Frequency of the TP53 p.R337H mutation in a Brazilian cohort of pediatric patients with solid tumors.

TP53 p.R337H germline mutation is highly prevalent in the Southern region of Brazil. We sought to investigate TP53 p.R337H mutation in pediatric tumor samples from a population settled in a geographic area of high prevalence for this variant. Mutation assessment and genetic counseling for carriers/relatives were provided. 6/57 tumor samples were heterozygous for TP53 p.R337H. As expected, a high frequency was observed within adrenocortical tumors (3/3) and choroid plexus carcinomas (2/2). Interestingly, the TP53 R337H mutation was found in one case of pediatric rhabdomyosarcoma with Li-Fraumeni pedigree. Our finding expands the spectrum of childhood cancer associated with this germline mutation.

The TP53 p.R337H mutation is uncommon in a Brazilian cohort of pediatric patients diagnosed with ependymoma.

BACKGROUND: Ependymoma (EPN) is the third most common childhood cancer of the central nervous system. RELA fusion-positive EPN accounts for approximately 70% of all childhood supratentorial tumors and shows the worst prognosis among the supratentorial EPNs. TP53 mutation is infrequent in RELA fusions EPNs. In the population from the Southern region of Brazil, there is a high incidence of the germline TP53 p.R337H mutation that predisposes carriers to develop early-onset tumors. However, despite this high incidence, the frequency of this mutation among EPN patients remains to be determined. Here, we investigated the presence of the TP53 p.R337H mutation in a larger cohort of pediatric EPNs of three institutions located in the state of São Paulo, Brazil. METHODS: The TP53 p.R337H mutation was screened by conventional RT-PCR and Sanger sequencing in 49 pediatric EPNs diagnosed during the period from 1995 to 2016. RESULTS: We described for the first time a case of a 5-year-old girl with RELA fusion EPN with a heterozygous TP53 p.R337H mutation. CONCLUSIONS: The present finding indicates that the TP53 p.R337H germline mutation is uncommon in patients with EPN in Brazil and screening of pediatric patients RELA fusion EPN may be informative to better understand the role of TP53 germline mutations in the development and prognosis of these tumors.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

BACKGROUND: Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). METHODS: Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. RESULTS: Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. CONCLUSION: The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.

WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.

Tetra-O-methyl nordihydroguaiaretic acid, an inhibitor of Sp1-mediated survivin transcription, induces apoptosis and acts synergistically with chemo-radiotherapy in glioblastoma cells.

Glioblastoma (GBM), one of the most malignant human neoplasias, responds poorly to current treatment modalities, with temozolomide (TMZ) being the drug most frequently used for its treatment. Tetra-O-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependent on the Sp1 transcription factor, such as Survivin and Cdk1. In the present study we evaluated the gene expression of Survivin, its spliced variants and Cdk1 in GBM samples and cell lines. Moreover, we investigated the effects of M4N combined or not with TMZ and/or radiation on GBM primary cultures and cell lines. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyses using different schedules of administration (simultaneous and sequential) were performed on GBM cell lines and primary cultures based on the Chou-Talalay method. For clonogenic survival, doses of 2, 4, and 6 Gy of gamma radiation. were used. All Survivin-spliced variants and the Cdk1 gene were expressed in GBM samples (n = 16) and cell lines (n = 6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and the Survivin-ΔEx3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, and enhanced the effects of radiation, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased the mitotic index and arrested the cell cycle mainly in the G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation for GB treatment.

Spindle assembly checkpoint gene expression in childhood adrenocortical tumors (ACT): Overexpression of Aurora kinases A and B is associated with a poor prognosis.

