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Aim: We studied the influence of coffee consumption on the therapeutic effect of methotrexate (MTX) in patients with rheumatoid arthritis (RA) sorted according to ADORA2A genotypes. Patients & methods: 82 RA patients were dichotomized according to caffeine intake with a threshold of 700 mg/week. Disease activity score 28 (DAS28) was applied (>3.2: high

Hepatocellular carcinoma (HCC) is a major contributor to the worldwide cancer burden. Recent studies on HCC have demonstrated dramatic alterations in expression of several cytochrome P450 (CYP) family members that play a crucial role in biotransformation of many drugs and other xenobiotics; however, the mechanisms responsible for their deregulation remain unclear.We investigated a potential involvement of miRNAs in downregulation of expression of CYPs observed in HCC tumors. We compared miRNA expression profiles (TaqMan Array Human MicroRNA v3.0 TLDA qPCR) between HCC human patient tumors with strong (CYP-) and weak/no (CYP+) downregulation of drug-metabolizing CYPs. The role of significantly deregulated miRNAs in modulation of expression of the CYPs and associated xenobiotic receptors was then investigated in human liver HepaRG cells transfected with relevant miRNA mimics or inhibitors.We identified five differentially expressed miRNAs in CYP- versus CYP+ tumors, namely miR-29c, miR-125b1, miR-505, miR-653 and miR-675. The two most-upregulated miRNAs found in CYP- tumor samples, miR-29c and miR-653, were found to act as efficient suppressors of CYP1A2 or AHR expression.Our results revealed a novel role of miR-653 and miR-29c in regulation of expresion of CYPs involved in crucial biotransformation processes in liver, which are often deregulated during liver cancer progression.

Colorectal cancer, one of the most frequent types of cancer worldwide, has a high mortality rate. Irinotecan (CPT-11) has been approved for the treatment of advanced or metastatic disease either as a single agent or, more commonly, as part of combined chemotherapeutic regimens. Treatment with irinotecan is often accompanied by severe toxicity (e.g. neutropenia and diarrhea) that can result in treatment interruption or cessation, thus jeopardizing the patient’s prognosis and quality of life. Irinotecan is bioactivated into its metabolite SN-38, which is subsequently detoxified by uridine diphosphate-glucuronosyl transferases (mainly UGT1A1). Further, ABC transporters (i.e. ABCB1, ABCC1–ABCC6, and ABCG2) are responsible for drug efflux into bile and urine whereas OATP transporters (SLCO1B1) enable its influx from blood into hepatocytes. Genetic polymorphisms in these enzymes/pumps may result in increased systemic SN-38 level, directly correlating with toxicity. Contemporary research is focused on the clinical implementation of genetic screenings for validated gene variations prior to treatment onset, allowing tailored individual doses or treatment regimens.

Cytochrome P450 family 1 (CYP1) enzymes contribute both to metabolism of xenobiotics and to the control of endogenous levels of ligands of the aryl hydrocarbon receptor (AhR). Their activities, similar to other CYPs, can be altered in tumor tissues. Here, we examined a possible role of proliferative/survival pathways signaling, which is often deregulated in tumor cells, and possible links with p300 histone acetyltransferase (a transcriptional co-activator) in the control of CYP1 expression, focusing particularly on CYP1A1. Using cell models derived from human liver, we observed that the induction of CYP1A1 expression, as well as other CYP1 enzymes, was reduced in exponentially growing cells, as compared with their non-dividing counterparts. The siRNA-mediated inhibition of proliferation/pro-survival signaling pathway effectors (such as β-catenin and/or Hippo pathway effectors YAP/TAZ) increased the AhR ligand-induced CYP1A1 mRNA levels in liver HepaRG cells, and/or in colon carcinoma HCT-116 cells. The activation of proliferative Wnt/β-catenin signaling in HCT-116 cells reduced both the induction of CYP1 enzymes and the binding of p300 to the promoter of CYP1A1 or CYP1B1 genes. These results seem to indicate that aberrant proliferative signaling in tumor cells could suppress induction of CYP1A1 (or other CYP1 enzymes) via competition for p300 binding. This mechanism could be involved in modulation of the metabolism of both endogenous and exogenous substrates of CYP1A1 (and other CYP1 enzymes), with possible further consequences for alterations of the AhR signaling in tumor cells, or additional functional roles of CYP1 enzymes.

