Krešimir Pavelić

Comparison of five different polymerase chain reaction methods for detection of human papillomavirus in cervical cell specimens

The polymerase chain reaction (PCR) methods enable the detection of large number of human papillomavirus (HPV) genotypes that infect the anogenital tract. In this study, two groups of cervical scrapes with abnormal cytomorphology were analysed. The first group was tested with three sets of consensus primers located within the L1 region of HPV genome, MY09/MY11 (i.e. MY), L1C1/L1C2-1/L1C2-2 (i.e. LC) and pI-1/pI-2 (i.e. pI) primer sets, while the second group of samples, which were all negative with the MY primers, was tested further with the LC primers, as well as with the GP5/GP6 (i.e. GP) primers. The GP primers were used in the nested PCR following amplification with the MY primers (i.e. MY/GP nested PCR). Samples from both groups were also tested with type-specific primers for HPV types 6/11, 16, 18, 31 and 33. In the first study group (N=164) there were 76.2% positive results obtained with at least one set of consensus primers. There were 62.2, 39, 62.2 and 59.1% positive results obtained with the MY, the pI, the LC and the HPV type-specific primer sets, respectively. The best results were obtained when both the MY and the LC primer sets were used, because in combination they detected 75% positive samples compared to 62.2% when used alone. There were 2. 4% samples negative with all consensus primers, but positive with one of the HPV type-specific primers, which increased the overall positivity rate to 78.6%. In the second study group (N=250) there were 8.4, 38.8 and 4% samples positive with the LC primers, the nested MY/GP and the HPV type-specific primer sets, respectively. Thus, the use of the MY/GP nested PCR increased significantly the positivity rate of HPV DNA detection and should be used for samples with a low copy number of HPV DNA. In conclusion, the following diagnostic protocol would be appropriate for detection of cancer-related HPVs: preselection of samples with the MY and the LC primers, additional amplification of the MY- and the LC-negative samples with the MY/GP nested PCR and HPV typing of consensus PCR-positive samples with the HPV type-specific primers.

Metastasis: new perspectives on an old problem

Many hypotheses have been postulated to explain the intricate nature of the metastatic process, but none of them completely accounted for the actual biological and clinical observations. Consequently, metastasis still remains an open issue with only few metastasis-inducing proteins experimentally validated so far. Recently proposed novel metastatic model, where serial and parallel metastatic processes are adequately integrated, might help to bridge the current gap between experimental results and clinical observations. In addition, the identification, isolation and molecular characterization of cancer stem cells, a population of the cells within the tumour mass able to proliferate, self-renew and induce tumorigenesis, will shed new light on the complex molecular events mediating metastasis, invasion and resistance to therapy. Understanding the molecular basis of these tumour characteristics will usher in a new age of individualized cancer therapy. In this review article, we will provide a current overview of molecular mechanisms underpinning metastasis, and discuss recent findings in this field obtained by global molecular profiling strategies such as proteomics.

Novel cyano- and amidinobenzothiazole derivatives: synthesis, antitumor evaluation, and X-ray and quantitative structure-activity relationship (QSAR) analysis.

Synthesis of a series of novel cyano- and amidinobenzothiazole derivatives 3-31 is described. All studied amidino derivatives showed noticeable antiproliferative effect on several tumor cell lines. Cyano derivatives 11-17 showed considerably less pronounced activity because of their poor solubility in aqueous cell culture medium, which was confirmed by the principal components (PC) analysis. Compounds 21, 22, 28, and 29 were tested for their effects on the cell cycle and apoptosis, whereby 22 and 29, having methyl group at the C-6 position in pyridine ring, showed drastic cell cycle perturbations that were both concentration- and time-dependent and induced apoptosis. The QSAR modeling, based on the physicochemical descriptors and on the measured biological activities, indicated the relevance of molecular polarizability and particular distribution of pharmacophores on the molecular surface for activity. In conclusion, benzothiazoles containing either isopropylamidino or imidazolyl groups will be considered as starting compounds for further investigation on lead identification.

