Selected E2F2 Polymorphisms in Oral and Oropharyngeal Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are subgroups of head and neck squamous cell carcinoma. E2F Transcription Factor 2 (E2F2) could contribute to cancer development, because it plays a critical role in many cellular processes, including the cell cycle, proliferation, differentiation, DNA damage response, and cell death. In the current study, we assessed the associations of five E2F2 polymorphisms (rs6667575, rs3218121, rs3218211, rs3218148, and rs3218203) with OSCC and OPSCC and influence on the TNM staging and grading. This is the first such survey to concern the European population. The study included 94 primary tumour samples following surgical resection from patients, whereas the control group consisted of 99 healthy individuals. We tried a matching of cases and controls for age and sample size. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination. Our results suggested that the most significant difference between the control group and the cancer group was the A/G heterozygote for rs3218121. Samples containing this genotype were mostly found in the control group. In our samples, rs6667575, rs3218121, rs3218211, and rs3218148 polymorphisms may affect the course of OSCC and OPSCC, while rs3218203 was not associated with OSCC and OPSCC. However, further studies are warranted to confirm our findings.

The levels of sICAM-1, sELAM-1, TNFα and sTNFR1 proteins in patients with colorectal adenocarcinoma in tumor and corresponding normal mucosa.

Colorectal cancer is a common malign disease of the gastrointestinal tract. The cancer survival rate depends on the stage of the disease at detection time. It is well known that several molecular mechanisms are involved in cancer and some molecules might affect or modulate cancerogenesis. The aim of the study was to assess the levels of sICAM-1, sELAM-1, TNFα and sTNFR1 protein in tumor and corresponding normal mucosa in a group of patients with colorectal adenocarcinoma and also associations of these parameters with demographic and clinical profiles of the patients. Tissue specimens were obtained during resection of neoplastic lesions. Protein levels were assayed in tissue homogenates by ELISA. The protein level of sICAM-1 in tumor was significantly increased in comparison to the corresponding normal mucosa (80.06 ng/mg vs 69.53 ng/mg, p=0.02). Furthermore, a significant positive correlation between sICAM-1 and sTNFR1 proteins levels in tumor (rs=0.58, p<0.001) and in corresponding normal mucosa (rs=0.48, p<0.001) was found. Also, significant correlations in corresponding normal mucosa were found between sELAM-1 and sICAM-1 (rs=0.58, p<0.001) and between sTNFR1 and sELAM-1 (rs=0.57, p<0.001). Significantly higher level of sTNFR1 in corresponding normal mucosa samples of patients with distant metastases was observed (p=0.04). Obtained results suggest that sICAM-1 protein could be considered as colorectal cancer marker. Furthermore, sTNFR1 also has the potential to become a good prognostic marker used during monitoring of the patients. Nevertheless, a further study in this area to confirm this correlation is required.

5α,6α-Epoxyphytosterols and 5α,6α-Epoxycholesterol Increase Nitrosative Stress and Inflammatory Cytokine Production in Rats on Low-Cholesterol Diet.

OBJECTIVE: Oxidized cholesterol derivatives are compounds with proven atherogenic and mutagenic effects. However, little is known about the effect of oxidized plant sterol derivatives (oxyphytosterols), whose structure is similar to the one of oxycholesterols. Our previous studies indicate that they have a similar profile of action, e.g., both exacerbate disorder of lipid metabolism and oxidative stress in experimental animals. The aim of the present study was to assess the effect of epoxycholesterol and epoxyphytosterols (mainly sitosterol) on the severity of nitrosative stress and the concentration of selected proinflammatory cytokines in blood and liver tissue of rats on a low-cholesterol diet. Material and Methods. Forty-five male Wistar rats were fed with feed containing 5α,6α-epoxyphytosterols (ES group, n: 15), 5α,6α-epoxycholesterol (ECh group, n: 15), and oxysterol-free feed (C group, n: 15) for 90 days (daily dose of oxysterols: 10 mg/kg). At the end of the experiment, nitrotyrosine, TNF-α, IL-1ß, IL-6, and lipid metabolism parameters were determined in blood serum. Furthermore, nitrotyrosine, TNF-α, cholesterol, and triglyceride content were determined in liver homogenates. RESULTS: Serum nitrotyrosine, IL-1ß, and TNF-α concentrations as well as TNF-α content in the liver were significantly higher in both groups exposed to oxysterols (ECh and ES groups) as compared to the C group. The serum IL-6 level and nitrotyrosine content in the liver were significantly higher in the ECh group, as compared to the C and ES groups. There was evidence to support the dyslipidemic effect of studied compounds. CONCLUSIONS: The results indicate that oxidized plant sterols have a similar toxicity profile to that of oxycholesterols, including nitrosative stress induction, proinflammatory effect, and impaired lipid metabolism.

