Neurochemistry International 80 (2015) 14–22

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Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i

The combination of adenosine deaminase inhibition and deoxyadenosine induces apoptosis in a human astrocytoma cell line Mercedes Garcia-Gil a, Maria Grazia Tozzi b, Stefano Varani a, Lorenza Della Verde a, Edoardo Petrotto a, Francesco Balestri b, Laura Colombaioni c, Marcella Camici b,* a

Dipartimento di Biologia, Unità Fisiologia Generale, Via S. Zeno 31, Pisa, Italy Dipartimento di Biologia, Unità Biochimica, Via S. Zeno 51, Pisa, Italy c CNR, Istituto di Neuroscienze, Via Giuseppe Moruzzi 1, Pisa, Italy b

A R T I C L E

I N F O

Article history: Received 14 April 2014 Received in revised form 6 November 2014 Accepted 17 November 2014 Available online 20 November 2014 Keywords: Adenosine deaminase Deoxycoformycin Human astrocytoma cell line Apoptosis Lactate production Baicalein

A B S T R A C T

Alterations in the functions of astrocytes contribute to the appearance of a variety of neurological pathologies. Gliomas, especially those of astrocytic origin, are particularly resistant to chemotherapy and are often characterized by a poor prognosis. Neuroblastoma is the tumour with the higher incidence in infants. Anticancer drugs can induce apoptosis and their cytotoxic effect is often mediated by this process. We have previously demonstrated that the combination of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine is toxic for a human astrocytoma cell line. In fact, after 15 h of treatment, this combination increases both mitochondrial reactive oxygen species and mitochondrial mass, induces apoptosis as indicated by cytochrome c release from mitochondria and activation of caspase-3. These events are preceded by reduction in lactate release in the medium. In this work we demonstrate that after 8 h of incubation with deoxyadenosine and deoxycoformycin, caspase-8 is activated, mitochondrial mass increases and mitochondrial reactive oxygen species decrease. The addition of baicalein to the incubation medium reduces cell death and caspase-3 activity induced by deoxycoformycin and deoxyadenosine in combination. This protective effect is correlated to an increase of lactate released in the medium, a decrease in the intracellular levels of dATP, and an increase in ATP levels, as compared with the cells subjected to the treatment with deoxycoformycin and deoxyadenosine without any further addition. The effect of baicalein appears to be related to an inhibition of deoxyadenosine phosphorylation, rather than or in addition to the well known antioxidant activity of the compound. This work indicates that an astrocytoma cell line, reported to be resistant to mitochondria-dependent pathways of apoptosis, is indeed very sensitive to a manipulation affecting the balance of cellular purine metabolite concentrations. The same treatment is also cytotoxic on a neuroblastoma cell line, thus suggesting long term implications for cancer therapy © 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: Ac-IETD-pNA, Ac-Ile-Glu-Thr-Asp-paranitroaniline; ADA, adenosine deaminase; ADF, human astrocytoma cell line; Ado, adenosine; AdoK, adenosine kinase; dAdo, deoxyadenosine; dATP, deoxyATP; dCF, deoxycoformycin; DMEM, Dulbecco’s modified Eagle’s medium; DEVD-pNA, Asp-Glu-Val-Asp-paranitroanilinine; DMSO, dimethylsulfoxide; FBS, foetal bovine serum; IAP, inhibitor of apoptosis proteins; LDH, lactic dehydrogenase; MT-ROS, MitoTracker Red CM-H2XROS; MTGreen, MitoTracker Green; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide); NDGA, nordihydroguaiaretic acid; NH2dAdo, 5’ amino-5’deoxyadenosine; PBS, phosphate buffered saline; pNA, paranitroanilinine; ROS, reactive oxygen species; SH-SY5Y, human neuroblastoma cell line; TMRM, tetramethylrhodamine methyl ester; ΔΨm, mitochondrial membrane potential. * Corresponding author. Dipartimento di Biologia, Unità di Biochimica, Via S.Zeno 51, Pisa 56127, Italy. Tel.: +390502211458; fax: +390502211460. E-mail address: [email protected] (M. Camici). http://dx.doi.org/10.1016/j.neuint.2014.11.005 0197-0186/© 2014 Elsevier Ltd. All rights reserved.

The deficiency of adenosine aminohydrolase (EC 3.5.4.4) (adenosine deaminase, ADA), the enzyme that catalyses the hydrolytic deamination of adenosine (Ado) and deoxyadenosine (dAdo), is associated with severe immunodeficiency and abnormalities in the functioning of many organs including nervous system (Camici et al., 2010). Neurological abnormalities, rarely reported in early infancy (Hershfield, 2001), include various degrees of motor coordination disorders, learning disability, hyperactivity, seizures, attention and hearing deficits (Hönig et al., 2007). Immune system impairments but not the neurological problems may be reverted by allogenic bone marrow transplantation (Hönig et al., 2007). Indeed, even though lower than in untreated patients, the level of Ado and dAdo appears to be higher than normal in ADA-deficient treated patients (Hirschhorn et al., 1981). It is conceivable that the described neurological manifestations arise from an adverse effect exerted by Ado,

