Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis.

OBJECTIVE AND DESIGN: Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. MATERIALS AND METHODS: Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 106 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. RESULTS: We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. CONCLUSIONS: In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

Carbon Tetrachloride Increases the Pro-inflammatory Cytokines Levels in Different Brain Areas of Wistar Rats: The Protective Effect of Acai Frozen Pulp.

Acai offers health benefits associated with its high antioxidante capacity, phytochemical composition, nutritional and sensory value. Therefore, the objective of this study was to evaluate the protective effect of acai frozen pulp on carbon tetrachloride (CCl4)-induced damage via modulation of anti- and pro-inflammatory cytokines in rat brain tissue. The rats were treated via oral (gavage) daily with water or acai frozen pulp for 14 days at a dose of 7 µL/g. On the 15th day, the animals in each group received a single intraperitoneal injection of CCl4 in a dose of 3.0 mL/kg or the same volume of mineral oil. After 4 h, the animals were euthanized by decapitation and the cerebral cortex, hippocampus and cerebellum were dissected and homogenated to evaluate the levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), interleukin 18 (IL-18), interleukin 6 (IL-6) and interleukin 10 (IL-10). Data were statistically analyzed by analysis of variance followed by the Tukey post hoc test. It was observed that CCl4 increased TNF-α, IL-1ß and IL-18 levels in all brain tissues, and that acai frozen pulp was able to prevent this increase. IL-6 and IL-10 brain tissue levels remained unchanged during all treatments. CCl4 experimental model was suitable to investigate brain tissue anti and pro-inflammatory cytokines. Acai frozen pulp prevented an increase in IL-1ß, IL-18 and TNF-α, while IL-6 and IL-10 levels remained unchanged. The precise pathway by which inflammation contribute to hepatic encephalopathy, as well as to how this pathway can be modulated, is still under investigation.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils.

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-ß2, PI3K, ERK, p38 and independent of Gαi protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

PRIMA-1, a mutant p53 reactivator, induces apoptosis and enhances chemotherapeutic cytotoxicity in pancreatic cancer cell lines.

TP53 mutation is a common event in many cancers, including pancreatic adenocarcinoma, where it occurs in 50-70 % of cases. In an effort to reactivate mutant p53 protein, several new drugs are being developed, including PRIMA-1 and PRIMA-1(Met)/APR-246 (p53 reactivation and induction of massive apoptosis). PRIMA-1 has been shown to induce apoptosis in tumor cells by reactivating p53 mutants, but its effect in pancreatic cancer remains unclear. Here we investigated the effects of PRIMA-1 on cell viability, cell cycle and expression of p53-regulated proteins in PANC-1 and BxPC-3 (mutant TP53), and CAPAN-2 (wild-type TP53) pancreatic cell lines. Treatment with PRIMA-1 selectively induced apoptosis and cell cycle arrest in p53 mutant cells compared to CAPAN-2 cells. The growth suppressive effect of PRIMA-1 was markedly reduced in p53 mutant cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1 induced cell death. Moreover, treatment with the thiol group donor N-acetylcysteine completely blocked PRIMA-1-induced apoptosis and reinforced the hypothesis that thiol modifications are important for PRIMA-1 biological activity. In combination treatments, PRIMA-1 enhanced the anti-tumor activity of several chemotherapic drugs against pancreatic cancer cells and also exhibited a pronounced synergistic effect in association with the Mdm2 inhibitor Nutlin-3. Taken together, our data indicate that PRIMA-1 induces apoptosis in p53 mutant pancreatic cancer cells by promoting the re-activation of p53 and inducing proapoptotic signaling pathways, providing in vitro evidence for a potential therapeutic approach in pancreatic cancer.

Tryptophan hydroxylase 2 (TPH2) gene polymorphisms and psychiatric comorbidities in temporal lobe epilepsy.