Novel Amidino-Substituted Thienyl-and Furylvinylbenzimidazole : Derivatives and Their Photochemical Conversion into Corresponding Diazacyclopenta[c]fluorenes. Synthesis, Interactions with DNA and RNA, and Antitumor Evaluation. 4

Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical cyclization into corresponding diazacyclopenta[ c]fluorenes is described. All studied compounds showed prominent growth inhibitory effect. The fused compounds showed stronger activity than nonfused ones, whereby imidazolyl-substituted compound 11 proved to be the most active one. Besides, it induced strong G2/M arrest of the cell cycle followed by drastic apoptosis, which is in accordance with the DNA intercalative binding mode determined by the spectroscopic studies. Nonfused derivatives induced strong S phase arrest of the cell cycle followed by apoptosis that together with DNA minor groove binding mode pointed to topoisomerase I inhibition. In addition, all nonfused compounds revealed pronounced selectivity toward tumor cells in comparison with nontumor cells. On the basis of the presented results, both nonfused and fused thiophene-containing imidazolyl derivatives should be considered as promising lead compounds for further investigation.

Biological and therapeutic effects of ortho-silicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy

Silicon (Si) is the most abundant element present in the Earths crust besides oxygen. However, the exact biological roles of silicon remain unknown. Moreover, the ortho-silicic acid (H4SiO4), as a major form of bioavailable silicon for both humans and animals, has not been given adequate attention so far. Silicon has already been associated with bone mineralization, collagen synthesis, skin, hair and nails health atherosclerosis, Alzheimer disease, immune system enhancement, and with some other disorders or pharmacological effects. Beside the ortho-silicic acid and its stabilized formulations such as choline chloride-stabilized ortho-silicic acid and sodium or potassium silicates (e.g. M2SiO3; M= Na,K), the most important sources that release ortho-silicic acid as a bioavailable form of silicon are: colloidal silicic acid (hydrated silica gel), silica gel (amorphous silicon dioxide), and zeolites. Although all these compounds are characterized by substantial water insolubility, they release small, but significant, equilibrium concentration of ortho-silicic acid (H4SiO4) in contact with water and physiological fluids. Even though certain pharmacological effects of these compounds might be attributed to specific structural characteristics that result in profound adsorption and absorption properties, they all exhibit similar pharmacological profiles readily comparable to ortho-silicic acid effects. The most unusual ortho-silicic acid-releasing agents are certain types of zeolites, a class of aluminosilicates with well described ion(cation)-exchange properties. Numerous biological activities of some types of zeolites documented so far might probably be attributable to the ortho-silicic acid-releasing property. In this review, we therefore discuss biological and potential therapeutic effects of ortho-silicic acid and ortho-silicic acid -releasing silicon compounds as its major natural sources.

Gastric cancer: the role of insulin-like growth factor 2 (IGF 2) and its receptors (IGF 1R and M6-P/IGF 2R).

Insulin‐like growth factor 2 (IGF 2) appears to be involved in the progression of many tumours. It binds to at least two different types of receptor: IGF type 1 (IGF 1R) and mannose 6‐phosphate/IGF type 2 (M6‐P/IGF 2R). Ligand binding to IGF 1R provokes mitogenic and anti‐apoptotic effects. M6‐P/IGF 2R has a tumour suppressor function—it mediates IGF 2 degradation. Mutation of M6‐P/IGF 2R causes both diminished growth suppression and augmented growth stimulation. The aim of this study was to investigate the role of IGF 2 and its receptors (IGF 1R and IGF 2R) in human gastric cancer. The expression of IGF 2 and its receptors was measured in order to analyse the possible correlation between the activity of these genes and cell proliferation in two different gastric tumour types: diffuse and intestinal. The effect of IGF 1 receptor blockage on cell proliferation and anchorage‐independent cell growth was also examined. Increased expression of IGF 2 and IGF 1R genes (at the mRNA and protein level) was found in gastric cancer when compared with non‐tumour tissue. Furthermore, there was a significant difference between IGF 2 expression in the more aggressive diffuse type and that in the intestinal type of gastric cancer. Moreover, the IGF 2 peptide level in the culture media obtained from the diffuse type of cancer cells was significantly higher when compared with the intestinal type. The level of IGF 2 peptide in the conditioned media strongly correlated with [3H]thymidine incorporation and cell proliferation. On the contrary, IGF 2R mRNA expression was much higher in the intestinal type of cancer than in the diffuse type. In addition, IGF 2R protein expression was substantially lower with progression of the diffuse cancer type to a higher stage. The αIR3 monoclonal antibody strongly inhibited [3H]thymidine incorporation and decreased the number of colonies in soft agar of cells overexpressing IGF 2. These findings suggest that members of the IGF family are involved in the pathogenesis of gastric cancer, probably by autocrine/paracrine stimulation of cell growth. Such tumours might be excellent candidates for therapeutic strategies aimed at interference with this pathway. Copyright

