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

BACKGROUND: Human 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. METHODS: Real 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. RESULTS: Overexpression 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. CONCLUSION: The 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.

LHX6 promoter hypermethylation in oncological pediatric patients conceived by IVF.

The multifactorial etiology of pediatric cancer is poorly understood. Environmental factors occurring during embryogenesis can disrupt epigenetic signaling, resulting in several diseases after birth, including cancer. Associations between assisted reproductive technologies (ART), such as in vitro fertilization (IVF), and birth defects, imprinting disorders and other perinatal adverse events have been reported. IVF can result in methylation changes in the offspring, and a link with pediatric cancer has been suggested. In this study, we investigated the peripheral blood methylomes of 11 patients conceived by IVF who developed cancer in childhood. Methylation data of patients and paired sex/aged controls were obtained using the Infinium MethylationEPIC Kit (Illumina). We identified 25 differentially methylated regions (DMRs), 17 of them hypermethylated, and 8 hypomethylated in patients. The most significant DMR was a hypermethylated genomic segment located in the promoter region of LHX6, a transcription factor involved in the forebrain development and interneuron migration during embryogenesis. An additional control group was included to verify the LHX6 methylation status in children with similar cancers who were not conceived by ART. The higher LHX6 methylation levels in IVF patients compared to both control groups (healthy children and children conceived naturally who developed similar pediatric cancers), suggested that hypermethylation at the LHX6 promoter could be due to the IVF process and not secondary to the cancer itself. Further studies are required to evaluate this association and the potential role of LHX6 promoter hypermethylation for tumorigenesis.

Copy Number Alterations in Hepatoblastoma: Literature Review and a Brazilian Cohort Analysis Highlight New Biological Pathways.

Hepatoblastoma (HB) is a rare embryonal tumor, although it is the most common pediatric liver cancer. The aim of this study was to provide an accurate cytogenomic profile of this type of cancer, for which information in cancer databases is lacking. We performed an extensive literature review of cytogenetic studies on HBs disclosing that the most frequent copy number alterations (CNAs) are gains of 1q, 2/2q, 8/8q, and 20; and losses at 1p and 4q. Furthermore, the CNA profile of a Brazilian cohort of 26 HBs was obtained by array-CGH; the most recurrent CNAs were the same as shown in the literature review. Importantly, HBs from female patients, high-risk stratification tumors, tumors who developed in older patients (> 3 years at diagnosis) or from patients with metastasis and/or deceased carried a higher diversity of chromosomal alterations, specifically chromosomal losses at 1p, 4, 11q and 18q. In addition, we distinguished three major CNA profiles: no detectable CNA, few CNAs and tumors with complex genomes. Tumors with simpler genomes exhibited a significant association with the epithelial fetal subtype of HBs; in contrast, the complex genome group included three cases with epithelial embryonal histology, as well as the only HB with HCC features. A significant association of complex HB genomes was observed with older patients who developed high-risk tumors, metastasis, and deceased. Moreover, two patients with HBs exhibiting complex genomes were born with congenital anomalies. Together, these findings suggest that a high load of CNAs, mainly chromosomal losses, particularly losses at 1p and 18, increases the tendency to HB aggressiveness. Additionally, we identified six hot-spot chromosome regions most frequently affected in the entire group: 1q31.3q42.3, 2q23.3q37.3, and 20p13p11.1 gains, besides a 5,3 Mb amplification at 2q24.2q24.3, and losses at 1p36.33p35.1, 4p14 and 4q21.22q25. An in-silico analysis using the genes mapped to these six regions revealed several enriched biological pathways such as ERK Signaling, MicroRNAs in Cancer, and the PI3K-Akt Signaling, in addition to the WNT Signaling pathway; further investigation is required to evaluate if disturbances of these pathways can contribute to HB tumorigenesis. The analyzed gene set was found to be associated with neoplasms, abnormalities of metabolism/homeostasis and liver morphology, as well as abnormal embryonic development and cytokine secretion. In conclusion, we have provided a comprehensive characterization of the spectrum of chromosomal alterations reported in HBs and identified specific genomic regions recurrently altered in a Brazilian HB group, pointing to new biological pathways, and relevant clinical associations.

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.

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.

BACKGROUND: Insulinomas 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). METHODS: Quantitative 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. RESULTS: Overexpression 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. CONCLUSION: The 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.

Thalidomide treatment down-regulates SDF-1alpha 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.

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

Apolipoprotein B (ApoB) plays a major role in the regulation of cellular cholesterol homeostasis and pathogenesis of atherosclerosis. This protein acts as a ligand for the cellular recognition and catabolism of low density lipoprotein (LDL) by the LDL receptor. Previous studies have shown that the expression of apoB in hepatic cells is regulated by the interaction of factors binding to enhancer elements in intron 2 and three elements designated III, IV and V. These elements lie within regions respectively -86 to -62, -72 to -53 and -53 to -33 from the ApoB promoter. In this study, we have suggested that transcription factor C/EBPalpha, which binds to the -53 to -33 region of the apoB, interacts with the HNF-4 synergistic complex and C/EBPalpha factors within -86 to -53 and may contribute to increase transcription of the ApoB gene.

A apolipoproteina B (apoB) tem um importante papel na regulação na homeostasia celular, do colesterol e na patogênese da aterosclerose. Esta proteína age como ligante para o reconhecimento e catabolismo lipoproteínas de baixa densidade (LBD) através do receptor de LDL. Estudos anteriores mostraram que a expressão do gene da apolipoproteína B (APOB) em células hepáticas é regulada pela interação de fatores ligados ao elemento enhancer no intron 2, e em 3 elementos denominados de III, IV e V localizados nas regiões -86 a -62, -72 a -53 e -53 a -33 , respectivamente, do promotor do gene da APOB. Neste trabalho, nós sugerimos que o fator de transcrição C/EBPalfa ligado a região -53 a -33 da APOB interage com o complexo HNF-4 e C/EBPalfa localizado dentro da região -86 a -53 do APO B e contribui para aumentar a transcrição 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 Al-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.