Psychiatric comorbidities are frequent in temporal lobe epilepsy (TLE). It is plausible that variance in serotonin-related genes is involved in the susceptibility of these associations. We report here the results on the association of tryptophan hydroxylase 2 (TPH2) gene polymorphisms with psychiatric comorbidities in TLE. A cohort study was conducted on 163 patients with TLE. We assessed the influence of the rs4570625 and rs17110747 polymorphisms in the TPH2 gene on psychiatric comorbidities in TLE. In patients with TLE, the presence of the T allele in the rs4570625 polymorphism was associated with psychotic disorders (OR=6.28; 95% CI=1.27-17.54; p=0.02), while the presence of the A allele in the rs17110747 polymorphism was associated with alcohol abuse (OR=20.33; 95% CI=1.60-258.46; p=0.02). Moreover, we identified male gender (OR=11.24; 95% CI=1.68-76.92; p=0.01) and family history of psychiatric disorder (OR=15.87; 95% CI=2.46-100; p=0.004) as factors also associated with alcohol abuse in TLE. Conversely, a family history of epilepsy was inversely associated with alcohol abuse (OR=0.03; 95% CI=0.001-0.60; p=0.02). Tryptophan hydroxylase 2 gene allele variants might be risk factors for psychiatric conditions in TLE. More specifically, we observed that the T allele in the rs4570625 polymorphism was associated with psychotic disorders, and the A allele in the rs17110747 TPH2 polymorphism was associated with alcohol abuse in patients with TLE. We believe that this study may open new research venues on the influence of the serotonergic system associated with psychiatric comorbidities in epilepsy.

The use of high-frequency audiometry increases the diagnosis of asymptomatic hearing loss in pediatric patients treated with cisplatin-based chemotherapy.

BACKGROUND: Cisplatin may cause permanent cochlear damage by changing cochlear frequency selectivity and can lead to irreversible sensorineural hearing loss. High-frequency audiometry (HFA) is able to assess hearing frequencies above 8,000 Hz; hence, it has been considered a high-quality method to monitor and diagnose early and asymptomatic signs of ototoxicity in patients receiving cisplatin. PROCEDURE: Forty-two pediatric patients were evaluated for hearing loss induced by cisplatin utilizing HFA, and its diagnostic efficacy was compared to that of standard pure-tone audiometry and distortion-product otoacoustic emissions (DPOAEs). The patient population consisted of those who signed an informed consent form and had received cisplatin chemotherapy between 1991 and 2008 at the Hospital de Clínicas de Porto Alegre Pediatric Unit, Brazil. RESULTS: Forty-two patients were evaluated. The median age at study assessment was 14.5 years (range 4-37 years). Hearing loss was detected in 24 patients (57%) at conventional frequencies. Alterations of DPOAEs were found in 64% of evaluated patients and hearing loss was observed in 36 patients (86%) when high-frequency test was added. The mean cisplatin dose was significantly higher (P = 0.046) for patients with hearing impairment at conventional frequencies. CONCLUSION: The results suggest that HFA is more effective than pure-tone audiometry and DPOAEs in detecting hearing loss, particularly at higher frequencies. It may be a useful tool for testing new otoprotective agents, beside serving as an early diagnostic method for detecting hearing impairment.

Ewing Sarcoma: influence of TP53 Arg72Pro and MDM2 T309G SNPs.

The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case-control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR-RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P=0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95% CI=1.22-9.21) with P=0.02. At the genotypic level, an association of the TT genotype with the control group (P=0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95% CI=1.03-8.58) with P=0.04 and 5.00 (95% CI=1.23-20.34) with P=0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.

Anti-EGFR therapy combined with neuromedin B receptor blockade induces the death of DAOY medulloblastoma cells.

PURPOSE: Medulloblastoma is the most common malignant childhood brain tumor for which the development of new molecularly targeted therapies is needed. Novel therapeutic targets under investigation include growth factor receptors. Here, we show that the combined inhibition of the epidermal growth factor receptor (EGFR) and neuromedin B receptor (NMBR, BB1) results in increased cell death in human medulloblastoma cell lines. METHODS: DAOY and D283 human medulloblastoma cells were treated with human recombinant neuromedin B (NMB, an NMBR agonist), the NMBR antagonist BIM-23127, the anti-EGFR monoclonal antibody cetuximab, or BIM-23127 combined with cetuximab. Cell death was examined with trypan blue cell counting. RESULTS: Both cell lines expressed mRNA for EGFR, NMB, and NMBR detected by reverse transcriptase polymerase chain reaction. Cetuximab at 10 µg/ml significantly reduced the number of DAOY cells, but did not affect D283 cells. NMB and BIM-23127 did not change cell number when used alone. However, cetuximab, at a dose that did not have an effect by itself, was able to reduce the number of DAOY cells when combined with BIM-23127. CONCLUSION: These results provide preliminary evidence that NMBR blockade can potentiate the antitumor effect of anti-EGFR therapy in medulloblastoma.

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition.

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.

The histone deacetylase inhibitor sodium butyrate in combination with brain-derived neurotrophic factor reduces the viability of DAOY human medulloblastoma cells.