5α,6α-Epoxyphytosterols and 5α,6α-Epoxycholesterol Increase Oxidative Stress in Rats on Low-Cholesterol Diet.

OBJECTIVE: Cholesterol oxidation products have an established proatherogenic and cytotoxic effect. An increased exposure to these substances may be associated with the development of atherosclerosis and cancers. Relatively little, though, is known about the effect of phytosterol oxidation products, although phytosterols are present in commonly available and industrial food products. Thus, the aim of the research was to assess the effect of 5α,6α-epoxyphytosterols, which are important phytosterol oxidation products, on redox state in rats. MATERIAL AND METHODS: The animals were divided into 3 groups and exposed to nutritional sterols by receiving feed containing 5α,6α-epoxyphytosterols (ES group) and 5α,6α-epoxycholesterol (Ech group) or sterol-free feed (C group). The levels of malondialdehyde (MDA), conjugated dienes (CD), and ferric reducing antioxidant potential (FRAP) were assayed in the plasma; anti-7-ketocholesterol antibodies and activity of paraoxonase-1 (PON1) were determined in serum, whereas the activity of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), S-glutathione transferase (GST), and superoxide dismutase (SOD) were assayed in RBCs. RESULTS: During the experiment, the levels of lipid peroxidation products increased, such as CD and anti-7-ketocholesterol antibodies. At the same time, the plasma levels of FRAP and serum activity of PON1 decreased alongside the reduced activity of GPx, GR, and SOD in RBCs. There was no effect of the studied compounds on the plasma MDA levels or on the activity of CAT and GST in RBCs. CONCLUSIONS: Both 5α,6α-epoxyphytosterols and 5α,6α-epoxycholesterols similarly dysregulate the redox state in experimental animal model and may significantly impact atherogenesis.

Expression profiles of selected genes in tumors and matched surgical margins in oral cavity cancer: Do we have to pay attention to the molecular analysis of the surgical margins?

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are associated with an interplay between genetics and the environment; they account for 3% of all diagnosed malignant tumors in men and 2% of those in women. OBJECTIVES: The aim of the study was to analyze the significance of TIMP3, SFRP1, SFRP2, CDH1, RASSF1, RORA, and DAPK1 gene expression in head and neck squamous cell carcinoma tumors, and in matching surgical margin samples. We also analyzed the association between clinical parameters and the expression of the selected genes. MATERIAL AND METHODS: Following surgical resection, 56 primary HNSCC tumors and matching surgical margin samples were collected from patients at the Clinic of Oncological and Reconstructive Surgery of Maria Sklodowska-Curie Memorial Cancer Center and the Institute of Oncology in Gliwice, Poland. The gene expression levels were analyzed by quantitative reverse transcription (qRT)-PCR. RESULTS: SFRP1 gene expression was statistically significantly lower in the tumor samples than in the surgical margins (0.30 ±0.36 vs 0.62 ±0.36; p < 0.01). No correlation was found between gene expression and clinical parameters, except DAPK1, where low expression correlated with alcohol abuse (0.85 ±1.19 vs 1.97 ±3.22; p = 0.074). Moreover, patients with G3 grade tumors, i.e., poorly differentiated tumors, had significantly higher values of DAPK1 gene expression than the G1 (well-differentiated tumors) and G2 (moderately differentiated) groups. CONCLUSIONS: There are many different reasons and concepts for altered gene expression in tumors and surgical margin tissue. Tumor heterogeneity and its microenvironment are undoubtedly linked to the biology of HNSCC. In order to understand specific tumor behavior and the microenvironment, further studies are needed. To find markers connected with cancer development and to provide insight into the earliest stages of cancer development, attention should also be focused on molecular analysis of the surgical margins.

Oxysterols Increase Inflammation, Lipid Marker Levels and Reflect Accelerated Endothelial Dysfunction in Experimental Animals.

