Estela Maria Novak

Synthesis and Characterization of a Metal Complex Containing Naringin and Cu, and its Antioxidant, Antimicrobial, Antiinflammatory and Tumor Cell Cytotoxicity

The antioxidant activity of flavonoids is believed to increase when they are coordinated with transition metal ions. However, the literature on this subject is contradictory and the outcome seems to largely depend on the experimental conditions. In order to understand the contribution of the metal coordination and the type of interaction between a flavonoid and the metal ion, in this study a new metal complex of Cu (II) with naringin was synthesized and characterized by FT-IR, UV-VIS, mass spectrometry (ESI-MS/MS), elemental analysis and 1H-NMR. The results of these analyses indicate that the complex has a Cu (II) ion coordinated via positions 4 and 5 of the flavonoid. The antioxidant, anti-inflammatory and antimicrobial activities of this complex were studied and compared with the activity of free naringin. The Naringin-Cu (II) complex 1 showed higher antioxidant, anti-inflammatory and tumor cell cytotoxicity activities than free naringin without reducing cell viability.

Thalidomide treatment down-regulates SDF-1α and CXCR4 expression in multiple myeloma patients

The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor CXCR4 are critically involved in directional migration and homing of plasma cells in multiple myeloma. Here, we show that the expression of SDF-1alpha and CXCR4 was significantly down-regulated in patients treated with thalidomide (n=10) as compared to newly diagnosed MM patients (n=31) and MM patients treated with other drugs (n=38). SDF-1 alpha and CXCR4 expression was also significantly decreased in a RPMI 8226 cell line treated with 10 and 20micromol/L of thalidomide. Our findings indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1alpha in multiple myeloma.

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

BackgroundHuman homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.MethodsReal time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.ResultsOverexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.ConclusionThe present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.

Synthesis, characterization and biological evaluation of Rutin-zinc(II) flavonoid -metal complex.

Synthesis of compounds analogous to natural products from secondary metabolites, such as flavonoids, is a promising source of novel drugs. Rutin (quercetin-3-O-rutinoside) is a natural flavone, which has, in its chemical structure, different sites for coordination with transition metals and the complexation with these metals enhances its biological properties. Rutin-zinc(II), a flavonoid-metal complex, was synthesized and characterized by UV-VIS, FT-IR, elemental analysis and (1)H NMR. The antioxidant and antitumor activities, as well as the cytotoxicity and in vivo toxicity of this complex were evaluated and compared with the free rutin. Rutin-zinc(II) has not shown any cytotoxicity against normal cells (fibroblasts and HUVECs) or toxicity in BALB/c mice, but has shown antioxidant activity in vitro and cytotoxicity against leukemia (KG1, K562 and Jurkat), multiple myeloma (RPMI8226) and melanoma (B16F10 and SK-Mel-28) cell lines in vitro. In Ehrlich ascites carcinoma model, Rutin-zinc(II) modulated the mitochondrial membrane potential and the expression of genes related to cell cycle progression, angiogenesis and apoptosis.

Differential expression of genes encoding proteins of the HGF/MET system in insulinomas

BackgroundInsulinomas are the most common functional pancreatic neuroendocrine tumors, whereas histopathological features do not predict their biological behaviour. In an attempt to better understand the molecular processes involved in the tumorigenesis of islet beta cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two inhibitors of HGF activator and of matriptase).MethodsQuantitative real-time reverse transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes in 24 sporadic insulinomas: 15 grade 1 (G1), six grade 2 (G2) and three hepatic metastases. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19.ResultsOverexpression of MET was observed in the three hepatic metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. A positive correlation was observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene.ConclusionThe final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoral progression and metastatization in insulinomas.

Differential expression of HOXB7 gene in multiple myeloma and extramedullary multiple myeloma patients.

