V-ATPase as an effective therapeutic target for sarcomas

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Research Article

V-ATPase as an effective therapeutic target for sarcomas Francesca Peruta,n, Sofia Avneta, Caterina Fotiaa, Serena Rubina Baglìoa, Manuela Salernoa, Shigekuni Hosogia,b, Katsuyuki Kusuzakib, Nicola Baldinia,c a

Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, Italy Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan c Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy b

article information

abstract

Article Chronology:

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton

Received 24 May 2013

efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chon-

Received in revised form

drosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor

30 September 2013

acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to

Accepted 18 October 2013

5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton

Available online 25 October 2013 Keywords: Osteosarcoma Rhabdomyosarcoma Chondrosarcoma V-ATPase

pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment.

Lysosomes

Introduction Musculoskeletal sarcomas are relatively rare, aggressive malignancies that show significant morbidity and mortality despite the use of intense multimodal therapies [1–3]. Exploring critical aspects of the metabolic pathways of sarcoma cells may offer novel possibilities to improve the prognosis and possibly reduce the side effects of conventional anticancer agents. The energetic metabolism of malignant cells is largely based on glycolysis, with increased lactate production and a high proton

& 2013 Elsevier Inc. All rights reserved.

efflux. This, in turn, is responsible for the development of a highly acidic extracellular environment [4]. Cancer cells typically show glycolytic rates that are up to 200 times higher than those of their normal counterparts, and upregulated glucose metabolism is a recognized hallmark of invasive cancer [5]. The hypoxic microenvironment that commonly develops in sarcomas is an additional factor that further contributes to tumor acidity. HIF signaling, that is specifically activated by hypoxic conditions, promotes the expression of glycolytic enzymes, that, in turn, increase the glycolytic metabolism and the acidification of extracellular

Abbreviations: ESOM, esomeprazole; OS, osteosarcoma; RS, rhabdomyosarcoma; CS, chondrosarcoma n Correspondence to: Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy. Fax: þ39 51 6366897. E-mail address: [email protected] (F. Perut). 0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.10.011

Effects of pH culture medium on cell viability Both primary cultures and cell lines were seeded (4  103 cells/ well for MG-63, HOS, SAOS-2, RD, and SW1353; 3  103 cells/well

Chondroblastic – grade 3/4 Fibroblastic – grade 4 Metastatic Alveolar Alveolar Myxoid – grade 2 Chondrosarcoma – grade 1 Distal tibia Scapula Proximal femur Leg Leg Proximal humerus Pelvis 44 71 18 10 7 73 78 F M M M F F F

Histological features Anatomical site Age

Osteosarcoma Osteosarcoma Osteosarcoma Rhabdomyosarcoma Rhabdomyosarcoma Chondrosarcoma Chondrosarcoma

Continuous cell lines of human osteosarcoma (Saos-2, MG-63, HOS), rhabdomyosarcoma (RD), and chondrosarcoma (SW1353) were used, all obtained from the American Type Culture Collection (ATCC, Rockville, MD). Human primary cell cultures of osteosarcoma (OS), rhabdomyosarcoma (RS), and chondrosarcoma (CS) were isolated from tissue samples and subjected to mechanical mincing before seeding. To guarantee the privacy of the donors a code number following the OS, CS or RS acronym was assigned. The clinical and pathological characteristics of the corresponding tumors is reported in Table 1. Before sample collection, approval from the institutional ethical committee and a signed informed consent were obtained. Both primary and continuous cell lines were maintained in Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen, Carlsbad, CA), with a 25 mM D-Glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate content and supplemented with 10% fetal bovine serum (FBS, Mascia Brunelli, Milan, Italy), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Invitrogen) at 37 1C and 5% CO2. Only cells in exponential growth phase were used.

