ORIGINAL RESEARCH ARTICLE
Journal of
Inorganic Phosphate Enhances Sensitivity of Human Osteosarcoma U2OS Cells to Doxorubicin via a p53-Dependent Pathway
Cellular Physiology
ANNAMARIA SPINA, LUCA SORVILLO, FRANCESCA DI MAIOLO, ANTONIETTA ESPOSITO, RAFFAELLA D’AURIA, DAVIDE DI GESTO, EMILIO CHIOSI, AND SILVIO NAVIGLIO* Department of Biochemistry and Biophysics, Medical School, Second University of Naples, Naples, Italy Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53-wild type U2OS cells (and not of p53-null Saos and p53-mutant MG63 cells) by slowing-down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin-induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub-G1 population, Bcl-2 downregulation, caspase-3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combinationinduced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine-alpha. Moreover, the doxorubicin-induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53-dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. ß 2012 Wiley Periodicals, Inc.
Osteosarcoma is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents (Chou et al., 2008). Surgery, radiotherapy, and high-dose chemotherapy (with agents such as doxorubicin, methotrexate, cisplatin, etoposide, and ifosfamide) are mainly effective in patients with localized disease and have improved overall survival over the last several years (Dai et al., 2011). However, clinically evident metastatic disease is present in 10–20% of patients at diagnosis. Despite aggressive treatment, more than one-third of patients develop recurrent high-grade osteosarcomas, with metastatic disease typically affecting the lung, liver, and bone itself, so that the 5-year survival rates are still not more than 60%. The frequent acquisition of drugresistant phenotypes and occurrence of second malignancies associated with chemotherapy remain serious problems (Kim and Helman, 2009). Moreover, toxic effects of chemotherapy still remain a major drawback in treatment of osteosarcoma patients. Thus, there is a pressing need for the development of new and alternative approaches to the treatment of osteosarcoma (Hattinger et al., 2010). Combination chemotherapy has received more attention in order to find compounds that could increase the therapeutic index of clinical anticancer drugs (Gutierrez et al., 2009). In this regard, dietary supplements, phytotherapeutic agents, and naturally occurring molecules (such as silibinin, resveratrol, plumbagin, benzyl isothiocyanate, 2-methoxyestradiol, DDTD) with antitumor activity and with the least toxicity to normal tissues are suggested as possible candidates to be investigated for their synergistic efficacy in combination with antineoplastic drugs (Raina and Agarwal, 2007; Chen et al., 2008; Maran et al., 2008; Kim et al., 2010; Szekeres et al., 2011; Tian et al., 2012). Inorganic phosphate (Pi) is an essential nutrient to living organisms. It plays a key role in diverse physiological functions, ß 2 0 1 2 W I L E Y P E R I O D I C A L S , I N C .
including osteoblast differentiation and skeletal mineralization (Yoshiko et al., 2007). Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption and depends mainly on the activity of Na/Pi cotransporters (Takeda et al., 2004). Pi is abundant in the diet, and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. Adequate control of Pi homeostasis is crucial, as a moderate increase in serum Pi Abbreviations: cAMP, 30 -50 -cyclic adenosine monophosphate; PKA, protein kinase A; ERK, extracellular signal-regulated kinases; MAPK, mitogen-activated protein kinases; MEK-1, mitogenactivated kinase kinase; Rap1, Ras-associated protein-1; Epac, exchange proteins activated by cAMP; Pi, inorganic phosphate; GSTP1, human glutathione S-transferase P1; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; mdm2, murine double minute 2; PARP, poly-(ADP-ribose) polymerase; DDTD, 30 ,40 -dichloro-3(3,4-dichlorophenylacetyl)-2,4,6-trihydroxydeoxybenzoin. Contract grant sponsor: PRIN 2009. *Correspondence to: Silvio Naviglio, Department of Biochemistry and Biophysics, Medical School, Second University of Naples, Via L. De Crecchio 7, 80138 Naples, Italy. E-mail:
[email protected] Manuscript Received: 2 March 2012 Manuscript Accepted: 22 May 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 1 June 2012. DOI: 10.1002/jcp.24124
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concentration and polymorphisms in genes involved in Pi metabolism may result in bone impairment and influence the aging process and lifespan (Prie´ et al., 2005). Relevantly, Pi is emerging as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression, and protein abundance in many cell types (Khoshniat et al., 2011). Recently, we have provided evidence that Pi inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi (Naviglio et al., 2006, 2011). Tumor suppressor pathways governed by p53 and Rb genes are known to be involved in the pathogenesis of osteosarcoma (Giordano et al., 2007; Ottaviani and Jaffe, 2009). Particularly, mutations or inactivation of p53 are found in almost 50% of OS patients and are thought to be associated with poor prognosis and drug resistance (Ognjanovic et al., 2012). Strategies aimed to restoration and up-regulation of p53 function are being actively investigated and considered a promising way to treat human tumors including osteosarcoma (Ganjavi et al., 2006; Ternovoi et al., 2006; Graat et al., 2007; Oshima et al., 2007; Yuan et al., 2007; Hedstro¨m et al., 2008). Moreover, available evidence suggests that the molecular profile may affect biological response of tumor cells, thereby suggesting to design combined therapies based on the disease molecular background (Le Tourneau et al., 2010). In this study, we asked whether Pi could affect chemosensitivity of osteosarcoma cells and investigated the possible involvement of p53 and the underlying mechanisms. Our data indicate that Pi is capable of inducing sensitization to doxorubicin in a p53-dependent manner and through a mechanism involving Erk1/2 down-regulation in U2OS osteosarcoma cells, implying that targeting Pi levels and its signaling might represent a novel simple way for therapeutic intervention in osteosarcoma. Materials and Methods Materials