To the Editor: Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of plasma cells in the hematopoietic bone marrow (1). Events such as alterations in the expression of transcription factors might be critically involved in MM (2). The deregulation of homeobox (HOX) genes is known to play an important role in fundamental processes in hematological malignancy (3, 4). Recently, Colla et al. (3) showed the presence of HOXB7 expression in 10% of MM patients studied. In this report, we compared HOXB7 expression in three cases classified as extramedullary spread of multiple myeloma (eMM) with 30 MM patients. Multiple myeloma and eMM patients were defined clinicopathologically and radiologically. All of them had received systematic chemotherapy. The MM patients were staged according to the criteria of Durie & Salmon (5) and eMM were with age of 55–58 yr and staged as IIIA; the M components was immunoglobulin G (n = 3). Informed consent was obtained from all patients, and all protocols were approved by the Ethics Committee of the Hospital das Clinicas, University of São Paulo Medical School. Plasma cells population from bone marrow aspirates of MM and eMM patients obtained for routine diagnostic or evaluation purposes after informed consent were enriched as described previously (6). Although our results are based on a small number of eMM patients, they were obtained by absolute quantitative real-time RT-PCR (qRT-PCR) and thus should provide an accurate quantification of gene expression. qRT-PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), TaqMan Gene Expression Assays probe and primer mix (Applied Biosystems), according to the manufacturer’s specifications. A dilution series of HOXB7 copies amplified in pTOPO-TA vector (Invitrogen, Carlsbad, CA, USA) with an insert of 123 bp fragment from exon 1-2 of HOXB7 gene (pHOXB7) was used as reference control. The volume of the purified linear dsDNA standards was adjusted to 10 copies ⁄ lL. This stock solution was serially diluted to obtain a standard series from 10 to 10 copies ⁄ lL with each step differing by 10-fold. As negative control was used a pool of mRNA from bone marrow of six healthy donors. Expression levels of HOXB7 were significantly higher (p < 0.02) in the three patients with eMM, developed 24 and 48 months after primary diagnosis, than in MM patients that showed HOXB7 expression. One patient developed eMM in pericardium 24 months and the two other developed plasmacytoma in inguinal lymph nodes 48 months after primary diagnosis. In the three eMM patients, 2.6 · 10 HOXB7copies ⁄ lL, 1.8 · 10 HOXB7 copies ⁄ lL and 3.8 · 10 HOXB7 copies ⁄ lL were found, respectively. The transcript levels of HOXB7 continued to increase during the 48-month follow-up period (median 36, range 12–60 months). Progression to eMM in the three patients demonstrated an aggressive phase of the disease and patients died because of disease progression. Within 30 MM patients tested, HOXB7 expression was found in 10 MM patients (six of them without and 4 with bone plasmacytoma). The median of HOXB7 gene expression in MM patients was 1 · 10 HOXB7 copies ⁄lL (range 10–10 copies ⁄ lL). Among the four MM patients with bone plasmacytoma, one case with 1.3 · 10 HOXB7 copies ⁄ lL and another patient with 2.4 · 10 HOXB7 copies ⁄lL showed bone tumor lesions in various sites, including vertebral column and upper and lower limbs. In the remaining two patients with 1.2 · 10 HOXB7 copies ⁄ lL and 2.8 · 10 HOXB7 copies ⁄lL, respectively, bone plasmacytoma were observed only in vertebral column. Two MM patients with bone plasmacytoma (4.6 · 10 HOXB7 copies ⁄lL) and four of the MM patients (median 10 copies ⁄ lL) without bone plasmacytoma died from the disease. Furthermore, in all patients studied, the transcript levels of HOXB7 continued to increase during the 48-month follow-up period (median 36, range 12–60 months). We also studied four patients with solitary plasmacytoma, but our results showed absence of HOXB7 expression (data no shown). In summary, the capacity of activating many angiogenic molecules makes HOXB7 an attractive target gene in MM.

Study of activity transcription factors C/EBPalpha in region - 53 to - 33 of promoter apolipoprotein B gene

A apolipoproteina B (apoB) tem um importante papel na regulacao na homeostasia celular, do colesterol e na patogenese da aterosclerose. Esta proteina age como ligante para o reconhecimento e catabolismo lipoproteinas de baixa densidade (LBD) atraves do receptor de LDL. Estudos anteriores mostraram que a expressao do gene da apolipoproteina B (APOB) em celulas hepaticas e regulada pela interacao de fatores ligados ao elemento enhancer no intron 2, e em 3 elementos denominados de III, IV e V localizados nas regioes -86 a -62, -72 a -53 e -53 a -33 , respectivamente, do promotor do gene da APOB. Neste trabalho, nos sugerimos que o fator de transcricao C/EBPa ligado a regiao -53 a -33 da APOB interage com o complexo HNF-4 e C/EBPa localizado dentro da regiao -86 a -53 do APO B e contribui para aumentar a transcricao do gene APOB.

Uncommon allele in APO AI-CIII-AIV gene cluster in a family with congenital generalized lipodystrophy

Congenital generalized lipodystrophy is a rare inherited disease. One of its features is a disturbance in lipid metabolism characterized by hypercholesterolemia and hypertriglyceridemia. A brother and a sister with congenital generalized lipodystrophy, an 8-year old male and a 12-year old female were studied. The mother and a 6-year old brother were healthy. The genetic analysis of Sstl RFLP of the apo AI-CIII-AIV gene cluster showed the presence of the rare Sstl allele (S2) in the patients but not in the healthy mother and brother. As this uncommon allele has been reported to be related to high plasma triglyceride levels, this association could be relevant in explaining in part the hypertriglyceridemia observed in these patients.