BACKGROUND: Pediatric adrenocortical tumors (ACT) are rare malignancies and treatment has a small impact on survival in advanced disease and the discovery of potential target genes could be important in new therapeutic approaches. METHODS: The mRNA expression levels of spindle checkpoint genes AURKA, AURKB, BUB, and BUBR1 were analyzed in 60 children with ACT by quantitative real time PCR. The anticancer effect of ZM447439, an experimental AURK inhibitor, was analyzed in a primary childhood ACT culture carrying the TP53 p.R337H mutation. RESULTS: A significant association was observed between malignancy as defined by Weiss score ≥3 and higher AURKA (2.0-fold, P = 0.01), AURKB (7.0-fold, P = 0.007), and BUBR1 (5.8-fold, P = 0.007) gene expression, and between unfavorable event (death or relapse) and higher expression of AURKA (6.0-fold, P = 0.034) and AURKB (17-fold, P = 0.013). Overexpression of AURKA and AURKB was associated with lower event-free survival in uni- (P < 0.001 and P = 0.006, respectively) and multivariate (P = 0.002 and P = 0.03, respectively) analysis. Significant lower Event free survival (EFS) was also observed in patients with moderate/strong immunostaining to AURKA (P = 0.012) and AURKB (P = 0.045). ZM447439 was able to induce inhibition of proliferation and colony formation in a primary childhood ACT culture carrying the TP53 p.R337H mutation. CONCLUSION: Our results suggest that AURKA and AURKB overexpression in pediatric ACT may be related to more aggressive disease and the inhibition of these proteins could be an interesting approach for the treatment of these tumors.

Inhibition of nuclear factor-κB by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-κB (NF-κB) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-κB blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases.

Panobinostat (LBH589) increase survival in adult xenografic model of acute lymphoblastic leukemia with t(4;11) but promotes antagonistic effects in combination with MTX and 6MP.

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice measuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).

The therapeutic potential of Aurora kinases targeting in glioblastoma: from preclinical research to translational oncology.

Glioblastoma is the most common aggressive primary brain tumor. Standard care includes maximal safe surgical resection, radiation, and chemotherapy with temozolomide. However, the impact of this therapeutic approach on patient survival is disappointing and poor outcomes are frequently observed. Therefore, new therapeutic targets are needed to treat this potentially deadly tumor. Aurora kinases are one of today's most sought-after classes of therapeutic targets to glioblastoma therapy. They are a family of proteins composed of three members: Aurora-A, Aurora-B, and Aurora-C that play different roles in the cell division through regulation of chromosome segregation. Deregulation of these genes has been reported in glioblastoma and a progressive number of studies have shown that inhibition of these proteins could be a promising strategy for the treatment of this tumor. This review discusses the preclinical and early clinical findings on the potential use of the Aurora kinases as new targets for the treatment of glioblastoma. KEY MESSAGES: GBM is a very aggressive tumor with limited therapeutic options. Aurora kinases are a family of serine/threonine kinases implicated in GBM pathology. Aurora kinases are critical for glioblastoma cell growth, apoptosis, and chemoresistance. Inhibition of Aurora kinases has a synergistic or sensitizing effect with chemotherapy drugs, radiotherapy, or with other targeted molecules in GBM. Several Aurora kinase inhibitors are currently in clinical trials.

SHOC2 scaffold protein modulates daunorubicin-induced cell death through p53 modulation in lymphoid leukemia cells.

SHOC2 scaffold protein has been mainly related to oncogenic ERK signaling through the RAS-SHOC2-PP1 phosphatase complex. In leukemic cells however, SHOC2 upregulation has been previously related to an increased 5-year event-free survival of pediatric pre-B acute lymphoid leukemia, suggesting that SHOC2 could be a potential prognostic marker. To address such paradoxical function, our study investigated how SHOC2 impact leukemic cells drug response. Our transcriptome analysis has shown that SHOC2 can modulate the DNA-damage mediated by p53. Notably, upon genetic inhibition of SHOC2 we observed a significant impairment of p53 expression, which in turn, leads to the blockage of key apoptotic molecules. To confirm the specificity of DNA-damage related modulation, several anti-leukemic drugs has been tested and we did confirm that the proposed mechanism impairs cell death upon daunorubicin-induced DNA damage of human lymphoid cells. In conclusion, our study uncovers new insights into SHOC2 function and reveals that this scaffold protein may be essential to activate a novel mechanism of p53-induced cell death in pre-B lymphoid cells.