All cell culture materials were from Gibco–Life Technologies (Gaithersburg, MD). Doxorubicin and p53 inhibitor pifithrine-a was purchased from Sigma (Sigma–Aldrich, St. Louis, MO). Antitubulin antibodies were obtained from Oncogene-Calbiochem (La Jolla, CA). Anti-procaspase-3 and anti-poly (ADP ribose) polymerase (PARP) antibodies were obtained from Upstate (Lake Placid, NY). Anti-p-ERK and anti-Bad antibodies were obtained from Cell Signaling Technology (Danvers, MA). All other antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). Cell culture and treatments
Human osteosarcoma U2OS, Saos-2, and MG-63 cell lines were obtained from the American Type Culture Collection (Rockville, MD). U2OS, Saos-2, and MG-63 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (FBS) and cultured at 378C in a 5% CO2 humidified atmosphere. Unless noted, all experiments were done in the above medium which contains 1 mM of Pi, and concentrations listed in the figures are final Pi medium concentrations. Added Pi was in the form of NaPO4, pH 7.4, from Sigma (Naviglio et al., 2006; Camalier et al., 2010). Doxorubicin was dissolved in ddH2O, stored at 48C and diluted with culture medium to final concentrations indicated in the figures (Zou et al., 2010). Typically, subconfluent cells were split (5 105/10 cm plate) and grown in 10% serum containing medium. After 24 h, the medium was removed, the cells were washed with PBS and incubated with 10% FBS fresh medium (time 0), supplemented or not with Pi and JOURNAL OF CELLULAR PHYSIOLOGY
doxorubicin, alone or in combination, and grown for the times and at concentrations indicated in the figures. Floating cells were recovered from culture medium by centrifugation, and adherent cells were harvested by trypsinization. Both floating and adherent cells were used in experiments aimed to study expression of proteins involved in apoptosis and to perform FACs analysis. Cell proliferation assay
Viable cells were determined by the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay, as previously described (Naviglio et al., 2010). Briefly, cells were seeded in 96-multiwell plates at the density of 5 103 cells/well. Cells were treated with Pi and doxorubicin alone or in combination for up to 72 h (see the figure legends). Before harvesting, 100 ml of MTT solution (5 mg/ml) was added to each well and incubated at 378C for 3 h, then the formazan product was solubilized by the addition of 100 ml 0.04 N HCl isopropanol. The optical density of each sample was determined by measuring the absorbance at 570 nm versus 650 nm using an enzyme-linked immunosorbent assay reader (Molecular Device Inc, Silicon Valley, CA). Cell proliferation assays were performed at least three times (in replicates of six wells for each data point in each experiment). Data are presented as means standard deviation (SD) for a representative experiment. Evaluation of apoptosis by flow cytometry
After drug treatment, cells were recovered as described in the above ‘‘cell culture and treatments’’ paragraph, fixed by resuspension in 70% ice-cold methanol/PBS and incubated overnight at 48C. After fixing, samples were pelleted at 400g for 5 min, and pellets were washed once with ice-cold PBS and centrifuged for a further 5 min. Pellets were resuspended in 0.5 ml DNA staining solution (50 mg/ml of propidium iodide, PI, and 100 mg RNase A in PBS), and incubated at 378C for 1 h in the dark. Samples were transferred to 5-ml Falcon tubes and stored on ice until assayed. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett-Packard computer (mod. 310) for data analysis. For the evaluation of intracellular DNA content, at least 20,000 events for each point were analyzed, and regions were set up to acquire quantitative data of cells with fragmented DNA (sub-G1 or apoptotic events) compared with the events that fell into the normal G1, S, G2 regions (Naviglio et al., 1998, 2009). Small interfering RNA transfection
The U2OS cells were plated onto six-well plates at a density of 3 105 cells/well with growth medium without antibiotics. After overnight incubation, transfection was performed at a confluency of 50% by using Opti-MEM media (Gibco-Life Technologies), Lipofectamine 2000 (Gibco-Life Technologies), and specific siRNA for p53 (Cell Signaling Technology) or non-specific siRNA (Santa Cruz Biotechnology), according to the manufacturer’s recommendations. Six hours later, the medium was replaced with growth medium without antibiotics. After transfection for 24 h, the cells were trypsinized, counted, and used, in part, to check p53 protein levels by immunoblotting analysis, or sub-seeded onto 96-well plates and subjected to MTT assay. Preparation of cell lysates
Cell extracts were prepared as follows. Briefly, 3–5 volumes of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 mg/ml aprotinin, leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added to recovered cells. After incubation on ice for 1 h, samples were centrifuged at 18,000g in an Eppendorf microcentrifuge for 15 min at 48C and the supernatant (SDS total extract) was recovered. Some aliquots were taken for protein quantification according to Bradford
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method (Bradford, 1976); others were diluted in 4 Laemmli buffer, boiled and stored as samples for immunoblotting analysis. Immunodetection of proteins
Typically, we employed 20–40 mg of total extracts for immunoblotting. Proteins from cell preparations were separated by SDS–PAGE and transferred onto nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus BioRad (Hercules, CA). II goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad), were used as a detection system (ECL) according to the manufacturer’s instructions Amersham Biosciences (Buckinghamshire, UK). Statistical analysis
Most of experiments were performed at least three times with replicate samples, except where otherwise indicated. Data are plotted as mean SD. The means were compared using analysis of variance (ANOVA) plus Bonferroni’s t-test. P-values of