M. Garcia-Gil et al./Neurochemistry International 80 (2015) 14–22

dAdo and their derivatives on the development, differentiation, and functioning of nervous system. In this regard, it is known that deficiency of ADA results in the accumulation of deoxyadenosine triphosphate (dATP), which affects normal methylation reactions, induces apoptosis in thymic lymphocytes, and inhibits ribonucleotide reductase thus interfering with normal DNA synthesis (Takeda et al., 1991). In a previous paper (Garcia-Gil et al., 2012) we have demonstrated that the inhibition of ADA in the presence of dAdo in a human astrocytoma cell line (ADF) induces apoptosis, interferes with glucose metabolism, since a reduction in lactate released in the medium is observed in treated cells as an event preceding caspase-3 activation and cellular death, and produces in longterm incubations an increase in mitochondrial ROS. Astrocytoma cells demonstrate both basic metabolic mechanisms of astrocytes as well as tumours in general, e.g. they show higher glycolytic rate, lactate production, ability to grow under hypoxia, suppression of cell death pathways. Therefore, on one hand, our cellular model may mimic a condition of ADA deficiency, on the other hand, ADF cells offer the possibility to study the effect of the inhibition of ADA activity, a manipulation affecting purine metabolism, on a cellular model deriving from a type of tumour reported to be particularly resistant to chemotherapeutic approaches and often characterized by a poor prognosis (Ohgaki and Kleihues, 2007). Indeed, over the past years, it has become evident that anticancer drugs can induce apoptosis and that their cytotoxic effects are often mediated by this process (Ferreira et al., 2002). However, the success of chemotherapy is often hampered by the appearance of broad drug resistance, which in several cases is due to defective common apoptosis-inducing pathways. These defects are probably at the basis of the initial expansion of the population of neoplastic cells eventually originating the tumour (Igney and Krammer, 2002). Thus, it is very important to identify the biochemical mechanisms leading to apoptosis of a specific tumour to predict the possible success of a given drug treatment and to plan more focused and effective therapies. In this respect, in this paper we have furthered our investigation, trying to understand the link between the production of lactate, therefore glucose metabolism, and the onset of the apoptotic program in astrocytoma cells. In addition, we have also tested the effect of the combination of ADA inhibition and dAdo on the viability of a neuroblastoma cell line, in order to assess whether this treatment could be effectively applied to a tumour of different origin.

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(glutamate–pyruvate transaminase) was from ICN Biomedicals (Irvine, CA, USA); deoxycoformicin (dCF) was from Tocris (Bristol, UK), the human astrocytoma cell line ADF and the human neuroblastoma cell line SH-SY5Y were generous gifts from Dr. W. Malorni (Istituto Superiore della Sanità, Rome, Italy) and Prof. A. Arcangeli (University of Florence, Italy), respectively. 2.2. Treatment of astrocytoma and neuroblastoma cells and viability assay The viability assay is based on the ability of metabolically active cells to reduce the MTT tetrazolium salt, water-soluble, into a purplecoloured, water-insoluble MTT formazan salt. Human neuroblastoma (SH-SY5Y) and astrocytoma (ADF) cell lines were routinely grown in DMEM and RPMI medium, respectively, containing 10% FBS, 2 mM glutamine, 200 UI/mL penicillin and 200 μg/mL streptomycin at 37 °C in a humidified 5% CO2/95% air atmosphere as described in Garcia-Gil et al. (2003, 2012). For the experiments, cells were seeded at 45,000 cell/cm2 in 96-well plates, containing 100 μL of medium. Three days after, medium was removed and cells were preincubated for 30 min with 1 μM dCF and/or the different effectors in serum-free DMEM medium plus 10 mM glucose and then incubated in the same medium containing 120 μM dAdo, plus effectors when indicated. In control cells both preincubation and incubation were performed in the absence of effectors (20 μM NDGA, 25 μM baicalein, 100 μM pepstatin A, 10 μM NH2dAdo, 50 μM calpain inhibitor III, 5 μM PD146176 for ADF and 10 μM NH2dAdo for SH-5Y5Y). After 24 h (for ADF) and 48 h (for SH-SY5Y) of treatment in the presence or absence of dCF and dAdo and the different effectors, 0.5 mg/ mL MTT in PBS was added to each well; cells were incubated at 37 °C in a humidified 5% CO2/95% air atmosphere for 30 min, the reaction was stopped by replacing the MTT solution with 100 μL DMSO and the formazan salts were dissolved by gentle shaking for about 5 min at room temperature. Formazan salts were quantified spectrophotometrically by reading the absorbance at 570 nm using the Ultramark Microplate Systems (Bio-Rad, Hercules, CA, USA). Each experiment was performed in triplicate and repeated at least two times. 2.3. Determination of lactate and quantification of adenylate pool in the incubation medium of ADF cells

2. Experimental procedures 2.1. Materials Asp-Glu-Val-Asp-paranitroanilinine (DEVD-pNA, caspase-3 substrate) Ac-Ile-Glu-Thr-Asp-paranitroaniline (Ac-IETD-pNA, caspase-8 substrate) and calpain inhibitor III were from Calbiochem, Merck (Nottingham, UK); Brilliant blu, protease inhibitor cocktail, 5′amino-5′-deoxyadenosine (NH2dAdo), baicalein, deoxyadenosine (dAdo), nordihydroguairetic acid (NDGA), 3-(4,5-dimethyll-thiazol2-yl)-2,5-diphenyltetrazolium bromide) (MTT), pepstatin A, [814 C] deoxyadenosine (10,000 dpm/nmol), L-lactate: NAD + oxidoreductase (EC 1.1.1.27) (lactic dehydrogenase, LDH), alcohol: NAD+ oxidoreductase (EC 1.1.1.1) (alcohol dehydrogenase), and PD 146176, were from Sigma (Milan, Italy); dimethylsulfoxide (DMSO) from Carlo Erba (Milan, Italy); Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, penicillin, streptomycin and trypsin from Euroclone (Pero, Milan, Italy); MitoTracker Green (MT-Green), tetramethylrhodamine methyl ester (TMRM) and MitoTracker Red CM-H2XROS (MT-ROS) from Molecular Probes, Invitrogen (San Giuliano Milanese, Milan, Italy); caspase-8 and caspase-3 activity kit were from Biovision Research Products (Mountain View, CA, USA); foetal bovine serum (FBS) and glutamine from Lonza (Basel, Switzerland); L-alanine: 2-oxoglutarate aminotransferase (EC 2.6.1.2)