Wnt/ß-catenin activation cooperates with loss of p53 to cause adrenocortical carcinoma in mice.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with limited therapeutic options. The lack of mouse models that recapitulate the genetics of ACC has hampered progress in the field. We analyzed The Cancer Genome Atlas (TCGA) dataset for ACC and found that patients harboring alterations in both p53/Rb and Wnt/ß-catenin signaling pathways show a worse prognosis compared with patients that harbored alterations in only one. To model this, we utilized the Cyp11b2(AS)Cre mouse line to generate mice with adrenocortical-specific Wnt/ß-catenin activation, Trp53 deletion, or the combination of both. Mice with targeted Wnt/ß-catenin activation or Trp53 deletion showed no changes associated with tumor formation. In contrast, alterations in both pathways led to ACC with pulmonary metastases. Similar to ACCs in humans, these tumors produced increased levels of corticosterone and aldosterone and showed a high proliferation index. Gene expression analysis revealed that mouse tumors exhibited downregulation of Star and Cyp11b1 and upregulation of Ezh2, similar to ACC patients with a poor prognosis. Altogether, these data show that altering both Wnt/ß-catenin and p53/Rb signaling is sufficient to drive ACC in mouse. This autochthonous model of ACC represents a new tool to investigate the biology of ACC and to identify new treatment strategies.

Notch pathway in ependymoma RELA-fused subgroup: upregulation and association with cancer stem cells markers expression.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

Molecular characterization of Wnt pathway and function of ß-catenin overexpression in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB is currently classified into four molecular subgroups (Wnt, Shh, Group 3, and Group 4). The wingless (Wnt) pathway is responsible for embryonic development and is deregulated in MB. We analyzed the activation of the Wnt pathway in MB cell lines and its correlation with the Shh pathway, with emphasis on the importance of cellular characterization. Transient ß-catenin transfection led to an increase in the ß-catenin gene and protein expression in MB cell lines. Wnt pathway activation resulted in a reduced number of colonies in all cell lines studied and a significant increase in the G2/M cell cycle phase only in ONS-76 cells. Regarding the Shh pathway, transfection caused a reduced expression of the PTCH1 and SMO genes only in the UW473 cells. Further studies are needed to understand the mechanism underlying the molecular events associated with the effects of Wnt activation in MB.

Antitumour activity of AMG 900 alone or in combination with histone deacetylase inhibitor SaHa on medulloblastoma cell lines.

OBJECTIVES: Medulloblastoma (MB) is the most common malignant childhood brain tumour. Aurora kinases are essential for cell division and are primarily active during mitosis. Recently, the combination of aurora kinases inhibitors (iAURK) and histone deacetylase inhibitors (iHDAC) has shown potential antitumour effects and had significant biological effects in preclinical cancer models. In this study, we analysed the effects of the pan-aurora kinases inhibitor AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on paediatric MB cell lines (UW402, UW473 and ONS-76). METHODS: Cell proliferation was measured by XTT assay, apoptosis was determined by flow cytometry and clonogenic capacity was studied. qRT-PCR assays were used to determine the mRNA expression in MB cell lines after treatment. Drug combination analyses were made based on Chou-Talalay method. RESULTS: AMG 900 caused the inhibition of cell proliferation, diminution of clonogenic capacity and increased the apoptosis rate in cell lines (P < 0.05). A synergistic effect in the AMG900-SaHa combination was evidenced on the inhibition of cell proliferation in all cell lines, especially in sequential drug treatment. Moreover, the combination of these drugs reached 100% of the inhibition in colony formation (synergistic effect). The treatment with AMG 900 increased the p21 and GDF15 expression, but did not alter the TP53 in one of the cell lines. CONCLUSIONS: These results indicate that AMG 900 may be a promising drug for the adjuvant treatment of MB, mainly when combined with iHDAC.

Chromosomal heterogeneity and instability characterize pediatric medulloblastoma cell lines and affect neoplastic phenotype.

Chromosomal heterogeneity is a hallmark of most tumors and it can drive critical events as growth advantages, survival advantages, progression and karyotypic evolution. Medulloblastoma (MB) is the most common malignant central nervous system tumor in children. This work attempted to investigate chromosomal heterogeneity and instability profiles of two MB pediatric cell lines and their relationship with cell phenotype. We performed GTG-banding and cytokinesis-block micronucleus cytome assays, as well as morphological characterization, cell population doubling time, colony-forming efficiency, and chemo-sensitivity assays in two pediatric MB cell lines (UW402 and UW473). Both MB cells showed a high chromosomal heterogeneity. UW473 cells showed ~2 fold higher both clonal- and non-clonal chromosomal alterations than UW402 cells. Besides, UW473 showed two clonal-groups well-differentiated by ploidy level (<2n> and <4n>) and also presented a significantly higher number of chromosomal instability biomarkers. These results were associated with high morphological heterogeneity and survival advantages for UW473 and proliferation advantages for UW402 cells. Moreover, UW473 was significantly more sensitive to methotrexate, temozolomide and cisplatin while UW402 cells were more sensitive to doxorubicin. These data suggest that distinct different degrees of karyotypic heterogeneity and instability may affect neoplasic phenotype of MB cells. These findings bring new insights into cell and tumor biology.

Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells.

BACKGROUND: Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that significantly improve the patient survival. Altered expression of the Aurora kinases was found in different malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the effects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma. MATERIALS AND METHODS: RT-PCR assays were used to determine the mRNA expression in glioblastoma tumor samples and in the cell lines. Cell proliferation was measured by XTT assay, and apoptosis was determined by flow cytometry. Drug combination analyses were made based in Chou-Talalay method. Gamma radiation for clonogenic survival used the doses of 2, 4 and 6 Gy. Changes in Aurora B level were assessed by Western blot analysis. RESULTS: Aurora A and B were expressed in glioblastoma samples as well as in the glioblastoma cell lines (n = 6). Moreover, ZM447439, a selective Aurora kinase inhibitor, decreased the proliferation separately and synergistically with temozolomide in primary cultures and cell lines of glioblastoma. ZM also enhanced the effects of radiation on the two cell lines studied (U343 and U251), mainly when associated with TMZ in U343 cells. Treatment with ZM induced apoptotic cell death and diminished Aurora B protein level. CONCLUSIONS: These data suggest that Aurora kinase inhibition may be a target for glioblastoma treatment and could be used as adjuvant to chemo- and radiotherapy.

Expression profile of apoptosis-related genes in childhood adrenocortical tumors: low level of expression of BCL2 and TNF genes suggests a poor prognosis.

BACKGROUND: Impaired apoptosis has been implicated in the development of childhood adrenocortical tumors (ACT), although the expression of apoptosis-related gene expression in such tumors has not been reported. METHODS: The mRNA expression levels of the genes CASP3, CASP8, CASP9, FAS, TNF, NFKB, and BCL2 were analyzed by quantitative real-time PCR in consecutive tumor samples obtained at diagnosis from 60 children with a diagnosis of ACT and in 11 non-neoplastic adrenal samples. BCL2 and TNF protein expression was analyzed by immunohistochemistry. RESULTS: A significant association was observed between tumor size ≥100 g and lower expression levels of the BCL2 (P=0.03) and TNF (P=0.05) genes; between stage IV and lower expression levels of CASP3 (P=0.008), CASP9 (P=0.02), BCL2 (P=0.002), TNF (P=0.05), and NFKB (P=0.03); Weiss score ≥3 and lower expression of TNF (P=0.01); unfavorable event and higher expression values of CASP9 (P=0.01) and lower values of TNF (P=0.02); and death and lower expression of BCL2 (P=0.04). Underexpression of TNF was associated with lower event-free survival in uni- and multivariate analyses (P<0.01). Similar results were observed when patients with Weiss score <3 were excluded. CONCLUSION: This study supports the participation of apoptosis-related genes in the biology and prognosis of childhood ACT and suggests the complex role of these genes in the pathogenesis of this tumor.

Antiproliferative effects of Tubi-bee propolis in glioblastoma cell lines.

Propolis is a resin formed by a complex chemical composition of substances that bees collect from plants. Since ancient times, propolis has been used in folk medicine, due to its biological properties, that include antimicrobial, anti-inflammatory, antitumoral and immunomodulatory activities. Glioblastoma is the most common human brain tumor. Despite the improvements in GBM standard treatment, patients' prognosis is still very poor. The aim of this work was to evaluate in vitro the Tubi-bee propolis effects on human glioblastoma (U251 and U343) and fibroblast (MRC-5) cell lines. Proliferation, clonogenic capacity and apoptosis were analyzed after treatment with 1 mg/mL and 2 mg/mL propolis concentrations for different time periods. Additionally, glioblastoma cell lines were submitted to treatment with propolis combined with temozolomide (TMZ). Data showed an antiproliferative effect of tubi-bee propolis against glioblastoma and fibroblast cell lines. Combination of propolis with TMZ had a synergic anti-proliferative effect. Moreover, propolis caused decrease in colony formation in glioblastoma cell lines. Propolis treatment had no effects on apoptosis, demonstrating a cytostatic action. Further investigations are needed to elucidate the molecular mechanism of the antitumor effect of propolis, and the study of its individual components may reveal specific molecules with antiproliferative capacity.