Antonella Muscella

Nadia Calabriso

Carla Vetrugno

Francesco Paolo Fanizzi

Sandra Angelica De Pascali

Carlo Storelli

Santo Marsigliante
email: santo.marsigliante@unile.it

Angelica Sandra

Carlo De Pascali

Santo Storelli

Marsigliante

O,O'-acac)(␥-acac)(DMS)] alters the intracellular calcium homeostasis in MCF-7 breast cancer cells

Keywords: Pt(O, O'-acac)(γ-acac)(DMS)], [Ca 2+ ] membrane calcium ATPase PVDF, polyvinylidene difluoride membrane ROS, reactive oxygen species SDS, sodium dodecyl sulphate

HAL is

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

Introduction

Free intracellular Ca 2+ , acting as a second messenger, is crucial for a diverse range of biological functions, such as fertilization, neurotransmission, muscle contraction, and gene transcription [START_REF] Carafoli | Generation, control, and processing of cellular calcium signals[END_REF]. Intracellular Ca 2+ signalling is also a key regulator of proliferation [START_REF] Lipskaia | Alteration in temporal kinetics of Ca 2+ signaling and control of growth and proliferation[END_REF], cell cycle progression [START_REF] Kahl | Regulation of cell cycle progression by calcium/calmodulin-dependent pathways[END_REF], and apoptosis [START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF].

Although the essential role of Ca 2+ signalling in maintaining homeostatic functions is well established, less is known about the role of aberrant Ca 2+ signalling in disease [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF][START_REF] Rizzuto | When calcium goes wrong: genetic alterations of a ubiquitous signaling route[END_REF]. In the past decade there has been a realization that particular cancers are associated with alterations in specific Ca 2+ channels or pumps. Ca 2+ signalling is involved in various tumorigenic pathways, and the inhibition or activation of specific Ca 2+ channels or pumps can reduce cellular proliferation and/or induce apoptosis. There is an increasing awareness that PMCA alterations, concurrent with an increase in Ca 2+ influx channel expression and TRPV6 [START_REF] Zhuang | Calciumselective ion channel, CaT1, is apically localized in gastrointestinal tract epithelia and is aberrantly expressed in human malignancies[END_REF], are associated with tumorigenesis, including that of the mammary gland [START_REF] Kamrava | Calcium signaling as a molecular target in cancer[END_REF]. Indeed, relative PMCA1 mRNA expression is greater in tumorigenic breast cancer MCF-7 and MDA-MB-231 cells than that in non-tumorigenic MCF-10A cells [START_REF] Lee | Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines[END_REF] . PMCA1 and PMCA4 expression is also lower in simian virus 40-transformed human skin fibroblasts [START_REF] Reisner | Analysis of plasma membrane Ca 2+ -ATPase expression in control and SV40-transformed human fibroblasts[END_REF]. Thus, perturbed PMCA regulation or function may be important in diseases such as cancer. Therapeutic modulation of PMCA expression and/or activity may have functional consequences in suppressing the tumorigenic phenotype. The appropriateness of PMCA as a possible drug target is also demonstrated by the prominence of other clinically used inhibitors of P-type ATPases, digoxin and omeprazole, as inhibitors of Na + /K + -ATPase and H + /K + -ATPase, respectively [START_REF] Chene | ATPases as drug targets: learning from their structure[END_REF][START_REF] Johnson | Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells[END_REF]. There are also studies indicating that modulators of Ca 2+ signalling may offer an effective cell death induction and confer another layer of selectivity for cancer cells with an aberration in multiple Ca 2+ channels or pumps. For example, tamoxifen alters Ca 2+ signalling in MCF-7 breast cancer cells, and in malignant glioma cell lines it increases cytosolic Ca 2+ responses to ATP-agonist activation [START_REF] Zhang | Tamoxifen-induced Enhancement of Calcium Signaling in Glioma and MCF-7 Breast Cancer Cells[END_REF]. In glioma cells tamoxifen also increases the radius of Ca 2+ wave propagation and the rate of Ca 2+ ionophore-mediated cell death.

Recently, this group has synthesized and studied a platinum complex containing two acetylacetonate (acac) ligands, one O,O'-chelate and the other sigma-linked by methionine in gamma position, and dimethyl-sulphide (DMS) in the metal coordination sphere, that has shown interesting biological activities [START_REF] Pascali | First examples of β-diketonate platinum II complexes with sulfoxide ligands[END_REF]. This compound ([Pt(O,O'acac)(γ-acac)(DMS)]) not only was able to induce apoptosis in endometrial cancer cells (HeLa), with activity up to about 100 times higher than that of cisplatin, but also showed for which the activity appears to be associated both with its intracellular accumulation and with the formation of DNA adducts [START_REF] Muscella | New platinum II complexes containing both an O,O′-chelated acetylacetonate ligand and a sulfur ligand in the platinum coordination sphere induce apoptosis in HeLa cervical carcinoma cells[END_REF][START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF], the cytotoxicity of this new compound is related to the intracellular accumulation only, showing a low reactivity with nucleobases and a specific reactivity with sulphur ligands, suggesting that the cellular targets could be amino acid residues of proteins.

The different action mechanism of the new complex, having different biological targets, with respect to cisplatin, may render it intrinsically able to evoke less chemo-resistance phenomena. Here we wanted to see whether [Pt(O,O'-acac)(γ-acac)(DMS)] was able to increase the level of free [Ca 2+ ] i in fura-2-loaded MCF-7 cells. The [Ca 2+ ] i increase was characterized and the underlying mechanisms were investigated; this study indicates that [Pt(O,O'-acac)(γ-acac)(DMS)] alters the homeostasis of Ca 2+ thus offering further therapeutic opportunities.

Materials and Methods

Determination of [Ca 2+ ] i

The medium was aspirated, and the cells were then harvested with 0.05% trypsin-EDTA solution. After washing the cells three times by pelleting (100X g for 5 min), the cells (4 X 106) were incubated with 5 µM fura 2-AM and 0.2% pluronic F-127 for 45 min at 37 • C with continuous shaking (100 cycles/min). Following the loading period, the cells were washed twice with a modified Krebs-Ringer buffer in which the bicarbonate was replaced by 20 mM Hepes, pH 7.4, incubated again for at least 10 min at room temperature to facilitate hydrolysis of the esterified probe, and washed once again. The cells were resuspended in 2 ml of the same buffer containing 0.1% BSA, and 20 µl of the cell suspension was added to a 2 ml fluorescence cuvette kept at 37 • C, and stirred throughout the experiment. The fluorescence intensity was measured with a JASCO FP 750 fluorimeter (Jasco Corporation, Japan). The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. The maximal fluorescence was determined at the end of the assay by adding 20 µl 10% SDS and the minimal fluorescence by adding 20 µl 0.5 M EGTA solution, pH 9.0. The cytoplasmic Ca 2+ concentration at the time t was calculated using the software of the fluorimeter and assuming a dissociation constant for the fura 2-Ca 2+ complex of 224 nM, according to the Grynkiewicz equation [START_REF] Grynkiewicz | A new generation of calcium indicators with greatly improved fluorescence properties[END_REF]. Mn 2+ strongly quenched the fura-2 fluorescence at 360 nm. Results are expressed as relative fluorescence quenching with respect to the maximal quenching induced by the addition of digitonin ( 80A c c e p t e d M a n u s c r i p t 5 µg/ml).

Ca 2+ -ATPase activity measurement

ATPase activities were determined by measuring inorganic phosphate (Pi) production by the method of Fiske and Subarrow [START_REF] Higgins | Separation and analysis of membrane lipid components[END_REF]. This method is based on the reaction between phosphate and molybdate, producing yellow molybdate phosphoric acid which contains a Mo(VI) that is then reduced to Mo(V) present in a blue-coloured heteropolyacid compound. This blue compound is directly measured by reading the absorbance at 700 nm. Ca 2+dependent ATPase was measured in crude MCF-7 cell homogenates in the presence of 0.5 mM ouabain and 5 mM sodium azide, potent inhibitor of mitochondrial Ca 2+ uptake.

Typically, 100 µg total cell lysates were incubated for 10 min at 37°C in a reaction mixture consisting of (in mM): 100 NaCl, 20 KCl, 15 Hepes, 12 Tris, 1.2 MgCl 2 , 2.5 ATP. This reaction buffer were supplemented with 200 µM CaCl 2 and the free Ca 2+ concentration (3 µM) has been calculated using the CHELATOR program (BMAXC STANDARD, http://www.stanford.edu). The further addition of 1 µM of thapsigargin (to inhibit SERCA activity, [START_REF] Lytton | Thapsigargin inhibits the sarcoplasmic and endoplasmic reticulum Ca 2+ -ATPase family of calcium pumps[END_REF]), and of 2 µM orthovanadate (a dose used to selectively inhibit PMCA activity freshly prepared to avoid its transformation to decameric vanadate species, [START_REF] Sepúlveda | Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca 2+ -ATPase isoform 1 SPCA1 in cerebellum relative to other Ca 2+ pumps[END_REF]) and of 800 µM EGTA (to measure the Mg 2+ -ATPase activity) were made in three different steps, and the activity was measured after each addition in order to evaluate the contribution of each ATPase to the total ATPase activity. The total Ca 2+ -ATPase activity (i.e. the sum of SERCA, PMCA, and SPCA) was obtained after subtraction of the Mg 2+ -ATPase activity from the (Ca 2+ + Mg 2+ )-ATPase activity obtained immediately after the addition of ATP. The SERCA activity was calculated by subtracting the activity measured in the presence of thapsigargin (which includes PMCA, SPCA, and Mg 2+ -ATPase activities) from the total (Ca 2+ + Mg 2+ )-ATPase activity. The SPCA activity was calculated by subtracting the Mg 2+ -ATPase activity from the activity in the presence of thapsigargin and vanadate. The PMCA activity was calculated by subtracting the Mg 2+ -ATPase activity and the SPCA activity from the activity in the presence of thapsigargin. Comparable results were obtained when PMCA activity was measured by using carboxyeosin, frequently mentioned as PMCA inhibitor [START_REF] Gatto | Eosin, a potent inhibitor of the plasma membrane Ca pump, does not inhibit the cardiac Na-Ca exchanger[END_REF] .

Preparation of subcellular fraction.

To obtain protein cell extracts and subcellular fractions we used a procedure previously described [START_REF] Muscella | Sublethal concentrations of the platinum II complex [Pt O,O'-acac -acac DMS ] alter the motility and induce anoikis in MCF-7 cells[END_REF]. Cells were washed twice in ice-cold PBS and harvested in 1 ml of PBS.