The human astrocytoma cell line (ADF) was routinely grown as described in Section 2.2. For the experiments, cells were seeded at 45,000 cell/cm2. Three days after, medium was removed and cells were preincubated for 30 min with 1 μM dCF in serum-free DMEM medium plus 10 mM glucose and then incubated in the same medium containing 120 μM dAdo (250 μL in 24-well plates). In control cells both preincubation and incubation were performed in the absence of effectors. In order to test the effect of 20 μM NDGA or 25 μM baicalein or 10 μM NH2dAdo, these compounds were added 30 min before dAdo. At different times of incubation, the medium was collected and subjected to the spectrophotometric assay at 340 nm for the determination of lactate (Martì et al., 1997). The reaction mixture contained in a final volume of 0.5 mL, 200 mM glycylglycine – 40 mM glutamate (pH 10), 4 mM NAD+, 27 U of lactic dehydrogenase, 7 U of glutamate–pyruvate transaminase, 20 μL of culture medium, and 20 mM Tris–HCl pH 7.4. For the determination of the intracellular adenylate pool (AMP, ADP, ATP, and dATP) the medium was removed, and the wells were washed twice with cold physiological solution, and incubated for 15 min with 0.1 mL perchloric acid 0.6 M. Cells were scraped and centrifuged 10,000 × g for 5 min. Supernatant was neutralized with 15 μL of 3.5 M K2CO3, centrifuged at 10,000 × g for 5 min and analysed by HPLC (Micheli et al., 1999).

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2.4. Enzyme activity assays ADF cell extracts for the determination of ATP: adenosine 5′phosphotransferase (EC 2.7.1.20) (adenosine kinase, AdoK) activity were prepared essentially according to Giorgelli et al. (2000). A radioenzymatic assay was applied for the determination of AdoK (Camici et al., 1995). The reaction mixture contained in a final volume of 50 μL, 5 μM dCF, 5 mM ATP, 8 mM MgCl2 and 60 μg of protein in 30 mM Tris–HCl, pH 7.4. After a 10 min preincubation at 37 °C with 20 μM NDGA or 25 μM baicalein or vehicle, the reaction was started by addition of 98 μM [8-14C] dAdo (10,000 dpm/nmol). At 0, 10, 20 and 30 min incubation, 10 μL aliquots were withdrawn and spotted on a DE-81 filter paper which was washed 15 min in 1 M ammonium formate and 10 min in two changes of water. The disks were dried and counted for radioactivity. For caspase-8 and caspase-3 assays, ADF cells were plated in 60 mm plates, incubated for different times both in the absence and presence of effectors, trypsinized and washed with PBS; pellets were resuspended in the Biovision lysis buffer incubated for 10 min at 4 °C and centrifuged at 10,000 g. Determination of caspase activity was carried out in a 96-well plate in a total volume of 100 μL by means of a spectrophotometric assay kit (Biovision) following manufacturer’s instructions using 30 μg protein/100 μM DEVD-pNA and 100 μg protein/100 μM Ac-IETD-pNA respectively. Extracts were incubated for 4 h at 37 °C in the presence of the corresponding tetrapeptide conjugated to paranitroaniline (pNA) and the released pNA was measured in a spectrophotometer at 405 nm every 60 min. One unit of enzyme activity represents the amount of enzyme which catalyses the formation of 1 nmol of product min−1 under the adopted experimental conditions.

laser line and the emission collected at 574 (±10 nm). The analysis of confocal images was made offline using both the image analysis software MetaMorph 5.0 (Universal Imaging, West Chester, PA, USA), or with MatLab routines (The MathWorks, Natick, MA, USA) adapted by our laboratory to cell density and mitochondrial fluorescence parameters evaluation (see for details Garcia-Gil et al., 2012). 2.6. Glucose determination in ADF cell culture medium The ADF culture medium collected at different times of incubation was subjected to glucose determination by a colorimetric assay upon reaction with the anthrone reagent. Sugars under acidic conditions are dehydrated to hydroxymethyl furfurals. These compounds form with anthrone a blue-green coloured product with a maximum absorption at 620 nm. The assay was performed mixing 250 μL of sample with 1250 μL of anthrone reagent (70% H2SO4, 2.5 mM anthrone, 130 mM thiourea) and then boiled 15 min until the reaction was completed. The solution was then allowed to cool and the absorbance was measured at 620 nm. A linear relationship exists between the absorbance and the amount of glucose present in the sample. The amount of glucose in the sample was evaluated using a calibration curve obtained with a series of standards of known glucose concentration ranging between 40 and 400 μg/mL. 2.7. Protein content Protein content was usually determined according to Bradford (1976), using the Bio-Rad protein assay kit (Bio-Rad Laboratories) and bovine serum albumin as standard. 2.8. Statistical analyses

2.5. Determination of ADF cell density, mitochondrial mass, mROS and mitochondrial potential ADF cells plated on 13 mm diameter glass coverslips in 24well plates, were incubated 8 h in serum-free DMEM medium plus 10 mM glucose both in the absence and in the presence of 120 μM dAdo, alone or in combination with 1 μM dCF. For confocal imaging, cells were loaded for 30 min at 37 °C in 5% CO2 with 2 nM MTGreen and 2 nM MT-ROS. MT-Green was used to determine the whole mitochondrial mass as it accumulates in mitochondrial matrix regardless of the transmembrane potential (Hoth et al., 1997). MTROS was used to measure the generation of ROS in mitochondria where it accumulates and is oxidized in proportion to the respiration rate, becoming fluorescent and detectable in living ADF cells by confocal microscopy. The double labelling of mitochondria with MT-ROS and MT-Green allowed the normalization of the ROS variation (F/F0) obtained following a given treatment to the total mitochondrial mass. To analyse the mitochondrial potential (ΔΨm), ADF cells were loaded with the cationic fluorescent dye TMRM (5 nM) that is readily sequestered by active mitochondria following the electrochemical gradient existing between cytoplasm and mitochondrial matrix. For confocal imaging, live ADF cells were mounted in a glass chamber on the stage of a TCS-NT Leica laser scanning confocal microscope (Leica, Nussloch, Germany). Laser power was kept at 10% of the maximal power to minimize cell photodamage and photobleaching of fluorescent probes. Images were acquired at 1024 × 1024 pixel resolution and averaged four times to improve the signal/noise ratio. Oil immersion objectives 63× (1.4NA) or 40× (1.2NA) (Leica) were used. For each field both fluorescent and transmitted light images were acquired on separate photomultipliers and merged in offline analysis. The MT-ROS probe was excited with the 543 laser line, the MT-Green probe was excited with the 488 laser line and the emissions were independently recorded at 600 and 516 (±10 nm). TMRM was excited with the 543