Hepatocellular carcinoma (HCC) remains a highly prevalent and deadly disease, being among the top causes of cancer-related deaths worldwide. Despite the fact that the liver is the major site of biotransformation, studies on drug metabolizing enzymes in HCC are scarce. It is known that malignant transformation of hepatocytes leads to a significant alteration of their metabolic functions and overall deregulation of gene expression. Advanced stages of the disease are thus frequently associated with liver failure, and severe alteration of drug metabolism. However, the impact of dysregulation of metabolic enzymes on therapeutic efficacy and toxicity in HCC patients is largely unknown. Here we demonstrate a significant down-regulation in European Caucasian patients of cytochromes P450 (CYPs), the major xenobiotic-metabolizing enzymes, in HCC tumour samples as compared to their surrounding non-cancerous (reference) tissue. Moreover, we report for the first time the association of the unique CYP profiles with specific transcriptome changes, and interesting correlations with expression levels of nuclear receptors and with the histological grade of the tumours. Integrated analysis has suggested certain co-expression profiles of CYPs with lncRNAs that need to be further characterized. Patients with large tumours with down-regulated CYPs could be more vulnerable to drug toxicity; on the other hand, such tumours would eliminate drugs more slowly and should be more sensitive to pharmacotherapy (except in the case of pro-drugs where activation is necessary).

OBJECTIVE The treatment of rheumatoid arthritis (RA) patients with methotrexate (MTX) is linked to the development or progression of rheumatoid nodules. The aim of this study was to determine whether folate and adenosine pathways-related single nucleotide polymorphisms might be predictive of increased nodule formation in RA patients treated with oral MTX. METHODS A total of 185 Caucasian RA patients were enrolled in this cross-sectional study, all of whom fulfilled the 1987 RA criteria of the American College of Rheumatology; each patient had a history of MTX treatment. RESULTS A higher frequency of the MTHFR 1298AA genotype was found in 17 (70.8%) of 24 patients with general nodules [odds ratio (OR)=3.08, 95% confidence interval (CI): 1.20-7.69] and in 14 (73.7%) of 19 patients who developed nodules during MTX treatment (OR=3.55, 95% CI: 1.22-10.32). In contrast, a negative association with nodules during MTX treatment (OR=0.29, 95% CI: 0.08-1.10) was found for 19 (79.2%) patients with the TT genotype (rs2298383) in the adenosine A2a receptor gene (ADORA2A). However, the significance did not remain upon correction for multiple testing. The combination of MTHFR 1298AA along with ADORA2A rs2298383 CC or CT genotypes occurring in one-third of RA patients showed a higher frequency of general nodules 15/59 (25.4%) as well as developing nodules during MTX treatment 13/59 (22.0%) in comparison with the overall studied group: 24/185 (13.0%) and 19/185 (10.3%), respectively. CONCLUSION This exploratory study indicates for the first time a plausible association of adenosine and folate pathways single nucleotide polymorphisms in nodules' etiopathogenesis.

Letter to the editor with respect to: Lima A, Bernardes M, Azevedo R, Seabra V and Medeiros R. Moving toward personalized medicine in rheumatoid arthritis: SNPs in methotrexate intracellular pathways are associated with methotrexate therapeutic outcome. Pharmacogenomics 17(15), 1649–1674 (2016).

Some single-nucleotide polymorphisms (SNPs) might be predictive of methotrexate (MTX) therapeutic outcome in rheumatoid arthritis (RA). The aim of this study was to determine whether SNPs in the methylenetetrahydrofolate reductase (MTHFR) gene are predictive of MTX response. Comparison was made using EULAR response criteria and according to the change of DAS28 (∆DAS28) after a 6-month MTX treatment in RA patient cohort. The two SNPs C677T (rs1801133) and A1298C (rs1801131) have been genotyped. A total of 120 patients were enrolled in the study, and all of them fulfilled the American College of Rheumatology 1987 RA criteria and are currently or previously taking MTX oral treatment, either as a monotherapy (n = 65) or in a combination with other disease-modifying antirheumatic drugs (n = 55). Genotyping was performed using qPCR allelic discrimination. We did not found any association of C677T and A1298C genotypes with MTX treatment inefficacy in dominant model (OR 1.23, 95 % CI 0.57–2.65, P = 0.697; and OR 0.98, 95 % CI 0.47–2.14, P = 1.0, respectively), or in recessive and codominant models. However, when ∆DAS28 after a 6-month therapy was used as a measure of treatment efficacy, the 677CT and 1298AC genotypes were found to be significantly associated with less favorable response to MTX (P = 0.025 and P = 0.043, respectively). In addition, even lower ∆DAS28 was determined for double-mutated 677CT–1298AC heterozygotes. It means that a synergistic effect of 677CT and 1298AC genotypes was observed. Nevertheless, the DAS28 baseline was lower here comparing to other genotypes. Unexpectedly, quite the opposite trend—i.e., better response to MTX—was found in genotypes 677CC–1298CC and 677TT–1298AA. It is an intriguing finding, because these double-mutated homozygotes are known for their low MTHFR-specific activity. Global significance was P = 0.013, η 2 = 0.160—i.e., large-size effect. Thus, our data show greater ability of 677CC–1298CC and 677TT–1298AA genotypes to respond to MTX treatment.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