OBJECTIVE: Oxidized cholesterol derivatives are thought to exert atherogenic effect thus adversely affecting vascular endothelium. The aim of the study was to assess the effect of 5α,6α-epoxycholesterol on experimentally induced hypercholesterolemia in rabbits, and the levels of homocysteine (HCY), asymmetric dimethylarginine (ADMA), paraoxonase-1 (PON-1), and inflammatory parameters (IL-6, TNF-α, CRP). MATERIAL AND METHODS: The rabbits were divided into 3 groups, 8 animals each, and fed with basic fodder (C), basic fodder plus cholesterol (Ch) or basic fodder plus 5α,6α-epoxycholesterol, and unoxidized cholesterol (ECh). Serum concentrations of studied parameters were determined at 45-day intervals. The study was continued for six months. RESULTS: We demonstrated that adding 5α,6α-epoxycholesterol to basic fodder significantly affected lipid status of the experimental animals, increasing total cholesterol and LDL cholesterol levels, as well as HCY and ADMA levels, whilst leaving the PON-1 activity unaffected. Additionally, the ECh group presented with significantly higher concentrations of inflammatory biomarkers (IL-6, TNF-α, and CRP). In the Ch group, lower yet significant (as compared to the C group) changes of levels of studied parameters were observed. CONCLUSION: Exposure of animals with experimentally induced hypercholesterolemia to 5α,6α-epoxycholesterol increases dyslipidaemia, endothelial dysfunction, and inflammatory response.

Hepatocyte growth factor and epidermal growth factor activity during later stages of rat liver regeneration upon interferon α-2b influence.

INTRODUCTION: Liver regeneration is a complex, highly coordinated process which can be disturbed by the impact of the anti-proliferative interferon α activity. In the model of partial hepatectomy (PH) in the rat the expression of HGF and EGF genes and their molecules' tissue concentrations were analyzed in the later stages of liver regeneration (48-120 h). MATERIAL AND METHODS: 40 three-month-old male Wistar rats were randomized to groups of 20 animals each. The rats of the study group (IFN/H) were injected subcutaneously with IFNα-2b, while the control group was injected with 0.5 ml of 0.9% NaCl (NaCl/H). In the liver tissue samples obtained during hepatectomy and autopsy (regenerating liver mass) the expression of HGF and EGF genes was estimated with the Q-PCR method and the analysis of HGF and EGF molecule concentrations in tissue homogenates was conducted with the ELISA method. RESULTS: HGF but not EGF expression was significantly higher at 48 h after PH, while EGF expression was higher in normal than in regenerating liver tissue at 120 h. The analyses of correlations between expression of HGF and EGF in regenerating liver tissue, both normal and upon IFNα-2b influence, together with correlations between those factors genes' expression and HGF and EGF tissue concentrations in analyzed samples, showed no significant differences. CONCLUSIONS: HGF and EGF are not significantly involved in regulation of later stages of rat liver regeneration. IFNα-2b does not impact expression of their genes or the presence of these growth factor molecules in regenerating liver tissue.

The Content of Reticulocyte Hemoglobin and Serum Concentration of the Soluble Transferrin Receptor for Diagnostics of Anemia in Chronically Hemodialyzed Patients.

BACKGROUND: Chronic renal disease constitutes a serious worldwide clinical problem. An important issue arising early during the treatment of renal failure is anemia. Patients in the end-stage of renal disease chronically treated with hemodialysis frequently suffer from anemia with iron deficiency. OBJECTIVES: The aim of the study was to evaluate the usefulness of determining the reticulocyte hemoglobin content and serum concentration of soluble transferrin receptor in the detection of anemia caused by iron deficiency in comparison with the classic markers of iron circulation in serum in chronic dialysis patients with ESRD. MATERIAL AND METHODS: 66 sets of hematologic results and iron turnover rates were analyzed, sampled from hemodialyzed patients (test group), as well as 34 sets of the same results taken from healthy people (control group). Statistically significant variables were found and a stepwise backward discriminant analysis was performed for them. RESULTS: The results showed that dialyzed patients have a significantly lower serum concentration of hemoglobin, CHr, HCT, TSAT, Fe and TIBC and significantly higher serum concentration of sTfR, ferritin and C-reactive protein compared to the control group. Based on the results of discriminant analysis, we proposed a scheme for assessing the risk of anemia. CONCLUSIONS: The concentrations of hemoglobin, soluble transferrin receptor, iron in the serum and C-reactive protein turned out to be the most useful for diagnostic purposes. Moreover, the concentration of soluble transferrin receptor confirmed its high diagnostic value in the detection of iron deficiency-based anemia in patients undergoing dialysis for chronic renal failure at the end-stage compared to conventional iron turnover ratios in the serum.

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from southern Poland.