Statistical analyses were performed using Student’s t test or the one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test with GraphPad InStat version 4.0 (GraphPad Software, San Diego, CA, USA) or Origin 8 (OriginLab Corporation, Northampton, MA, USA). 3. Results 3.1. Effect of different compounds on viability We have previously demonstrated that dCF and dAdo in combination are cytotoxic for ADF cells (Garcia-Gil et al., 2012). Here we confirm this result (Fig. 1A), and extend our analysis to a human neuroblastoma cell line (SH-SY5Y). As shown in Fig. 1B, treatment with both compounds in combination but not with dAdo or dCF alone decreases viability in SH-SY5Y cells. For a better understanding of the mechanism underlying the cytotoxic action of the combination of dCF and dAdo, we have tested the effect of NH2dAdo, an AdoK inhibitor (Miller et al., 1979) on ADF and SH-SY5Y cell viability (Fig. 1A, B). This compound does not affect cell viability, and completely reverts the effect of 1 μM dCF and 120 μM dAdo both in ADF and SH-SY5Y cell lines, indicating that dAdo must be phosphorylated in order to induce cell death. ADF cells were also incubated with pepstatin A, a cathepsin D inhibitor. This treatment did not modify dCF plus dAdo effect on viability (Fig. 1A). Calpain inhibitor III had a cytotoxic effect on ADF control cells but it appeared to protect against the effect exerted by dCF plus dAdo (Fig. 1A). In addition, we have also tested the effect of NDGA and baicalein, known to be lipoxygenase inhibitors and powerful antioxidants (Czapski et al., 2012), and PD 146176, a 15-lipoxygenase inhibitor without antioxidant properties (Sendobry et al., 1997) (Fig. 1A). Both NDGA and baicalein, but not PD 146176 had a protective effect on ADF cells treated with dCF plus dAdo (Fig. 1A).

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Fig. 1. Effect of different compounds on ADF and SH-SY5Y cell viability. (A) ADF cells were preincubated in the absence (white columns) or presence (grey columns) of 1 μM dCF for 30 min and then treated for 24 h both without (white columns) and with (grey columns) 120 μM dAdo. Where indicated, cells were also preincubated with the following effectors: 20 μM NDGA, 25 μM baicalein, 5 μM PD146176, 100 μM pepstatin A, 10 μM NH2dAdo and 50 μM calpain inhibitor III. Cell viability was measured as % of MTT reduction. Results are expressed as mean ± SD of at least two experiments performed in octuplicate. Significance: *in the absence vs in the presence of dCF plus dAdo in the different experimental conditions; #in the presence of the different effectors vs no addition (in the absence of dCF plus dAdo, white columns); §in the presence of different effectors vs no addition (in the presence of dCF plus dAdo, grey columns). *,#p < 0.05; **,§§,##p < 0.01; ***,§§§,###p < 0.001. (B) SH-SY5Y cells were preincubated in the absence (white columns) or presence (grey columns) of 10 μM NH2dAdo and 1 μM dCF (when indicated) for 30 min and then treated for 48 h with 120 μM dAdo (when indicated). Viability was measured as % of MTT reduction. Results are expressed as mean ± SD of two experiments performed in octuplicate. Significance: in the absence (control) vs in the presence of dCF plus dAdo: ###p < 0.001; in the absence vs in the presence of NH2dAdo (with dCF plus dAdo): ***p < 0.001.

We have previously demonstrated that the treatment of ADF cells with dCF plus dAdo decreases both viability and extracellular lactate concentration (Garcia-Gil et al., 2012). In order to know whether the compounds that increase viability are able to modify lactate formation, ADF cells were preincubated for 30 min with 25 μM baicalein, 20 μM NDGA or 10 μM NH2dAdo both in the absence and in the presence of 1 μM dCF, then 120 μM dAdo was added to the samples containing dCF and the incubation was extended for 8 h. Lactate has been measured in the medium as described in Section 2, Experimental procedures (Fig. 2). Our previous results demonstrated that, after 8 h treatment, ADF cell viability was not affected and that the incubation with dCF or dAdo alone had no effect on lactate production (Garcia-Gil et al., 2012). The effect of dCF plus dAdo on the formation of lactate was counteracted by the addition of the three compounds. Lactate concentration was 20% higher in cells treated with dCF plus dAdo in the presence of baicalein or NDGA than in their absence, whereas the presence of NH2dAdo completely reverted the effect of dCF plus dAdo on lactate concentration. 3.3. ADA inhibition increases caspase-8 activity in ADF cells We have previously demonstrated that ADA inhibition in ADF cells increases caspase-3 activity without activating caspase-2 or caspase-9 (Garcia-Gil et al., 2012). We have assayed the activity of caspase-8 in cell lysates obtained from cells incubated for 6–16 h in the presence or the absence of the combination of dCF and dAdo. The caspase-8 is activated after 8 h of treatment and remains active for at least 16 h (Fig. 3). 3.4. Effect of baicalein and NDGA on caspase-8 and caspase-3 activity in ADF cells The protective effect of baicalein and NDGA on ADF cell viability could be due to a modification of the activation of initiator and/ or effector caspases. To assess this issue we have incubated control and treated cells in the presence and absence of baicalein or NDGA.