Multiple myeloma still remains incurable in the majority of cases prompting a further search for new and better prognostic markers. Emerging evidence has suggested that circulating microRNAs can serve as minimally invasive biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance. In this study, a global analysis of serum microRNAs by TaqMan Low Density Arrays was performed, followed by quantitative real-time PCR. The analyses revealed five deregulated microRNAs: miR-744, miR-130a, miR-34a, let-7d and let-7e in monoclonal gammopathy of undetermined significance, newly diagnosed and relapsed multiple myeloma when compared to healthy donors. Multivariate logistical regression analysis showed that a combination of miR-34a and let-7e can distinguish multiple myeloma from healthy donors with a sensitivity of 80.6% and a specificity of 86.7%, and monoclonal gammopathy of undetermined significance from healthy donors with a sensitivity of 91.1% and a specificity of 96.7%. Furthermore, lower levels of miR-744 and let-7e were associated with shorter overall survival and remission of myeloma patients. One-year mortality rates for miR-744 and let-7e were 41.9% and 34.6% for the ‘low’ expression and 3.3% and 3.9% for the ‘high’ expression groups, respectively. Median time of remission for both miR-744 and let-7e was approximately 11 months for the ‘low’ expression and approximately 47 months for the ‘high’ expression groups of myeloma patients These data demonstrate that expression patterns of circulating microRNAs are altered in multiple myeloma and monoclonal gammopathy of undetermined significance and miR-744 with let-7e are associated with survival of myeloma patients.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3 % of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

The aim of this study is to evaluate the biologic importance and prognostic significance of selected clinicopathological parameters in patients with oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma, with emphasis on smoking, protein p16(INK4a) (p16) expression, and human papillomavirus (HPV) status.The study sample consisted of 48 patients with OSCC and 44 patients with OPSCC. Half of the patients were nonsmokers and the other half were gender-, age- and tumor localization-matched smokers. p16 expression was detected in 17/48 (35 %) OSCCs and in 36/44 (82 %) OPSCCs and HPV DNA was present in 7/48 (15 %) OSCCs and in 35/44 (80 %) OPSCCs. The sensitivity and specificity of p16 expression for HPV DNA presence were 0.74 and 0.88, respectively. The OPSCCs were more frequently basaloid (p0.001) while the OSCCs were more frequently conventional (p0.000001). The OSCCs were more likely to recur locally and to be the cause of death (p = 0.009 in both parameters).The HPV-positive tumors were more frequently localized in oropharynx, were basaloid SCCs and were p16- and HPV-positive (p0.000001 in all 4 parameters). The HPV-negative tumors were more frequently localized in oral cavity (p0.000001), more frequently asociated with local, regional and locoregional recurence (p = 0.011, p = 0.019 and p = 0.030, respectively) and with tumor-related death (p = 0.003). There was no significant difference with regard to smoking history (p0.05). The survival of patients with HPV-positive tumors was significantly longer (median 112 months; 95% CI 54 - 112 months) than that of patients with HPV-negative tumors (median 17 months; 95% CI 12 - 39 months) (p0.001). The HPV status of OSCC/OPSCC is an important biological and prognostic parameter and should be examined in all cases, using PCR or immunohistochemical detection of surrogate marker p16. Smoking itself does not seem to be an important prognostic factor.

