Differential microRNA expression profile in blood of children with Down syndrome suggests a role in immunological dysfunction.

Down syndrome (DS), caused by trisomy of chromosome 21 (HSA21), results in a broad range of phenotypes. However, the determinants contributing to the complex and variable phenotypic expression of DS are still not fully known. Changes in microRNAs (miRNAs), short non-coding RNA molecules that regulate gene expression post-transcriptionally, have been associated with some DS phenotypes. Here, we investigated the genome-wide mature miRNA expression profile in peripheral blood mononuclear cells (PBMCs) of children with DS and controls and identified biological processes and pathways relevant to the DS pathogenesis. The expression of 754 mature miRNAs was profiled in PBMCs from six children with DS and six controls by RT-qPCR using TaqMan® Array Human MicroRNA Cards. Functions and signaling pathways analyses were performed using DIANA-miRPath v.3 and DIANA-microT-CDS software. Children with DS presented six differentially expressed miRNAs (DEmiRs): four overexpressed (miR-378a-3p, miR-130b-5p, miR-942-5p, and miR-424-3p) and two downregulated (miR-452-5p and miR-668-3p). HSA21-derived miRNAs investigated were not found to be differentially expressed between the groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed potential target genes involved in biological processes and pathways pertinent to immune response, e.g., toll-like receptors (TLRs) signaling, Hippo, and transforming growth factor ß (TGF-ß) signaling pathways. These results suggest that altered miRNA expression could be contributing to the well-known immunological dysfunction observed in individuals with DS.

Anti-EGFR treatment effects on laryngeal cancer stem cells.

Laryngeal cancer (LC) is one of the common head and neck neoplasms and is characterized by resistance to conventional therapy and poor prognosis. This may result from the presence of cancer stem cells (CSCs), which form a small population in tumors with metastatic potential, high invasive capacity, self-renewal, and differentiation. This study aimed to evaluate the effectiveness of 5-fluorouracil and cisplatin individually, as well as the combination of cetuximab and paclitaxel in a CSC subpopulation separated with biomarkers related to tumoral growth (CD44, CD117, and CD133). In addition, expression of TrkB, KRAS, HIF-1α, and VEGF-A genes and proteins related to cell proliferation were evaluated in this subpopulation. The CD44, CD133, and CD117 biomarkers were used to analyze the identification and separation of both subpopulations using FACSAria Fusion. Subpopulations positive for CD44, CD133, and CD117 or lacking these biomarkers were classified as laryngeal cancer stem cells (LCSCs) or laryngeal cancer non-stem cells (non-LCSCs), respectively. Matrigel invasion and colony forming assays were performed to confirm CSC presence. Subpopulations were cultured and exposed to 5-fluorouracil, cisplatin, and cetuximab/paclitaxel drugs for 24 h. Cell proliferation was determined using MTS assay. KRAS and TrkB gene expression levels were evaluated using quantitative real time PCR with TaqMan® Assay in both subpopulations. The non-LCSC subpopulation was considered as the control for relative expression. We found that the LCSC subpopulation demonstrated more resistance to cetuximab and paclitaxel combination chemotherapy when compared with the non-LCSC subpopulation of the cell line. These LCSC subpopulations presented up-regulated expression of KRAS, HIF-1α, and VEGF-A genes and proteins and no TrkB gene expression, but TrkB protein expression was up-regulated in the LC cell line when compared to the non-CSC subpopulation. "In conclusion, the combination of CD44, CD133, and CD117 biomarkers has stem cell properties. Moreover, LCSCs, are capable of resisting treatment and present high KRAS, HIF-1α, and VEGF-A gene expression".

Glutathione S-transferase Polymorphisms in Head and Neck Squamous Cell Carcinoma Treated with Chemotherapy and/or Radiotherapy.