The samples were centrifuged for 30 s at 10,000 g, and cell pellets were resuspended in After a 10 min incubation on ice, cells were passed several times through a 20 gauge syringe and then centrifuged at 13,000 g for 10 min at 4 °C. For preparation of subcellular fractions cells were ruptured in homogenization buffer (mM): 20 Tris HCl, pH 7.5, conteining 250 sucrose, 2 EDTA, 0.5 EGTA, 0.2 phenylmethylsulfonyl fluoride and a cocktail of protease inhibitors, by Dounce homogenization and centrifuged immediately at 2,000 g for 10 min. The supernatant was collected and centrifuged at 100,000 g for 1 hr to separate cytosolic and membrane fractions. The membrane fraction was subsequently resuspended in extraction buffer (mM): 20 Tris HCl, pH 7.5, containing 150 NaCl, 1 EDTA, 1 EGTA, and a cocktail of protease inhibitors (containing 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor and 10 μg/ml leupeptin) with 1% (vol/vol) Nonidet P-40. Nuclei were pelleted by centrifugation at 2,000 g for 15 min at 4°C and resuspended in high-salt buffer (mM): 20 Tris-HCl, pH 7.9, 420 NaCl, 10 KCl, 0.1 Na 3 VO 4 , 1 EDTA, 1 EGTA, 20% glycerol, supplemented with a cocktail of protease inhibitors, and sonicated until no nuclei remained intact. Samples were then centrifuged at 13,000 g for 10 min at 4 °C, and the resultant supernatant was used as the nuclear extract.

The purity of fractions was tested by immunoblotting with antibodies specific to α-Na + /K + ATPase subunity (membrane protein) and β-actin (cytoplasmic protein). Proteins in the homogenates and cellular fraction were determined using the Bio-Rad protein assay kit 1.

Lyophilized bovine serum albumin was used as a standard.

Western blot analysis.

Proteins in homogenates and cellular fraction were determined using the Bio-Rad protein assay kit 1 (Milan, Italy). Lyophilised bovine serum albumin was used as a standard. Total cell proteins or proteins of the distinct sub-cellular fractions were dissolved in SDS sample buffer and separated on 10% or 15% SDS gels. Separated proteins were transferred electrophoretically onto polyvinylidene difluoride membrane (PVDF) (Amersham International). Equal protein loading was confirmed by Ponceau S staining. Blots were incubated with specific primary antibodies and the immune complexes were detected using appropriate peroxidase conjugated secondary antibodies and enhanced chemiluminescent detection reagent ECL (Amersham International). Blots were stripped and used for sequential incubations with relative control antibody (α-Na + /K + ATPase subunity for membrane protein and β-actin for cytoplasmic protein). Densitometric analysis was carried out on Western blots using the NIH Image (v1.63) software. The pixel intensity A c c e p t e d M a n u s c r i p t 7 for each region was analyzed, the background was subtracted, and the protein expressions were normalized to β-actin loading control for each lane.

Intracellular reactive oxygen species (ROS) formation.

ROS generation was detected by nitroblue tetrazolium (NBT) assay as previously described [START_REF] Muscella | Sublethal concentrations of the platinum II complex [Pt O,O'-acac -acac DMS ] alter the motility and induce anoikis in MCF-7 cells[END_REF]. NBT (1 mg/ml ) was added to medium of treated MCF-7 and incubations were carried out at 37 °C for 15-60 min. NBT is reduced by ROS to a dark blue insoluble form of NBT called formazan. Cells were then carefully washed and lysed in a 90% dimethylsulfoxide solution containing 0.01 N NaOH and 0.1% SDS. Absorbance of formazan was measured at 550 nm against lysis buffer blank. Data are expressed as % of control untreated MCF-7 cells.

Statistical analysis.

Experimental points represent means ± standard deviation (S.D.) of 3-6 replicates.

Statistical analysis was carried out using the ANOVA. When indicated, post hoc tests (Bonferroni/Dunn) were also performed. A p value less than 0.05 was considered to achieve statistical significance.

Reagents and MCF-7 cell line.

The PKC inhibitors GF109203X and Gö6976 were obtained from Calbiochem (Darmstadt, Germany). Fura 2-AM and thapsigargin were from Molecular Probes (Eugene, OR, USA).

Antibodies used in western analyses to PKC-, -ε, -δ, - and -, β-actin and -subunit of Na+/K+ATPase, goat anti-rabbit IgG conjugated with peroxidase, as well as control

RESULTS

Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on [Ca 2+ ] i

We showed previously that the new Pt complex containing an O,O'-chelated acetylacetonate ligand (acac) and a dimethyl-sulphide in the Pt coordination sphere,

Effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on Mn 2+ uptake in MCF-7 cells

To test whether the changes of the [Ca 2+ ] i response caused by [Pt(O,O'-acac)(γacac)(DMS)] were due to modulation of the plasma membrane permeability, we measured Mn 2+ influx after the addition of [Pt(O,O'-acac)(γ-acac)(DMS)]. Mn 2+ is a good Ca 2+ -entry tracer, since it is not pumped out of the cell [START_REF] Merritt | Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry[END_REF]. Mn 2+ uptake was estimated by the quenching of fura-2 fluorescence when excited at 360 nm, which is an isosbestic wavelength and is insensitive to variations in [Ca 2+ ] i concentration. entry was drastically decreased by the impermeant cation La 3+ (100 µM), an effect similar to that obtained with 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] (Fig. 2). The effect of 10 µM

[Pt(O,O'-acac)(γ-acac)(DMS)]
or 100 µM La 3+ on Mn 2+ entry was not augmented were both were used in combination (Fig. 2B).

Inhibitory effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on of Ca 2+ -ATPases activity

First, Ca 2+ -dependent ATPase was measured in crude MCF-7 cell homogenates in the presence of 0.5 mM ouabain. The method used to measure Ca 2+ -dependent ATPase in MCF-7 cells was sensitive and reproducible. In standard culture conditions, the coefficient of variation of a single assay was 3.2% (n=8) and Ca 2+ -dependent ATPase activities (PMCA, SERCA and SPCA) increased linearly with increasing protein concentrations.

Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on ATP-induced Ca 2+ response in MCF-7 cells

In order to understand whether the effects provoked by [Pt(O,O'-acac)(γ-acac)(DMS)] had an effective role in controlling the homeostasis of intracellular calcium, we provoked physiological disturbances in [Ca 2+ ] i using ATP, an agonist able to induce intracellular calcium signalling in MCF-7 cells, due to stimulation of phospholipase C through P2Y2

receptors [START_REF] Bowler | Signalling in human osteoblasts by extracellular nucleotides-their weak induction of the c-Fos proto-oncogene via Ca 2+ mobilisation is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogenactivated protein kinase[END_REF]. The [Ca 2+ ] i transient evoked by ATP consisted of a rapid peak followed by a sustained phase of elevated [Ca 2+ ] i , which persisted for over 3 min (Fig. 4A). The maximum increase in [Ca 2+ ] i was observed at 100 µM ATP, with a [Ca 2+ ] i of 509 ± 21 nM.

ATP-induced [Ca

Effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on thapsigargin-evoked extracellular

Ca 2+ entry.

Thapsigargin, an inhibitor of the SERCA, allows Ca 2+ to leak out from the ER pool [START_REF] Lytton | Thapsigargin inhibits the sarcoplasmic and endoplasmic reticulum Ca 2+ -ATPase family of calcium pumps[END_REF] causing complete store depletion also in MCF-7 cells [START_REF] Anantamongkol | Regulation of Ca 2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca 2+ -ATPase type 2[END_REF]. The simultaneous administration of thapsigargin and [Pt(O,O'-acac)(γ-acac)(DMS)] to MCF-7 cells, kept in Ca 2+ -free medium, increased the [Ca 2+ ] i peak, probably as a consequence of the decreased PMCA activity (Fig. 6B andC). In fact, a similar [Ca 2+ ] i increase was also obtained by pretreatment of cells with orthovanadate ([VO 3 (OH)] 2 ), a PMCA inhibitor [START_REF] Sepúlveda | Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca 2+ -ATPase isoform 1 SPCA1 in cerebellum relative to other Ca 2+ pumps[END_REF] (Fig. 6B andC). Furhermore, differences in extracellular Ca 2+ entry obtained by thapsigargin, in the presence and in the absence of [Pt(O,O'-acac)(γ-acac)(DMS)], were also observed (Fig. 6B andD). However, the effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on extracellular Ca 2+ entry due to ATP or thapsigargin were different (Fig. 6A andD). The effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on the extracellular Ca 2+ entry were also evaluated by using the Mn 2+ quenching technique, since this cation is not transported by the major calcium pumps, SERCA and PMCA. Indeed, the entry of Mn 2+ increased in both thapsigargin-and ATP-treated cells, resulting however significantly higher when using ATP (Fig. 6 E and F), probably because of the opening of second messengers-operated Ca 2+ channels, in addition to store-operated channels. When 10 µM [Pt(O,O'-acac)(γacac)(DMS)] was administered together with thapsigargin or ATP, the quenching of Mn 2+ significantly decreased, reaching the same value (Fig. 6F). Taken together, these results suggest that the Ca 2+ /Mn 2+ -permeable plasma membrane channels opened by ATP only are targets of [Pt(O,O'-acac)(γ-acac)(DMS)]. Conversely, the Ca 2+ /Mn 2+ -permeable plasma membrane store-operated channels opened by both ATP and thapsigargin, may not be affected by [Pt(O,O'-acac)(γ-acac)(DMS)].

Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on Na + /Ca 2+ -Exchanger activity

In order to understand the involvement of a Na + /Ca 2+ Since the difference between the early peak rise in [Ca 2+ ] i obtained in physiological and Na + -free buffers in the absence or presence of [Pt(O,O'-acac)(γ-acac)(DMS)] is practically identical (Fig. 7C), we may conclude that [Pt(O,O'-acac)(γ-acac)(DMS)] has no effects on the activity of the Na + /Ca 2+ exchanger. A c c e p t e d M a n u s c r i p t 14

Effects of [Pt(O,O'-acac)(γ-acac)(DMS)]-activated PKC-α on extracellular Ca

ROS are responsible for the [Pt(O,O'-acac)(γ-acac)(DMS)]-induced decrease of the PMCA activity.

[Pt(O,O'-acac)(γ-acac)(DMS)] increased the level of ROS in a time-dependent manner with ROS generation significantly different respect to control (ANOVA: p<0.005) as soon as 1 min (Fig. 9A). [Pt(O,O'-acac)(γ-acac)(DMS)]-provoked ROS production was partially inhibited by diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase (Fig. 9D).

Since another important sources of intracellular ROS are mitochondria, we studied the specific production of mitochondrial O -2 by MitoSOX red. We found that [Pt(O,O'-acac)(γacac)(DMS)] increased the mitochondrial synthesis of O -2 (Fig. 9B), in a time-dependent manner (Fig. 9C), and that such effect was blocked by the antioxidant apocynin (10 µM).

Apocynin also reversed the [Pt(O,O'-acac)(γ-acac)(DMS)]-provoked inhibition of PMCA activity (Fig. 9E).

Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on other human breast cell lines

To determine whether the observations made on MCF-7 cells are also valid for other cell lines, we tested two other breast cancer cell lines, MDA-MB-231 and SKBR-3.