Background: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. Methods: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. Results: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient’s age at the time of diagnosis. Conclusions: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Adefovir (PMEA) and tenofovir (PMPA) and their prodrugs, adefovir dipivoxil (bisPOM-PMEA) and tenofovir disoproxil (bisPOC-PMPA), were subjected to a detailed study of their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The inhibition of marker enzyme activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 was examined with high-performance liquid chromatography (HPLC) or spectroscopic (fluorescence, luminescence) detection. Adefovir and adefovir dipivoxil did not significantly influence activities of most CYP enzymes. The activity of CYP3A4 was inhibited by adefovir dipivoxil at concentrations over 100 microM. Adefovir and its prodrug inhibited CYP2C9 at concentrations below 100 microM; inhibition by adefovir was of the uncompetitive (at the lower inhibitor concentrations) or of the competitive nature with a Ki = 420 microM. Tenofovir and tenofovir disoproxil influenced the activity of CYP2C9, and competitive inhibition was found with Ki = 580 and 395 microM, respectively. Tenofovir disoproxil was shown to inhibit microsomal CYP2E1 activities by a mixed-type inhibition with Ki values at about 140 microM. The results indicate the possibility of an influence of the compounds tested on the respective CYP activities when used at high doses.

MicroRNAs are short non-coding ribonucleic acid molecules that regulate gene expression at the post-transcriptional level thus affecting important physiological as well as pathophysiological processes in the organism, for example cell differentiation, proliferation, apoptosis, and metabolism. They are involved in pathogenesis of many diseases including cancer. Many microRNAs are tissue or organ-specific which implies their possible potential as biomarkers or maybe even therapeutical agents as documented by microRNA research interest rising exponentially during last years. Among all, microRNAs are important also for physiological function of the kidney and they are involved in various renal disorders. Today research is focused mainly on renal and urinary tract carcinogenesis, acute kidney injury, chronic renal diseases (polycystic kidney disease) or renal complications of systemic diseases such as diabetic or hypertension nephropathy and autoimmune kidney injury including acute allograft rejection after kidney transplantation. The review summarizes current information about microRNA effect on kidney development and function and also on the most common kidney diseases.

Clinical behavior of neuroblastoma (NBL) is remarkably heterogeneous, as it ranges from spontaneous regression to aggressive clinical phenotype and death. There is increasing body of evidence demonstrating that microRNAs could be considered the potential biomarkers for clinical applications in NBL. In this report, we focus on molecular characterization of high-risk as well as low-risk and intermediate-risk NBL cases in the context of the microRNA expression profile that is specific for the given risk category of the disease. We investigated a total of 30 NBL patients, out of whom there were 19 patients with low- to intermediate-risk and 11 with high-risk NBLs as defined by the Clinical Oncology Group. We determined the expression profiles of 754 microRNAs (miRNAs), whereas the miRNA expression levels were normalized to RNU44, mean expression levels were calculated, and data were analyzed by use of the microarray biostatistical approaches. We identified the signature of 38 miRNAs differentially expressed between these groups of NBL patients (P < 0.05): 17 miRNAs were upregulated and 21 miRNAs were downregulated in the tumors of high-risk NBL patients. We confirm some of the previous observations and we report several new microRNAs associated with aggressive NBL, both being relevant subjects for further translational validation and functional studies.

Background Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients. Methods We selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling. Results Expression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified. Conclusions Our pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established.