PURPOSE: Histone deacetylase inhibitors (HDACis) are a promising class of anticancer agents for the treatment of brain tumors. HDACis can increase the expression of brain-derived neurotrophic factor (BDNF) in brain cells. We have previously shown that BDNF reduces the viability of medulloblastoma cells. The aim of the present study was to examine the effect of the HDACi sodium butyrate (NaB) combined with human recombinant BDNF (hrBDNF), on the viability of human medulloblastoma cell lines. METHODS: DAOY and ONS76 medulloblastoma cells were treated with NaB, hrBDNF, or NaB combined with hrBDNF. Cell viability was measured with the MTT assay. RESULTS: NaB combined with hrBDNF significantly reduced the viability of DAOY medulloblastoma cells. In ONS76 cells, NaB alone reduced viability, but the effect was not potentiated by hrBDNF. CONCLUSION: These findings provide early evidence for a rationale supporting further evaluation of HDACis and BDNF as a new combinatorial approach to inhibit the growth of medulloblastoma.

Methylation of BDNF and SLC6A4 Gene Promoters in Brazilian Patients With Temporal Lobe Epilepsy Presenting or Not Psychiatric Comorbidities.

The relationship between epilepsy and psychiatric comorbidities has been recognized for centuries, but its pathophysiological mechanisms are still misunderstood. It is biologically plausible that genetic or epigenetic variations in genes that codify important neurotransmitters involved in epilepsy as well as in psychiatric disorders may influence the development of the latter in patients with epilepsy. However, this possibility remains poorly investigated. The aim of this study was to evaluate the methylation profile of the BDNF and SLC6A4, two genes importantly involved in neuroplasticity, in patients with temporal lobe epilepsy (TLE) regarding the development or not of psychiatric comorbidities. One hundred and thirty-nine patients with TLE, 90 females and 45 males, were included in the study. The mean age of patients was 44.0 (+12.0) years, and mean duration of epilepsy was 25.7 (+13.3) years. The Structured Clinical Interview for DSM-IV shows that 83 patients (59.7%) had neuropsychiatric disorders and 56 (40.3%) showed no psychiatric comorbidity. Mood disorders were the most common psychiatric disorder observed, being present in 64 (46.0%) of all 139 patients. Thirty-three (23.7%) patients showed anxiety disorders, 10 (7.2%) patients showed history of psychosis and 8 (5.8%) patients showed history of alcohol//drug abuse. Considering all 139 patients, 18 (12.9%) demonstrated methylation of the promoter region of both BDNF and SLC6A4 genes. A significant decreased methylation profile was observed only in TLE patients with mood disorders when compared with TLE patients without a history of mood disorders (O.R. = 3.45; 95% C.I. = 1.08-11.11; p = 0.04). A sub-analysis showed that TLE patients with major depressive disorder mostly account for this result (O.R. = 7.20; 95% C.I. = 1.01-56.16; p = 0.042). A logistic regression analysis showed that the independent factors associated with a history of depression in our TLE patients was female sex (O.R. = 2.30; 95% C.I. = 1.02-5.18; p = 0.044), not controlled seizures (O.R. = 2.51; 95% C.I. = 1.16-5.41; p = 0.019) and decreased methylation in BDNF and SLC6A4 genes (O.R. = 5.32; 95% C.I. = 1.14-25.00; p = 0.033). Our results suggest that BDNF or SLC6A4 genes profile methylation is independently associated with depressive disorders in patients with epilepsy. Further studies are necessary to clarify these matters.

BDNF/TrkB content and interaction with gastrin-releasing peptide receptor blockade in colorectal cancer.

OBJECTIVE: Neurotrophin and neuropeptide pathways are emerging targets in cancer. Here we show that brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are present in colorectal cancer and that BDNF levels are increased in tumors compared to nontumor tissue. In addition, we investigate the role of BDNF in influencing the response of colorectal cancer cells to inhibition of gastrin-releasing peptide receptors (GRPR). METHODS: Fresh-frozen sporadic colorectal adenocarcinoma specimens and adjacent nonneoplastic tissue from 30 patients, as well as paraffin-embedded colorectal cancer samples from 21 patients, were used in this study. Cell proliferation and mRNA and protein levels were examined in HT-29 or SW620 cells treated with a GRPR antagonist, human recombinant BDNF (hrBDNF), a Trk antagonist K252a, or cetuximab. RESULTS: Expression of BDNF and TrkB was detected in tumor samples and cell lines. BDNF levels were higher in tumor samples compared to nonneoplastic tissue. BDNF expression and secretion were increased by GRPR blockade in HT-29 cells through a mechanism dependent on epidermal growth factor receptors. Treatment with hrBDNF prevented the effect of GRPR blockade on cell proliferation, whereas a Trk inhibitor reduced proliferation. CONCLUSIONS: BDNF and TrkB are present in colorectal cancer and might contribute to resistance to GRPR antagonists.

