-100 lM) and MCF10A (triangles-30 lM and circles-100 lM) cell lines. Experiments were executed in triplicate while ICP MS measures were done in quintuplicate

aggregation of the used mixture, containing the various possible isomers, is surely lower, compared with that of a single isomer derivative (Edrei et al. 1998) (note that the Weber and Bush template synthetic procedure leads to a mixture of positional isomers, which, if necessary, can be separated only by sophisticated technologies). As matter of fact, among the two polyanionic tested drugs (PtPcS and RuPcS), only PtPcS showed interesting anticancer properties, in vitro. As demonstrated, low doses of PtPcS (5–30 lM) gave rise to a significant reduction of cell proliferation in all tested breast cancer cell lines, without affecting the cell growth of normal cells (MCF10A).

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Interestingly, as demonstrated by flow cytometry, PtPcS induced a significant accumulation of cancer cells on S phase of the cell cycle. For these reasons and because PtPcS did not induce cell death on our models (as confirmed by image flow cytometry), it may be considered a cytostatic agent, as reported for the cisplatinum (Ting Chang et al. 2011). Of note, as we demonstrated, confirming previously published results, cis-platinum determines its cytostatic effect by inducing the accumulation of breast cancer cells in G2/M phase of the cell cycle (Ting Chang et al. 2011). These data may give rise to different action mechanisms of cis-platinum and PtPcS. Even if further investigation will be need, it is possible to hypothesize that the biological activity of phthalocyanines is associated to the planarity of these aromatic molecules, which may intercalate within the doublestranded DNA structure, thus interfering with the cellular machinery. Between Ru and Pt, the former is more selective; this finding could be associated to metals coordination properties. The octahedral RuPcS can be destacked inside the extracellular environment more efficiently than the respective square planar platinum derivative; this may contribute to explain the better selectivity showed by platinum compounds. Regarding the possible side effects, it must be noted that PtPcS was less effective to induce cell cycle perturbations on normal cells than cis-platinum, thus resulting less cytotoxic. On the other hand, since the mortality of more than 90 % of cancer patients is related to the metastases development (Entschladen et al. 2004), cell movement through tissues plays a crucial role in cancer progression. Therefore, the targeting of cell motility and invasiveness in anticancer drug development has recently attracted increasing interest. For these reasons, the effects of PtPcS on cell motility were also investigated by the scratch test method and results demonstrated that PtPcS was able to effectively reduce cell locomotion in breast cancer cells more than in normal cells. Even if further investigations will be need to clarify the PtPcS action mechanism, taken together, these findings shed light on promising therapeutic effects produced by this drug. In fact, PtPcS is able, when administered in vitro at low doses, to selectively slowdown cell growth and motility of breast cancer cells, but not of normal cells, thus possibly providing new perspectives for the characterization of novel therapies for breast cancer.

587 Acknowledgments The authors are grateful for financial support to the ‘‘Ministero dell’Universita` e della Ricerca’’ (MIUR) for PRIN Project: 2008F5A3AF_004, (Metalphthalocyanines as potential antitumor drugs) and FIRB Project 2010. The present research was also supported by ‘‘Carichieti’’ foundation, Chieti, Italy.

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