Ljubica Glavaš-Obrovac

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

Hashimotos 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 (Pc) and Akaike Information Criteria were used for model comparisons. The best‐fit [global Pc = 7.2 × 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), Pc = 8 × 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), Pc = 4 × 10−4]. Two extended BsmI‐ApaI‐TaqI RFLP haplotypes, the common baT [35.7 vs. 47.3%, OR = 1.63 (1.17–2.27), Pc = 0.012] and rare BaT variants [6.5 vs. 1.2%, OR = 0.17 (0.06–0.55), Pc = 1.2 × 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 Pc = 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), Pc = 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.

Tumor-Cell-Targeted Methionine-enkephalin Analogues Containing Unnatural Amino Acids: Design, Synthesis, and in Vitro Antitumor Activity

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

An overview of coagulation disorders in cancer patients

A diversity of coagulation disorders in cancer patients arise from tumor-specific growth characteristics, neoangiogenesis with impaired endothelial lining, defective myelopoiesis, hypoproteinemia or metastatic lesions growth with organ dysfunction. Recent investigations have found a clinically relevant correlation of coagulation disorders and tumor growth. These prompted new therapeutic strategies focused on growth factors with the aim to control tumor metastasis, particularly if used for the treatment of micrometastatic disease. However, such treatment may lead to the life threatening coagulation imbalance. A coagulation homeostasis may become further impaired after nonsurgical cancer therapy, especially after preoperative irradiation, which produces lesions precipitating both bleeding and thrombosis. Anticancer chemotherapy may affect liver function and decrease the synthesis of both procoagulation and anticoagulation factors. The most of chemotherapeutic protocols affect platelet synthesis, which arises as a principal dose-limiting side effect. It was observed both during combined systemic chemotherapy and local antitumor therapy. Although the side effects produced by chemotherapy are reversible, endothelial lesions may persist for many years after the anticancer treatment. Instead of cancer patients, theres a growing cohort of patients with nonmalignant diseases who use cytostatics in the perioperative period, and are candidates for surgical procedures not related to their malignant disease, i.e. hernia repair. In this patient population a special attention must be paid to the preoperative evaluation of coagulation status and thromboprophylaxis. This overview reminds the most common coagulation disorders in cancer patients in the perioperative period. It emphasizes the need for proper patient monitoring which may facilitate the diagnostics and treatment of cancer-related coagulation disorders in the perioperative setting.

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 [3H]-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.

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4-6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4-6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC(50)=1 x 10(-6)M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC(50)=4.3 x 10(-6)M).

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 patients 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.

Hh‐Gli signaling pathway activity in oral and oropharyngeal squamous cell carcinoma

The aim of this study was to examine the role of Hh‐Gli signaling in oral and oropharyngeal squamous cell carcinoma (SCC). The role of this signaling pathway in SCC formation has not yet been elucidated.

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.

Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene.

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5-10 and 15) has been synthesized. Compounds 5-10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA, whereby most of them form stacked dimers along the RNA double helix. The only exception is compound 15 which, due to its structural features, showed no interaction with DNA or RNA. Compounds 5-10 have shown a moderate to strong cytotoxic effect (GI50=1.5-9.0 μM) on a panel of seven tumour cell lines. The diimidazoline derivative 9, due to its highest inhibitory potential on the growth of all tested tumour cell lines, was investigated in more detail by testing its ability to enter into cells and influence the cell cycle. Compound 9 (5 μM) was internalized successfully in cell cytoplasm during a 30-min incubation period, followed by nuclear localization upon 90-min incubation. Significant arrest in HeLa cells in the G2/M phase, shown by cell cycle analysis at an equitoxic (50 μM) concentration, suggests interaction of a studied compound with cellular DNA as the main mode of biological action.

Probing the Structural Properties of DNA/RNA Grooves with Sterically Restricted Phosphonium Dyes: Screening of Dye Cytotoxicity and Uptake

To explore in greater detail the recently reported rare kinetic differentiation between homo‐polymeric and alternating AT‐DNA sequences by using sterically restricted phosphonium dyes that form dimers within the DNA minor groove, new analogues were prepared in which the quinolone phosphonium moiety was kept constant, while the size and hydrogen bonding properties of the rest of the molecule were varied. Structure–activity relationship studies revealed that a slight increase in length by an additional methylene unit results in loss of kinetic AT selectivity, but yielded an AT‐selective fluorescence response. These DNA/RNA‐groove‐bound dyes combine very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. In contrast to longer analogues, a decrease in length (by methylene unit removal) and rearrangement of positive charge resulted in dyes that had switched to the intercalative binding mode to GC DNA/dsRNA but that still form dimers in the minor groove of AT sequences, consequently yielding a significantly different chiro‐optical response. The latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cells.