MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

DNA repair mechanisms protect our genome from carcinogenesis

Human cells are constantly exposed to DNA damage. Without repair, damage can result in genetic instability and eventually cancer. The strong association between the lack of DNA damage repair, mutations and cancer is dramatically demonstrated by a number of cancer-prone human syndromes, such as xeroderma pigmentosum (XP), ataxia-telangiectasia (AT) and Fanconi anemia (FA). This review focuses on the historical discoveries related with these three diseases and describes their impact on the understanding of DNA repair mechanisms and the causes of human cancer. As deficiencies in DNA repair are also often related with progeria symptoms, unrepaired damage and aging are somehow related. Several other pathologies associated with DNA repair defects, genetic instability and increased cancer risk are also discussed. In fact, studies with cells from these many syndromes have helped in understanding important levels of protection against cancer and aging, although little help has actually been conferred to the patients in terms of therapy. Finally, the recent advances in combined basic and translational research on DNA repair and chemotherapy are presented.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Trends in socioeconomic inequalities in cancer mortality in Barcelona: 1992–2003

Abstract Background The objective of this study was to assess trends in cancer mortality by educational level in Barcelona from 1992 to 2003. Methods The study population comprised Barcelona inhabitants aged 20 years or older. Data on cancer deaths were supplied by the system of information on mortality. Educational level was obtained from the municipal census. Age-standardized rates by educational level were calculated. We also fitted Poisson regression models to estimate the relative index of inequality (RII) and the Slope Index of Inequalities (SII). All were calculated for each sex and period (1992–1994, 1995–1997, 1998–2000, and 2001–2003). Results Cancer mortality was higher in men and women with lower educational level throughout the study period. Less-schooled men had higher mortality by stomach, mouth and pharynx, oesophagus, larynx and lung cancer. In women, there were educational inequalities for cervix uteri, liver and colon cancer. Inequalities of overall and specific types of cancer mortality remained stable in Barcelona; although a slight reduction was observed for some cancers. Conclusion This study has identified those cancer types presenting the greatest inequalities between men and women in recent years and shown that in Barcelona there is a stable trend in inequalities in the burden of cancer.

Global gene expression profiling of oral cavity cancers suggests molecular heterogeneity within anatomic subsites

Abstract Background Oral squamous cell carcinoma (OSCC) is a frequent neoplasm, which is usually aggressive and has unpredictable biological behavior and unfavorable prognosis. The comprehension of the molecular basis of this variability should lead to the development of targeted therapies as well as to improvements in specificity and sensitivity of diagnosis. Results Samples of primary OSCCs and their corresponding surgical margins were obtained from male patients during surgery and their gene expression profiles were screened using whole-genome microarray technology. Hierarchical clustering and Principal Components Analysis were used for data visualization and One-way Analysis of Variance was used to identify differentially expressed genes. Samples clustered mostly according to disease subsite, suggesting molecular heterogeneity within tumor stages. In order to corroborate our results, two publicly available datasets of microarray experiments were assessed. We found significant molecular differences between OSCC anatomic subsites concerning groups of genes presently or potentially important for drug development, including mRNA processing, cytoskeleton organization and biogenesis, metabolic process, cell cycle and apoptosis. Conclusion Our results corroborate literature data on molecular heterogeneity of OSCCs. Differences between disease subsites and among samples belonging to the same TNM class highlight the importance of gene expression-based classification and challenge the development of targeted therapies.

