Histaminergic System and Inflammation-Related Genes in Normal Large Intestine and Adenocarcinoma Tissues: Transcriptional Profiles and Relations.

Transcriptional analyses such as microarray data have contributed to the progress in the diagnostics and therapy of colorectal cancer (CRC). The need for such research is still present because of the disease being common in both men and women with a high second position in cancer rankings. Little is known about the relations between the histaminergic system and inflammation in the large intestine and CRC. Therefore, the aim of this study was to evaluate the expression of genes related to the histaminergic system and inflammation in the CRC tissues at three cancer development designs: all tested CRC samples, low (LCS) and high (HCS) clinical stage, and four clinical stages (CSI-CSIV), to the control. The research was carried out at the transcriptomic level, analysing hundreds of mRNAs from microarrays, as well as carrying out RT-PCR analysis of histaminergic receptors. The following histaminergic mRNAs: GNA15, MAOA, WASF2A, and inflammation-related: AEBP1, CXCL1, CXCL2, CXCL3, CXCL8, SPHK1, TNFAIP6, were distinguished. Among all analysed transcripts, AEBP1 can be considered the most promising diagnostic marker in the early stage of CRC. The results showed 59 correlations between differentiating genes of the histaminergic system and inflammation in the control, control and CRC, and CRC. The tests confirmed the presence of all histamine receptor transcripts in both the control and colorectal adenocarcinoma. Significant differences in expression were stated for HRH2 and HRH3 in the advanced stages of CRC adenocarcinoma. The relations between the histaminergic system and inflammation-linked genes in both the control and the CRC have been observed.

Effect of static magnetic fields and phloretin on antioxidant defense system of human fibroblasts.

The available evidence from in vitro and in vivo studies is deemed not sufficient to draw conclusions about the potential health effects of static magnetic field (SMF) exposure. Therefore, the aim of the present study was to determine the influence of static magnetic fields and phloretin on the redox homeostasis of human dermal fibroblasts. Control fibroblasts and fibroblasts treated with phloretin were subjected to the influence of static magnetic fields. Three chambers with static magnetic fields of different intensities (0.4, 0.55, and 0.7 T) were used in the study. Quantification of superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPX1), microsomal glutathione S-transferase 1 (MGST1), glutathione reductase (GSR), and catalase (CAT) messenger RNAs (mRNAs) was performed by means of real-time reverse transcription PCR (QRT-PCR) technique. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were measured using a commercially available kit. No significant differences were found in SOD1, SOD2, GPX1, MGST1, GSR, and CAT mRNA levels among the studied groups in comparison to the control culture without phloretin and without the magnet. There were also no changes in SOD, GPx, and CAT activities. In conclusion, our study indicated that static magnetic fields generated by permanent magnets do not exert a negative influence on the oxidative status of human dermal fibroblasts. Based on these studies, it may also be concluded that phloretin does not increase its antioxidant properties under the influence of static magnetic fields. However, SMF-induced modifications at the cellular and molecular level require further clarification.

[Expression of melatonin receptors genes and genes associated with regulation of their activity in endometrial cancer]. / Ekspresja genów receptorów melatoninowych i genów zwiazanych z regulacja ich aktywnosci w gruczolakoraku endometrium.

OBJECTIVES: The aim of the study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in endometrial adenocarcinoma to identify probable diagnostic and prognostic molecular markers. MATERIAL AND METHODS: The material included endometrial adenocarcinoma tissue samples of histopathological grades G1, G2, G3, and normal endometrium. The molecular analysis was performed on 37 patient samples. Total RNA was extracted and used for the microarray HG-U133A analysis. Among 22 283 ID mRNA, only entities of genes associated with regulation of melatonin receptors activity were selected. qRT-PCR was employed for validation, what allowed to compare melatonin receptor genes activation in endometrial cancer tissues to the normal endometrium. RESULTS: The results of the microarray experiments showed that only 18 ID mRNA were differential in endometrial cancer samples as compared to the control at p-value<0.05 and FC(log2)>1.5. These genes were identified as differentially expressed in grade G2-ASMTL, GNA 11, PER2, PTGDS and in grade G3-GNA12, GNA 11. Silencing of RGS4 encoding RGP4, which regulates signal transmission by G protein, was observed in all cancer groups, independently of the histopathological grade. CONCLUSIONS: The profile expression of genes associated with regulation of melatonin receptors activity was different and dependent on the histopathological grade of endometrial cancer and can be an additional diagnostic and prognostic marker Statistically significant was the down-regulation of melatonin biosynthesis genes (ASMTL) and melatonin signal transmitters (GNA 11, GNA 12, RTGS).

[Expression profile of genes associated with the histaminergic system estimated by oligonucleotide microarray analysis HG-U133A in women with endometrial adenocarcinoma]. / Profil ekspresji genów zwiazanych z ukladem histaminergicznym wyznaczony technika mikromacierzy oligonukleotydowych HG-U133A u kobiet z gruczolakorakiem endometrium.

