Genome-wide association analysis suggests novel loci for Hashimoto's thyroiditis.

PURPOSE: Hashimoto's thyroiditis (HT) is the most common form of autoimmune thyroid diseases. Current knowledge of HT genetics is limited, and not a single genome-wide association study (GWAS) focusing exclusively on HT has been performed to date. In order to decipher genetic determinants of HT, we performed the first GWAS followed by replication in a total of 1443 individuals from Croatia. METHODS: We performed association analysis in a discovery cohort comprising 405 cases and 433 controls. We followed up 13 independent signals (P < 10-5) in 303 cases and 302 controls from two replication cohorts and then meta-analyzed results across discovery and replication datasets. RESULTS: We identified three variants suggestively associated with HT: rs12944194 located 206 kb from SDK2 (P = 1.8 × 10-6), rs75201096 inside GNA14 (P = 2.41 × 10-5) and rs791903 inside IP6K3 (P = 3.16 × 10-5). Genetic risk score (GRS), calculated using risk alleles of these loci, accounted for 4.82% of the total HT variance, and individuals from the top GRS quartile had 2.76 times higher odds for HT than individuals from the lowest GRS quartile. CONCLUSIONS: Although discovered loci are implicated with susceptibility to HT for the first time, genomic regions harboring these loci exhibit good biological candidacy due to involvement in the regulation of the thyroid function and autoimmunity. Additionally, we observe genetic overlap between HT and several related traits, such as hypothyroidism, Graves' disease and TPOAb. Our study adds a new knowledge of underlying HT genetics and sets a firm basis for further research.

Different apoptosis ratios and gene expressions in two human cell lines after sevoflurane anaesthesia.

BACKGROUND: The aim of this study was to determine the effect of a single exposure of carcinoma cells (Caco-2 and HEp-2) to an anaesthetic gas mixture containing sevoflurane 3%, applied for a period of either 1 or 2 h, on the induction of apoptosis, propapototic gene expression and sphingomyelinase activity. METHODS: Apoptosis was determined by flow cytometry. p53, caspase 3 and CYP2E1 gene expression was determined using reverse transcriptase polymerase chain reaction. Activities of acid (aSMase) and neutral sphingomyelinases (nSMase) were measured using methyl-(14)C sphingomyeline, and for de novo ceramide and lipid synthesis [(3)H] palmitic acid was used. All results were compared with controls and analysed by Mann-Whitney and Kruskal-Wallis tests. RESULTS: In the treated Caco-2 cells, the apoptotic ratio increased 24 h after anaesthesia (16.9%; P=0.04). The expression of both p53 and caspase-3 genes increased in Caco-2 and decreased in HEp-2 cells. The CYP2E1 gene expression was observed only in the Caco-2 cells. In control cells, the catalytic activity of aSMase was 2.3 times higher than that of nSMase activity. Decreased aSMase and nSMase activities were observed in Caco-2 cells 24 h after exposition. aSMase activity was halved (54.2%; P=0.06) in HEp-2 cells 24 h after anaesthesia. De novo ceramide synthesis correlated with SMase activity in Caco-2 cells. CONCLUSION: Sevoflurane anaesthesia induces late apoptosis in the colonic and laryngeal cancer cells investigated. Although the results obtained may indicate that an anaesthetic gas mixture containing sevoflurane induces p53-dependent apoptosis in the Caco-2 cells, the mechanism of apoptosis induction is unclear and remains to be elucidated.

Association of vitamin D receptor gene 3'-variants with Hashimoto's thyroiditis in the Croatian population.