The activities of caspase-8 and caspase-3 have been measured in lysates prepared after 8 and 24 h of treatment, respectively (Fig. 4A, B). There is no activation of caspase-3 following 8 h of treatment with dCF plus dAdo (data not shown). The presence of either 25 μM baicalein or 20 μM NDGA increases caspase-8 and caspase-3 activity in untreated cells. In dCF plus dAdo-treated cells, baicalein decreases significantly caspase-3 activation, while its effect on caspase-8, although hinting at a decrease, is not significant. The

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Fig. 2. Effect of NDGA, baicalein and NH2dAdo on lactate level in the medium of ADF cells. ADF cells were preincubated in the absence (white columns) or presence (grey columns) of 1 μM dCF for 30 min and then treated for 8 h both without (white columns) and with (grey columns) 120 μM dAdo. Where indicated, cells were also preincubated with the following effectors: 20 μM NDGA, 25 μM baicalein, 10 μM NH2dAdo. The medium has been collected and lactate concentration measured as described in Section 2, Experimental procedures. Data are expressed as average ± SD of at least three experiments performed in triplicate. Significance: *in the absence vs in the presence of dCF plus dAdo in the different experimental conditions; §in the presence of different effectors vs no addition (in the presence of dCF plus dAdo, grey columns). *p < 0.05; ***,§§§p < 0.001.

M. Garcia-Gil et al./Neurochemistry International 80 (2015) 14–22

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time (h) Fig. 3. Time course of caspase-8 activation in ADF cells. ADF cells have been incubated in the absence (control cells) or the presence of 1 μM dCF plus 120 μM dAdo for the indicated times and caspase-8 activity measured as described in Section 2, Experimental procedures. Data are expressed as fold activity ± SD with respect to control cells of at least two experiments performed in quadruplicate. Significance: ***p < 0.001.

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Fig. 5. Effect of NDGA, baicalein and NH2dAdo on adenylate pool of ADF cells. ADF cells were preincubated in the absence or presence of 1 μM dCF for 30 min and then treated for 8 h both without and with 120 μM dAdo. Where indicated, cells were also preincubated with the following compounds: 20 μM NDGA, 25 μM baicalein and 10 μM NH2dAdo. The levels of ATP (Panel A) and dATP (Panel B) have been measured as described in Section 2, Experimental procedures. Data are expressed as average ± SD of five experiments performed in quadruplicate. Significance: §in the absence vs in the presence of different effectors; *between the indicated columns. ***,§§§p < 0.001; **p < 0.01; *,§p < 0.05.

15 addition of NDGA to treated cells does not alter activation of caspase8, but unexpectedly increases caspase-3 activity.

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Fig. 4. Effect of NDGA and baicalein on caspase-8 and caspase-3 activity in ADF cells. (A) ADF cells were preincubated in the absence (white columns) or presence (grey columns) of 1 μM dCF for 30 min and then treated for 8 h both without (white columns) and with (grey columns) 120 μM dAdo. Where indicated, cells were also preincubated with the following effectors: 20 μM NDGA and 25 μM baicalein. The activity of caspase-8 has been measured as described in Section 2, Experimental procedures. (B) ADF cells have been treated as in (A) for 24 h and the activity of caspase-3 has been measured as described in Section 2, Experimental procedures. Data are expressed as average ± SEM of at least three experiments performed in quadruplicate. Significance: *in the absence vs in the presence of dCF and dAdo in the different experimental conditions; #in the presence of the different effectors vs no addition (in the absence of dCF plus dAdo, white columns); §in the presence of different effectors vs no addition (in the presence of dCF plus dAdo, grey columns). *,§,#p < 0.05; ##p < 0.01; ***,###p < 0.001.

Eight hour-treatment of ADF cells with dCF plus dAdo caused the formation of intracellular dATP, with a reduction in ATP levels. The addition of baicalein or NDGA to cells treated with dCF plus dAdo brings about an increase in ATP levels, and a decrease in dATP concentrations. The addition of NH2dAdo to cells treated with dCF plus dAdo, by inhibiting the phosphorylation of dAdo ensures that dATP is not formed and the levels of ATP are as those found in control cells (Fig. 5). The levels of AMP and ADP were not significantly affected as compared with the control in all the adopted experimental conditions (results not shown). 3.6. Effect of baicalein and NDGA on AdoK activity of ADF cells In order to investigate the possibility that baicalein or NDGA could decrease dATP levels by inhibiting the activity of AdoK, we have prepared lysates ADF cells and we have measured the activity of this

M. Garcia-Gil et al./Neurochemistry International 80 (2015) 14–22

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time (min) Fig. 6. Effect of NDGA and baicalein on AdoK activity in ADF cells. Time course of AdoK activity measured as described in Section 2, Experimental procedures. In the absence of effectors (●), in the presence of NDGA (▲) and baicalein (Ë). Data are expressed as average ± SD of two experiments performed in triplicate.

enzyme in the presence and absence of 25 μM baicalein or 20 μM NDGA. Fig. 6 shows the time-course of AdoK activity. Baicalein appears to inhibit AdoK activity, while NDGA has no significant effect on the enzyme activity. AdoK specific activity is 0.132 ± 0.022 U mg−1 (n = 9) in cell extracts without any addition; in the presence of 20 μM NDGA, the enzyme specific activity is 0.117 ± 0.008 U mg−1 (n = 6, ns vs control), while in the presence of baicalein, the specific activity is 0.057 ± 0.019 U mg−1 (n = 6, p < 0.001 vs. control). 3.7. Effect of ADA inhibition on mitochondrial mass, mROS and ΔΨm in ADF cells Since mitochondria use glycolysis-derived pyruvate for oxidative phosphorylation, we considered whether an increase of mitochondrial activity could explain the observed reduction of extracellular lactate following the treatment for 8 h with dCF plus dAdo of ADF cells. As changes of the mitochondrial mass appear to be highly related to the cellular metabolic needs and are an early sign of energetic unbalance or cellular stress, we started evaluating the effect of ADA inhibition on the mitochondrial mass of ADF cells. Mitochondria were stained with the fluorescent probe MT-Green and the percentage of the intracellular area occupied by them was quantified and expressed as percentage of the control value. The treatment with dAdo did not produce any significant change of the mitochondrial mass, while increases larger than +20% were observed after treatments with dCF or dCF plus dAdo (Fig. 7A). We also evaluated the two main parameters related to the mitochondrial activity: the level of mROS and the ΔΨm. The fluorescent probe MT-ROS was used to label functional mitochondria and to quantify the changes in mROS (Fig. 7B). The MT-ROS signal in the different experimental conditions was normalized to the respective values of mitochondrial mass to obtain, for each treatment, a mean value of mROS production independent of the mitochondrial mass changes. The MT-ROS signal significantly decreased after treatment for 8 h with dAdo (−43.9% ± 12,81), dCF (−58.4% ± 10.12) and dCF plus dAdo (−17.7% ± 14.16). To evaluate the impact of ADA inhibition on ΔΨm we used the fluorescent probe TMRM. Also in this case the TMRM-fluorescence signal obtained by confocal microscopy was normalized to the corresponding value of mitochondrial mass, to have an assessment of the mean ΔΨm/mitochondrion, excluding that the variation in TMRM fluorescence could be ascribed to variations in the mitochondrial