BACKGROUND/AIM: The Glutathione S-transferases (GSTs) are important carcinogen-metabolizing enzymes. Polymorphisms involved in these enzymes can modulate the development and treatment of head and neck cancer. To investigate the association of GSTs polymorphisms with head and neck cancer and risk factors, clinical-pathological features, and survival time of the patients treated with chemotherapy and/or radiotherapy. METHODS: The GST gene polymorphisms were evaluated in 197 cases and 514 controls by PCR-RFLP-Polymerase Chain Reaction Restriction Fragment Length Polymorphism. RESULTS: The GSTP-313 was associated with a decreased risk for HNSCC (p=0.050). The GSTP1 haplotype analysis revealed a higher frequency of the AC and AT haplotypes in the case group than in the control group (p=0.013 and p=0.019, respectively), and the opposite for G-C haplotype (p = 0.015). Yet, the different combinations between the genotypes were associated with an increased risk of cancer. The study showed no association between the polymorphisms and primary tumor site, clinical-pathological characteristics, treatment (chemotherapy and/or radiotherapy) and survival time of the patients. CONCLUSION: The GST polymorphisms combination showed an increased risk for carcinogenesis, and studies with larger casuistry can contribute to the clarification of the role in individual patient differences for the response to chemotherapy and/or radiotherapy and identify biomarkers of susceptibility.

Atorvastatin increases oxidative stress and inhibits cell migration of oral squamous cell carcinoma in vitro.

OBJECTIVE: This study aimed to evaluate the effect of atorvastatin treatment on reactive oxygen species (ROS) production and tumor angiogenesis in oral squamous cell carcinomas. MATERIAL AND METHODS: An HN13 cell line was treated with 1 µM, 5 µM, and 10 µM of atorvastatin. VEGF-A gene expression was evaluated by quantitative real time PCR. VEGF-A protein expression was quantified from total protein and conditioned media by ELISA. Cellular oxidative stress was measured using 2',7'-dichlorfluorescein-diacetate (DCFH-DA). Angiogenesis assay was performed using human umbilical vein endothelial cells (HUVEC). The effect of atorvastatin on cell migration was evaluated by wound healing assay. RESULTS: 5 µM and 10 µM of atorvastatin significantly increased VEGF-A gene expression in the HN13 cell line. Intracellular expression of the VEGF-A protein was higher in the cells treated with 5 µM and 10 µM than in the control cells. VEGF-A protein expression was also higher in the conditioned media from the atorvastatin-treated cells than in the media from the DMSO-treated cells. 5 µM and 10 µM of atorvastatin increased oxidative stress. Regarding angiogenesis assay, 5 µM of atorvastatin resulted in higher numbers of branch points, compared to the solvent. 10 µM of atorvastatin treatment resulted in significantly reduced cell migration. CONCLUSIONS: This study showed that atorvastatin increases the oxidative stress and angiogenesis in oral squamous cell carcinomas. The decrease of cell migration indicates atorvastatin's inhibitory effect in oral tumors. These results suggest that atorvastatin could increase the intracellular oxidative stress in these cells, leading to a toxic microenvironment and inhibiting their metastasis.

Molecular evaluation of glutathione S transferase family genes in patients with sporadic colorectal cancer.