A novel set of ss-N-biaryl ether sulfonamide hydroxamates as potential MMPs inhibitors: Molecular dynamics simulations and molecular properties evaluation

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases involved in the extracellular matrix degradation. MMP-2 and MMP9 are overexpressed in several human cancer types, including melanoma, thus the development of new compounds to inhibit MMPs' activity is desirable. Molecular dynamic simulation and molecular properties calculations were performed on a set of novel beta-N-biaryl ether sulfonamide-based hydroxamates, reported as MMP-2 and MMP-9 inhibitors, for providing data to develop an exploratory analysis. Thermodynamic, electronic, and steric descriptors have significantly discriminated highly active from moderately and less active inhibitors of MMP-2 whereas apparent partition coefficient at pH 1.5 was also significant for the MMP-9 data set. Compound 47 was considered an outlier in all analysis, indicating the presence of a bulky substituent group in R3 is crucial to this set of inhibitors for the establishment of molecular interactions with the S1 subsite of both enzymes, but there is a limit. (C) 2012 Wiley Periodicals, Inc.

Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness. (C) 2012 Elsevier Inc. All rights reserved.

Systematic UPLC-ESI-MS/MS Study on the Occurrence of Staurosporine and Derivatives in Associated Marine Microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

Antimicrobial and antiproliferative activities of stingless bee Melipona scutellaris geopropolis

Background Geopropolis is a type of propolis containing resin, wax, and soil, collected by threatened stingless bee species native to tropical countries and used in folk medicine. However, studies concerning the biological activity and chemical composition of geopropolis are scarce. In this study, we evaluated the antimicrobial and antiproliferative activity of the ethanolic extract of geopropolis (EEGP) collected by Melipona scutellaris and its bioactive fraction against important clinical microorganisms as well as their in vitro cytotoxicity and chemical profile. Methods The antimicrobial activity of EEGP and fractions was examined by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six bacteria strains as well as their ability to inhibit Streptococcus mutans biofilm adherence. Total growth inhibition (TGI) was chosen to assay the antiproliferative activity of EEGP and its bioactive fraction against normal and cancer cell lines. The chemical composition of M. scutellaris geopropolis was identified by reversed-phase high-performance liquid chromatography and gas chromatography–mass spectrometry. Results EEGP significantly inhibited the growth of Staphylococcus aureus strains and S. mutans at low concentrations, and its hexane fraction (HF) presented the highest antibacterial activity. Also, both EEGP and HF inhibited S. mutans biofilm adherence (p < 0.05) and showed selectivity against human cancer cell lines, although only HF demonstrated selectivity at low concentrations. The chemical analyses performed suggest the absence of flavonoids and the presence of benzophenones as geopropolis major compounds. Conclusions The empirical use of this unique type of geopropolis by folk medicine practitioners was confirmed in the present study, since it showed antimicrobial and antiproliferative potential against the cancer cell lines studied. It is possible that the major compounds found in this type of geopropolis are responsible for its properties.

Bioactive extracts and chemical constituents of two endophytic strains of Fusarium oxysporum

Ethyl acetate extracts of cultures grown in liquid Czapek and on solid rice media of the fungal endophyte Fusarium oxysporum SS46 isolated from the medicinal plant Smallanthus sonchifolius (Poepp.) H. Rob., Asteraceae, exhibited considerable cytotoxic activity when tested in vitro against human cancer cells. Chromatographic separation yielded anhydrofusarubin (1) and beauvericin (2) that were identified based on their ¹H and 13C NMR data. Compounds 1 and 2 showed the strongest cytotoxic activity against different cancer cell lines. Compound 2 also showed promising activity against Leishmania braziliensis. Hexanic extract of F. oxysporum SS50 grown on solid rice media also afforded a mixture of compounds that displayed cytotoxic activity against different cancer cell lines. Chemical analysis of the mixture of compounds, investigated by gas chromatography-mass spectrometry (GC-MS), showed that there was a predominance of methyl esters of fatty acids and alkanes.