Hashimoto's thyroiditis (HT) is the most frequent autoimmune thyroid disease with strong genetic background. Vitamin D receptor (VDR) endocrine system affects immunosuppressive, regulatory and tolerogenic decisions required for induction and maintenance of peripheral immune tolerance. With respect to the biological function of the VDR and functionally plausible gene-expression data, we sought to test whether particular 3'-restriction fragment length polymorphisms (RFLP) and haplotypes previously directly or indirectly associated with VDR mRNA 3'-allelic imbalance phenotype and differences in total VDR mRNA expression are implicated in HT susceptibility. Thus, 145 Croatian HT patients and 145 age-, sex- and ethnically matched euthyroid controls were genotyped for VDR rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI) polymorphisms by polymerase chain reaction-RFLP method. Covariate-adjusted single-locus and haplotype-phenotype regression analyses were performed. Permutation corrections (P(c)) and Akaike Information Criteria were used for model comparisons. The best-fit [global P(c) = 7.2 x 10(-4)]BsmI-TaqI BT haplotype was found significantly more often in subjects without HT [12.2% vs. 3.7%; odds ratio (OR, 95% confidence intervals) = 0.28 (0.14-0.56), P(c) = 8 x 10(-4)], whereas the bT haplotype was significantly more frequent in individuals with HT [45.7% vs. 61.8%; OR = 1.91 (1.37-2.65), P(c) = 4 x 10(-4)]. Two extended BsmI-ApaI-TaqI RFLP haplotypes, the common baT [35.7 vs. 47.3%, OR = 1.63 (1.17-2.27), P(c) = 0.012] and rare BaT variants [6.5 vs. 1.2%, OR = 0.17 (0.06-0.55), P(c) = 1.2 x 10(-3)] were associated with HT, representing predisposing and protective haplotypes, respectively. In single-RFLP association analyses, only rs1544410 polymorphism was associated with HT phenotype (allelic P(c) = 0.0078) and appeared to function under the recessive model, with decreased risk of HT among the BB homozygotes [OR = 0.39 (0.21-0.7), P(c) = 0.0052] when compared to the reference b(+)-genotypes. These data suggest that common haplotypic variants within the VDR gene 3'-region previously linked to VDR mRNA expression and allelic imbalance could be associated with HT in the general population, and thus, may be involved in the pathogenesis of HT.

Antitumor efficiency of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo.

The purpose of this study was to investigate antitumor activity of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo. A novel series of hydrochloride or dihydrochloride salts of the novel 2-(fluoro-substituted phenyl)-6-aminobenzothiazoles (5-7) have been prepared in multistep synthesis starting from 3- or 4-fluorobenzaldehydes and 2-amino-5-nitrothiophenol and evaluated for their antiproliferative activity against human cervical (HeLa), breast (MCF-7), colon (CaCO-2), and laryngeal (Hep-2) carcinomas and against fibroblast cell lines (WI-38). Also, antitumor activity of these compounds was evaluated in vitro and in vivo against murine melanoma (B16-F10), fibrosarcoma (FsaR), and squamous cell carcinoma (SCCVII). The tested compounds were found to exert good cytotoxic activity in vitro. The cytotoxic effect was selective, cell specific, and dose dependent, between 33 microM for MCF-7 and 110 microM for WI-38. Benzothiazoles reduced de novo protein and DNA synthesis up to 75%. All examined benzothiazoles had significant antitumor activity in vivo against melanoma B16-F10, fibrosarcoma, and squamous cell carcinoma. The best therapeutic results were achieved when therapy started 7 days after tumor cell implantation and when benzothiazoles were given repeatedly five times every 2 days, i.e., on day 7, 9, 11, 13, and 15 after transplantation of tumor cells.

Anaesthetic implications of anticancer chemotherapy.

In anaesthetic practice we deal with cancer patients who are scheduled for operations on tumours or other manifestations of malignant disease. Those patients are often debilitated and have significant weight loss accompanied with hypoproteinaemia, anaemia and coagulation disorders. Oncological patients usually present to the anaesthetist before tumour disease surgery, but they are also candidates for elective operations (e.g. hernia repair) and urgent/emergency surgery (e.g. trauma, fractures and ileus). Chemotherapeutic agents given to these patients are potentially noxious, can affect the conduct of anaesthesia and, furthermore, may aggravate the patient's condition. In this review the most commonly used cytostatic drug regimens and their common side-effects are listed. Some preclinical studies on anaesthetic and cytostatic drug metabolism and interactions are emphasized, as well as clinically relevant perioperative alterations that may affect anaesthetic management in cancer patients. An anaesthetist may have to modify a routine anaesthetic regimen in cancer patients especially if anticancer chemotherapeutics were given. Clinically silent toxic drug effects may become apparent during operation, trauma or in the early postoperative course in such patients. Altered reactions to commonly used anaesthetics in patients receiving chemotherapeutics and an impaired stress reaction may occur in such patients. Special attention must be drawn to protection against opportunistic infections.