AIM: To evaluate the association between polymorphisms in glutathione S transferases (GSTs) and the risk of sporadic colorectal cancer (SCRC), tumor progression and the survival of patients. METHODS: A case-control study of 970 individuals from the Brazilian population was conducted (232 individuals from the case group with colorectal cancer and 738 individuals from the control group without a history of cancer). PCR multiplex and PCR-RFLP techniques were used to genotype the GST polymorphisms. The tumors were categorized according to the TNM classification: tumor extension (T), affected lymph nodes (N), and presence of metastasis (M). Logistic regression, multiple logistic regression and survival analysis were used to analyze the data. The results are presented in terms of odds ratio (OR) and 95% confidence interval (CI). The level of significance was set at 5% (P ≤ 0.05). RESULTS: Age equal to or over 62 years (OR = 8.79; 95%CI: 5.90-13.09, P < 0.01) and female gender (OR = 2.91; 95%CI: 1.74-4.37; P < 0.01) were associated with increased risk of SCRC. Analysis of the polymorphisms revealed an association between the GSTM1 polymorphisms and a risk of SCRC (OR = 1.45; 95%CI: 1.06-2.00; P = 0.02), as well as between GSTT1 and a reduced risk of the disease (OR = 0.65; 95%CI: 0.43-0.98; P = 0.04). An interaction between the presence of the wild-type allele of GSTP1 Ile105Val polymorphism and tobacco consumption on risk of SCRC (OR = 2.33; 95%CI: 1.34-4.05; P = 0.05) was observed. There was an association between the GSTM1 null genotype and the presence of advanced tumors (OR = 2.33; 95%CI: 1.23-4.41; P = 0.009), as well as increased risk of SCRC in the presence of a combination of GSTT1 non-null/GSTM1 null genotypes (OR = 1.50; 95%CI: 1.03-2.19; P = 0.03) and GSTT1 non-null/GSTM1 null/GSTP1 Val* (OR = 1.85; 95%CI: 1.01-3.36, P = 0.04). Combined GSTT1 non-null/GSTM1 null genotypes (OR = 2.40; 95%CI: 1.19-4.85; P = 0.01) and GSTT1 non-null/GSTM1 null/GSTP1 Val* (OR = 2.92; 95%CI: 1.05-8.12; P = 0.04) were associated with tumor progression. Polymorphisms were not associated with the survival of patients with SCRC. CONCLUSION: Females aged 62 years or older are more susceptible to SCRC. Polymorphisms of GSTT1 and GSTM1 null genotypes modulated the susceptibility to SCRC in the population studied.

Relationship between CD44high/CD133high/CD117high cancer stem cells phenotype and Cetuximab and Paclitaxel treatment response in head and neck cancer cell lines.

Recent evidence suggests that cancer stem cells (CSCs), a small population of cancer cells that are highly tumourigenic, capable of self-renewal and have the ability to differentiate into cells that constitute the tumor, are the "drivers" of local recurrence and metastatic spread and may be associated with resistant to conventional therapy. The objectives of the study are to identify and characterize two head and neck cancer cell lines with regard CD44high/CD133high/CD117high profile (CSCs) and CD44low/CD133low/CD117low profile (Non-CSCs); to investigate the influence of chemotherapy treatment in CSCs and compare with Non-CSCs; to evaluate CD44 and EGFR gene expression in CSCs. Fluorescent-activated cell sorting (FACS) using specific cell surface marker combination (CD44, CD117 and CD133) was performed to isolate CSCs of Non-CSCs from cell lines. The Wound Healing assay was performed to confirm the presence of CSCs. After, the CSCs subpopulation and Non-CSCs were cultured and exposed for 24 h to Cetuximab and Paclitaxel treatment, separately. Cell proliferation was determined by MTS assay. CD44 and EGFR gene expression was quantified by quantitative real time PCR (qPCR) using TaqMan® Assay in both subpopulations. CSCs subpopulation untreated were considered as relative expression control. We firstly characterized CSCs in HN13 and HEP-2 cell lines with CD44, CD133 and CD117 biomarkers. We treated CSCs and Non-CSCs subpopulations with Cetuximab and Paclitaxel treatment and found that CSCs subpopulations demonstrated more resistance to Paclitaxel chemoterapy, when compared with Non-CSCs subpopulations of oral cancer cell line. These CSCs subpopulations presented up-regulation of CD44 gene and down-regulation of EGFR gene in oral cancer cell line, and down-regulation of CD44 gene and up-regulation of EGFR gene in laryngeal cancer cell line when compared with Non-CSCs subpopulations. We conclude that the combination of CD44, CD133 and CD117 biomarkers have stem cell properties in both cell lines. CSCs has ability to resist to Paclitaxel treatment in oral cancer cell line. CSCs present high expression of CD44 gene and down expression of EGFR gene in oral cancer cell line. CSCs in laryngeal cell line present down expression of CD44 gene and high expression of EGFR gene when compared with cells without characteristics of cancer stem cells.

Interleukin 6 and 10 Serum Levels and Genetic Polymorphisms in Children with Down Syndrome.