Background There is no standard serum biomarker used for diagnosis or early detection of recurrence for renal cell carcinoma (RCC) patients. MicroRNAs (miRNAs) are abundant and highly stable in blood serum, and have been recently described as powerful circulating biomarkers in a wide range of solid cancers. Our aim was to identify miRNA signature that can distinguish the blood serum of RCC patients and matched healthy controls and validate identified miRNAs as potential biomarkers for RCC. Methods In the screening phase of the study, blood serum of 15 RCC patients and 12 matched healthy controls were analyzed by use of the TaqMan Low-Density Arrays enabling parallel identification of expression levels of 667 miRNAs through qRT-PCR-based approach. In the validation phase, identified miRNAs were further evaluated on the independent group of 90 RCC patients and 35 matched healthy controls by use of individual qRT-PCR assays and statistically evaluated. Results We identified 30 miRNAs differentially expressed between serum of RCC patients and healthy controls: 19 miRNAs were up-regulated and 11 miRNAs were down-regulated in RCC patients. MiR-378, miR-451 and miR-150 were further evaluated in the independent group of patients, and two of them were successfully validated: levels of miR-378 were increased (p = 0.0003, AUC = 0.71), miR-451 levels were decreased (p < 0.0001, AUC = 0.77) in serum of RCC patients. Combination of miR-378 and miR-451 enable identification of RCC serum with the sensitivity of 81%, specificity 83% and AUC = 0.86. Conclusions Circulating miRNAs in serum are promising biomarkers in RCC.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the important unmet medical needs in RCC is prognostic biomarker enabling identification of patients at high risk of relapse after nephrectomy. MicroRNAs (miRNAs) constitute a robust regulatory network with posttranscriptional regulatory efficiency for almost one-half of human coding genes, including oncogenes and tumor suppressors. To identify potential prognostic miRNAs, we analyzed expression profiles in tumors of different prognostic groups of RCC patients. Seventy-seven patients with clear cell RCC and detailed clinicopathological data were enrolled in a single-center study. Global miRNA expression profiles were obtained by use of TaqMan Low Density Arrays (754 parallel quantitative reverse-transcriptase polymerase chain reactions (qRT-PCR) reactions). For validation of identified miRNAs individual miRNA TaqMan assays were performed in an independent group of patients. We identified tumor relapse-signature based on the expression of 64 miRNAs differentially expressed between relapse-free RCC patients and RCC patients who developed relapse (20 miRNAs were increased, 44 miRNAs were decreased). In the validation phase of the study, we successfully confirmed that expression levels of miR-143, miR-26a, miR-145, miR-10b, miR-195, and miR-126 are lower in the tumors of RCC patients who developed tumor relapse, moreover, the lowest levels of these miRNAs we observed in primary metastatic tumors. By using Kaplan-Meier analysis, we identified that miR-127-3p, miR-145, and miR-126 are significantly correlated with relapse-free survival of nonmetastatic RCC patients. If further validated, we suggest that identified miRNAs might be used for identification of RCC patients at high risk of early relapse after nephrectomy in clinical practice.

The aim of the study was to investigate the role of high-risk human papillomavirus (HR-HPV) infection in the etiopathogenesis of oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma in non-smoking and non-drinking patients (NSNDP). Twenty-four OSCCs and 22 OPSCCs were analyzed by immunohistochemistry for p16INK4a protein (p16) expression and by chromogene in situ hybridization (CISH) and polymerase chain reaction (PCR) for HR-HPV DNA presence. The series included 23 males and 23 females aged 35–93 years. p16 expression was seen in 7 out of 24 (29%) OSCCs and in 22 out of 22 (100%) OPSCCs. Using CISH, HR-HPV DNA was observed in 6 out of 24 (25%) OSCCs and in 21 out of 22 (95%) OPSCCs. HPV DNA was found in 3 out of 24 (13%) OSCCs and in 18 out of 22 (82%) OPSCCs using PCR. HPV 16 and 33 were detected in 16 and in two cases, respectively. Compared with OSCCs, OPSCCs more frequently showed basaloid morphology (p

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK-STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (-583/-574-bp). However, inhibition of the JAK-STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (-1604/-1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (-1551/-1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK-STAT6 and IRS1/2 and NFATc1.

CYP2C9 is an important drug-metabolizing enzyme that metabolizes, e. g., warfarin, antidiabetics, and antiphlogistics. However, the endogenous regulation of this enzyme is largely unknown. In this study, we examined the role of GATA transcription factors in the gene expression of CYP2C9. We investigated four putative GATA binding sites within the first 200 base pairs of CYP2C9 promoter at the positions I: -173/-170, II: -167/-164, III: -118/ -115, and IV: -106/-103. Luciferase activity driven by a wildtype CYP2C9 promoter construct was strongly up-regulated in Huh-7 cells upon cotransfection with expression plasmids for GATA-2 and GATA-4, whereas mutations introduced into GATA binding site III or I and II reduced this induction to a significant extent. Electrophoretic mobility shift assays revealed specific binding of GATA-4 and GATA-6 to the oligonucleotides containing GATA binding sites I and II. Furthermore, the association of GATA-4 with CYP2C9 promoter was confirmed by chromatin immunoprecipitation assays in HepG2 cells. Taken together, these data strongly suggest an involvement of liver-specific transcription factor GATA-4 in the transcriptional regulation of CYP2C9. Swedish Research Council; Stockholm County Council; Danish Agency of Science, Technology and Innovation; Lundbeck Foundation; Portuguese Foundation for Science and Technology [SFRH/BPD/34152/2006 IBB/CBME]