Systematic UPLC-ESI-MS/MS study on the occurrence of staurosporine and derivatives in associated marine microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

A rigorous approach to facilitate and guarantee the correctness of the genetic testing management in human genome information systems

Abstract Background Recent medical and biological technology advances have stimulated the development of new testing systems that have been providing huge, varied amounts of molecular and clinical data. Growing data volumes pose significant challenges for information processing systems in research centers. Additionally, the routines of genomics laboratory are typically characterized by high parallelism in testing and constant procedure changes. Results This paper describes a formal approach to address this challenge through the implementation of a genetic testing management system applied to human genome laboratory. We introduced the Human Genome Research Center Information System (CEGH) in Brazil, a system that is able to support constant changes in human genome testing and can provide patients updated results based on the most recent and validated genetic knowledge. Our approach uses a common repository for process planning to ensure reusability, specification, instantiation, monitoring, and execution of processes, which are defined using a relational database and rigorous control flow specifications based on process algebra (ACP). The main difference between our approach and related works is that we were able to join two important aspects: 1) process scalability achieved through relational database implementation, and 2) correctness of processes using process algebra. Furthermore, the software allows end users to define genetic testing without requiring any knowledge about business process notation or process algebra. Conclusions This paper presents the CEGH information system that is a Laboratory Information Management System (LIMS) based on a formal framework to support genetic testing management for Mendelian disorder studies. We have proved the feasibility and showed usability benefits of a rigorous approach that is able to specify, validate, and perform genetic testing using easy end user interfaces.

Germline DNA copy number variation in familial and early-onset breast cancer

Introduction: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." Methods: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. Results: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. Conclusions: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.

Non-classical HLA-E gene variability in Brazilians: a nearly invariable locus surrounded by the most variable genes in the human genome

The non-classical human leukocyte antigen (HLA) class I genes present a very low rate of variation. So far, only 10 HLA-E alleles encoding three proteins have been described, but only two are frequently found in worldwide populations. Because of its historical background, Brazilians are very suitable for population genetic studies. Therefore, 104 bone marrow donors from Brazil were evaluated for HLA-E exons 14. Seven variation sites were found, including two known single nucleotide polymorphisms (SNPs) at positions +424 and +756 and five new SNPs at positions +170 (intron 1), +1294 (intron 3), +1625, +1645 and +1857 (exon 4). Haplotyping analysis did show eight haplotypes, three of them known as E*01:01:01, E*01:03:01 and E*01:03:02:01 and five HLA-E new alleles that carry the new variation sites. The HLA-E*01:01:01 allele was the predominant haplotype (62.50%), followed by E*01:03:02:01 (24.52%). Selective neutrality tests have disclosed an interesting pattern of selective pressures in which balancing selection is probably shaping allele frequency distributions at an SNP at exon 3 (codon 107), sequence diversity at exon 4 and the non-coding regions is facing significant purifying pressure. Even in an admixed population such as the Brazilian one, the HLA-E locus is very conserved, presenting few polymorphic SNPs in the coding region.

Genome-Wide Analysis in Brazilian Xavante Indians Reveals Low Degree of Admixture

Characterization of population genetic variation and structure can be used as tools for research in human genetics and population isolates are of great interest. The aim of the present study was to characterize the genetic structure of Xavante Indians and compare it with other populations. The Xavante, an indigenous population living in Brazilian Central Plateau, is one of the largest native groups in Brazil. A subset of 53 unrelated subjects was selected from the initial sample of 300 Xavante Indians. Using 86,197 markers, Xavante were compared with all populations of HapMap Phase III and HGDP-CEPH projects and with a Southeast Brazilian population sample to establish its population structure. Principal Components Analysis showed that the Xavante Indians are concentrated in the Amerindian axis near other populations of known Amerindian ancestry such as Karitiana, Pima, Surui and Maya and a low degree of genetic admixture was observed. This is consistent with the historical records of bottlenecks experience and cultural isolation. By calculating pair-wise F-st statistics we characterized the genetic differentiation between Xavante Indians and representative populations of the HapMap and from HGDP-CEPH project. We found that the genetic differentiation between Xavante Indians and populations of Ameridian, Asian, European, and African ancestry increased progressively. Our results indicate that the Xavante is a population that remained genetically isolated over the past decades and can offer advantages for genome-wide mapping studies of inherited disorders.