Antineoplastic activity of novel N-1-sulfonypyrimidine derivatives.

We evaluated the potential activity of novel N-1-sulfonyl derivatives of pyrimidine bases uracil and cytosine on pancreatic carcinoma cells (MIAPaCa2), colon carcinomas cells (HT-29, CaCo2), cervical carcinoma cells (HeLa) and poorly-differentiated cells from lymph node metastasis of colon carcinoma (SW-620). The cytotoxicity of N-1-sulfonylpyrimidine derivatives was analyzed with the MTT cell survival assay and their antiproliferative activity was measured via radioactive precursors incorporation assay. The N-1-sulfonylpyrimidine derivatives affected the growth of all examined cell lines at concentrations of 10(-8)-10(-5) M, by 25-70%. Growth inhibition depended on the tumor cell line type and the concentration of investigated compounds. The compounds 2, 4, 7, 8 and 9 inhibited DNA, RNA and protein synthesis in CaCo2, MIAPaCa2 and HeLa cells. The exposure of tumor cells in vitro to compounds 2, 4, 7, 8, 9, 10 and 11, at the 10(-6) M concentration, caused both morphological (condensation of chromatin, cell shrinkage), as well as biochemical changes (ladder pattern of DNA fragmentation and exposure of phosphatidylserine on outer lipid bilayer plasma membrane) characteristic of apoptosis. After 24 hours of the N-1-sulfonylpyrimidine derivative application, the p53 oncoprotein expression could not be detected by immunocytochemical analysis. On the basis of present results it can be concluded that novel N-1-sulfonylpyrimidine derivatives are promising antitumor agents with a strong antiproliferative activity and an ability to induce apoptosis in treated tumor cells.

In vitro cytotoxicity of three 4,9-diazapyrenium hydrogensulfate derivatives on different human tumor cell lines.

DNA intercalating agents interfere with DNA's role as a template in replication and transcription by inserting an intercalator molecule between adjacent base pairs. We synthesized three potential novel intercalators, 4,9-diazapyrenium hydrogensulfate derivatives: 5, 10-diphenyl-4,9-dimethyl-4,9-diazapyrenium hydrogensulfate (FDAP), 4, 9-dimethyl-4,9-diazapyrenium hydrogensulfate (GDAP) and 2,4,7, 9-tetramethyl-4,9-diazapyrenium hydrogensulfate (MDAP) and tested their biological effects in vitro on four human tumor cell lines (SKBr3: breast carcinoma, HeLa: cervical carcinoma, CaCo2: colon carcinoma and SW620: poorly differentiated cells from lymph node metastasis of colon carcinoma). Cytotoxic effects on cell growth and viability were determined using tetrazolium dye (MTT) assay. DNA synthesis and proliferation of treated cells were studied by the [(3)H]-thymidine incorporation test. DNA fragmentation was analyzed by agarose gel electrophoresis. The growth inhibitory effect was cell-specific and dose-dependent. The most pronounced antiproliferative effect was observed on SKBr3 cells for FDAP (10(-5) M) 91.8%, for MDAP (10(-5) M) 85.3% and on SW620 cells for GDAP (10(-5) M) 65.3%. The DNA ladder fragmentation of treated HeLa and SKBr3 cells, as a hallmark of apoptosis, was observed. Based on specific DNA fragmentation, morphological changes (reduced cell volume, round cell shape, condensed chromatin) and growth inhibition of treated human tumor cells we conclude that tested substances induced apoptotic cell death.

4,9-diazapyrenium dications induce apoptosis in human tumor cells.

We investigated the antiproliferative effects of two planar 4,9-diazapyrenium hydrogenasulphates against human malignant MiaPaCa 2 (pancreatic carcinoma), Hep 2 (laryngeal carcinoma) and human normal fibroblasts (WI 38) cell lines. The tested compounds were very potent in inhibiting the growth of the treated cell lines. Treatment with molar concentrations of the substances (10(-4)-10(-7) M) caused growth inhibition by more than 50%. The morphological changes of treated cells were also observed. Cells became smaller, with condensed chromatin and fragmented nuclei, the characteristics of dying cells. The identification of DNA-fragmentation and the appearance of chromatin aggregation leads us to assume the tested substances induced apoptosis of the investigated tumor cell lines.