**

100

B

***

75

* *** 50 25 0 ***

TMRM /mitochondrion

0

MT- R ROS /mitochond drion

0.0

200

***

***

C

150 100 50 0 Control

dAdo

dC CF

dCF+dAdo

Fig. 7. Mitochondrial changes induced by ADA inhibition in ADF cells. Graphs show the quantification of the fluorescence signals collected from mitochondria of living ADF cells by confocal microscopy. The data are expressed as percentage relative to the fluorescence of the control cells, considered as 100%. Cells were pre-incubated in the absence or in the presence of 1 μM dCF for 30 min and then treated for 8 h both without and with 120 μM dAdo. (A) Quantification of the mitochondrial mass determined by the fluorescent probe MT-Green, expressed as ratio between area occupied by mitochondria vs total cellular area and normalized to the control value. (B) mROS production determined by the mROS-sensitive probe MT-ROS. (C) Mitochondrial potential, ΔΨm. The degree of the mitochondrial membrane polarization was measured by the changes of the TMRM fluorescence intensity. Data were obtained from four independent experiments. Significance: control vs treated cells: **p < 0.01; ***p < 0.001.

mass. We treated actively respiring mitochondria with dAdo, dCF or dCF plus dAdo taking the TMRM fluorescence as a quantitative measure for transmembrane polarization (Fig. 7C). ΔΨm significantly increased after treatments compared with control: dAdo +53.9% ± 16.22; dCF +52.2% ± 35.32; dCF plus dAdo +85.8% ± 27.32. 3.8. Effect of ADA inhibition on extracellular glucose in ADF cells In order to clarify whether glucose uptake could be modified during the treatment with dCF plus dAdo we have measured the glucose concentration in the extracellular medium of ADF cells at different times of incubation. Up to 6 h incubation, the

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10

glucose (mM)

8

**

6

4

0 0

2

4

6

8

time (h) Fig. 8. Effect of ADA inhibition on extracellular glucose concentration in ADF cells. ADF cells have been incubated in the absence (○) or presence of dCF plus dAdo (●) for the indicated times and the levels of glucose in the medium have been measured as described in Section 2, Experimental procedures. Data are expressed as average ± SD of two experiments performed in quadruplicate. Significance: **p < 0.01.

concentration of glucose is apparently unchanged in the medium of both control and treated cells. After 8 h of treatment, a decrease in the glucose concentration is observed, which is more pronounced in the medium of control cells (Fig. 8). 4. Discussion Astrocytes play an important role in the nervous system providing trophic factors and energetic substrates to neurons (Sofroniew and Vinters, 2010). Alterations in their functions contribute to the appearance of a variety of neurological pathologies. Gliomas, especially those of astrocytic origin, are particularly resistant to chemotherapy and are often characterized by a poor prognosis (Ohgaki and Kleihues, 2007). Neuroblastoma is the tumour with the higher incidence in infants. Around half of all cases are currently classified as high-risk for disease relapse, with overall survival rates less than 40% despite intensive multimodal therapy (Maris et al., 2007). Anticancer drugs can induce apoptosis and their cytotoxic effect is often mediated by this process (Ferreira et al., 2002). In a previous paper we have demonstrated that treatment with dCF plus dAdo in a human astrocytoma cell line (ADF) induces apoptosis, interferes with glucose metabolism, since a reduction in lactate released in the medium is observed in treated cells as an event preceding caspase-3 activation and cellular death, and produces in longterm incubations an increase in mitochondrial ROS. The incubation with antioxidants such as melatonin, Mito-Q or N-acetylcysteine did not increase survival (Garcia-Gil et al., 2012). In the present paper we have further investigated the mechanism of apoptosis induced by dCF plus dAdo in ADF cells. We are aware that this cell model might respond to the treatment in a different manner as compared with normal astrocytes. Nonetheless, our results may provide some hints on the mechanisms by which ADA deficiency affects brain metabolism. On the other hand, our cell model is the best suited for the understanding of the molecular pathways leading to apoptosis in a glioma of astrocytic origin. In addition, in order to ascertain whether the cytotoxic effect of ADA inhibition is restricted to ADF cells or can be extended to other tumour cells, we have used SHSY5Y cells, derived from a human neuroblastoma and we have observed that the combination of dAdo and dCF decreases viability also in this neuronal cell line. In the present work we have demonstrated that the treatment with dCF plus dAdo increases caspase-8 activity in astrocytoma cells.