Immunological impairment is a condition that is often observed in individuals with Down syndrome (DS). The immune response is modulated by pro- and anti-inflammatory cytokines whose expressions could be influenced by genetic polymorphisms. The present study was aimed at evaluating the frequencies of -174G>C, -572G>C, and -597G>A polymorphisms in the interleukin 6 (IL-6) gene and -592C>A, -1082A>G, and -819C>T polymorphisms in the IL-10 gene and the IL-6 and IL-10 serum levels in healthy individuals with and without DS. The authors also aimed to investigate the impact of the genotypes on the interleukin concentrations. The genetic polymorphisms were investigated in 200 DS individuals and 200 controls without DS. The serum measurement of IL-6 and IL-10 was performed in a subgroup (54 cases and 54 controls) by enzyme-linked immunosorbent assay (ELISA). The frequencies of the polymorphisms and haplotypes evaluated were not different between individuals with and without DS. IL-10 concentration was higher in DS children but was not influenced by IL-10 gene polymorphisms. IL-6 genotypes had no influence on IL-6 serum levels. The IL-10 serum levels are increased in DS individuals, but IL-10 polymorphisms are not the main factors that influence the IL-10 expression in DS.

Candidate Biomarkers for Oral Squamous Cell Carcinoma: Differential Expression of Oxidative Stress-Related Genes

Background: Alteration in the biotransformation of exogenous compounds can result in production of reactive oxygen species (ROS), which can predispose cells to malignant transformation in the head and neck. This study aimed to evaluate the expression of genes involved in antioxidant metabolism in the oral squamous cell carcinoma (OSCC). Methods: The expression of eighty-four genes was evaluated in OSCC and non-tumor tissues by quantitative real-time polymerase chain reaction using the TaqMan Gene Expression Array. The biological mechanisms related to the differentially expressed genes were investigated using Gene ­ NCBI, KEGG, UNIPROT and REACTOME databases. Results: Twenty-one genes encoding enzymes involved in antioxidant metabolism were differentially expressed in the OSCC case. Four genes (ATOX1, PRDX4, PRNP, and SOD2) were up-regulated, and seventeen (ALOX12, CAT, CSDE1, DHCR24, DUOX1, DUOX2, EPHX2, GLRX2, GPX3, GSR, GSTZ1, MGST3, PRDX1, OXR1, OXSR1, SOD1, and SOD3) were down-regulated. We identified 14 possible novel biomarkers for OSCC. The differentially expressed genes appeared related to important biological processes involved in carcinogenesis, such as inflammation, angiogenesis, apoptosis, genomic instability, invasion, survival, and cell proliferation. Conclusions: Our study identified novel biomarkers which might warrant further investigation regarding OSCC pathogenesis since the altered expression in the genes can modulate biological processes related to oxidative stress and predispose cells to malignant transformation in the oral cavity.

Overexpression of Antiangiogenic Vascular Endothelial Growth Factor Isoform and Splicing Regulatory Factors in Oral, Laryngeal and Pharyngeal Squamous Cell Carcinomas

Background: Overexpression of proangiogenic vascular endothelial growth factor A family VEGFAxxx is associated with tumor growth and metastasis. The role of the alternatively spliced antiangiogenic family VEGFAxxxb is poorly investigated in head and neck squamous cell carcinomas (HNSCCs). The antiangiogenic isoform binds to bevacizumab and its expression level could influence the treatment response and progression-free survival. In this study, the relative expression of VEGFAxxx and VEGFA165b isoforms and splicing regulatory factors genes was investigated in a series of HNSCCs. Methods: VEGFAxxx, VEGFA165b, SRSF6, SRSF5, SRSF1 and SRPK1 gene expression was quantified by quantitative real time PCR in 53 tissue samples obtained by surgery from HNSCC patients. Protein expression was evaluated by immunohistochemistry. Results: VEGFAxxx and VEGFA165b were overexpressed in HNSCCs. Elevated protein expression was also confirmed. However, VEGFA isoforms demonstrated differential expression according to anatomical sites. VEGFAxxx was overexpressed in pharyngeal tumors while the VEGFA165b isoform was up-regulated in oral tumors. The VEGFA165b isoform was also positively correlated with expression of the splicing regulatory genes SRSF1, SRSF6 and SRSF5. Conclusions: We concluded that VEGFAxxx and VEGFA165b isoforms are overexpressed in HNSCCs and the splicing regulatory factors SRSF1, SRSF6, SRSF5 and SRPK1 may contribute to alternative splicing of the VEGFA gene. The findings for the differential expression of the antiangiogenic isoform in HNSCCs could facilitate effective therapeutic strategies for the management of these tumors.