Furthermore, we also included the MCF-10A, a nontumorigenic breast epithelial cell that appeared to be less sensitive to [Pt(O,O'-acac)(γ-acac)(DMS)] than MCF-7 cells in cytotoxicity assays [START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF]. Thus, a selected set of crucial experiments (effects of 10 µM

DISCUSSION

Antineoplastic drugs resistance poses a major impediment to the successful treatment of breast cancer. About 70% of breast cancers are oestrogen receptor positive (ER+ve). The remaining 30% are ER-ve [START_REF] Osborne | The value of estrogen and progesterone receptors in the treatment of breast cancer[END_REF]. Oestrogen hormone promotes breast cancer cell proliferation through ER, therefore, oestrogen antagonists such as tamoxifen are used to block ER activation in ER+ve breast cancer patients [START_REF] Thorpe | Estrogen and progesterone receptor determinations in breast cancer, technology, biology and clinical significance[END_REF]. We have previously synthesized a new Pt complex, [Pt(O,O'-acac)(γ-acac)(DMS)], in the attempt to find novel strategies for a better management of ER-ve breast cancer. This compound may be a promising new decreases Bcl-2 expression In MCF-7 cells and contributes to the formation of pores by facilitating the translocation of Bax, a proapoptotic factor, from the cytosol to the outer mitochondrial membrane [START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF]. This mechanism, allows the dissipation of mitochondrial membrane potential and the release of cytochrome c and is Ca 2+ independent [START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF].

Furthermore, the dissipation of membrane potential minimizes the contribution of mitochondria to Ca 2+ homeostasis and causes ATP depletion which further increases the [Ca 2+ ] i by decreasing the Ca 2+ pump activities. It is well known that in apoptosis, the appropriateness of Ca 2+ handling is of fundamental importance [START_REF] Orrenius | Regulation of cell death: the calciumapoptosis link[END_REF] with Ca 2+ playing different roles in this process; for example, Ca 2+ from the extracellular space, or released from endoplasmic reticulum, leads to an increase in the mitochondrial Ca 2+ concentration, which results in the increase of transition pore permeability which activates apoptosis [START_REF] Rizzuto | When calcium goes wrong: genetic alterations of a ubiquitous signaling route[END_REF]. It is well established that altered expression of specific Ca 2+ channels and pumps are characteristic features of some cancers and anticancer drugs such as cisplatin, are able to increase [Ca 2+ ] i through various mechanisms [START_REF] Florea | Occurrence, use and potential toxic effects of metals and metal compounds[END_REF][START_REF] Tomaszewski | SnCl2 reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats[END_REF][START_REF] Kawai | Relationship of intracellular calcium and oxygen radicals to cisplatin-related renal cell injury[END_REF]. We here show that [Pt(O,O'-acac)(γacac)(DMS)] evokes an early increase in [Ca 2+ ] i (Fig. 1A). This supports the hypothesis that homeostatic mechanisms involved in the regulation of [Ca 2+ ] i may play a role during and6). Indeed, [Pt(O,O'-acac)(γ-acac)(DMS)] decreased the permeability of the plasma membrane, an effect that is comparable to that caused by La 3+ (Fig. 2). This increase could be caused by the action of [Pt(O,O'-acac)(γ-acac)(DMS)] on at least one of the various systems that regulate Ca 2+ homeostasis, such as Ca 2+ transport pumps (the plasma membrane Ca 2+ -ATPases PMCAs, the sarco/endoplasmic reticulum Ca 2+ -ATPases SERCAs, and the secretory pathway Ca 2+ -ATPases SPCAs), and secondary Na + /Ca 2+ antiporter. We have shown that both SPCA and SERCA activities were not affected by the treatment with [Pt(O,O'-acac)(γ-acac)(DMS)] (Fig. 3). The PMCA removes the Ca 2+ through the plasma membrane and is important for the maintenance of basal levels of cytosolic Ca 2+ and lowering of [Ca 2+ ] i after the generation of Ca 2+ transients [START_REF] Carafoli | Generation, control, and processing of cellular calcium signals[END_REF]. Measuring the ATPase activity gave a direct indication that PMCA was inhibited by Pt(O,O'-acac)(γacac)(DMS)] (Fig. 3). This inhibition resulted in changes in the ATP-induced [Ca 2+ ] i profile. Clearly, PMCA represents a potential therapeutic target, since its inhibition affects the efflux of Ca 2+ by increasing its concentration that may eventually lead to the induction of apoptosis. In this regard it has been shown that even the partial inhibition of PMCA-mediated Ca 2+ efflux is sufficient to reduce proliferation of MCF-7 cells [START_REF] Kawai | Relationship of intracellular calcium and oxygen radicals to cisplatin-related renal cell injury[END_REF]. Furthermore, an antisense construct directed towards PMCA leads to changes in cellcycle kinetics, such as lengthening the G2 phase and simultaneously reducing the Ca 2+ efflux [START_REF] Lee | Antisense-mediated Inhibition of the Plasma Membrane Calcium-ATPase Suppresses Proliferation of MCF-7 Cells[END_REF]. PMCA is a very sensitive target for oxidative stress as occurs in pancreatic acinar cells [START_REF] Pariente | Release of calcium from mitochondrial and non mitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide[END_REF] and in neuronal and liver membranes [START_REF] Zaidi | Effects of reactive oxygen species on brain synaptic plasma membrane Ca 2+ -ATPase[END_REF][START_REF] Bruce | Oxidant-impaired intracellular Ca 2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca 2+ -ATPase[END_REF]. We have noticed that addition of [Pt(O,O'-acac)(γ-acac)(DMS) to MCF-7 cells induces rapid production of ROS (Fig. 9A andC). It is known that ROS can be produced through the activation of NAD(P)H oxidase, or mitochondrial pathways [START_REF] Fariss | Role of mitochondria in toxic oxidative stress[END_REF]. Therefore, it is expected that the origin of the ROS in cells treated with [Pt(O,O'-acac)(γ-acac)(DMS)] is twofold: primarily by NAD(P)H oxidase, but also by mitochondria (with apocinyn inhibiting this effect, Fig. 9B). In fact, whilst the inhibition of NAD(P)H oxidase by DPI only partially prevented [Pt(O,O'-acac)(γacac)(DMS)]-induced ROS generation, the use of the antioxidant apocinyn inhibited this effect (Fig. 9D) and restored the PMCA activity due to ROS production (Fig. 9E). The effects of ROS on PMCA could be due to either the aggregation of PMCA molecules, as a result of Cys oxidation to disulphides and increased hydrophobic interactions [START_REF] Zaidi | Effects of reactive oxygen species on brain synaptic plasma membrane Ca 2+ -ATPase[END_REF], or to the increase of [Ca 2+ ] i due to the oxidative stress which activates Ca 2+ -dependent proteases, such as caspases, known to make PMCA inactive [START_REF] Pászty | Cleavage of the plasma membrane Ca 2+ ATPase during apoptosis[END_REF][START_REF] Schwab | Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis[END_REF]. A somewhat similar mechanism has been described in neurons, whereby cleavage of the Na + /Ca 2+ exchanger by caspases was shown to mediate the Ca 2+ overload during the excitotoxicity associated with brain ischemia [START_REF] Bano | Cleavage of the plasma membrane Na + / Ca 2+ exchanger in excitotoxicity[END_REF]. However, we show that the effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on [Ca 2+ ] i are not due to modulation of the Na + /Ca 2+ exchanger activity (Fig. 7). It seems that the mechanism dependent on caspase activation does not occur in our case. This conclusion is based upon previous results [START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF] which show that the time frame over which ] i can be due to Ca 2+ -influx from the extracellular space or Ca 2+ -release from intracellular stores [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF][START_REF] Clapham | Calcium signaling[END_REF]. Channels mediating Ca 2+ -influx from the extracellular space are voltage-gated and ligand-gated channels, TRP and store-operated channels. In general, non-excitable tissues, including the epithelium, do not express voltage gated Ca 2+ channels. However, recent studies have shown that T-type Ca 2+ channels are expressed in cancerous cells, MCF-7 included [START_REF] Taylor | Selective blockade of T-type Ca 2+ channels suppresses human breast cancer cell proliferation[END_REF]. MCF-7 cells also express TRPM7 [START_REF] Guilbert | Evidence that TRPM7 is required for breast cancer cell proliferation[END_REF] and 8 [START_REF] Chodon | Estrogen regulation of TRPM8 expression in breast cancer cells[END_REF],

TRC1 [START_REF] Hiani | Extracellular Signal-Regulated Kinases 1 and 2 and TRPC1 Channels are Required for Calcium-Sensing Receptor-Stimulated MCF-7 Breast Cancer Cell Proliferation[END_REF] and 6 [START_REF] Guilbert | Expression of TRPC6 channels in human epithelial breast cancer cells[END_REF]. TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue [START_REF] Zhuang | Calciumselective ion channel, CaT1, is apically localized in gastrointestinal tract epithelia and is aberrantly expressed in human malignancies[END_REF], suggesting that it may play some role in tumor development. Tamoxifen inhibits TRPV6 activity in TRPV6-expressing MCF-7 breast cancer cells [START_REF] Bolanz | Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells[END_REF] and alters Ca 2+ signalling in MCF-7 breast cancer cells. In malignant glioma cell lines tamoxifen increases cytosolic Ca 2+ responses to ATP-agonist activation [START_REF] Zhang | Tamoxifen-induced Enhancement of Calcium Signaling in Glioma and MCF-7 Breast Cancer Cells[END_REF]. Some metals (e.g. Pb 2+ [START_REF] Büsselberg | Actions of metals on membrane channels, calcium homeostasis and synaptic plastivity[END_REF] and methyl-mercury [START_REF] Leonhardt | Voltage-activated calcium channel currents of rat DRG neurons are reduced by mercuric chloride HgCl 2 and methylmercury CH 3 HgCl[END_REF]), directly block membrane channels leading to a decrease in [Ca 2+ ] i . [Pt(O,O'-acac)(γ-acac)(DMS)] reduced the Ca 2+influx from the extracellular space, as demonstrated by the fluorescence quenching due to Mn 2+ influx (Fig. 2 and6E). Mn 2+ enters the cell using Ca 2+ channels but it is not transported by either the SERCA nor by PMCA. [Pt(O,O'-acac)(γ-acac)(DMS)] decreases the entry of Mn 2+ in cells untreated (Fig. 2) and treated with either ATP or thapsigargin (Fig. 6E). Since the effects were greatest in ATP-treated cells, it is likely that [Pt(O,O'-acac)(γacac)(DMS)] acts also on channels coupled to G proteins or second messengers produced by agonists such as ATP. In this regard, it is known that the PKCs are intracellular transducers also involved in regulating Ca 2+ influx [START_REF] Mchugh | Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain[END_REF]. PKC-α is the first isoform activated in [Pt(O,O'-acac)(γ-acac)(DMS)]-treated cells. This activation is temporally compatible with the rapid decrease in the plasmamembrane permeability (Fig. 8A). The involvement of PKC-α is highlighted by the effects elicited by its pharmacological inhibition (Fig. 8B andC). Contrary to other metals, [Pt(O,O'-acac)(γ-acac)(DMS)] does not interact directly with channels but it activates intracellular signalling leading to the control of some Ca 2+ channels. We wanted to investigate if the effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on Ca 2+

homeostasis also had an impact on the cell ability to handle the complex changes during

A c c e p t e d M a n u s c r i p t 4 high

 4 cytotoxicity in cisplatin resistant MCF-7 breast cancer cells. Differently from cisplatin,

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

 6 following buffer (mM): 20 Tris-HCl, pH 8, containing 100 NaCl, 2 EDTA, 2 Na 3 VO 4 , 0.2% Nonidet P-40 and 10% glycerol, supplemented with a cocktail of protease inhibitors.

  free radical produced in mitochondria, was measured using the cellpermeable probe MitoSOX Red. The reduced probe is taken up by cells and in the presence of O 2 .-is oxidized and becomes fluorescent, emitting a red fluorescence after 488 nm laser excitation. After treatment with the drug, cells were incubated for 10 minutes with MitoSOX Red reagent 5 μM in 0.5 ml of Hank's Balanced Salt Solution with calcium and magnesium (CaCl 2 140 mg/L, MgCl 2 -6H 2 O 10 mg/L and MgSO 4 -7H 2 O 10 mg/L) at 37 °C. After washing twice, adhered cells were observed (maximum excitation/emission of MitoSOX reagent: 510/580nm) and images and values of fluorescence were obtained by fluorescence microscopy as mean fluorescence intensities (MFI/cell ).

