Augusto Faria Andrade

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.

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.

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.

The aurora kinase inhibitor Amg 900 increases apoptosis and induces chemosensitivity to anticancer drugs in the Nci-h295 adrenocortical carcinoma cell line

Adrenocortical tumor (ACT) is a malignancy with a low incidence rate and the current therapy for advanced disease has a limited impact on overall patient survival. A previous study from our group suggested that elevated expression of aurora-A and aurora-B is associated with poor outcome in childhood ACT. Similar results were also reported for adult ACTs. The present in-vitro study shows that AMG 900 inhibits aurora kinases in adrenocortical carcinoma cells. AMG 900 inhibited cell proliferation in NCI-H295 cells as well as in the ACT primary cultures and caused apoptosis in the cell line NCI-H295. Furthermore, it potentialized the mitotane, doxorubicin, and etoposide effects on apoptosis induction and acted synergistically with mitotane and doxorubicin in the inhibition of proliferation. In addition, we found that AMG 900 activated Notch signaling and rendered the cells sensitive to the combination of AMG 900 and Notch signaling inhibition. Altogether, these data show that aurora kinases inhibition using AMG 900 may be an adjuvant therapy to treat patients with invasive or recurrent adrenocortical carcinomas.

HG-17THE HISTONE DEACETYLASE INHIBITOR PCI-24781 SHOWS ANTIPROLIFERATIVE, PRO-APOPTOTIC AND RADIOSENSITIZING EFFECTS ON PEDIATRIC GLIOBLASTOMA CELL-LINES

The acetylation of DNA can modulate the expression of genes involved in the development and progression of malignancies. Histone deacetylase (HDACs) inhibitors are clinically active and well tolerated in the treatment of a wide variety of tumors. HDACs inhibitors can sensitize cell response to ionizing radiation, potentially reducing treatment doses and side effects. OBJECTIVES: To assess the potential radiosensitizer effect of abexinostat (PCI-24781), a new and potent pan-HDAC inhibitor in two pediatric glioblastoma cell-lines (SF188 and KNS42). METHODS: The effect of PCI-24781 on proliferation rates, clonogenic capacity and apoptosis were compared prior and following cell irradiation. The clonogenic assay was performed without irradiation (48h of incubation) at the doses of 0.25, 0.5, 1 and 2uM of PCI-24781. The clonogenic assay combined with irradiation was performed at 48h using the inhibitor IC30 for each cell-line in combination with irradiation doses of 0.5, 1, 2 and 4Gy. For cell proliferation assays, the intervals of 24h, 48h, 72h and 96h at the doses of 0.5, 1, 2, 4, 8 and 16uM of PCI-24781 were selected for study. Apoptosis were evaluated at 48h-cultures at the doses of 2, 4, 8 and 16uM. RESULTS: The HDAC inhibitor repressed cell proliferation, displaying a more significant effect at 48h in the dose of 2uM (p < 0.05). PCI-24781 also induced apoptosis (p < 0,05); the percentage of cell death for SF188 and KNS42 (at 16uM) was 40% and 55% respectively. Cells demonstrated massive decrease in colony formation with PCI-24781; this effect was more pronounced when drug was combined with irradiation (p < 0.001). CONCLUSION: These data demonstrate potential radiosensitizer and antiproliferative effects of PCI- 24781 on pediatric glioblastoma cell-lines. This study will now focus on key proteins responsible for the double-strand breaks repair caused by irradiation, trying to elucidate epigenetic pathways potentially involved in the therapy with PCI-24781. Financial Support: Fapesp (process no. 2013/15891-8).

Novel histone deacetylase inhibitors for the treatment of pediatric brain tumors.

Pediatric brain tumors (BT) represent a broad group of malignancies that affect children, displaying different degrees of aggressiveness and prognosis. Current studies demonstrate a crosslink between genetic and epigenetic changes within these tumors. Histone modifications are key elements in the pathogenesis of cancer in general and in brain tumors in particular. It is well documented that at least two classes of enzymes control acetylation of histones: histone acetyltransferases (HATs) and histone deacetylase (HDACs). Transformed HAT or HDAC action was identified in a number of human tumors. It has been hypothesized that HDACs regulate gene expression by deacetylating important genes for cell maintenance. Several HDACs inhibitors have been characterized in the last years and have been shown to promote growth blockage, differentiation and apoptosis in various types of tumors, including glioblastomas, medulloblastomas, neuroblastomas, melanomas, and leukemias. Some of these inhibitors are currently under clinical investigation for different cancer treatments. This review summarizes important mechanisms of histone modifications and discusses recent discoveries with impact on the pre-clinical and clinical field of pediatric brain tumor treatment.