INTRODUCTION: Influence of histamine on tumor development remains obscure. The exact mechanism of this action is not known. Different data indicate high concentrations of histamine in tumor tissues, such as malignant melanoma, breast cancer, colon carcinoma, lymphomas and leukemia. To the best of our knowledge, the literature offers no reports about the role of histamine and of differences between expression patterns of histamine-related genes in endometrial cancer AIM: The aim of the study was a comparative analysis of the gene expression profile involved with histaminergic system in endometrioid endometrial cancer in relation to histologically normal endometrium, and identification of differentiation genes whose transcriptional activity significantly differs in pathomorphological grades G1,G2, G3 of endometrial cancer as compared to the control group. MATERIAL AND METHODS: Total RNA was extracted from 24 endometrial probes using TRIzol reagent (Invitrogen). The expression profile of 119 transcripts associated with histaminergic system was assessed using oligonucleotide microarrays of HG-U 133A (Affymetrix). After normalization of the results with RMA Express software, differentiation genes were mined by the use of one-way analysis ANOVA and U Mann-Whitney test carried out in Gene Spring 11.5 software. RESULTS: Among 119 transcripts, 14 expressed more than 1.5-fold change and were significant at p<0.05 in endometrioid endometrial cancer in relation to the normal endometrium. Further analysis led to the identification of differentially expressed genes in grades G1, G2 and G3 of endometrial adenocarcinoma as compared to the control group, which were specific for each of the studied groups in grade G1 (CPA3), in grade G2 (HNMT LYN, DPT ITPKB, RASA4, APR RAB1 1FIP1, YWHAZ, VAMP8, RAB25) and in grade G3 (HRH3). CONCLUSIONS: Our results confirmed the role of the histaminergic system in the pathogenesis of endometrial adenocarcinoma. The observed differences in the expression of those genes, depending on the grade of adenocarcinoma, may indicate an important role of the isolated differentiation genes in endometrial tumorigenesis.

The influence of chain microstructure of biodegradable copolyesters obtained with low-toxic zirconium initiator to in vitro biocompatibility.

Because of the wide use of biodegradable materials in tissue engineering, it is necessary to obtain biocompatible polymers with different mechanical and physical properties as well as degradation ratio. Novel co- and terpolymers of various composition and chain microstructure have been developed and applied for cell culture. The aim of this study was to evaluate the adhesion and proliferation of human chondrocytes to four biodegradable copolymers: lactide-coglycolide, lactide-co-ε-caprolactone, lactide-co-trimethylene carbonate, glycolide-co-ε-caprolactone, and one terpolymer glycolide-colactide-co-ε-caprolactone synthesized with the use of zirconium acetylacetonate as a nontoxic initiator. Chain microstructure of the copolymers was analyzed by means of ¹H and ¹³C NMR spectroscopy and surface properties by AFM technique. Cell adhesion and proliferation were determined by CyQUANT Cell Proliferation Assay Kit. After 4 h the chondrocyte adhesion on the surface of studied materials was comparable to standard TCPS. Cell proliferation occurred on all the substrates; however, among the studied polymers poly(L-lactide-coglycolide) 85 : 15 that characterized the most blocky structure best supported cell growth. Chondrocytes retained the cell membrane integrity evaluated by the LDH release assay. As can be summarized from the results of the study, all the studied polymers are well tolerated by the cells that make them appropriate for human chondrocytes growth.

Evaluation of melanogenesis in A-375 melanoma cells treated with 5,7-dimethoxycoumarin and valproic acid.

Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10-150 µM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.

Expression patterns of kinin-dependent genes in endometrial cancer.

OBJECTIVE: The present study has focused on the identification of the differences between expression patterns of kinin-dependent genes in endometrial cancer with the use of real-time quantitative reverse transcription polymerase chain reaction and oligonucleotide microarray. MATERIALS AND METHODS: The study group consisted of 50 endometrium samples collected from women with endometrial cancer. Gene expression of kinin receptors BR1 and BR2 was evaluated with real-time quantitative reverse transcription polymerase chain reaction. The analysis of the expression profile of genes related to the kinin mitogenic signal transduction pathway was performed using HG-U133A oligonucleotide microarrays. RESULTS: The transcriptional activity of the B1 receptor for kinins increased in patients with grade 1 (G1) and grade 2 (G2) endometrial cancer when compared to the control group, whereas it decreased in patients with grade 3 (G3) endometrial cancer. The expression of the B2 receptor showed a growing trend reaching the peak in the G2, whereas G3 was characterized by a decrease in the gene transcriptional activity. Significant differential gene expression was recorded for GNB1, PRKAR1A, KRAS, MAP2K2, GNG5, MAPK1, ADCY9, GNG11, JUN, PRKCA, PRKACB, FOS, PLCB4, ADCY8, and GNG12. CONCLUSION: The expression changes in kinin-dependent genes might cause disturbance in the underlying biological processes, which could be important for the pathogenesis of endometrial cancer. This will eventually help to improve treatment strategies for patients with endometrial cancer in the future.

Evaluation of melanogenesis in A-375 cells in the presence of DMSO and analysis of pyrolytic profile of isolated melanin.