A c c e p t e d M a n u

  s c r i p t antibodies, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The inhibitors of NADPH oxidase, diphenyleneiodonium (DPI) and apocynin, nitroblue tetrazolium (NBT), MitoSOX Red and all the inorganic salts were obtained from Sigma (Milan, Italy). MCF-7 cell line was cultured in DMEM (Celbio, Pero, Milan, Italy) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells were grown to 70% confluence and then treated with [Pt(O,O'-acac)(γ-acac)(DMS)] at various concentrations and for different incubation periods.

[

  Pt(O,O'-acac)(γ-acac)(DMS)], induces apoptosis in MCF-7 breast cancer cell line, relatively insensitive to cisplatin. In contrast to cisplatin, the cytotoxicity of [Pt(O,O'acac)(γ-acac)(DMS)] correlated with cellular accumulation but not with DNA binding, as it activates the mitochondrial apoptotic pathway [16]. In many cases, an early and pivotal event in apoptosis is a cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) increase. To determine whether this was an early event during [Pt(O,O'-acac)(γ-acac)(DMS)], induced apoptosis, we first examined whether intracellular [Ca 2+ ] i levels increased in MCF-7 cells after [Pt(O,O'-acac)(γ-acac)(DMS)] exposure, using fura-2-AM in a spectrofluorometric assay. In MCF-7 cells, the basal level of [Ca 2+ ] i was 102 ± 7 nM (mean ± S.D.; n=30). MCF-7 cells were incubated for different times (1 -20 min) with crescent concentrations of [Pt(O,O'acac)(γ-acac)(DMS)]. The treatment with [Pt(O,O'-acac)(γ-acac)(DMS)] increases [Ca 2+ ] i in dose-dependent manner (Fig. 1A); between 0.5 and 10 µM, the increase in [Ca 2+ ] i ranged from 145 ± 10 nM to 250 ± 24 nM. Concentrations above 10 µM were not tested because they are clinically irrelevant. In MCF-7 cells, the increase in [Ca 2+ ] i was restricted to [Pt(O,O'-acac)(γ-acac)(DMS)] since cisplatin and carboplatin (up to a concentration of 100 µM), did not elevate [Ca 2+ ] i (Fig. 1B and C). While transient elevations of [Ca 2+ ] i are important signals within cells, prolonged elevations of [Ca 2+ ] i could be lethal. A [Ca 2+ ] i overload may activate cell death via apoptosis [4]. Theoretically, there are at least two possibilities for altering [Ca 2+ ] i homeostasis in MCF-7 cells by [Pt(O,O'-acac)(γacac)(DMS)]. Elevation of the [Ca 2+ ] i can result: a) from Ca 2+ -influx from the extracellular A c c e p t e d M a n u s c r i p t space and/or from Ca 2+ -release from intracellular stores; b) from Calcium-ATPases (PMCA/SERCA/SPCA) and/or the Na + -Ca 2+ -Exchanger (NCX) activity inhibition.

  Fig. 2A shows the fluorescence quenching by Mn 2+ influx: the membrane permeability was significantly decrease by [Pt(O,O'-acac)(γ-acac)(DMS)] in a dose-dependent manner (Fig 2B). Mn 2+

Figure

  Figure3Apresents data on ouabain-insensitive ATP hydrolysis in the presence and absence (obtained adding 800 µM EGTA) of Ca 2+ . MCF-7 cells clearly catalysed Ca 2+sensitive ATP hydrolysis. Approximately 35% of ouabain-insensitive ATPase activity (~ 7.1 ± 0.3 μmol Pi/h/mg/protein as previously shown,[START_REF] Muscella | Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells[END_REF]) was Ca 2+ dependent. This activity consisted of the following activities: PMCA (1.12 ± 0.11 μmol Pi/h/mg/protein), SERCA (0.63 ± 0.11 μmol Pi/h/mg/protein and SPCA (0.16 ± 0.13 μmol Pi/h/mg/protein) (all the activities were mean ± S.D. of five independent experiments, Fig.3A). [Pt(O,O'-acac)(γacac)(DMS)] (0.1-10 µM) had no effects on SERCA activity (Fig.3B), nor on that of the SPCA (data not shown). Conversely, [Pt(O,O'-acac)(γ-acac)(DMS)] produced a significant decrease in PMCA activity in a dose-and time-dependent manner (ANOVA: P<0.0001; Fig.3B and C). The maximal effect, in sub-confluent conditions, was obtained after 10

  2+ ] i transients were then examined in the presence of increasing [Pt(O,O'acac)(γ-acac)(DMS)] concentrations (0.5 -10 µM). As shown in Fig. 4A, in a first phase lasting about 1 min, 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] significantly decreased the peak of [Ca 2+ ] i (from 509 ± 21 nM to 440 ± 18 nM, n = 7; p<0.001); afterwards, it increased, in a dose-dependent manner, both the rate of the Ca 2+ decline and the post-stimulatory [Ca 2+ ] i level, calculated after 3 min from ATP administration. In order to quantify the effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on such ATP-evoked Ca 2+ transient, the variations in Ca 2+profile were also evaluated both as b/a ratio and as Ca 2+ rate of decline. Precisely, we expressed the [Ca 2+ ] i observed after 2 min from stimulation (b), as a fraction of [Ca 2+ ] i peak (a) (Fig.4Bin set), and the b/a ratio was calculated for the different concentrations (0.5-10 µM). Figure4Bshows that under control conditions (i.e., exposure to ATP alone) (b) was 30% of peak [Ca 2+ ] i . When applied together with ATP, [Pt(O,O'-acac)(γacac)(DMS)] increased, in a dose dependent manner, the b/a ratio (Fig.4B). The effect provoked by the channel blocker La 3+ (100 µM), was very different from the effect of 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] (Fig.4B and C), inasmuch as, while they both were able to strongly reduce the Ca 2+ entry, La 3+ , differently from [Pt(O,O'-acac)(γ-acac)(DMS)], had no effect on PMCA activity. This suggests that the increase in the b/a ratio caused by [Pt(O,O'-acac)(γ-acac)(DMS)] is mainly due to inhibition of the pump rather than the decrease of plasma membrane permeability. Obviously, [Pt(O,O'-acac)(γ-acac)(DMS)]treated cells had a slower rate of PMCA-mediated free intracellular Ca 2+ efflux. The rate of decline, assayed in a time interval of 1 min after the peak, could be a relative measure of PMCA-mediated free intracellular Ca 2+ efflux. The rates of decline in [Ca 2+ ] i obtained with A c c e p t e d M a n u s c r i p t 11 the various [Pt(O,O'-acac)(γ-acac)(DMS)] concentrations were plotted against the relative [Ca 2+] i peak values, for comparisons of PMCA-mediated Ca 2+ efflux rates (Fig.4D). In order to discriminate between the effects on plasma membrane channels and PMCA inhibition provoked by [Pt(O,O'-acac)(γ-acac)(DMS)] and those due to the channel closure only, the rate of decline in [Ca 2+ ] i obtained with 100 µM La 3+ was also evaluated; as shown in Fig.4D, La 3+ provoked a rate of Ca 2+ decline and a Ca 2+ peak value significantly different from those provoked by [Pt(O,O'-acac)(γ-acac)(DMS)]. Since PMCA activity is known to be Ca 2+ -dependent, MCF-7 cells were treated or not, for the indicated times, with 100 µM ATP and then Ca 2+ -ATPase activity was assayed. ATP stimulated PMCA activity by about 50% after 2.5 min incubation and [Pt(O,O'-acac)(γ-acac)(DMS)] significantly decreased the activity of the ATP-stimulated PMCA (Fig.3D). The origin of the ATPinduced Ca 2+ sustained phase was studied using a Ca 2+ -free medium obtained substituting 2 mM MgCl 2 for 2 mM CaCl 2 in the standard experimental medium previously passed through a Chelex 100 column. In these experimental conditions, the effect of ATP on peak height was much less pronounced with respect to that obtained in the presence of Ca 2+containing solution ([Ca 2+ ] i peak decreased from 509 ± 21 nM to 286 ± 12 nM (p< 0.001).

FigureM a n u s c r i p t 12 CaCl 2

 122 Figure 5A shows an example of a [Ca 2+ ] i transient evoked by bath application of ATP (100 µM) in Ca 2+ -free external solution. The ATP-induced [Ca 2+ ] i transient was characterized by a rapid and pronounced initial rise that then declined completely to the pre-stimulatory level. When ATP was applied simultaneously with [Pt(O,O'-acac)(γ-acac)(DMS)], the [Ca 2+] i transient appeared significantly wide and decelerated (Fig.5B). In MCF-7 cells, the pathway that links purinoceptors to intracellular Ca 2+ release involves the InsP3 signalling cascade[START_REF] Rossi | Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol[END_REF]; such Ca 2+ release then results in external Ca 2+ entry. Thus, we investigated whether the InsP3-dependent Ca 2+ release was involved in the [Pt(O,O'-acac)(γacac)(DMS)]-induced [Ca 2+ ] i increase by using 2-aminoethoxydiphenyl borate (2-APB), a membrane-permeant InsP3R blocker[START_REF] Maruyama | 2APB, 2aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins 1,4,5 P3induced Ca 2+ release[END_REF]. Cells incubated with 20 µM 2-APB 10 min before the stimulation with 100 µM ATP, in Ca 2+ -free conditions, led to a significant reduction (by 62 ± 6%, n=5, P <0.05) of the amplitude of the ATP-induced [Ca 2+ ] i transient (Fig.5A and C), confirming prior observations showing that the ATP induced [Ca 2+ ] i transient is due to Ca 2+ release through InsP3Rs[START_REF] Peppiatt | 2-Aminoethoxydiphenyl borate 2-APB antagonises inositol 1,4,5trisphosphate-induced calcium release, inhibits calcium pumps and has a usedependent and slowly reversible action on store-operated calcium entry channels[END_REF]. Afterwards, cells were incubated with 20 µM 2-APB for 10 min and then stimulated with ATP together with [Pt(O,O'-acac)(γacac)(DMS)]: again, a significant inhibition of the [Ca 2+ ] i transient (by 68 ± 5%, n=5, P <0.05) was obtained (Fig. 5B and C), similar to that obtained in the absence of [Pt(O,O'acac)(γ-acac)(DMS)], therefore suggesting that [Pt(O,O'-acac)(γ-acac)(DMS)] caused an InsP3-independent Ca 2+ increase. Extracellular Ca 2+ entry was generated by adding 2 mM

  exchanger in the [Pt(O,O'-acac)(γacac)(DMS)]-induced [Ca 2+ ] i changes, we studied the effects of [Pt(O,O'-acac)(γ-A c c e p t e d M a n u s c r i p t 13 acac)(DMS)] on Ca 2+ influx using buffers with Na + concentrations being 0 and 140 mM.First, the effects of ATP were dependent on Na + concentration, since the replacement of extracellular Na + with choline, significantly increased the early peak rise in [Ca 2+ ] i from 509 ± 21 nM to 585 ± 30 nM (ANOVA; p<0.001; n = 6, Fig.7A). Secondly, cells co-stimulated with ATP and [Pt(O,O'-acac)(γ-acac)(DMS)], displayed a lower [Ca 2+ ] i peak in the presence of 140 mM Na + (450 ± 15 nM, n = 6) than in the absence (525 ± 21 nM, n=6, Fig.7B).

