Caroline Brunetto de Farias

Mangiferin, a naturally occurring glucoxilxanthone improves long-term object recognition memory in rats

Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a xanthone widely distributed in higher plants showing antioxidative, antiviral, anticancer, antidiabetic, immunomodulatory, hepatoprotective, and analgesic effects. In the present study, we have investigated the effects of systemic administration of mangiferin on behavioral outcomes of neurological function in normal rats. A single intraperitoneal injection of mangiferin (10, 50, or 100mg/kg body weight) enhanced novel object recognition (NOR) memory when given immediately post-training. The administration of mangiferin 6h post-training did not affect NOR memory. There were no significant differences between groups in the total time exploring both objects, indicating that mangiferin did not affect locomotion or motivation. Mangiferin stimulated cell proliferation and induced a significant increase in the supernatant levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-alpha in vitro in human U138-MG glioblastoma cells. The results indicate that mangiferin enhances recognition memory through a mechanism that might involve an increase in neurotrophin and cytokine levels.

Stimulation of Proliferation of U138-MG Glioblastoma Cells by Gastrin-Releasing Peptide in Combination with Agents That Enhance cAMP Signaling

Increasing evidence indicates that gastrin-releasing peptide (GRP) acts as an autocrine growth factor for brain tumors. However, it remains unclear whether the cAMP/protein kinase A (PKA) signaling pathway plays a role in mediating the mitogenic effects of GRP. We show here that GRP combined with agents that stimulate the cAMP/PKA pathway promotes proliferation of human gliobastoma cells. Treatment with GRP combined with the adenylyl cyclase activator forskolin, the cAMP analog 8-Br-cAMP or the phosphodiesterase type IV inhibitor rolipram increased proliferation of U138-MG cells in vitro measured by MTT assay. None of the compounds had an effect when given alone. GRP receptor (GRPR) mRNA and protein expression in U138-MG cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The results suggest that GRP and the GRPR interact with the cAMP/PKA signaling pathway in stimulating cancer cell proliferation.

Gastrin-Releasing Peptide Receptors Regulate Proliferation of C6 Glioma Cells through a Phosphatidylinositol 3-Kinase-Dependent Mechanism

Gastrin-releasing peptide (GRP) has been proposed as a major growth factor in brain tumors, and GRP receptor (GRPR) antagonists show antiproliferative effects in experimental gliomas. However, the underlying molecular events downstream of GRPR activation remain poorly understood. In the present study, we examined the role of the GRPR in regulating proliferation of glioma cells in vitro and its possible interaction with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Expression of GRPR mRNA and protein in C6, U-87MG, and U-373MG glioma cells was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Proliferation of C6 and U-87MG, but not U-373MG cells was significantly inhibited by the GRPR antagonist RC-3095, whereas the GRPR agonist bombesin (BB) significantly enhanced proliferation of C6 cells. The BB-induced stimulatory effect on cell proliferation was prevented by either RC-3095 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Our results provide the first evidence that the GRPR regulates proliferation of C6 glioma cells and suggest that PI3K is required for GRPR-mediated stimulation of glioma growth.

BDNF and PDE4, but not the GRPR, Regulate Viability of Human Medulloblastoma Cells

Medulloblastoma is the most common brain tumor of childhood. Emerging molecular targets in medulloblastoma include neurotrophin and neuropeptide receptors. In the present study, we have examined the influence of brain-derived neurotrophic factor (BDNF)/TrkB receptor- and gastrin-releasing peptide receptor (GRPR)-mediated signaling on the viability of human medulloblastoma cells. The expression of TrkB and GRPR was confirmed by immunohistochemistry and mRNA for both BDNF and GRPR was detected by reverse transcriptase polymerase chain reaction in Daoy, D283, and ONS76 cells. Treatment with BDNF significantly inhibited the viability of Daoy and D283, but not ONS76 cells, measured with the MTT assay. Neither the GRPR agonists GRP and bombesin nor the GRPR antagonist RC-3095 affected cell viability. Because previous findings have indicated that the viability of glioma cells might be enhanced by GRP when combined with the cAMP phosphodiesterase-4 (PDE4) inhibitor rolipram, we also examined the effects of rolipram alone or combined with GRP on cell viability. Rolipram significantly reduced the viability of all three cell lines, and the inhibitory effect of rolipram in Daoy cells was not modified by cotreatment with GRP. The results suggest that BDNF/TrkB and PDE4, but not the GRPR, regulate the viability of medulloblastoma cells.

