Kleiton Silva Borges

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.

Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. Despite the advances in treatment, about 20% of patients relapse and/or die, indicating the need for different therapies for this group. Zebularine (ZB) is a potent DNA methyltransferase (DNMT) inhibitor and has been associated with gene demethylation and enhancement of tumor chemosensitivity. This study aimed to evaluate the effects of ZB, alone or combined with chemotherapeutics (methotrexate and vincristine), on childhood ALL cell lines. Cell proliferation, apoptosis, and clonogenic capacity were studied in Jurkat and ReH cell lines. Bisulfite modification, followed by methylation-specific PCR was carried out to evaluate aryl hydrocarbon receptor (AhR) methylation status. Gene expression of DNMT1, DNMT3a, DNMT3b, and AhR was assessed using qRT-PCR. Both cell cultures were sensitive to ZB, showing a dose-dependent and time-dependent response (P<0.05). ZB induced apoptosis and decreased clonogenic capacity in both cell lines. Combination with methotrexate resulted in a strong synergistic effect, whereas combination with vincristine led to an antagonistic response in both cell lines. ZB treatment decreased gene expression of the three DNMTs and induced AhR gene promoter demethylation and its re-expression. These results indicate that ZB may be a promising drug for the adjuvant treatment of ALL, mainly when combined with methotrexate.

Spindle Assembly Checkpoint Gene Expression in Childhood Adrenocortical Tumors (ACT): Overexpression of Aurora Kinases A and B Is Associated With a Poor Prognosis

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.

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-&kgr;B (NF-&kgr;B) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-&kgr;B 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.

Update on the Use of l-Asparaginase in Infants and Adolescent Patients with Acute Lymphoblastic Leukemia

Great improvements have been made in acute lymphoblastic leukemia (ALL) treatment in the past decades, especially due to the use of L-asparaginase (L-ASP). Despite the significant success rate, several side effects mainly caused by toxicity, asparaginase silent inactivation, and cellular resistance, encourage an open debate regarding the optimal dosage and formulation of L-ASP. Alternative sources of asparaginases have been constantly investigated in order to overcome hypersensitivity clinical toxicity. Additionally, genomic modulation as gene expression profiling, genetic polymorphisms, and epigenetic changes is also being investigated concerning their role in cellular resistance to L-ASP. Understanding the mechanisms that mediate the resistance to L-ASP treatment may bring new insights into ALL pathobiology and contribute to the development of more effective treatment strategies. In summary, this review presents an overview on L-ASP data and focuses on cellular mechanisms underlying resistance and alternative therapies for the use of asparaginase in childhood ALL treatment.

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.

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

BackgroundGlioblastoma 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).MethodsCell 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.ResultsViability 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.ConclusionThe 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.

Hypoxia-related gene expression profile in childhood acute lymphoblastic leukemia: prognostic implications

Abstract A cellular hypoxic condition is a key event in several human cancers, but knowledge about its role in childhood acute lymphoblastic leukemia (ALL) is very limited. In the present study, the gene expression profile of hypoxia-related genes (HIF1A, CA9, VEGF and SCL2A1) was evaluated in bone marrow samples of 113 pediatric patients. HIF1A mRNA up-regulation was significantly associated with higher 5-year event-free survival rates in patients with B-ALL as well as in the overall ALL population in both univariate and multivariate analysis (p = 0.023 and p = 0.041, respectively). In gene expression analysis, low oxygen levels promoted HIF1A activation in a time-dependent manner, in both ALL cell lines. In vitro cytotoxic assays suggested an initial trend toward hypoxia-related resistance in the first 24 h, but evaluation at later time points (48–72 h) clearly showed that there was no relevant difference in drug response when comparing hypoxic and normal oxygen level conditions. Modulation of mRNA expression of several hypoxia-related genes was also observed after hypoxic exposure in a cell specific manner, suggesting that HIF1A mRNA expression could play a different role in specific subtypes of leukemia. Despite the remaining questions regarding hypoxia-mediated mechanisms, these findings could be helpful to provide new insights into the role of hypoxia in childhood ALL.

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.

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

Abstract 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.