OS 3844 OS 3947 OS 3210 RS 4028 RS 4098 CS 4465 CS 4082

Cells

Sex

Materials and methods

Histotype

environment [6]. This phenomenon, also known as Warburg effect [7], claims that cancer development is closely associated with a dysfunctionality of mitochondrial metabolism. Interestingly, cancer cells are, unlike normal cells, able to live in the acidic microenvironment that develops as a consequence of high lactic acid production by glycolysis [8,9]. Previous in vivo studies have shown that the extracellular pH levels (pHe) of different tumor types, including sarcomas, ranges from 6.4 to 7.3, whereas the pHe level of normal tissues is neutral to alkaline (7.2–7.5) [10,11]. pH homeostasis is finely tuned by a number of ion/proton pumps, including V-ATPase, Naþ/Hþ exchanger, carbonic anhydrases (CA) II, IX, and XII, protonlinked monocarboxylate transporters (MCTs), Cl-/HCO3-exchangers, and ATP synthase [9,12,13]. A variation in the pHi/pHe ratio of 0.1 pH units may disrupt crucial biochemical and biological processes, such as ATP synthesis, enzyme functions, proliferation, migration, invasion, and metastasis [14,15]. On this basis, proton dynamics has been recently considered as a potential target for the treatment of several malignancies [16–18], and proton pump inhibitors (PPIs), such as omeprazole or esomeprazole (ESOM), have shown antineoplastic activity towards human hematopoietic and solid tumors [19–21]. We have recently demonstrated the fundamental role of V-ATPase and the potential therapeutic activity of omeprazole in Ewing sarcoma [22]. In this study, we extended our observations to a larger panel of sarcomas, and demonstrated that in other malignancies of mesenchymal origin the PPI ESOM is able to inhibit the cell growth in a dose-dependent manner, thus providing a novel therapeutic approach to be combined to conventional regimens.

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Table 1 – Clinical and pathological characteristics of patients derived biopsies used to obtain osteosarcoma, rhabdomyosarcoma and chondrosarcoma primary cell cultures.

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for OS 3844, OS 3947, OS 3210, RS 4028, RS 4098 CS 4465, and CS 4402) on 96w plates and incubated for the population doubling time at different pH in buffered IMDM medium. Cell viability was measured by the Hoechst 33342 assay. Briefly, after washing with PBS, cells were incubated with 25 μg/ml bisBenzimide Hoechst 33342 (Sigma-Aldrich, St Louis, MO) for 30 min. After washing with PBS, 200 μl/w of PBS were distributed on cells and the fluorescence was read at an excitation/emission wavelength of 360/460 nm using the Cytofluor 2350 fluorimeter (Millipore Corp, Bedford, MA). The results were reported as percentage of growth in respect to the maximum growth detected (100%). The experiment was repeated three times, four replicates for each condition.

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roche-applied-science.com): CA II-f 5′-GGGGTACGGCAAACACAA-3′; CA II-r 5′-TCGATGTCAACAGGGGACT-3′; NHE-1-f 5′-TCCACCTGT CAGGCATCAT-3′; NHE-1-r 5′-GATGTTGGCCTCCACATAGG-3′; MCT1-f 5′-GGATTGGTGACCATTGTGG-3′; MCT-1-r 5′-CATGTCATTGAGCC GACCTA-3′; MCT-2-f 5′-CCATGTTTCTAAACACCTGTCG-3′; MCT-2-r 5′-ACAATCCAACCCCATCCTC-3′; V-ATPase VoC-f 5′-TTCGTTTTTCGCCGTCAT-3′; V-ATPase VoC-r 5′-CCACTGGGATGATGGACTTC-3′. The protocol of amplification was: 95 1C for 10 min; 95 1C for 10 s, 60 1C for 30 s, and 72 1C for 1 s for 45 cycles; 40 1C for 30 s. The results were expressed as the ratio between gene of interest and β-actin as reference gene (NM_001101.2; ACTB-f 5′-CCAACCGCGA GAAGATGA-3′; ACTB-r 5′-CCAGAGGCGTACAGGGATAG-3′) according to the 2-ΔΔCT method [24].