Molecular mechanisms of anticancer activity of natural dietetic products.

Abstract. The efficiency of dietetic supplements in cancer prevention and treatment is a popular and controversial subject of research. New in vitro and in vivo research results indicate that some dietetic supplements do indeed show anticancer activity. The strongest anticancer action has been demonstrated by natural compounds with multifunctional activity. For instance, antioxidants, which also bind to and modulate the activity of protein kinases involved in signal transduction cascades show both cytostatic and cytotoxic activity towards cancer cells. Other activities such as angiogenesis inhibition, nitric oxide synthase inhibition, and pro-oxidants production have also been observed. Catechins and polyphenols from plant extracts such as green tea show the strongest anticancer activity. The initial clinical trials with some flavonoid molecules are already underway.

Medicine on a small scale

Over the past few years, nanotechnology has emerged as a new and exciting research field that deals with the design, synthesis and fabrication of structures at the molecular scale. Smallness is not in itself the prime goal; it is rather the expectation that, by manipulating matter at the molecular level, new intrinsic material properties can be created. Because living matter itself is basically composed of biological ‘nanomachines’ and nanostructures, researchers recognized quite early on that biology and medicine could be prime fields for the application of nanotechnology. In general, nanomedicine can be defined as the monitoring, repairing, construction and control of human biological systems at the cellular level by using materials and structures engineered at the molecular level. It encompasses much more than just being an extension of ‘molecular medicine’, and future products and developments may have extraordinary and far‐reaching implications for the definition, diagnosis and treatment of disease, and for how medicine is practised (Freitas, 2002). The main interests currently lie in improving diagnostic methods and in developing better drug delivery systems to improve disease therapy. More generally, the scientific community is increasingly focusing its attention on the novel chemical and physical properties of nano‐sized materials to develop new applications in regard to human health. > Nanomedicine can be defined as the monitoring, repairing, construction and control of human biological systems at the cellular level by using materials and structures engineered at the molecular level Nanotechnology today deals mainly with two rather different but complementary types of material: nano‐sized structures (or nanoparticles) and nanoporous materials. There are already some exciting developments in the field of diagnostics based on the use of nanoparticles, in particular fluorescent semiconductor quantum dots (QDs). QDs are monodisperse inorganic nanocrystalline particles made from semiconducting material and are typically 2–10 nm in size—about the size of a …

Synthesis, spectroscopic characterization and antiproliferative evaluation in vitro of novel Schiff bases related to benzimidazoles.

A series of novel benzimidazole substituted Schiff bases were synthesized by reaction of aromatic aldehydes with corresponding 2-aminobenzimidazoles. Their structure has been studied by 1H and 13C NMR, IR and UV/Vis spectroscopy. Majority of prepared Schiff bases were tested on their antiproliferative activity in vitro and exerted non-specific antiproliferative activity on the tested cell lines at the highest tested concentration. Compounds 18 and 19 exerted the strongest non-specific antiproliferative effect on all cell lines and a concentration-dependent effect on HeLa and MCF-7 cell lines at micromolar concentrations but simultaneously being highly cytotoxic on human fibroblasts as well.

Expand Long PCR for fragile X mutation detection.

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full‐mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin‐labeled 5′‐(CGG)5–3′ probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.