Abstract A294: Antitumor activity of AMG900, an Aurora kinase inhibitor, alone or in combination with chemotherapeutic agents on H295A adrenocortical carcinoma cell line.

Pediatric adrenocortical tumors (ACT) are rare malignancies and in advanced disease the treatment has a small impact on overall survival. Previous study from our group suggests that AURKA and AURKB overexpression in pediatric ACT may be related to more aggressive disease. These genes are involved in the maintenance of the genome integrity during cell cycle division and they have been considered as new targets to cancer treatment. The present study shows the previous results of the effects of the new Aurora kinase inhibitor AMG900, associated or not with standard chemotherapeutic agents on adrenocortical carcinoma cell line H295A. Cell proliferation was assessed by Giemsa staining and apoptosis was performed by flow cytometry. Quantitative RT-PCR assays were used to determine the mRNA expression after AMG900 exposition. Drug combination analysis was made based on Chou-Talalay method. Hormones dosage assay was carried out to evaluate the effects of the Aurora kinase inhibitor on hormone secretion. AMG900 inhibited cell proliferation and caused apoptosis in a dose dependent manner in H295A cells. Moreover, it acted synergistically with doxorubicin, cisplatin and etoposide in the apoptosis induction, but only with doxorubicin in the proliferation inhibition. AMG900 treatment induced the mRNA expression of the genes p53, p21, and GDF15 and effectively inhibited dehydroepiandrosterone, androstenedione, cortisol and testosterone secretion. These data suggest that Aurora kinase inhibition by AMG900 may be a new therapeutic approach to adrenocortical carcinoma treatment. Financial Support: FAPESP (10/07020-9 and 12/09391-0) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A294. Citation Format: Kleiton S. Borges, Augusto F. Andrade, Vanessa S. Silveira, David S.M Antonio, Elton J.R. Vasconcelos, Sonir R.R. Antonini, Luiz G. Tone, Carlos A. Scrideli. Antitumor activity of AMG900, an Aurora kinase inhibitor, alone or in combination with chemotherapeutic agents on H295A adrenocortical carcinoma cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A294.

Abstract A128: Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines.

Medulloblastoma (MB) is the most common malignant brain tumors in childhood, accounting for 20 % of all primary pediatric intracranial tumors. Although conventional therapies can cure a large proportion of patients with medulloblastoma, the majority of survivors suffer from long-term side effects, including developmental and neurological deficits. Thus, new approaches and therapeutic drugs are needed. Epigenetic drugs such as inhibitors of DNA methyltransferases (iDNMTs) have shown antineoplastic effects in different tumors. Zebularine (ZB) is a potent inhibitor of DNA methylation and has been associated with induction of apoptosis and enhancing tumor chemo- and radiosensitivity. However, its effects on childhood MB have not been previously reported. Herein, this study shows the preliminary data from the effects of ZB on cell proliferation, cell cycle, clonogenic capacity and apoptosis of childhood MB cells. DAOY, ONS-76, UW-402 and UW-473 MB cell lines were used in all functional studies. Cell proliferation by XTT® assay, clonogenic capacity, cell cycle and apoptosis by flow cytometry using propidium iodide and anexin-V staining were performed using different doses of ZB (50-800µM). Statistical analysis was performed by one or two-way ANOVA and Bonferoni post-hoc. ZB decreased cell proliferation and clonogenic capacity in all cell lines in a time- and dose-dependent manner, respectively (P< 0.05). ZB also caused apoptotic cell death in MB cells, where the percentage of apoptotic cells significantly increased after treatment compared control (P<0.05), reaching 60% on DAOY cells. Since the dose of 200uM, the drug showed a cell cycle arrest in S-phase in UW-473 and UW-402 cell lines and in G2/M phase in ONS-76 and DAOY cells after 72h of exposure. These results indicate that ZB may be a promising drug for MB treatment and it confirms the potential of demethylating drugs in cancer treatment. Others studies and novel assays are being conducted to better understand these findings. Financial Support: FAPESP (process no. 2011/22440-7) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A128. Citation Format: Augusto F. Andrade, Kleiton S. Borges, Veridiana K. Suazo, Angel Mauricio Castro-Gamero, Rosane G. P. Queiroz, Carlos A. Scrideli, Luiz Gonzaga Tone. Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A128.