Lysosome and acidic vesicle staining Three different stainings were used for lysosomes and acidic vesicles. Cells were incubated with 0.4% neutral red dye (Carlo Erba, Arese, Milano, Italy) in PBS for 2 h at 37 1C. After washing with PBS, the cultures were examined under a phase-contrast inverted microscope. Lysosomes stained red. Alternatively, labeling and tracking of acidic organelles in live cells was performed by incubating cells with 1 μM LysoSensor Green DND-189 (LS) and 50 nM LysoTracker Red DND-99 (LT) (Invitrogen) stains in IMDM complete medium. After 30 min at 37 1C, cells were washed with fresh medium and observed by confocal microscopy (Nikon Eclipse E600). According to the method of Millot et al. [23], the emission spectra of the pH-sensitive dye acridine orange (AO) was used to measure slight pH variations in acidic organelles of different sarcoma cell cultures. Cells in exponential growth phase were incubated with AO (Sigma, St Louis, MO ) at a concentration of 0.5 μg/ml in IMDM complete medium for 15 min. After one wash with IMDM complete medium, x,y emission spectra from a confocal section within a living cell were recorded using a confocal laser microspectrofluorimeter (Nikon, TI), equipped with an argon-ion laser. Cells were focussed with a  40, 1.3 NA (S Fluor, Nikon) and excited at 457 nm, and the resulting fluorescence emission in the 500–700 nm range was collected. For intracellular measurements of AO emission the pinhole size was fixed to a diameter of 54 μm. To characterize the profile of AO emission spectra, the red band contribution (R%) within the whole emission spectrum has been calculated as follows: R%¼100I655/ (I655þI530) where I655 and I530 are the green (520–540 nm) and the red (645–665 nm) integrated emission intensities, respectively. The R% was calculated for all the acidic organelles within a single cells, and the average R% of acidic organelles of one single cells was considered.

Expression of ion/proton pumps by quantitative real-time PCR Total RNA was extracted from semi-confluent cells, both primary and continuous cell lines, using the RNeasy Mini Kit (Qiagen GmbH). Total mRNA was reverse transcribed by the Advantage RT-for-PCR Kit (Roche). The expression of CA II (NM_000067.2), NHE1 (NM_003047.3), MCT-1 (NM_003051.3), MCT-2 (NM_004731.3), and V-ATPase VoC (NM_001694.2) was evaluated by Real-Time PCR using the Light Cycler instrumentation (Roche Diagnostics) by amplifying 1 μg of cDNA and the Universal Probe Library system (Roche Applied Science). Probes and primers were selected using a web-based assay design software (ProbeFinder https://www.

Expression and localization of V-ATPase by western blotting and immunofluorescence Whole-cell extracts from confluent sarcoma cell lines and primary cultures were lysated with RIPA buffer (Tris pH 7.6 50 mM, NaCl 150 mM, Triton-X 100 5%, sodium deoxycholate 0.25%, EGTA pH 8 1 mM, NaF 1 mM) (Sigma-Aldrich) supplemented with protease inhibitors (Roche). Equal amount of lysate were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting with an anti-ATP6V0c (Abcam), anti-ATP6V1B2 (SigmaAldrich), and anti-actin (Cell Signaling) antibodies, as reference. The quantification of signals from each band was performed by a dedicated software (Quantity One, Biorad Laboratories Headquarters, Hercules, Ca). For the immunofluorescence staining of V-ATPase, cells were washed with PBS, fixed in 3% paraformaldehyde in PBS containing 300 mM sucrose for 30 min at 22 1C. After washing in PBS, permeabilization was performed with 0.1% Triton X-100 for 5 min. Then, cells were incubated with anti-V-ATPase V0a1 antibody (Sigma) 1:30 or 1:100, and secondary anti-rabbit antibody Alexa green 488 nm (1:1000, Molecular Probes, Life Technologies). Different amount of primary antibody has been used to detect V-ATPase localized on cell membrane or in the intracellular compartment. Actin cytoskeleton was stained by using rhodamine–phalloidin fluorescent dye 0.06 mM (Molecular Probes, Eugene, USA). Cells were then analyzed by a confocal microscope (Nikon TI-E).

SiRNA transfection Specific gene silencing was obtained by siRNA technology associated with pipette-type electroporation. MG63, RD, and SW1353 cells were trypsinized at semi-confluence, and counted after erythrosine dye staining. 15 μl of cell suspension containing 150,000 cells and 2 pmol of specific siRNA (ON-TARGETplus Human ATP6V0C siRNA Smart poll, Dharmacon, Thermo Scientific, Waltham, MA) or control siRNA (siRNA ctr, ON-TARGETplus Non-targeting Control Pool, Dharmacon) were transferred into a 1-mm cuvette (Neons Transfection System, Invitrogen, Life Technologies). Electroporated cells were transferred into 2 ml of complete medium, and seeded in 12-well plates (20,000 cell/ well) for RNA isolation or cell counting. After one day (T0), medium was changed with complete medium at pH 6.5 or unbuffered medium. At T0 and after additional two days (T1) cells were counted by using erythrosine dye. At T1, cells were also

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used to isolate RNA, as previously described. Both assays were repeated four times in duplicate.