In 2008-2011 ticks were collected from southern Poland. Out of 6336 individuals collected and identified as Ixodes ricinus, 768 (2 larvae, 84 nymphs, 417 females, 265 males) were included in molecular study. The aim of this study was to investigate the prevalence and types of genospecies of Borrelia burgdorferi sensu lato in ticks. The polymerase chain reaction (PCR) was applied to detect the presence of pathogens in ticks. Subsequently the amplified DNA was digested with TasI enzyme. The infection rate was 15% (116) of examined ticks. PCR-RFLP analysis allowed distinguishing three genospecies of B. burgdorferi s.l.: B. burgdorferi sensu stricto, B. afzelii, and B. garinii. RFLP analyses of 116 positive samples revealed 96 (83%) monoinfections and 13 (11%) coinfections, whereas unidentified genospecies were present in 7 (6%) of positive samples. In the case of monoinfections, B. burgdorferi s.s. was the predominant species of pathogen in infected ticks - 61.4%. Other genospecies: B. garinii and B. afzelii were detected in 22.9% and 15.6% of the samples, respectively. To sum up, 15 % of ticks were infected by B. burgdorferi s.l which increases the risk of human infections in the recreational areas of southern Poland. Furthermore, there is a need to increase public awareness and implement more preventive measures concerning Lyme disease.

Potential use of histone deacetylase inhibitors in cancer therapy.

Epigenetics is a branch of science that focuses on mechanisms related to control and modification of expression of genetic material without any changes to its sequences. Such mechanisms include post-translational modifications of histones. It is widely known that carcinogenesis is related to hypoacetylation of genes that influence apoptosis, the cell cycle, cell signaling, the immunologic response, angiogenesis and occurrence of metastasis. Currently conducted research focuses on several strategies related to epigenetic therapy. One such strategy is based on the use of histone deacetylase inhibitors. This paper presents mechanisms through which these compounds work and a summary of their characteristics. It also includes a review of clinical tests related to histone deacetylase inhibitors, as well as their relationship with other chemotherapeutic methods. A better understanding of the involved mechanisms will provide a rational basis to improve the therapeutic outcome of available antitumor agents.

8-hydroxy-2'-deoxyguanosine in colorectal adenocarcinoma--is it a result of oxidative stress?

BACKGROUND: 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidatively modified lesions in DNA and is a marker of the oxidative stress. 8-OHdG is a mutagenic lesion and it can mispair with adenine, causing G:C→T: A transversion. Our task was to determine the 8-OHdG level in patients with colorectal adenocarcinoma directly in tumor tissues and corresponding normal mucosa. MATERIAL/METHODS: Samples of tumor tissues and corresponding normal mucosa of 47 patients undergoing surgery for colorectal cancer were analyzed. DNA was isolated from both tumor and normal tissues. Then, DNA was hydrolyzed to nucleotides using nuclease P1 and alkaline phosphatase. The 8-OHdG and 2'-dG (2'-deoxyguanosine) were determined in hydrolysates by high-performance liquid chromatography (HPLC) with electrochemical (EC) and UV detector. RESULTS: The levels of 8-OHdG in colorectal adenocarcinoma tissues were higher than in corresponding normal mucosa. No significant differences were shown in 8-OHdG levels in the cancerous and cancer-free tissues between age and sex and stages A/B and C/D of Duke's classification. CONCLUSIONS: 8-OHdG reflects the local oxidative stress in colon adenocarcinoma tissue together with ageing processes, but not the intensity of tumorigenesis itself. Because of many factors that could influence the oxidative modification of DNA bases, its role as a diagnostic and/or prognostic factor in colon adenocarcinoma seems to be limited.

Methylation of the MGMT and p16 genes in sporadic colorectal carcinoma and corresponding normal colonic mucosa.

BACKGROUND: Colorectal cancer, one of the most aggressive cancers, occurs with a high incidence in most countries. The usual treatment is surgery and subsequent chemotherapy and radiotherapy. Cancer development and progression is dictated by series of alterations in genes such as tumor suppressor genes, DNA repair genes, oncogenes and others. In colorectal carcinogenesis disturbances different from mutations called an epigenetic regulation are also taken into consideration. Epigenetics is defined as a modifications of the genome, heritable during cell division, which do not involve a change in the DNA sequence. In our study we analyzed methylation of CpG islands in the MGMT and p16 genes in sporadic colorectal cancers and normal corresponding colonic mucosa. MATERIAL/METHODS: Fresh tissue samples were obtained from 68 patients (age of 23 to 81 years) with primary colorectal adenocarcinoma and corresponding normal tissues. We used methylation-specific polymerase chain reaction (MSP) for analysis of the methylation status of MGMT and p16. RESULTS: Methylation of MGMT and p16 was detected in 59% and 53% of tumors, respectively. In corresponding normal colonic mucosa methylation of MGMT was detected in 20% and p16 in 18%. The normal colon mucosa obtained from younger patients (age of <65 years) showed less methylation frequency as compared with the normal mucosa from the older ones (age of >65 years). CONCLUSIONS: The older age and female gender are generally associated with higher methylation levels for most CpG islands in normal colonic mucosa. These results indicate that MGMT and/or p16 aberrant methylation may play an important role in colorectal cancer.