A gastrin-releasing peptide receptor antagonist stimulates Neuro2a neuroblastoma cell growth: prevention by a histone deacetylase inhibitor.

Gastrin-releasing peptide (GRP) acts as an autocrine growth factor for neuroblastoma and other types of cancer, and its cell-surface receptor, GRPR, is overexpressed in advanced-stage human neuroblastoma. GRPR knockdown and GRPR antagonism inhibit the growth of experimental neuroblastoma. Here we show that a GRPR antagonist promotes rather than inhibits the growth of neuroblastoma cells. The GRPR antagonist, RC-3095, at 0.1 nM inhibited, whereas at 100 nM stimulated proliferation of Neuro2a murine neuroblastoma cells in vitro. The stimulatory effects were prevented by the histone deacetylase inhibitor (HDACi), sodium butyrate (NaB). Expression of GRPR mRNA in Neuro2a cells was analyzed by RT-PCR. These findings provide evidence that a GRPR antagonist can stimulate the growth of cancer cells, and suggest that GRPR might interact with epigenetic mechanisms in regulating neuroblastoma cell growth.

Changes in IDH2, TET2 and KDM2B Gene Expression After Treatment With Classic Chemotherapeutic Agents and Decitabine in Myelogenous Leukemia Cell Lines.

BACKGROUND: Hematological malignancies are a heterogeneous group of tumors with increased proliferative and auto-replicative capacity. Despite treatment advances, post-treatment quality of life remains highly affected. Studies addressing the molecular mechanisms of these diseases are critical for the development of effective, rapid and selective therapies, since few therapeutic strategies succeed in being effective without triggering high-grade toxicities or debilitating late effects. Our aim of this study was to verify changes in the expression of genes involved in the malignant phenotype of hematological malignancies, by treating human cell lines in vitro with classic chemotherapeutic agents and the demethylating agent, decitabine. METHODS: KASUMI-1 and K-562 human myeloid leukemia cell lines were plated at a density of 3 × 104 cells/well and treated with increasing concentrations of different chemotherapeutic agents commonly used in the clinical setting. After 24 and 48 h of treatment, cell viability was tested, and RNA was extracted. Complementary DNA (cDNA) was synthesized and quantitative real-time polymerase chain reaction (qPCR) was performed to evaluate the gene expression of IDH2, TET2 and KDM2B. RESULTS: A modulation in gene expression was observed before and after treatment with classic chemotherapeutic agents. It was possible to demonstrate a difference in gene expression when cells were treated with chemotherapeutic agents or decitabine alone when compared to chemotherapeutic agents in association with decitabine. CONCLUSIONS: The genes tested, and the modulation of their expression during in vitro treatments suggest that IDH2, TET2, and KDM2B should be further investigated as potential biomarkers for ongoing treatment response and follow-up for patients diagnosed with hematological malignancies of the myeloid lineage.

Targeting Histone Deacetylase Activity to Arrest Cell Growth and Promote Neural Differentiation in Ewing Sarcoma.

There is an urgent need for advances in the treatment of Ewing sarcoma (EWS), an aggressive childhood tumor with possible neuroectodermal origin. Inhibition of histone deacetylases (HDAC) can revert aberrant epigenetic states and reduce growth in different experimental cancer types. Here, we investigated whether the potent HDAC inhibitor, sodium butyrate (NaB), has the ability to reprogram EWS cells towards a more differentiated state and affect their growth and survival. Exposure of two EWS cell lines to NaB resulted in rapid and potent inhibition of HDAC activity (1 h, IC50 1.5 mM) and a significant arrest of cell cycle progression (72 h, IC50 0.68-0.76 mM), marked by G0/G1 accumulation. Delayed cell proliferation and reduced colony formation ability were observed in EWS cells after long-term culture. NaB-induced effects included suppression of cell proliferation accompanied by reduced transcriptional expression of the EWS-FLI1 fusion oncogene, decreased expression of key survival and pluripotency-associated genes, and re-expression of the differentiation neuronal marker ßIII-tubulin. Finally, NaB reduced c-MYC levels and impaired survival in putative EWS cancer stem cells. Our findings support the use of HDAC inhibition as a strategy to impair cell growth and survival and to reprogram EWS tumors towards differentiation. These results are consistent with our previous studies indicating that HDis can inhibit the growth and modulate differentiation of cells from other types of childhood pediatric tumors possibly originating from neural stem cells.