Cell proliferation assay after esomeprazole treatment Cells were seeded in duplicate in 12-well plates (3  104 cells/well for Saos-2, RD and SW1353; 2  104 cells/well for MG63) under acidic (pH 6.5) or not-acidic conditions (pH 7.4), or in unbuffered medium. After 24 h the culture medium was replaced with fresh medium containing 30–60–120 μM esomeprazole (ESOM) (SigmaAldrich) dissolved in DMSO. As controls, cells were incubated with medium at the same concentration of DMSO. After 72 h, cells were harvested and the number of viable cells was evaluated by the erythrosin B dye vital staining [25]. Results were expressed as percentage of growth inhibition in respect to cells in control medium. The experiment was repeated three times, two replicates for each conditions.

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regions. The emission ratio was calibrated using solutions (110 mM KCl, 25 mM KHCO3, 11 mM glucose, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES) with varying pH that contained 10 μM nigericin (Kþ/Hþ ionophore). The fluorescence emission ratio (644 nm/594 nm) was calculated and used to estimate pHc from the calibration curve.

Statistical analysis Statistical analysis was performed using the StatView™ 5.0.1 software for Windows (SAS Institute, Cary, NC). Results were reported as mean7standard deviation and the differences were analyzed using the non-parametric Mann–Whitney test for the difference between group and the Wilcoxon Rank for differences of paired values, in the pHe evaluation. Only po0.05 were considered significant.

Intracellular emission spectra of AO after esomeprazole treatment MG63, SW1353 and RD cells maintained under acidic (pH 6.5) medium, were treated with 120 μM esomeprazole (ESOM) for 30 min and the acidity of organelles was quantified by measuring the intracellular emission spectra of AO, as previously described.

Extracellular and cytosolic pH measurementy after ESOM treatment MG63, Saos-2, RD, and SW1353 cells (2  10  6 cells) were treated with 60 μM ESOM (Sigma-Aldrich) in unbuffered medium (1 ml) for 30 min The cells were then harvested, washed in pHMed (80% normal saline, 10% unbuffered RPMI-1640 (Invitrogen) and 10% FBS), and incubated in pHMed for 3 h at 37 1C. The cells were then collected by centrifugation (10 min at 500 g) and the supernatant was harvested for pH measurement. pH was immediately measured by a digital pH-meter (pH 301, HANNA Instruments). The difference of pHe of treated cells vs untreated cells was analyzed. The experiment was replicated three times in duplicate. Cytosolic pH (pHc) of RD cells was measured by using carboxyseminaphthorhodafluor-1 (carboxy-SNARF-1) (Molecular Probes, Eugene, OR, USA), a pH-sensitive fluorescent dye, with a confocal laser microspectrofluorimeter (Nikon, TI). Cells were seeded into glass chamber slides incubated for 48 h at different pH (6.5 or 7.4) in buffered RPMI1640 medium. Then cells were incubated with 60 μM ESOM or DMSO, as control, in acidic medium (pH 6.5). The pHc has been measured every 10 min for 1 h and after 3 h from ESOM treatment. Cells were then were equilibrated at 37 1C in air with 5% CO2 for 30 min in saline solutions (115 mM NaCl, 25 mM NaHCO3, 11 mM glucose, 4.4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, pH 7.4) containing 10 μM carboxy-SNARF-1 of the acetoxymethyl ester form. The chamber slides were placed in an incubator, OkoLab (Napoli, Italy), on the stage of the confocal microscope and allowed to adapt for at least 20 min before starting pHc measurements. The excitation laser beam of 514 nm (Ar laser) was directed to the sample via S Plan Fluor ELWD 40X lens (Nikon). The resulting fluorescence emission was collected centered at 644 nm and 594 nm. Several regions of interest (ROI) with a diameter of 1 μm were then randomly selected excluding nuclear

Fig. 1 – Effects of culture medium pH on cell viability. Cells were incubated for the population doubling time, at different pH in buffered medium. (a) Osteosarcoma (Saos-2,MG63, HOS, OS 3844, OS 3947, OS 3210), (b) rhabdomyosarcoma (RD, RS 4028, RS 4098), and (c) chondrosarcoma (SW1353, CS 4465, CS 4402 cell lines and primary cells, respectively. Sarcoma cells maintained a high viability (475%) in acidic medium.