7 (

 7 2+ entryProtein kinase C (PKC) is known to regulate Ca 2+ homeostasis through negative feedback mechanisms[START_REF] Mchugh | Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain[END_REF]. Inasmuch as activated PKCs translocate from the cytosol to the cellular membranes, we analysed, by Western blots, the distribution of PKCs expressed by MCF-PKC-α, -δ, -ε, -ι and -δ[START_REF] Muscella | PKC-zeta is required for angiotensin II-induced activation of ERK and synthesis of C-FOS in MCF-7 cells[END_REF]) in cells treated with 10 μM Pt(O,O'-acac)(γ-acac)(DMS)] for different incubation times (0.5, 1, 5, and 10 min). As shown in Fig.8A, a cytosol-tomembrane translocations of PKC-α, PKC-δ and PKC-ε were observed. The involvement of translocating PKCs in the effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on extracellular Ca 2+ entry was investigated by using the PKC inhibitors GF109203X (able to inhibit all isoforms) and Gö6976 (and inhibitor of conventional, calcium-dependent PKC-α; note that PKC-α is the only conventional PKC expressed by MCF-7 cells)[START_REF] Qatsha | Gö 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro[END_REF]. Pre-incubation of cells with GF109203X (0.1 and 1 µM for 15 min) (data not shown), or with Gö6976 (1 µM) for 15 min, reversed the effects of 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] on extracellular Ca 2+ entry (Fig.8B), suggesting that such effects may be mediated by PKC-α. The role of PKCα in modulating plasma membrane permeability can also be seen in experiments of Mn 2+ quenching, where the use of Gö6976 in cells treated with both [Pt(O,O'-acac)(γacac)(DMS)] and ATP significantly increased the Mn 2+ influx (from 5.77 ± 0.22 to 14.70 ± 0.46  360 /min, p<0.05; n=6), back to values obtained in cells treated with ATP only (Fig.8C). Moreover, Gö6976 reversed the effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on plasma membrane permeability in untreated cells (from 2.19 ± 0.13 to 6.52 ± 0.26  360 /min, p<0.05; n=6, Fig.8B). These results suggest that [Pt(O,O'-acac)(γ-acac)(DMS)]-activated PKC-α changed the plasmalemma Ca 2+ permeability interfering with some Ca 2+ plasma membrane channels.

[

  Pt(O,O'-acac)(γ-acac)(DMS)] on (a) intracellular [Ca 2+ ] i levels, (b) membrane permeability, (c) PMCA activity and (d) ROS generation)) were performed and the results shown in TableI. The results show that the effect of [Pt(O,O'-acac)(γ-acac)(DMS)] are not significantly different in the three tumor cell lines while, on the contrary, such effects are much less evident, although significant compared with controls, in normal MCF-10A cells.

A c c e p t e d M a n u s c r i p t 15 therapeutic

 15 agent because it induces apoptosis in MCF-7 breast cancer cell line, relatively insensitive to cisplatin[START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF]. We have demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)]

[

  Pt(O,O'-acac)(γ-acac)(DMS)]-induced apoptosis. The increase of [Ca 2+ ] i provoked by [Pt(O,O'-acac)(γ-acac)(DMS)] does not depend on the external Ca 2+ concentration (Figs. 5

  These changes are clearly due mainly to the combined effects of [Pt(O,O'-acac)(γacac)(DMS)] on permeability and PMCA activity. Since the effects of inhibition of PMCA occurred slowly (Fig. 3C), compared to changes in permeability (Figs. 2, 6E and 8C),A c c e p t e d M a n u s c r i p t 16 these latter changes became more important in the second phase of the transient, causing an overall rise in Ca 2+ level.

  caspases became significantly activated by [Pt(O,O'-acac)(γ-acac)(DMS)] occurred much later compared to the inactivation of PMCA, which is already evident after 30-sec exposure to [Pt(O,O'-acac)(γ-acac)(DMS)] (Fig. 3C). Consequently, the anti-neoplastic potential of [Pt(O,O'-acac)(γ-acac)(DMS)] might also be based on its ability to produce changes in Ca 2+ homeostasis, like others pump inhibitors. We have tried to estimate the possible effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on the membrane permeability to Ca 2+ . A raise in the [Ca 2+

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

CaFig. 1 .

 1 Fig. 1. Effects of Pt(II) compounds on [Ca 2+ ] i . Intracellular Ca 2+ concentration was quantified by fluorescence after loading the cells with the calcium indicator fura-2-AM. Time-course of the effects of various concentrations of (A) [Pt(O,O'-acac)(γ-acac)(DMS)], (B) Cisplatin and (C) 100 µM Carboplatin on [Ca 2+ ] i in MCF-7 cells. Results shown are representative of six separate experiments. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.

Fig. 2 .

 2 Fig. 2. Comparison of the permeabilities of the plasma membrane to Mn 2+ in absence and in presence of [Pt(O,O'-acac)(γ-acac)(DMS)]. Mn 2+ entry was measured by the quenching of fluorescence with an excitation wavelength of 360 nm, and an emission wavelength of 510 nm. The curves represent fluorescence relative to total quenching attained by the addition of digitonin to saturate fura-2 with Mn 2+ . (A) Cells were kept in nominally Ca 2+ -free medium, as 2 mM Mn 2+ was applied to the medium (arrow), it entered cells, with Mn 2+ entry much slower in [Pt(O,O'-acac)(γacac)(DMS)]-treated cells. Mn 2+ entry was blocked by the impermeant cation La 3+ (100 µM), and [Pt(O,O'-acac)(γ-acac)(DMS)] had, on Mn 2+ influx, effects similar to those obtained by La 3+ . Each tracing is representative of five separate experiments. (B) The permeability of MCF-7 cell plasma membrane was significantly decreased by [Pt(O,O'acac)(γ-acac)(DMS)] in a dose-dependent manner. Mn 2+ entry in cells treated with 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] was the same in the presence or in the absence of La 3+ . Average data are from experiments shown in A (n=5). Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Pt(acac) 2 DMS stands for Pt(O,O'-acac)(γ-acac)DMS.

Fig. 3 .

 3 Fig. 3. Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on Ca 2+ -ATPases activities.(A) ATPase activity was assessed in the presence of 0.5 mM ouabain, as inorganic phosphate (Pi) liberated from ATP during a 10 min incubation at 37°C. Ca 2+ refers to incubation in the presence of 80 µM CaCl 2 , while EGTA refers to incubation with 800 µM EGTA. Ca 2+ -sensitive ATPase activity is the difference between the two conditions. In order to identify the relative contributions of the PMCA, SPCA and SERCA, Ca 2+ -sensitive ATPase activity was assessed in the presence of the SERCA inhibitor thapsigargin or/and in presence of eosin and orthovanadate, as detailed in "Material and Method". The Ca 2+ -ATPase activities were expressed as µM of Pi liberated per mg of protein per hour, based

Fig. 4 .

 4 Fig. 4. Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] and La 3+ on the ATP-induced Ca 2+ transients. (A) MCF-7 cells, loaded with fura-2-AM, were stimulated with 100 µM ATP in the presence of extracellular Ca 2+ . The effects of ATP on [Ca 2+ ] i were also assessed in the presence of increasing concentrations of [Pt(O,O'-acac)(γ-acac)(DMS)]. Each tracing is representative of seven separate experiments. (B) Inhibitory action of [Pt(O,O'-acac)(γ-acac)(DMS)] on ATP-induced [Ca 2+ ] i transients was quantified by the b/a ratio, in which a represents the maximum peak amplitude (see B, inset) and b is the amplitude of the sustained component evaluated after 2 min from ATP administration. The b/a ratio was calculated for [Pt(O,O'-acac)(γ-acac)(DMS)], and for 100 µM La 3+ , and then plotted for the various concentrations of [Pt(O,O'-acac)(γ-acac)(DMS)]. Each bar represents mean ± S.D. of seven separate experiments. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (C) Effect of lanthanum on ATP-mediated [Ca 2+ ] i increases in MCF-7 cells; cells were incubated with 100 µM ATP or pre-treated with 100 µM La 3+ for 30 sec prior stimulation with 100 µM ATP. Each tracing is representative of seven separate experiments. (D) The rate of decline of [Ca 2+ ] i transient assayed in a time interval of 1 min after the peak, is a relative measure of PMCA-mediated free intracellular Ca 2+ efflux. The rates of decline in [Ca 2+ ] i obtained with the various [Pt(O,O'-acac)(γ-acac)(DMS)] concentrations and with 100 µM La 3+ were plotted against the relative [Ca 2+ ] i peak values for comparisons of PMCA-mediated Ca 2+ efflux rates in [Pt(O,O'-acac)(γ-acac)(DMS)]-treated and non MCF-7 cells. Pt(acac) 2 DMS stands for Pt(O,O'-acac)(γ-acac)DMS.

A c c e p t e d M a n u s c r i p t 29 or

 29 ATP, cells were exposed to 2 mM Mn 2+ , in the presence or in the absence of [Pt(O,O'acac)(γ-acac)(DMS)]. Each tracing is representative of five separate experiments. (F) Average data are from experiments shown in (E) (n=5). Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.

Fig. 7 .

 7 Fig. 7. Effects of [Pt(O,O'-acac)(γ-acac)(DMS)] on Na + /Ca 2+ -Exchanger activity (A) Ca 2+ influx in cells stimulated with ATP, in buffers with Na + concentrations of 0 and 140 nM, in the presence (B) or not (A) of [Pt(O,O'-acac)(γ-acac)(DMS)]. Results shown are representative of four experiments. (C) [Ca 2+ ] i values obtained from A and B after 30 sec (peak) stimulation with ATP in normal and Na + -free buffers in the presence or not of [Pt(O,O'-acac)(γ-acac)(DMS)]. The data are means ± S.D. of four different experiments run in triplicate. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. Pt(acac) 2 DMS stands for [Pt(O,O'-acac)(γ-acac)DMS].

Fig. 8 .

 8 Fig. 8. The effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on extracellular Ca 2+ entry is due to PKC- activation (A) MCF-7 cells were treated without or with 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] for the indicated times. Cytosol (cyt) and membrane (mem) fractions for translocation studies, were analyzed by western blotting with specific anti-PKCs antibodies. The purity of fractions was tested by immunoblotting with anti β-actin and anti-α subunit of Na + /K + ATPase monoclonal antibodies. The figures are representative of four independent experiments. (B, C) MCF-7 cells, loaded with fura-2-AM, and kept in nominally Ca 2+ -free medium, were stimulated with ATP or ATP plus 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)], with or without 1 µM Gö 6976. (C) Mn 2+ entry was measured by the quenching of fluorescence with an excitation wavelength of 360 nm, and an emission wavelength of 510 nm. The curves represent fluorescence relative to total quenching attained by the addition of digitonin to saturate fura-2 with Mn 2+ . Each tracing is representative of six separate experiments. Arrows indicate time points at which 100 µM ATP, 2 mM CaCl 2 or 2 mM Mn 2+ were added. Pt(acac) 2 DMS stands for [Pt(O,O'-acac)(γ-acac)DMS].