Aims Cytochrome P4502C19 (CYP2C19) is an important enzyme involved in the metabolism of antiulcer drugs and antidepressants. However, despite the well documented drug-dependent variability of CYP2C19 expression, the mechanisms underlying the regulation of the enzyme remain unknown. In this study we investigated whether the transcription factor family GATA is involved in the regulation of CYP2C19 gene expression. Main methods We identified a novel putative GATA binding site at position –165/–156 within the CYP2C19 gene promoter. 5′-Deletion fragments of the CYP2C19 promoter containing wild type or mutant variants of this GATA binding site were co-transfected with expression vectors encoding the transcription factors GATA-4 or GATA-2 and analyzed using dual luciferase gene reporter assays in HepG2 and Huh-7 hepatoma cells. Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitations (ChIP) were performed to proof a sequence-specific interaction of GATA proteins with the putative GATA binding site. Key findings The wild type fragments of CYP2C19 promoter were highly upregulated by GATA-4 and GATA-2 in luciferase gene reporter assay, whereas mutations introduced into the GATA binding sites caused a significant activity loss. Similar attenuation was observed upon co-transfection of GATA-4 with a known co-regulator of GATA activity, FOG-2. EMSA analysis revealed a sequence-specific binding of GATA-4 and GATA-6 to the wild type GATA binding site. In addition, the association of GATA-4 with the CYP2C19 promoter was confirmed by ChIP analysis. Significance These data indicate that GATA-4 plays an important role in the transcriptional regulation of CYP2C19 expression.

We demonstrated for the first time an outstanding ability of rosiglitazone to mediate a profound enhancement of LA-12-induced apoptosis associated with activation of mitochondrial pathway in human colon cancer cells. This effect was preferentially observed in the G1 cell cycle phase, independent on p53 and PPARγ proteins, and accompanied with significant changes of selected Bcl-2 family protein levels. Further stimulation of cooperative synergic cytotoxic action of rosiglitazone and LA-12 was demonstrated in the cells deficient for PTEN, where mitochondrial apoptotic pathway was more stimulated and G1-phase-associated dying was reinforced. Our results suggest that combined treatment with rosiglitazone and LA-12 might be promising anticancer strategy in colon-derived tumours regardless of their p53 status, and also favourable in those defective in PTEN function.

Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without such manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p

To determine a possible influence of acyclic nucleoside phosphonates on FMO3 and CYP activity at molecular level in vitro.Inhibition of individual CYP and FMO3 activities in the reconstituted system and in artificial membranes (bactosomes) was studied.Activity of FMO3 was inhibited by PMPA and bisPOC-PMPA even at low levels of these drugs (below 100 microM). In reconstituted system with CYP2C9, no inhibition of CYP2C9 activity was observed. On the other hand, experiments based on membrane coexpressed system showed a modest extent of inhibition (for PMEA, PMPA, bisPOM-PMEA and bisPOC-PMPA the level of inhibition was 77.8%; 74.1%; 64.2% and 68.6%, respectively at 400 microM).PMPA and bisPOC-PMPA are able to inhibit FMO3 activity at relatively low levels (10-100 microM) indicating a relatively specific interaction. Activity of CYP2C9 was affected when using the membranes coexpressed with NADPHcytochrome P450 reductase. This is probably due to more natural conditions for maintaining the CYP activity in bacterial membranes. As the inhibitor concentration in the systemic circulation does not exceed 2 microM, the probability of a significant in vivo effect of adefovir, tenofovir and the respective prodrugs on the microsomal system of cytochromes P450 and FMO3 is relatively low.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. The prognosis of GBM patients varies considerably and the histopathological examination is not sufficient for individual risk estimation. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and were repeatedly proved to play important roles in pathogenesis of GBM. In our study, we performed global miRNA expression profiling of 58 glioblastoma tissue samples obtained during surgical resections and 10 non-tumor brain tissues. The subsequent analysis revealed 28 significantly deregulated miRNAs in GBM tissue, which were able to precisely classify all examined samples. Correlation with clinical data led to identification of six-miRNA signature significantly associated with progression free survival [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.33-2.94, P < 0.001] and overa+ll survival (HR 2.86, 95% CI 1.91-4.29, P < 0.001). O(6)-methylguanine-DNA methyltransferase methylation status was evaluated as reference method and Risk Score based on six-miRNA signature indicated significant superiority in prediction of clinical outcome in GBM patients. Multivariate Cox analysis indicated that the Risk Score based on six-miRNA signature is an independent prognostic classifier of GBM patients. We suggest that the Risk Score presents promising prognostic algorithm with potential for individualized treatment decisions in clinical management of GBM patients.