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Fig. 2 – Lysosomes and acidic vesicles staining. (a) Cells were incubated with Neutral Red that stains lysosomes red; (b) Evaluation of acidic vesicles in sarcoma cells by using LysoSensor Green and LysoTracker Red dyes. Numerous acidic lysosomes (red to yellow staining according to a decrease pH gradient) were observed in all sarcoma cell types. (c) Representative pictures of acridine orange (AO) uptake. Red staining is associated with a higher concentration of AO within acidic organelles (low pH), whereas green vacuolar staining is associated with a lower AO concentration AO (high pH). (d) AO emission spectra and (e) quantification of the red band contribution (R%). Acidic organelles of RD cells are significantly more acidic than that of MG63 or SW1353 cells (n ¼12; significant level n for po0.05 and nnn for po0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Results Effects of pH culture medium on cell viability All sarcoma cells were able to survive in acidic medium. The viability was over 75% for a pH ranging from 5.9 to 6.8 (Fig. 1A) and 5.9 to 7.4 in OS cell lines and primary cultures, respectively, from 6.1 to 7.4 and 6.0 to 7.4 in RS cell lines and primary cultures, respectively (Fig. 1B), and from 6.0 to 7.4 in CS cell lines and primary cultures (Fig. 1C).

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As shown by representative pictures of MG63, RD and SW1353 cells (Fig. 2C), the size and the number of acidic organelles differed among different sarcomas. The intracellular emission spectra of AO (green and red emission intensities) was different among OS, RS and CS cell lines, showing a different local concentration of AO (Fig. 2D). By the quantification of the red band contribution of AO, corresponding to oligomeric AO, lysosomes of RD cells were found to be significantly more acidic than lysosomes of MG63 and SW1353 cells (Fig. 2E).

Expression of V-ATPase and other ion/proton pumps Lysosome and acidic vesicle staining OS, RS and CS cells showed a high number of lysosomes as revealed by neutral red staining (Fig. 2 A). LT and LysoSensor LS stainings were used to confirm the high acidity of lysosomes in sarcoma cells. LT stains red acidic organelles, while LS indicates the pH of acidic organelles, as its fluorescence intensity is higher in acidic environments. Numerous acidic lysosomes (red to yellow staining according to a lower pH gradient) were observed in sarcoma cell lines (Fig. 2B). To quantify different levels of lysosome acidity, the measurement of intracellular emission spectra of AO were used. AO is a fluorescent small cationic molecule that accumulates within lysosomes and other acidic organelles of living cells. OS, RS and CS cells were all able to uptake AO. The red staining of AO is associated with a high concentration of the dye within acidic organelles, indicating a low pH, whereas the green vacuolar staining is associated with a low AO concentration, indicating a high pH.

Since human V-ATPase may be targeted by PPI [26], we evaluated the expression of this proton pump as a preliminary evidence of its therapeutic potential in sarcomas. Real-Time PCR analysis showed a specific histotype pattern of V-ATPase VoC expression (Fig. 3A ), with significantly higher levels of expression in RS cells compared to CS and OS cells (p¼ 0.004). The expression of other ion/proton pumps was assessed by qRT-PCR, and comparable levels of Naþ/Hþ exchanger 1, carbonic anhydrase II and proton-linked monocarboxylate transporter 1 has been found in OS, RS, and CS cell lines and primary cultures (Fig. 3A). MCT-2 expression was undetectable in all samples. The expression of V-ATPase was confirmed at the protein level in all sarcoma cell lines and primary cultures (Fig. 3B). Two different subunits were analyzed, the VoC, part of the Vo integral domain, that translocates protons, and the V1B2, a subunit of the V1 peripheral domain, that is responsible for ATP hydrolysis [27]. The expression of VoC subunit was significantly higher in RS cells compared to CS and OS cells (p ¼0.004), while comparable amount of V1B2 subunit were detected in RS, OS and CS cells.