Fig. 9 .

 9 Fig. 9. The effect of [Pt(O,O'-acac)(γ-acac)(DMS)] on PMCA is due to Reactive Oxygen Species (ROS) generation. (A) Cells were treated or not with 10 µM [Pt(O,O'-acac)(γ-acac)(DMS)] for the indicated

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

Figure

  Figure

Figure 4 A

 4 Figure 4

Figure

  Figure

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

  Figure

Figure

  Figure

Acknowledgements

The research was supported by University of Salento (Fondi di Ateneo per la Ricerca di Base, 2009).

A c c e p t e d M a n u s c r i p t 18 especially, in those of the sustained phase of elevated [Ca 2+ ] i (Fig. 4B). Experiments with La 3+ , a Ca 2+ channel blocker that has no effects on PMCA activity, corroborated the idea that the changing in the ATP-provoked [Ca 2+ ] i profile due to [Pt(O,O'-acac)(γ-acac)(DMS)],

were due to PMCA activity inhibition and plasma membrane channels closure (Fig. 4B,C and D). The involvement of these channels, and the importance of PMCA inhibition, was observed in experiments performed with ATP in buffer without Ca 2+ , where [Pt(O,O'acac)(γ-acac)(DMS)] increased slightly, but significantly, the ATP-provoked Ca 2+ transient (Fig. 5A andC). This increase may be due to the inhibition of PMCA. We noticed that the addition of external Ca 2+ causes a significant extracellular Ca 2+ entry that is in part blocked by [Pt(O,O'-acac)(γ-acac)(DMS)] (Figs. 5 and6). [Pt(O,O'-acac)(γ-acac)(DMS)] does not seem to affect the Ca 2+ released from the intracellular stores because no significant effect on Ca 2+ transient provoked by ATP was evident in cells with the InsP3Rs blocked by 2-APB (with the exception of a slight increase in the plateau). This may also be due to the decreased PMCA activity (Fig. 5B andC). Consistently, [Pt(O,O'-acac)(γ-acac)(DMS)] had effect on Ca 2+ peak provoked by ATP in Ca 2+ -free buffers and on the [Ca 2+ ] i returning to control values, a phase that resulted to be slower due to reduced calcium efflux (Fig. 6B andC). Finally, it is important to note that the results we obtained (Tab. I) on the various breast cell lines provide evidence that [Pt(O,O'-acac)(γ-acac)(DMS)] may be a promising new anticancer agent, as it exhibited higher calcium homeostasis perturbation in cancer cell lines than in non-cancerous MCF-10A cells, which also appeared to be less sensitive to the cytotoxic effect of the Pt-compound [START_REF] Muscella | Pt O,O'-acac -acac DMS], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway[END_REF].

In conclusion, we demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] alters the [Ca 2+ ] i and that this change is due both to the PKC-α-mediated closure of some channels and to the inhibition of PMCA. The key points of this model are summarized in Fig. 10. The overall effect of [Pt(O,O'-acac)(γ-acac)(DMS)] is to increase the [Ca 2+ ] i , an effect that is linked to its ability to trigger rapid apoptosis in MCF-7 cells.

Conflict of interest

The authors state no conflict of interest.


Mariëlle J Van Breemen

Saskia M Rombach

Nick Dekker

Ben J Poorthuis

Gabor E Linthorst

Aeilko H Zwinderman

Frank Breunig

Christoph Wanner

Johannes M Aerts
email: j.m.aerts@amc.uva.nl

Carla E Hollak

Reduction of elevated plasma globotriaosylsphingosine in patients with classic Fabry disease following enzyme replacement therapy

Keywords: Fabry disease, globotriaosylsphingosine, lysoGb3, Enzyme replacement therapy (ERT)

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Introduction

The lysosomal storage disorder Fabry disease (OMIM 301500) results from deficient activity of the lysosomal hydrolase α-galactosidase A (EC 3.2.1.22) [START_REF] Brady | Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency[END_REF][START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF]. Since the α-galactosidase A (GLA) gene is located on Xq22.1, hemizygous males suffer from Fabry disease. However, many female heterozygotes also display symptoms despite considerable amounts of circulating residual enzyme that varies due to random X-inactivation [START_REF] Macdermot | Natural history of Fabry disease in affected males and obligate carrier females[END_REF][START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF]. In both males and females different phenotypic manifestations of Fabry disease occur [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF]. The classical manifestations in males involve angiokeratoma, anhidrosis and acroparesthesias at puberty, followed by renal insufficiency, left ventricular hypertrophy (LVH) and cerebrovascular disease in the fourth or fifth decade of life. In females the disease manifestations are usually, but not always, attenuated. A complete lack or severely reduced α-Gal A activity results in classical manifestations of Fabry disease. During the past decades α-Gal A deficient patients with relatively few and isolated symptoms, such as LVH or renal failure at later stages in life, have been described [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF][START_REF] Elleder | Cardiocyte storage and hypertrophy as a sole manifestation of Fabry's disease. Report on a case simulating hypertrophic non-obstructive cardiomyopathy[END_REF][START_REF] Nakao | Fabry disease: detection of undiagnosed hemodialysis patients and identification of a "renal variant" phenotype[END_REF]. These patients are generally referred to as atypical Fabry patients, to distinguish them from those with multi-organ symptoms at a young age. Relatively high residual α-Gal A activity is often noted in these atypical cases [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF]. The precise causes for the remarkable heterogeneous manifestations of Fabry disease are still poorly understood [START_REF] Elleder | Cardiocyte storage and hypertrophy as a sole manifestation of Fabry's disease. Report on a case simulating hypertrophic non-obstructive cardiomyopathy[END_REF][START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry Outcome Survey[END_REF][START_REF] Eng | Fabry disease: baseline medical characteristics of a cohort of 1765 males and females in the Fabry Registry[END_REF][START_REF] Schiffmann | Fabry disease[END_REF]. The nature of mutations in the GLA gene and the corresponding degree of residual α-Gal A activity, undoubtedly influence disease manifestations [START_REF] Saito | Prediction of the clinical phenotype of Fabry disease based on protein sequential and structural information[END_REF][START_REF] Wu | Cardiovascular manifestations of Fabry disease: relationships between left ventricular hypertrophy, disease severity, and alpha-galactosidase A activity[END_REF][START_REF] Branton | Natural history of Fabry renal disease: influence of alpha-galactosidase A activity and genetic mutations on clinical course[END_REF]. There is growing evidence for other modifiers beyond the GLA gene. For example, a modulating role may be envisioned for α-galactosidase B, another α-galactosidase arisen by gene duplication with low activity towards the lipid substrate of α-Gal A [START_REF] Hamers | Characterization of alpha-galactosidase isoenzymes in normal and Fabry human-Chinese Hamster somatic cell hybrids[END_REF][START_REF] Tajima | Use of a modified alpha-N-acetylgalactosaminidase in the development of enzyme replacement therapy for Fabry disease[END_REF]. In addition, polymorphisms c.174G>C of interleukin-6, c.894G>T of endothelial nitric oxide synthase (eNOS), factor V c.1691G>A mutation (factor V Leiden), and the c.13A>G and c.79G<A of protein Z are all significantly associated with the presence of cerebral white matter lesions on brain MRI [START_REF] Altarescu | Effect of genetic modifiers on cerebral lesions in Fabry disease[END_REF]. Two polymorphisms in the NOS3 gene, encoding eNOS, have also been shown to influence hypertrophic cardiomyopathy as measured by left posterior wall thickness of the heart [START_REF] Rohard | Association between polymorphisms of endothelial nitric oxide synthase gene (NOS3) and left posterior wall thickness (LPWT) of the heart in Fabry disease[END_REF].

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endothelial, perithelial, and smooth muscle cells of the vascular system, as well as renal epithelial cells, myocardial cells, and cells of the autonomic nervous system [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF]. The accumulating glycosphingolipids contain terminal α-galactosyl moieties, such as globotriaosylceramide (Gb3; also named ceramidetrihexoside); galabiosylceramide (Gb2); and, to a lesser extent, blood group B, B1, and P 1 antigens [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF][START_REF] Sweeley | Fabry's disease : classification as a sphingolipidosis and partial characerization of a novel glycolipid[END_REF]. The prominent lysosomal storage of Gb3 in cells of the arterial wall is generally thought to cause a systemic vasculopathy with progressive renal insufficiency, cardiac involvement and CNS pathology in Fabry patients [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF][START_REF] Schiffmann | Fabry disease[END_REF][START_REF] Rombach | Vasculopathy in patients with Fabry disease: current controversies and research directions[END_REF]. Symptomatic Fabry hemizygotes, but not most heterozygotes, show increased plasma Gb3 levels [START_REF] Desnick | α-Galactosidase A deficiency: Fabry disease[END_REF][START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF]. Mass spectrometry-based and HPLCbased procedures have become available allowing accurate quantification of the globoside [START_REF] Mills | Monitoring the clinical and biochemical response to enzyme replacement therapy in three children with Fabry disease[END_REF][START_REF] Fauler | Rapid determination of urinary globotriaosylceramide isoform profiles by electrospray ionization mass spectrometry using stearoyl-d35-globotriaosylceramide as internal standard[END_REF][START_REF] Fuller | Urinary lipid profiling for the identification of Fabry hemizygotes and heterozygotes[END_REF][START_REF] Auray-Blais | Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease[END_REF][START_REF] Groener | HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma[END_REF].

Plasma Gb3 has been found to poorly reflect Fabry disease manifestations and therapeutic outcome [START_REF] Vedder | The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels[END_REF][START_REF] Whitfield | Monitoring enzyme replacement therapy in Fabry disease--role of urine globotriaosylceramide[END_REF][START_REF] Young | Is globotriaosylceramide a useful biomarker in Fabry disease?[END_REF][START_REF] Bekri | The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature[END_REF]. Recently, another striking lipid abnormality has been noted in patients with classical Fabry disease manifestations [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. Globotriaosylsphingosine is deacylated globotriaosylceramide (designated as lysoGb3). The relative increase in the plasma lysoGb3 exceeds that of Gb3 by more than an order of magnitude. High nanomolar lysoGb3 concentrations occur in plasma samples from symptomatic Fabry males with classical disease manifestations. Also in symptomatic Fabry females, clearly increased levels of lysoGb3 occur whilst concomitantly Gb3 concentrations are in the normal range [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF].

Two different recombinant α-Gal A preparations are in use for the treatment of Fabry disease [START_REF] Eng | Safety and efficacy of recombinant human alpha-galactosidase A--replacement therapy in Fabry's disease[END_REF][START_REF] Schiffmann | Enzyme replacement therapy in Fabry disease: a randomized controlled trial[END_REF].