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.

The histone deacetylase inhibitor sodium butyrate promotes cell death and differentiation and reduces neurosphere formation in human medulloblastoma cells.

Increasing evidence suggests that alterations in epigenetic mechanisms regulating chromatin state play a role in the pathogenesis of medulloblastoma (MB), the most common malignant brain tumor of childhood. Histone deacetylase (HDAC) inhibitors, which increase chromatin relaxation, have been shown to display anticancer activities. Here we show that the HDAC inhibitor sodium butyrate (NaB) markedly increases cell death and reduces colony formation in human MB cell lines. In addition, NaB increased the mRNA expression of Gria2, a neuronal differentiation marker, in D283 and DAOY cells and reduced the number of neurospheres in D283 cell cultures. Finally, NaB reduced the viability of D283 cells when combined with etoposide. These data show that NaB displays pronounced inhibitory effects on the survival of human MB cells and suggest that NaB might potentiate the effects of etoposide. In addition, our study suggests that HDAC inhibition might promote the neuronal differentiation of MB cells and provides the first evidence that an HDAC inhibitor might suppress the expansion or survival of MB cancer stem cells.

Current and emerging molecular targets in glioma.

Gliomas are the most common and lethal neurological cancers. Despite research efforts, the prognosis for patients with malignant gliomas remains poor. Advances in the understanding of cellular and molecular alterations in gliomas have led to the emergence of experimental molecularly targeted therapies. This article summarizes recent progress in the development of targeted therapies for glioma, focusing on emerging molecular targets, including neuropeptide and neurotrophin pathways, glutamate receptors, epigenetic mechanisms and glioma stem cell targets. Recent clinical trials of small molecules and antibodies targeted at growth factor pathways and intracellular signaling cascades are also discussed.

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.

Trk inhibition reduces cell proliferation and potentiates the effects of chemotherapeutic agents in Ewing sarcoma

Ewing sarcoma (ES) is a highly aggressive pediatric cancer that may arise from neuronal precursors. Neurotrophins stimulate neuronal devlopment and plasticity. Here, we found that neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as their receptors (TrkA and TrkB, respectively) are expressed in ES tumors. Treatment with TrkA (GW-441756) or TrkB (Ana-12) selective inhibitors decreased ES cell proliferation, and the effect was increased when the two inhibitors were combined. ES cells treated with a pan-Trk inhibitor, K252a, showed changes in morphology, reduced levels of β-III tubulin, and decreased mRNA expression of NGF, BDNF, TrkA and TrkB. Furthermore, combining K252a with subeffective doses of cytotoxic chemotherapeutic drugs resulted in a decrease in ES cell proliferation and colony formation, even in chemoresistant cells. These results indicate that Trk inhibition may be an emerging approach for the treatment of ES.

Design of Novel Potent Inhibitors of Human Uridine Phosphorylase-1: Synthesis, Inhibition Studies, Thermodynamics, and in Vitro Influence on 5-Fluorouracil Cytotoxicity

Uridine (Urd) is a promising biochemical modulator to reduce host toxicity caused by 5-fluorouracil (5-FU) without impairing its antitumor activity. Elevated doses of Urd are required to achieve a protective effect against 5-FU toxicity, but exogenous administration of Urd is not well-tolerated. Selective inhibitors of human uridine phosphorylase (hUP) have been proposed as a strategy to increase Urd levels. We describe synthesis and characterization of a new class of ligands that inhibit hUP type 1 (hUP1). The design of ligands was based on a possible SN1 catalytic mechanism and as mimics of the carbocation in the transition state of hUP1. The kinetic and thermodynamic profiles showed that the ligands here presented are the most potent in vitro hUP1 inhibitors developed to date. In addition, a lead compound improved the antiproliferative effects of 5-FU on colon cancer cells, accompanied by a reduction of in vitro 5-FU cytotoxicity in aggressive SW-620 cancer cells.