Fig.3 – Expression of V-ATPase. (a) Expression of V-ATPase, carbonic anhydrase II, Naþ/Hþ exchanger, and proton-linked monocarboxylate transporters MCT –1 in human sarcomas cell lines and primary cultures (n¼18 for OS; n¼9 for CS and RS cells) by real time PCR. A significative higher level of V-ATPase is expressed by RS cells compared to CS and OS cells (p¼0.004); (b) Western blotting of V0c and V1B2 subunits of V-ATPase (representative images) and (c) densitometric analysis (n¼ 4). A significative higher level of V-ATPase VoC protein is expressed by RS cells compared to CS and OS cells (po0.05).

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Fig. 4 – V-ATPase localization. Immunofluorescence of V-ATPase V0a1 subunit (green) in permeabilized cells. Actin cytoskeleton was stained using rhodamine–phalloidin fluorescent dye (red). Representative. images of an xy field observed by confocal microscope. V-ATPase is expressed in the Golgi apparatus in the perinuclear region (a), in the cell membrane (b) and in some vesicles (c). White arrows, co-localization of V-ATPase V0a1 and Actin staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The immunofluorescence assay was performed to elucidate V-ATPase localization in RS, OS and CS cells. V-ATPase has been found localized in the Golgi apparatus in the perinuclear region (Fig. 4A), on the plasmalemma (Fig. 4B) and in some intracellualr vesicles (Fig. 4C).

(p¼0.02) cells at acidic pHe (Fig. 5B–D). The ability of the siRNA against the VoC subunit of V-ATPase to affect cell growth was confirmed in unbuffered medium for MG63 (p¼0.02), RD (p¼0.02) and SW1353 (p¼0.03) cells (Fig. 5E–G). The percentage of inhibition at acidic pH was higher for RD cells that express the higher level of V-ATPase V0C, when compared to OS and CS.

Effect of V-ATPase inhibition Cell proliferation after ESOM treatment We demonstrated the specific and significant inhibition of V-ATPase mRNA expression in MG63, RD and SW1353 cells treated with a siRNA against the V0c subunit of V-ATPase (Fig. 5A). Afterwards, we showed that treatment with V-ATPase VoC siRNA decreased the number of cells of MG63 (p¼ 0.04), RD (p¼ 0.02) and SW1353

ESOM inhibited OS and RS cell proliferation in a dose dependent manner in buffered, pH 6.5, and unbuffered medium (Saos-2: p¼ 0.02 for 30 μM vs 120 μM and 60 μM vs 120 μM at pH 7.4, 6.5 and unbuffered; MG63: p ¼0.02 for 30 μM vs 120 μM and p ¼0.03

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Fig. 5 – V-ATPase silencing. (a) Specific gene silencing effect was observed by qRT-PCR in MG63, RD and SW1353 cells; (b) V-ATPase silencing affect MG63, (c) RD and (d) SW1353 cells growth in a acidic medium (pH 6.5) (significant level n for po0.05) and in unbuffered medium (e), (f) and (g).

for 60 μM vs 120 μM at pH 7.4, p¼ 0.004 for 30 μM vs 120 μM and for 60 μM vs 120 μM at pH 6.5 and in unbuffered medium; RD: p¼ 0.02 for 30 μM vs 120 μM and 60 μM vs 120 μM at pH 7.4, 6.5 and unbuffered). A significative difference in CS inhibition between low and high ESOM dosage was observed in acidic and unbuffered medium (SW1353: p¼ 0.02 for 30 μM vs 120 μM and 60 μM vs 120 μM at pH 7.4, 6.5 and unbuffered) (Fig. 6). In all sarcoma models, the inhibitory effect of ESOM on cell proliferation was significantly higher in acidic (pH 6.5) medium, a condition that fully activates the drug (Fig. 7), and in unbuffered medium, whose pH level, after 72 h, was found to be acidic (pH: 6.8570.02, 6.8270.03, 6.7270.01 and 6.9770.03 for Saos-2, MG63, RD and SW1353, respectively). According to the results obtained with the specific silencing with the anti-V-ATPase siRNA, the highest inhibitory effect was observed in RD cells.

Effect of ESOM on lysosome pH The addition of ESOM induced a significant decrease in the % R (red band contribution) of AO in MG63, RD and SW1353 cells (Fig. 8A and B). This decrease in the local accumulation of AO was related to alkalinization of acidic organelles, possibly due to the inteference of ESOM on V-ATPase activity.