The activation of caspase-8 precedes that of caspase-3. The mechanism of caspase-8 activation in our experimental conditions remains unknown. At present, we cannot exclude the involvement of the extrinsic pathway of apoptosis. We have previously demonstrated that cytochrome c is released after 15 h of treatment with dCF plus dAdo (Garcia-Gil et al., 2012). The release of other mitochondrial factors could be necessary to activate caspase-3 and/or caspase-8. It is known that Omi/HtrA2 and Smac/Diablo bind to inhibitor of apoptotic proteins (IAP), promoting the release of active caspases, including caspase-3. In addition, the protease Omi/HtrA2 is able to cleave some IAPs, such as the caspase-8 inhibitor Pea-15, a phosphoprotein enriched in astrocytes (Fiory et al., 2009). The activation of caspase-8 will then activate caspase-3 (Vande Walle et al., 2008). However, preliminary experiments (results not shown) have not demonstrated modification in Pea-15 expression between control and treated cells for 8 h. Since cathepsin D has been demonstrated to stimulate caspase-8 activation in neutrophils and pancreatic cells (Conus and Simon, 2008), we have incubated the cells in the presence of the cathepsin D inhibitor pepstatin A (up to 100 μM), but this treatment did not modify cell viability or caspase-8 activation in treated cells (data not shown). The involvement of calpains in cell death and the calpain crosstalk with caspases have been demonstrated in several models including astrocytes and glioblastoma cells (Das et al., 2010; Smith and Schnellmann, 2012; Takuma et al., 2004), but evidence for prosurvival/anti-apoptotic roles of calpains has also been obtained (Cervia et al., 2007; Tan et al., 2006). Our results do not exclude an involvement of this apoptotic pathway, because a slight, but significant, increase in viability, as compared with treated ADF cells, is observed in the presence of calpain inhibitor III. However, the inhibitor itself significantly decreases cell viability. For this reason, we decided to omit this compound from our further study. Lipoxygenase inhibitors such as NDGA and baicalein, able to protect different cells from oxidative stress (Chen et al., 2006; Guzmán-Beltrán et al., 2008) increase viability in ADF cells treated with dCF plus dAdo. Our results are in agreement with those reporting a protective effect of both NDGA and baicalein in several models of degeneration in the nervous system (Guzmán-Beltrán et al., 2008; Pallast et al., 2010; Yu et al., 2012). We have investigated whether the protective effect of baicalein and NDGA occurs at the initial or at the executive phases of apoptosis, measuring their effects on caspase-8 and caspase-3 activation. Both substances added alone damage control cells (approximately 40% and 20% decrease in cell viability with NDGA and baicalein alone, respectively) and increase caspase-8 and -3 activity. Such an antiproliferative and/ or proapoptotic effect has also been described in other cell types (Meyer et al., 2007). When added in the presence of dCF plus dAdo, baicalein decreases significantly caspase-3 but not caspase-8 activation. NDGA does not reduce caspase-3 or -8 activation in cells incubated with dCF plus dAdo, instead, caspase-3 activity is surprisingly higher when NDGA is present, despite the increased viability. NDGA could reduce a caspase-independent death, possibly preventing the nuclear localization of the apoptosis inducing factor (AIF) as described for other lipoxygenase inhibitors (Pallast et al., 2010). The lack of effect of and PD 146176, a 15-lipoxygenase inhibitor without antioxidant properties (Sendobry et al., 1997) on dCF plus dAdo treated cells appears to point to an antioxidant activity of NDGA and baicalein. However, the incubation with baicalein or NDGA partially reverts other effects induced by dCF plus dAdo such as reduction of lactate levels in the culture medium, increase in intracellular dATP and reduction of ATP. The formation of dATP is strictly related to the cytotoxic effect of dCF plus dAdo both in astrocytoma and neuroblastoma cells. Indeed, the addition of NH2dAdo to cells treated with the combination, by inhibiting the phosphorylation of dAdo and therefore the formation of dATP, brings about a complete recovery in cell viability. We have demonstrated

M. Garcia-Gil et al./Neurochemistry International 80 (2015) 14–22

that baicalein inhibits AdoK activity in ADF cells and indeed the reduction in dATP level reflects in an increase in cell viability. Therefore, the effect of baicalein could be due to this hitherto unknown action rather than or in addition to its antioxidant effect. The modification of the levels of lactate, ATP and dATP appears to be strictly correlated, but the link between these phenomena is not clear at the moment. Several mechanisms could explain the extracellular lactate reduction observed in ADF cells: decrease in glucose uptake, glycolysis inhibition or increased utilization of pyruvate by mitochondria. The decrease of ATP observed in cells treated with dCF plus dAdo might be caused by the ATP-consuming sequential steps of dAdo phosphorylation resulting in the formation of dATP. However, we do not observe an increase in ADP and AMP, the products of ATP dephosphorylation. We might speculate therefore that the adenylate pool is converted to Ado that, being ADA inhibited, should accumulate and leave the cell through an equilibrative transporter (Parkinson et al., 2006). Ado could bind to its specific receptors in ADF astrocytoma cells (Ceruti and Abbracchio, 2013) and, even though this hypothesis needs to be validated by experimental results, one may speculate that this event may result in a decrease in glycolysis rate, as described in the heart following treatment with Ado and with a receptor A1 agonist (Finegan et al., 1996). On the other hand, we may also hypothesize that the greater requirement of ATP for dATP synthesis may need a continuous regeneration of ATP through oxidative phosphorylation, therefore channelling pyruvate to mitochondria. Then, the greater use of glucose-derived pyruvate in the oxidative pathway rather than in lactate formation could result in the observed decrease of lactate released to the culture medium by ADF cells. In this regard, both the early decrease in mROS production and the hyperpolarization of the ΔΨm point to a lower rate of pyruvate utilization in dCF plus dAdo treated cells, instead of a channelling of pyruvate to oxidative metabolism. Also the increase in mitochondrial mass might reflect an attempt of the cell to cope with the decrease in mitochondrial activity. However, it must be noted that all these effects occur in all the experimental conditions, whereas only the combination of dCF plus dAdo results in lactate decrease and later in ADF cell death, thus indicating that the observed changes in mitochondrial activity appear unrelated to the effect on glucose metabolism, but rather linked to some early and specific dysfunction of mitochondrial physiology caused by dAdo unbalance. Interestingly the measure of the intracellular ROS by 2′,7′dichlorofluorescein, another widely used fluorescent probe, does not detect any alteration in cytoplasmic ROS after 8 h treatment with dAdo and dCF alone or in combination (data not shown), indicating that the observed mROS decrease in ADF cells is a very early event concerning only the mitochondrial compartment. On the other hand, both MT-ROS and dichlorofluorescein detect the larger and widespread ROS increase appearing after 15 h of treatment with dCF plus dAdo, associated with irreversible cellular dysfunction and typical of the late phases of cell damage, in accord to our previously reported results (Garcia-Gil et al., 2012). In order to have an insight regarding glucose utilization in our experimental conditions, we have measured the variation of glucose concentration in the extracellular medium both in control and treated ADF cells. We observe that glucose decreases significantly only after 8 h of incubation. This could indicate that ADF cells are using glycogen reserves first and only later do they use extracellular glucose. This is in agreement with the observations indicating that astrocytes mobilize glycogen even in the presence of glucose (Sickmann et al., 2009). In cells treated with dCF plus dAdo there is a minor decrease in glucose concentration in the medium, thus suggesting that the lower level of lactate in the medium of treated cells might be related to a decreased uptake of glucose. Overall, our results indicate that the treatment of ADF cells with dAdo and dCF in combination causes a decrease of lactate in the