DNA Methylation Events as Markers for Diagnosis and Management of Acute Myeloid Leukemia and Myelodysplastic Syndrome.

During the onset and progression of hematological malignancies, many changes occur in cellular epigenome, such as hypo- or hypermethylation of CpG islands in promoter regions. DNA methylation is an epigenetic modification that regulates gene expression and is a key event for tumorigenesis. The continuous search for biomarkers that signal early disease, indicate prognosis, and act as therapeutic targets has led to studies investigating the role of DNA in cancer onset and progression. This review focuses on DNA methylation changes as potential biomarkers for diagnosis, prognosis, response to treatment, and early toxicity in acute myeloid leukemia and myelodysplastic syndrome. Here, we report that distinct changes in DNA methylation may alter gene function and drive malignant cellular transformation during several stages of leukemogenesis. Most of these modifications occur at an early stage of disease and may predict myeloid/lymphoid transformation or response to therapy, which justifies its use as a biomarker for disease onset and progression. Methylation patterns, or its dynamic change during treatment, may also be used as markers for patient stratification, disease prognosis, and response to treatment. Further investigations of methylation modifications as therapeutic biomarkers, which may correlate with therapeutic response and/or predict treatment toxicity, are still warranted.

Combined Treatments with a Retinoid Receptor Agonist and Epigenetic Modulators in Human Neuroblastoma Cells.

Neuroblastoma (NB) is the most common extracranial solid childhood tumor accounting for around 15% of pediatric cancer deaths and most probably originates from a failure in the development of embryonic neural crest cells. Retinoids can inhibit the proliferation and stimulate differentiation of NB cells. In addition, epigenetic events involving changes in chromatin structure and DNA methylation can mediate the effects of retinoids; hence, the scope of this study is to investigate the use of retinoids and epigenetic drugs in NB cell lines. Here, we demonstrate that the combination of retinoid all trans-retinoic acid (ATRA) with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase is more effective in impairing the proliferation of human SH-SY5Y and SK-N-BE(2) NB cells than any drug given alone. Treatments also induced differential changes on the messenger RNA (mRNA) expression of retinoid receptor subtypes and reduced the protein content of c-Myc, the neuronal markers NeuN and ß-3 tubulin, and the oncoprotein Bmi1. These results suggest that the combination of retinoids with epigenetic modulators is more effective in reducing NB growth than treatment with single drugs.

In vitro antitumor effect of sodium butyrate and zoledronic acid combined with traditional chemotherapeutic drugs: a paradigm of synergistic molecular targeting in the treatment of Ewing sarcoma.

Histone deacetylase inhibitors and bisphosphonates have a promising future in the treatment of cancer as targeted anticancer drugs, particularly when used together or in combination with other cytotoxic agents. However, the effects of these combined treatments have not yet been systematically evaluated in Ewing sarcoma. The in vitro effects on cellular proliferation, viability and survival were investigated in two Ewing sarcoma cell lines, SK-ES-1 and RD-ES. The cell lines were treated with sodium butyrate, a histone deacetylase inhibitor and zoledronic acid, a bisphosphonate, alone, together or in combination with chemotherapeutic drugs recommended for clinical treatment of Ewing sarcoma. The data demonstrated that the combination of sodium butyrate and zoledronic acid had a synergistic cytotoxic effect at 72 h following treatment, persisting for 10-14 days post-treatment, in both cell lines tested. All combinations between sodium butyrate or zoledronic acid and the traditional antineoplastic drugs (doxorubicin, etoposide and vincristine) demonstrated a synergistic cytotoxic effect at 72 h in SK-ES-1 and RD-ES cells, except for the combinations of sodium butyrate with vincristine and of zoledronic acid with doxorubicin, which showed only an additive effect in RD-ES cell lines as compared to each agent alone. These acute effects observed in both Ewing sarcoma cell lines were confirmed by the clonogenic assay. The present data suggest that combining histone deacetylase inhibitors and bisphosphonates with traditional chemotherapeutic drugs is a promising therapeutic strategy for the treatment of Ewing sarcoma, and provides a basis for further studies in this field.