Effect of ESOM on pHe and pHc ESOM treatment impaired the ability of tumor cells to acidify the extracellular medium (Fig. 8C). The pH increase after ESO treatment ranges from 0.15 for OS cells to 0.29 for RD cells, and the increment of pHe in respect to untreated condition was significative for OS (p¼ 0.03), RS (p¼ 0.03) and CS cells (p¼ 0.04). Exposure to acidic pHe was previously shown to induce acidification of pHc [28] in cancer cells. Therefore, we first investigated the effect of acidic pHe (6.5) on pHc in RD cells. Exposure to acidic pHe strongly decreased the pHc (p¼ 0.0002) (Fig. 8D). The pHc value of RD cells exposed to pHe 6.5 did not significantly change during the observation time, while after the

treatment with ESOM the pHc of RD cells dropped to more acidic values within 50 min (p¼ 0.02).

Discussion The ability of tumor cells to acidify the extracellular microenvironment and survive in these conditions has been extensively demonstrated in several human malignancies [29,30]. In the context of mesenchymal tumors, the pHe level of malignant lesions appears to be quite acid, ranging from 6.2 to 7.4, and significantly lower than that of benign tumors [10,31]. In this study, for the first time we provide evidence that OS, CS, and RS cells are able to survive in a same range of acidic microenvironment. Such an aberrant pH homeostasis, critical for cancer cell survival, results from an active proton extrusion by proton pumps or from proton compartimentalization within lysosomes. Sarcoma cells are characterized by the presence of numerous highly acidic organelles, as shown by AO and LS stainings. Among different sarcoma variants, acidic organelles from RS cells showed the most acidic pH levels, as shown by the analysis of intracellular emission spectra of AO accumulation. The activity of different ion/proton transporters is a major mechanism that tumor cells exploits to counteract cytosolic acidification resulting from an increased glycolytic metabolism [9]. Among different ion/proton transporters, the V-ATPase has been extensively studied as an important therapeutic target [32–34] and a key effector of proton secretion and lysosome activity. In this study, we demonstrated a histotype-specific pattern of V-ATPase expression, that was significantly higher is RS compared to CS and OS cells, while other ion/proton transportes, such as Naþ/Hþ exchanger 1, carbonic anhydrase II and proton-linked monocarboxylate transporter 1 were expressed at comparable levels. V-ATPase expression was confirmed in the lysates of both primary and continuous sarcoma cell lines by using two different antibodies against the VoC and V1B2 subunits, that are

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Fig. 7 – Comparison of ESOM treatment of sarcoma cells at different pH. The activity of ESOM (120 μM) is significantly increased in acidic or unbuffered medium compared to conventional buffered medium (significant level n for po0.05 ).

Fig. 6 – Effect of ESOM treatment on sarcoma cell viability. Percentages of inhibition at 72 h of cells treated with ESOM (30–120 μM) in respect to untreated cells. Sarcoma cells were maintained in medium with different pH 7.4, 6.5, and unbuffered medium (n po0.05 for 120 μM vs 30 μM and 60 μM in all medium; # po0.05 for 120 μM vs 30 μM and 60 μM in unbuffered and pH 6.5 medium).

representative isoforms of the two domain V0 and V1, which reversible dissociation regulates V-ATPase activity. VoC subunit, that is part of the V0 integral domain that translocates protons, was found to be expressed at significantly higher levels in RS cells when compared to OS and CS cells. V-ATPase subunits exist in multiple isoforms, that may associated and dissociates reversibly. VoC subunit may represent the limiting subunit, that could explain the higher sensitivity of RS vs OS and CS to esomeprazole-induced proliferation inhibition. V-ATPase localization was explored by using different concentration of anti-V0a1 transmembrane domain antibody in immunofluorescence staining. The V0a1 subunit is localized mostly in the Golgi apparatus in the perinuclear region. However, using higher amount of antibody, that produced an oversaturated signal in Golgi apparatus region, it was possible to detect V-ATPase in some vesicles and on the a cell membrane. The critical role of V-ATPase in OS, RS, and CS cells survival at acidic pH conditions was confirmed by siRNA treatment. V-ATPase V0c mRNA expression was strongly and significantly decreased by siRNA silencing and OS, RS, and CS cells viability was dramatically affected by this treatment. The role of V-ATPase on cell survival at acidic pH conditions had been previously shown in other cancers [33,35]. However, even if V-ATPase seems to be a good target for sarcoma treatment, V-ATPase inhibitors are also highly toxic for normal cells and tissues due to their ubiquitous expression underlying the physiological activity of these transporters [36]. Proton pump inhibitors (PPI) are acid activated pro-drugs, that have been successfully used for the treatment of peptic disease [37]. Besides targeting the gastric proton pump, PPI have been shown to also be able to inhibit the activity of V-ATPase, although at much higher concentrations [26,38]. PPI are chiral drugs and may be administered as racemates with the exception of omeprazole, whose S-enantiomer (esomeprazole) is also widely used in clinical applications. In fact, ESOM is metabolized to a lesser degree and at lower rate than omeprazole, resulting in higher plasma levels and, as a consequence, in a better therapeutic efficacy [39]. Since PPI are prodrugs which need acidity to be transformed into their active molecule, we investigated the antiproliferative effect of PPI in buffered and acidic culture conditions (i.e. unbuffered medium and medium at pH 6.5), comparable to those present in the microenvironment of sarcomas. Under these conditions, ESOM treatment