One enzyme is produced using Chinese hamster ovary cells with classic recombinant technology (agalasidase beta, Fabrazyme) and the other enzyme is produced using cultured human skin fibroblasts with an activated promoter of the α-gal A gene (agalasidase alfa, Replagal). Both recombinant enzymes are comparable in properties and differ only slightly in glycan composition [START_REF] Blom | Recombinant enzyme therapy for Fabry disease: absence of editing of human alpha galactosidase A mRNA[END_REF]. The two enzyme preparations have independently been examined in clinical investigations and are both registered in Europe for the treatment of Fabry patients. Although both enzyme replacement therapies (ERTs) were found to result in the desired clearance of Gb3 from the endothelium, the clinical effects are not as robust as anticipated based on the impact of ERT in type 1 Gaucher disease [START_REF] De Fost | Superior effects of high-dose enzyme replacement therapy in type 1 Gaucher disease on bone marrow involvement and chitotriosidase levels: a 2-center retrospective analysis[END_REF]. In some

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patients stabilization of renal function and improvement in cardiac hypertrophy occurs upon therapy, but a considerable number experience progressive complications [START_REF] Vedder | Treatment of Fabry disease: outcome of a comparative trial with agalsidase alfa or beta at a dose of 0.2 mg/kg[END_REF]. From a recent analysis of the Fabry Outcome Survey observational database, it was concluded by comparison with historical natural history data for patients who were not treated with ERT, that long-term treatment with agalsidase alfa leads to substantial and sustained clinical benefits [START_REF] Mehta | Enzyme replacement therapy with agalsidase alfa in patients with Fabry's disease: an analysis of registry data[END_REF]. However, discerning the specific effect of ERT from that of other interventions, for example the use of renin-angiotensin inhibitors, remains a challenge [START_REF] Knebelmann | Enzyme therapy for Fabry's disease: registered for success?[END_REF]. It has been documented that a large portion of Fabry hemizygotes develops antibodies against the recombinant enzymes during ERT [START_REF] Linthorst | Enzyme therapy for Fabry disease: neutralizing antibodies toward agalsidase alpha and beta[END_REF][START_REF] Ohashi | Influence of antibody formation on reduction of globotriaosylceramide (GL-3) in urine from Fabry patients during agalsidase beta therapy[END_REF][START_REF] Vedder | Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3[END_REF]. In addition, it has been hypothesized that the formation of antibodies possibly affects outcome [START_REF] Bénichou | A retrospective analysis of the potential impact of IgG antibodies to agalsidase beta on efficacy during enzyme replacement therapy for Fabry disease[END_REF][START_REF] Hollak | Immune response to enzyme replacement therapy in Fabry disease: impact on clinical outcome?[END_REF].

Since monitoring of efficacy of ERT is hampered by the heterogeneous manifestations of Fabry disease as well as the irreversible nature of many symptoms, we investigated the impact of therapy on plasma lysoGb3 levels in patients with classical Fabry disease manifestations. Plasma lysoGb3 can be accurately quantified and offers an objective parameter to assess whether the underlying biochemical deficiency in Fabry patients is corrected by therapy. In a previous study, we investigated outcomes of Gb3 levels in relation to different ERTs [START_REF] Vedder | Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3[END_REF]. We here report the outcome of the investigation in which plasma Gb3 as well as lysoGb3 levels were monitored in patients receiving either agalasidase alfa (0.2 mg) or agalasidase beta (0.2 or 1.0 mg) per kg bodyweight every two weeks. The impact of formation of neutralizing anti-(α-Gal A) antibodies on plasma lipid corrections is also described.

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Materials and Methods

Classical Fabry disease patients and controls

Control subjects for Gb3 analysis consisted of 10 males and 15 females. Plasma lysoGb3 levels were measured in 9 male and 9 female healthy volunteers (age range 5-77 year). Forty-three Fabry patients (22 males and 21 females; age range 18-71 year) from the Academic Medical Center (AMC) and from the Universitätsklinikum Würzburg (UKW) who presented with classical Fabry disease and received a minimum of 12 months of ERT were eligible for this study. This is a subset of patients described in an earlier study [START_REF] Vedder | Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3[END_REF] excluding patients with an atypical variant of disease and with incomplete sample collection. All patients were treatment naïve prior to enrolment. Classical Fabry disease is defined by the presence of characteristic symptoms and elevated plasma lysoGb3 and/or Gb3 in hemizygotes, and in case of heterozygotes, having a mutation known to be associated with a classical phenotype (through literature or a male family member with classical symptoms). Patients received three different ERT treatments: infusions every two weeks with agalsidase alfa at 0.2 mg/kg (n=14; 7 males and 7 females), agalsidase beta at 0.2 mg/kg (n=11; 6 males and 5 females), or agalsidase beta at 1.0 mg/kg (n=18; 9 males and 9 females). All patients had been diagnosed by means of a reduced α-Gal A activity in leukocytes (males) and/or a mutation in the α-Gal A gene (females) [START_REF] Vedder | Treatment of Fabry disease: outcome of a comparative trial with agalsidase alfa or beta at a dose of 0.2 mg/kg[END_REF]. None of the investigated male Fabry patients showed a residual enzyme activity exceeding 5% of the normal mean.

Stored blood samples from Fabry disease patients were analyzed. Informed consent and approval of the institutional review board had been obtained as part of a previous study [START_REF] Vedder | Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3[END_REF].

Antibodies

Serum samples were evaluated for the presence of anti-(α-Gal A) antibodies as previously described [START_REF] Linthorst | Enzyme therapy for Fabry disease: neutralizing antibodies toward agalsidase alpha and beta[END_REF]. To assess neutralizing activity, different volumes of serum (0.1-10 µl of serum, corresponding to a dilution of 1/150-1/1.5) were incubated with a standard amount of recombinant α-galactosidase A

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(agalsidase beta, 2.1 ng). Enzyme activity was determined after 10 minutes of incubation at room temperature. The serum dilution that resulted in 50% reduction of the enzyme activity was recorded (IC50). Assuming a linear relationship, the amount of enzyme that would be inhibited by 1 µl of serum was calculated.

Plasma Gb3 and lyso-Gb3

Plasma Gb3 and lysoGb3 were determined at t=0, t=3 and t=12 months of ERT. If available, lipid concentrations were also measured in plasma specimens obtained after 6 and 9 months of treatment.

Quantitation of Gb3 in plasma samples was performed as described previously [START_REF] Groener | HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma[END_REF]. Quantitative measurements of plasma lysoGb3 were performed as described with minor modifications [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF][START_REF] Rombach | Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease[END_REF].

Briefly, 100 µl of plasma was extracted with 600 µl of chloroform/methanol 1/2 (vol/vol). The extract was centrifuged for 10 min. at 14,000×g and the pellet was discarded. To the supernatant, 500 µl chloroform/MQ-H 2 O 1/1.5 (vol/vol) was added, mixed, and centrifuged for 2 min. at 14,000×g to separate the phases. The upper phase was collected, and the lower chloroform phase was re-extracted with 500 µl of methanol/ MQ-H 2 O 1/1 (vol/vol) to quantitatively extract all lysoGb3. The combined upper phases were dried under nitrogen flow, taken up in 500 µl MQ-H 2 O, and extracted twice with 500 µl of water-saturated 1-butanol. LysoGb3 was recovered from the butanol phase with an overall recovery of >90%. The butanol phase was dried, dissolved in 120 µl (hemizygotes) or 90 µl (heterozygotes) of freshly prepared 0.1 M NaOH in methanol and incubated at 37°C for 1 hour. Of this solution, 50 µl was derivatized with 25 µl of o-phtaldialdehyde (OPA) reagent (5 mg of OPA, 100 µl of ethanol, 5 µl of 2-mercaptoethanol, and 10 ml of 3% boric acid, pH 9.0). The OPA-derivatized lyso-Gb3 was separated by HPLC and quantified by fluorescence detection as described previously [START_REF] Groener | HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma[END_REF].

All plasma samples were extracted in duplicate. Quantification was performed by addition of lysoGb3 (Sigma-Aldrich) to normal plasma at concentrations ranging from 0 to 1 mM. The limit of detection of plasma lysoGb3 is 3 nM.

Statistical methods

Results are given as median and range. Differences in lipid levels between groups were assessed by the Mann-Whitney U-test (2 groups) and the Kruskal-Wallis test (>2 groups). To compare lipid levels between patients before and after therapy, data were analyzed using the Wilcoxon matched pairs test (2 time points). To compare lipid levels between patients before and after one year of therapy, data were analyzed using repeated measures analysis of variance (>2 time points). For this analysis lipid data of individuals were expressed as percentage of t=0 (pre-treatment). Correlations were tested by the rank correlation test (Spearman coefficient, ρ). Results were considered to be statistically significant when two-tailed P-values were <0.05.

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Results

Pre-treatment plasma concentrations of lysoGb3 and Gb3.

Patients with classical manifestations of Fabry disease receiving ERT for a minimum period of one year were analyzed on plasma concentrations of lysoGb3 and Gb3. All patients (22 males and 21

females) showed increased plasma lysoGb3 levels prior to ERT (Fig. 1). One female patient formed an exception, having a very low lysoGb3 level. All males showed elevated plasma Gb3, but only in 3 females the concentration of Gb3 was above the normal range (Fig. 1).

Effect of different ERT regimens on plasma lipids in hemizygotes.

Fig. 2 shows the effect of ERT on lysoGb3 and Gb3 levels for hemizygotes. Plasma lysoGb3 and Gb3 concentrations of male patients were not significantly different at t=0 in any of the 3 treatment groups (P=0.8175 and P=0.1223, respectively). Already after 3 months of ERT, independent of the treatment regimen, a significant reduction (P=0.0313) in plasma lysoGb3 concentration was reached. Thereafter lysoGb3 levels did not decrease further up to 12 months of treatment (P-values for each treatment regimen >0.05; see also Fig. 2, Table 1). Reductions in plasma Gb3 by ERT were noted within 3 months of ERT (P-values for each treatment regimen <0.01). Comparison of the changes in plasma lysoGb3 (or Gb3) concentration induced by the 3 different treatments revealed no clear difference (see Fig. 2). The use of repeated measures analysis of variance revealed that the reduction in plasma lysoGb3 was significantly larger for patients treated with agalsidase beta at 1.0 mg/kg compared with patients treated with agalsidase alfa (P=0.003) and beta (P=0.046) at a dose of 0.2 mg/kg.

Formation of anti-(α-Gal A) antibodies and impact on plasma lipid reduction.

We examined the influence of the treatment regimens on formation of neutralizing antibodies. Table 2 shows that 16 of the 22 hemizygotes had detectable antibodies. The lowest percentage of antibody

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ACCEPTED MANUSCRIPT 10 formation (43%) was noted for patients receiving agalsidase alfa. The dose of agalsidase beta did not seem to strongly determine the chance on antibody formation in hemizygotes (0.2 mg/kg: 83%, 1.0 mg/kg: 89%). None of the female patients developed anti-(α-Gal A) antibodies.