Hepatocellular Carcinoma: a Comprehensive Review of Biomarkers, Clinical Aspects, and Therapy

Hepatocellular carcinoma (HCC) is a cause of several deaths related to cancer worldwidely. In early stage, curative treatments such as surgical resection, liver transplant and local ablation can improve the patient ´s survival. However, the disease is detected in advanced stage; moreover some available therapies are restricted to palliative care and local treatment. Early detections of HCC and adequate therapy are crucial to increase survival as well as to improve the patient´s quality of life. Therefore, researchers have been investigating molecular biomarkers with high sensibility and reliability as Golgi 73 protein (GP73), Glypican-3 (GPC3), Osteopontin (OPN), microRNAs and others. MicroRNAs can regulate important pathways on carcinogenesis, as tumor angiogenesis and progression. So, they can be considered as possible markers of prognosis in HCC, and therapeutic target for this tumor type. In this review, we discuss the recent advances related to the cause (highlighting the main risk factors), treatment, biomarkers, clinic aspects, and outcome in hepatocellular carcinoma.

CYP1A1, CYP2E1 and EPHX1 polymorphisms in sporadic colorectal neoplasms.

AIM: To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer (SCRC) risk. METHODS: Six hundred forty-one individuals (227 patients with SCRC and 400 controls) were enrolled in the study. The variables analyzed were age, gender, tobacco and alcohol consumption, and clinical and histopathological tumor parameters. The CYP1A1*2A, CYP1A1*2C CYP2E1*5B and CYP2E1*6 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The EPHX1 Tyr113His, EPHX1 His139Arg and CYP1A1*2C polymorphisms were detected by real-time PCR. Chi-squared test and binary logistic regression were used in the statistical analysis. Haplotype analysis was conducted using the Haploview program, version 2.05. RESULTS: Age over 62 years was a risk factor for SCRC development (OR = 7.54, 95%CI: 4.94-11.50, P < 0.01). Male individuals were less susceptible to SCRC (OR = 0.55, 95%CI: 0.35-0.85, P < 0.01). The CYP2E1*5B polymorphism was associated with SCRC in the codominant (heterozygous genotype: OR = 2.66, 95%CI: 1.64-4.32, P < 0.01), dominant (OR = 2.82, 95%CI: 1.74-4.55, P < 0.01), overdominant (OR = 2.58, 95%CI: 1.59-4.19, P < 0.01), and log-additive models (OR = 2.84, 95%CI: 1.78-4.52, P < 0.01). The CYP2E1*6 polymorphism was associated with an increased SCRC risk in codominant (heterozygous genotype: OR = 2.81, 95%CI: 1.84-4.28, P < 0.01; homozygous polymorphic: OR = 7.32, 95%CI: 1.85-28.96, P < 0.01), dominant (OR = 2.97, 95%CI: 1.97-4.50, P < 0.01), recessive (OR = 5.26, 95%CI: 1.35-20.50, P = 0.016), overdominant (OR = 2.64, 95%CI: 1.74-4.01, P < 0.01), and log-additive models (OR = 2.78, 95%CI: 1.91-4.06, P < 0.01). The haplotype formed by the minor alleles of the CYP2E1*5B (C) and CYP2E1*6 (A) polymorphisms was associated with SCRC (P = 0.002). However, the CYP1A1*2A, CYP1A1*2C, EPHX1 Tyr113His and EPHX1 His139Arg polymorphisms were not associated with SCRC. CONCLUSION: In conclusion, the results demonstrated that CYP2E1*5B and CYP2E1*6 minor alleles play a role in the development of SCRC.