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medium, a decrease of ATP, an increase of dATP, an increase in mitochondrial mass, and the activation of caspase-8 preceding that of caspase-3, resulting in apoptosis. Compounds that increase viability (NH2dAdo, NDGA, baicalein) also induce an increase of lactate and ATP and a decrease of dATP. The modification of the levels of lactate, ATP and dATP and the onset of the apoptotic program appear to be strictly correlated, but the link between these phenomena is not clear at the moment. Since baicalein inhibits AdoK, its protective effect on treated ADF cells could be due to this previously unknown action rather than or in addition to its antioxidant effect. In conclusion, this work demonstrates that a perturbation of purine metabolism induced by ADA inhibition leads to cell death in tumour cell models of astrocytic and neuronal origin, thus providing data which might be important to improve therapeutical approaches to these types of tumour. Conflict of interest Authors declare no conflicts of interest. The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements This work was supported by grants from University of Pisa. We would like to thank Dr. Grazia Della Sala and Marco Cicchini for their valuable help in adapting MatLab routines to the analysis of cell density and mitochondrial functions. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. doi:10.1016/0003-2697(76)90527-3. Camici, M., Turriani, M., Tozzi, M.G., Turchi, G., Cos, J., Alemany, C., et al., 1995. Purine enzyme profile in human colon-carcinoma cell lines and differential sensitivity to deoxycoformycin and 2-deoxyadenosine in combination. Int. J. Cancer 62, 176–183. doi:10.1002/ijc.2910620212. Camici, M., Micheli, V., Ipata, P.L., Tozzi, M.G., 2010. Pediatric neurological syndromes and inborn errors of purine metabolism. Neurochem. Int. 56, 367–378. doi:10.1016/j.neuint.2009.12.003. Ceruti, S., Abbracchio, M.P., 2013. Adenosine signaling in glioma cells. Adv. Exp. Med. Biol. 986, 13–30. doi:10.1007/978-94-007-4719-7_2. Cervia, D., Garcia-Gil, M., Simonetti, E., Di Giuseppe, G., Guella, G., Bagnoli, P., et al., 2007. Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases. Apoptosis 12, 1349–1363. doi:10.1007/s10495-007-0075-7. Chen, Y.C., Chow, J.M., Lin, C.W., Wu, C.Y., Shen, S.C., 2006. Baicalein inhibition of oxidative-stress-induced apoptosis via modulation of ERKs activation and induction of HO-1 gene expression in rat glioma cells C6. Toxicol. Appl. Pharmacol. 216, 263–273. doi:10.1016/j.taap.2006.05.008. Conus, S., Simon, H.U., 2008. Cathepsins: key modulators of cell death and inflammatory responses. Biochem. Pharmacol. 76, 1374–1382. doi:10.1016/ j.bcp.2008.07.041. Czapski, G.A., Czubowicz, K., Strosznajder, R.P., 2012. Evaluation of the antioxidative properties of lipoxygenase inhibitors. Pharmacol. Rep. 64, 1179–1188. Das, A., Banik, N.L., Ray, S.K., 2010. Flavonoids activated caspases for apoptosis in human glioblastoma T98G and U87MG cells but not in human normal astrocytes. Cancer 116 (1), 164–176. doi:10.1002/cncr.24699. Ferreira, C.G., Epping, M., Kruyt, F.E., Giaccone, G., 2002. Apoptosis: target of cancer therapy. Clin. Cancer Res. 8, 2024–2034. Finegan, B.A., Lopaschuk, G.D., Gandhi, M., Clanachan, A.S., 1996. Inhibition of glycolysis and enhanced mechanical function of working rat hearts as a result of adenosine A1 receptor stimulation during reperfusion following ischaemia. Br. J. Pharmacol. 118, 355–363. doi:10.1111/j.1476-5831.1996.tb15410.x. Fiory, F., Formisano, P., Perruolo, G., Beguinot, F., 2009. PED/PEA-15, a multifunctional protein controlling cell survival and glucose metabolism. Am. J. Physiol. Endocrinol. Metab. 297, E592–E601. doi:10.1152/ajpendo.00228.2009. Garcia-Gil, M., Pesi, R., Perna, S., Allegrini, S., Giannecchini, M., Camici, M., et al., 2003. 5’-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells. Neuroscience 117, 811–820. doi:10.1016/S03064522(02)00836-9. Garcia-Gil, M., Tozzi, M.G., Allegrini, S., Folcarelli, S., Della Sala, G., Voccoli, V., et al., 2012. Novel metabolic aspects related to adenosine deaminase inhibition in a human astrocytoma cell line. Neurochem. Int. 60, 523–532. doi:10.1016/ j.neuint.2012.02.008.

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