The impact of the presence of neutralizing antibodies on ERT-induced reductions in plasma lysoGb3 and Gb3 was examined. Plasma lysoGb3 and Gb3 levels at t=0 were comparable in hemizygotes, whether developing antibodies during ERT or not (P=0.0972 and P=0.3568, respectively). Plasma lysoGb3 concentrations tended to be higher in hemizygotes with antibodies when treated for 12 months with agalsidase alfa or beta at a dose of 0.2 mg/kg, compared with hemizygotes without antibodies 1 (Fig. 3). This was not the case for patients treated for 12 months with agalsidase beta at a dose of 1.0 mg/kg 1 (Fig. 3). The same picture emerges when analyzing lipid data of individuals expressed as percentage of t=0 (pre-treatment) using repeated measures analysis of variance: the reduction in plasma lysoGb3 is significantly less for patients with antibodies receiving a dose of 0.2 mg/kg agalsidase alfa (P=0.008) compared to those without antibodies. The effect was less for patients with antibodies receiving a dose of 0.2 mg/kg agalsidase beta (P=0.192). Of note, this analysis indicated that the plasma lysoGb3 reduction in patients receiving a dose of 0.2 mg/kg agalsidase alfa was just not significantly different from that in patients receiving a dose of 0.2 mg/kg agalsidase beta (P=0.082). The lysoGb3 reduction in patients receiving a dose of 1.0 mg/kg agalsidase beta was comparable to that in patients without antibodies (P=0.497).

Effect of different ERT regimens on plasma lipids in heterozygotes.

Finally, we investigated the response of plasma lipid concentrations in heterozygotes to different treatment regimens. Plasma lysoGb3 and Gb3 concentrations of female patients were not significantly different at t=0 in any of the 3 treatment groups (P=0.5398 and P=0.4138, respectively; see also Table 3). During ERT plasma Gb3 concentrations hardly changed, which is not surprising given the fact that these are mostly within the normal range (upper limit of normal range 3.18 µM). In female patients, plasma lysoGb3 concentrations at t=0 were rather low, compared with males. The range of plasma

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lysoGb3 levels below the detection limit at start of ERT whereas another heterozygote, also receiving 1.0 mg/kg agalsidase beta, showed a relatively high lysoGb3 (143 nM). Thus, a direct comparison of the effect of different treatments on plasma lysoGb3 in female patients is difficult given the heterogeneity among the studied patients; however most patients (n=17) showed a reduction with ERT. A minority (n=4) showed stabilization at low level with ERT. None of the female patients showed an increase in plasma lysoGb3 with ERT.

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Discussion

ERT of Fabry disease is extremely costly and demonstration of its clinical efficacy is difficult.

Reductions in the storage lipid Gb3 in skin, kidney and cardiac biopsies have been documented earlier [START_REF] Eng | Safety and efficacy of recombinant human alpha-galactosidase A--replacement therapy in Fabry's disease[END_REF], substantiating the ability of recombinant α-Gal A preparations to effectively supplement cells of Fabry patients with degradative capacity. Unfortunately, plasma Gb3 concentration has not been found to be a useful surrogate marker and the use of sequential plasma Gb3 measurements for monitoring efficacy of ERT has not been recommended [START_REF] Young | Is globotriaosylceramide a useful biomarker in Fabry disease?[END_REF][START_REF] Bekri | The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature[END_REF]. Recently it has become clear that plasma lysoGb3 elevation is a characteristic feature of patients with classical manifestations of Fabry disease [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. The increase in the plasma concentration of this lipid is most striking in hemizygotes. The relative extent of the increase in plasma concentration of lysoGb3 in Fabry patients is far larger than that of Gb3. The aim of our investigation was to establish whether plasma lysoGb3 concentrations of Fabry patients are changed by ERT, and may have potential as a surrogate marker to monitor therapeutic intervention. For this purpose, patients with classical manifestations of Fabry disease were examined that were treated by two-weekly intravenous administration of agalsidase alfa at 0.2 mg/kg, agalsidase beta at 0.2 mg/kg, or agalsidase beta at 1.0 mg/kg.

In all investigated male patients plasma lysoGb3 was high prior to ERT (102-397 nM; control subjects: <3 nM). The 3 different treatment regimens all resulted in marked reductions in lysoGb3 within 3 months, after which the levels tended to stabilize. No clear differences were noted between the different treatments. Examination of the occurrence of anti-(α-Gal A) antibodies, determined by an in vitro neutralizing assay, revealed a high incidence, particularly among patients receiving agalsidase beta. Treatment with a high dose agalsidase beta led to a similar reduction in plasma lysoGb3 as observed in patients without antibodies. This suggests that a dose increase can compensate for reduced efficacy of recombinant α-Gal A in individuals with anti-(α-Gal A) antibodies. Negative effects of antibodies on ERT-induced reductions in urinary Gb3 were reported earlier, which also could be compensated by the use of high dose enzyme infusion [START_REF] Linthorst | Enzyme therapy for Fabry disease: neutralizing antibodies toward agalsidase alpha and beta[END_REF][START_REF] Ohashi | Influence of antibody formation on reduction of globotriaosylceramide (GL-3) in urine from Fabry patients during agalsidase beta therapy[END_REF][START_REF] Vedder | Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3[END_REF].

In female patients, plasma lysoGb3 levels are much lower than in male patients. A direct comparison of the effect of different treatments on plasma lysoGb3 concentration was hampered by the relatively

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13 large variability in its concentration in the investigated females prior to treatment. However, it is noteworthy that in all heterozygotes, with each of the treatment regimen, a reduction in plasma lysoGb3 level or at least stabilization at low level was observed. None of the female patients developed anti-(α-Gal A) antibodies.

The high plasma concentration of lysoGb3 is characteristic for patients with classical manifestations of Fabry disease. A role for lysoGb3 in pathogenesis of Fabry disease, as suggested for deacylated galactosylceramide in Krabbe disease [START_REF] Kobayashi | Globoid cell leukodystrophy is a generalized galactosylsphingosine (psychosine) storage disease[END_REF][START_REF] Giri | The role of AMPK in psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease[END_REF][START_REF] White | Psychosine accumulates in membrane microdomains in the brain of Krabbe patients, disrupting the raft architecture[END_REF], is presently under investigation. Of note, at concentrations occurring in plasma of hemizygotes, lysoGb3 has been found to promote smooth muscle cell proliferation [START_REF] Aerts | Elevated globotriaosylsphingosine is a hallmark of Fabry disease[END_REF]. In addition, lysoGb3 has been reported to stimulate cultured human podocytes to express TGF-β1, extracellular matrix proteins (fibronectin and type IV collagen) and the macrophage inhibitory factor receptor CD74 [START_REF] Sanchez-Niño | Globotriaosylsphingosine actions on human glomerular podocytes: implications for Fabry nephropathy[END_REF]. These factors have been implicated in glomerular injury in diabetic nephropathy. Recent data suggest that high plasma lysoGb3 correlates with increased risk for cerebrovascular disease in Fabry males and LVH in Fabry females. Life-time exposure to lysoGb3, assessed as plasma lipid concentrations multiplied by age, was found to correlate with disease severity in male as well as female patients [START_REF] Rombach | Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease[END_REF]. These observations suggest, but certainly do not prove, that lysoGb3 plays a direct role in the pathogenesis of Fabry disease. Of note, only partial corrections in plasma lysoGb3 concentrations of male patients receiving ERT were observed.

Apparently, even high dose ERT is insufficient to completely correct the plasma lysoGb3 abnormality.

Thus, it might be possible that ERT of male Fabry patients results in an ameliorated progression of disease and not a complete halt or reversal of disease manifestation. We noted no correlation of plasma lysoGb3 content of male Fabry patients with disease manifestation prior to, or after one year of therapy. Among female Fabry patients, the combined disease severity score (Mainz severity scoring index (MSSI) [START_REF] Whybra | The Mainz Severity Score Index: a new instrument for quantifying the Anderson-Fabry disease phenotype, and the response of patients to enzyme replacement therapy[END_REF]) showed no significant correlation with plasma lysoGb3 levels prior to treatment (Spearman ρ 0.4421; P=0.099). Changes in plasma lysoGb3 within one year of ERT were no obvious predictor for changes in some clinical parameter.

At present plasma lysoGb3 does not qualify as surrogate marker of Fabry disease. Long-term followup of Fabry patients receiving ERT has to reveal whether plasma lysoGb3 levels correlate with

A C C E P T E D M A N U S C R I P T

meaningful clinical outcomes such as progression of cardiac, renal or cerebrovascular complications.

It will be of particular interest to analyze whether a poor response in plasma lysoGb3 following ERT is associated with a poor clinical response. Since it has become clear that advanced disease cannot be reversed by ERT, correction of lysoGb3 is presumably of little value in this group. However, when early treatment has been installed, measurement of plasma lysoGb3 concentration may prove to be a valuable tool in clinical management of Fabry patients before irreversible damage has occurred.

In conclusion, our investigation has rendered a valuable new insight. ERT of patients with classical manifestations of Fabry disease with recombinant α-Gal A preparations results in correction of plasma lysoGb3. This finding further substantiates that ERT is capable of correcting a characteristic biochemical abnormality in Fabry patients.

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Legends

Fig. 1 .Fig. 2 .

 12 Fig. 1. Plasma levels of lysoGb3 and Gb3 in controls and Fabry patients prior to therapy. (A) Plasma

Fig. 3 .

 3 Fig. 3. Effect of different treatment regimens on plasma lipid levels in male Fabry patients with and

Footnotes 1

 1 Fig.1

Table 1 .

 1 Effect of different treatment regimens on plasma levels of lysoGb3 and Gb3 in male Fabry

	patients. The data reflect absolute numbers (median and range) and sample size (n). R0.2, treatment
	with agalsidase alfa at 0.2 mg/kg; F0.2, treatment with agalsidase beta at 0.2 mg/kg; F1.0, treatment
	with agalsidase beta at 1.0 mg/kg. Normal values for lysoGb3 <3 nM, normal values for Gb3 <3.18
	µM.

Table 2 .

 2 Antibody formation in male Fabry patients treated with different treatment regimens. The data reflect absolute and relative (%) numbers. R0.2, treatment with agalsidase alfa at 0.2 mg/kg; F0.2, treatment with agalsidase beta at 0.2 mg/kg; F1.0, treatment with agalsidase beta at 1.0 mg/kg. AB+,

patients with neutralizing antibodies; AB-, patients without neutralizing antibodies.

Table 3 .

 3 Effect of different treatment regimens on plasma levels of lysoGb3 and Gb3 in female Fabry

	patients. The data reflect absolute numbers (median and range) and sample size (n). R0.2, treatment
	with agalsidase alfa at 0.2 mg/kg; F0.2, treatment with agalsidase beta at 0.2 mg/kg; F1.0, treatment
	with agalsidase beta at 1.0 mg/kg. Normal values for lysoGb3 <3 nM, normal values for Gb3 <3.18
	µM.

Table 2

 2

	Males	Total	AB+	AB-	% AB+
	Total	22	16	6	73
	R0.2	7	3	4	43
	F0.2	6	5	1	83
	F1.0	9	8	1	89

Acknowledgements

We would like to acknowledge Dave Speijer and Ans Groener for useful suggestions during the preparation of the manuscript. Els Ormel is acknowledged for her excellent support in the Fabry outpatient clinic. We would like to thank Symen Kuiper for measuring plasma Gb3 and Wilma Donker-Koopman for measuring antibodies. We are very grateful to the patient members of the Dutch Fabry patient society (FSIGN) and to all other Fabry patients for their cooperation.