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Fig. 8 – ESOM treatment effect on lysosomal and extracellular pH. (a) Representative pictures of MG63, RD, and SW1353 cells treated with ESOM in acidic medium; (b) quantification of the red band contribution (R%) of AO emission spectra after ESOM treatment. ESOM treatment significantly increased the pH of acidic organelles in all sarcoma cells (significant level nn for po0.01 and nnn for po0.001). (c) Difference on pHe after ESOM treatment. ESOM treatment induced a significant increment of pHe in all cell lines in respect to the untreated cells (CTR) (p¼ 0.03 for OS and RS cells; p¼ 0.04 for CS cells). (d) Effect of acidic medium on cytosolic pH (pHc) on RD cells (n¼ 16). Acidic pHe induced significantly acidification of pHc. (e) Effect of ESOM on pHc (n¼ 4). ESOM treatment significantly decreased the pHc of RD cells within 50 min (significant level n for po0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

induced a dose-dependent inhibition of cell proliferation. The inhibitory activity of ESOM on cells seeded in “physiological” pH medium (7.4) is in keeping with a previous report in pancreatic cancer [21]. Culture medium acidification, resulting from an intensive ion transporter activity of tumor cells, may be responsible for PPI activation and cytotoxicity. In fact, this activity was significantly increased in an acidic microenvironment, both in unbuffered and in 6.5 pH medium, where the percentage of growth inhibition obtained with the highest ESOM dose was over 70% for OS, over 90% for RS, and over 80% for CS, respectively. ESOM treatment was particularly effective in RD cells, that also showed the highest level of V-ATPase expression, and whose lysosomal compartment is particularly acidic. To evaluate the role of ESOM on lysosomal compartment of sarcoma cells, the AO emission spectra after ESOM treatment were analyzed. We demonstrated a lower red band contribution in ESOM treated sarcoma cells, interpreted as a lower local accumulation of the dye that would derive from a low level of acidity in these organelles. Thus, ESOM treatment directly interfere with the ability of V-ATPase to protect sarcoma cells in a acidic microenvironment by means of proton compartimentalization or extrusion. In fact, ESOM treatment strongly interfered also with extracellular pH, as shown by the significative increment of pHe detected for all cell lines after ESOM treatment. Moreover, we found that exposure to acidic pHe induce acidification of pHc, as already demonstrated in other tumors [28], and after exposure to ESOM the pHc of cells dropped to more

acidic values within 50 min. Thus, the crucial role of V-ATPase in proton dynamics was assessed by the analysis the effect of ESOM treatment on the pHe, pHc and on the pH of acidic organelles. Moreover, since most anticancer treatments are weak acids or bases, ESOM, interfering with pHe and lysosomal pH, could have a chemosensitizing activity, as already described for other PPI, as omeprazole and lansoprazole, in different malignancies [40,41]. Further investigation could support this novel therapeutic approach in association with conventional therapies, opening new possibilities for the treatment of drug-resistant tumors or to reduce the chemotherapy-related toxicity.

Conclusions In summary, this study provides consistent evidence that acidic microenvironment is a constant feature of sarcomas and that PPI administration is able to induce remarkable cytotoxicity, therefore uncovering a novel therapeutic target to be combined to conventional anticancer agents, as previously suggested in other malignancies [42].

Acknowledgments This study has been supported by AIRC IG 11426 “5 per mille Welfare 2009”.

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