The increase of a skin malignant melanoma (melanoma malignum) incidence in the world has been observed in recent years. The tumour, especially in advanced stadium with metastases, is highly resistant to conventional treatment. One of the strategies is to modulate melanogenesis using chemical compounds. In this study, the processes of differentiation and melanogenesis induced by dimethylsulfoxide (DMSO) in human melanoma cells (A-375) were investigated. Natural melanin isolated from A-375 melanoma cell line treated with 0.3% DMSO was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) method. The products derived from pheomelanin have not been stated in the pyrolytic profile of analyzed melanin. Within all products derived from eumelanins, 1,2-benzenediol has been predominated. It has been shown that in the melanoma cells stimulated with 0.3% and 1% DMSO, the increase of transcriptional activity of the tyrosinase gene took place. It was accompanied by the rise of tyrosinase activity and an accumulation of melanin in the cells. The better knowledge about the structure of melanins can contribute to establish the uniform criteria of malignant melanoma morbidity risk.

Aldehydic lipid peroxidation products in human brain astrocytomas.

Among mediators of oxidative stress, highly reactive secondary aldehydic lipid peroxidation products can initiate the processes of spontaneous mutagenesis and carcinogenesis and can also act as a growth-regulating factors and signaling molecules. We explored whether these aldehydes and histone H3 mRNA levels could serve as biomarkers of malignancy and predictive factor in human brain astrocytomas. Histone H3 mRNA, a biomarker of cellular proliferation, was analyzed by QRT-PCR (TaqMan). Aldehydic lipid peroxidation products were determined as their dinitrophenylhydrazone derivatives in specimens obtained from 26 adult patients with brain astrocytomas. RP-HPLC with diode array detector and MSMS spectrometer were used for the analysis. H3 mRNA, 2-hydroxyhexanal, and 4-hydroxynonenal levels were higher in high-grade astrocytomas compared to low-grade astrocytomas and showed negative correlation with survival. Higher levels of 2-hydroxyhexanal and 4-hydroxynonenal, and lower levels of n-hexanal were associated with poorer patient prognosis. Our data suggest that tissue concentrations of aldehydic lipid peroxidation products can assist grading and predicting the clinical outcome in patients with astrocytic brain tumors. Possibly, this parameter will enhance optimal selection of patients for individualized treatment protocols, tailored to unique biochemical and molecular profile of the tumor.

H3 mRNA level as a new proliferative marker in astrocytomas.

Replication-dependent H3.1 and H3.2 histones are encoded by 11 genes. The H3 mRNA levels in brain astrocytomas using real-time RT-PCR assay was examined. The sequence of primers and probe used in amplification was designed basing on the reference sequence GenBank accession no. The H3 mRNA levels correlated with tumor grade (R=0.56, P=0.0012), Ki-67 proliferative antigen labeling index (R=0.58, P=0.0008) and patient survival time (R=-0.50, P=0.005), discriminating low-grade and high-grade tumors. Quantification of H3 mRNA with real-time RT-PCR using the proposed pair of primers may supplement classic proliferative tests and predictive factors in brain astrocytomas.

[Significance of the DNA-HPV research in cervical cancer prevention]. / Znaczenie badan wykrywajacych DNA-HPV w profilaktyce raka szyjki macicy.

The purpose of our research was to assess DNA-HPV frequention observation and evaluation of the diagnostic value mRNA E6 and E7 HPV16 and HPV18 profile concentration in prognostic risk of intraepithelial lesions and cervical cancer progression in women with cytological screening. Human papilloma virus (HPV) infection were detected in 13.8% normal samples, presence HPV16 and 18 in 7.5% samples were detected. HPV 16, 18 or HPV16 and 18 infection were detected in 85% HSIL (high-grade squamous intraepithelial lesion) samples. HPV16 or HPV18 infection in 100% cancer samples were detected. In samples from control group expression of E6 and E7 genes were not detected. In LSIL (low-grade squamous intraepithelial lesion) group HPV16 E7 gene in 2.6% samples, in HSIL group E7 gene in 9.5% samples were detected. In all cancer samples E7 or E6 HPV16 and/or HPV18 were detected.

Immunolocalization and expression of kallistatin and tissue kallikrein in human inflammatory bowel disease.

The distribution of tissue kallikrein (TK) and its plasma inhibitor, kallistatin in plasma and intestinal tissue, was studied in patients with active ulcerative colitis (UC) and Crohn's disease (CD). TK was localized to goblet cells and kallistatin to epithelial cells of normal human intestine. Both proteins are visualized in macrophages inside granulomas in CD as well as in plasmocytes in both CD and UC. Intestinal tissue kallikrein (ITK) and kallistatin are significantly decreased in inflamed intestine compared to noninflammatory controls. TK mRNA is significantly decreased in intestinal biopsy samples from active UC patients compared with inactive patients or controls. Immunoreactive TK is present in plasma in very low concentrations in patients and did not differ in normal subjects. Plasma kallistatin was significantly decreased in patients with active disease compared to normal controls. Our data suggest that release of TK during inflammation plays a role in inflammatory bowel disease.