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The aim of this study was to identify the volatile compounds in hydrosols of different Veronica species (Veronica cymbalaria, V. anagalloides- aquatica, V. agrestis, V. jacquinii, V. officinalis, and V. beccabunga) and to examine their cytotoxic and apoptotic activities on the bladder cancer cell line T24. Hydrosols were obtained by microwave-assisted extraction (MAE) using the Milestone ETHOS X apparatus for 30 min at 800 W (98 °C). The volatile compounds contained in them were analyzed by gas chromatography and mass spectrometry (GC-MS). Cell viability and proliferation were assessed by measuring cell metabolism using the MTT assay. Hydrosols were tested at different dilutions (50%, 40%, 30%, 20%, and 10%) for cytotoxic activity at 4, 24, 48, and 72 hours. For apoptosis, cells were treated with hydrosols for 48 hours. The FITC Annexin V Apoptosis Detection Kit with PI was used to detect cell apoptosis by flow cytometry. The major compounds in all investigated Veronica species were phenylacetaldehyde, caryophyllene oxide and hexadecanoic acid. The hydrosols of the analyzed Veronica species showed inhibitory activity on the bladder cancer cell line T24. The results of the study show that Veronica species are an excellent source of volatile compounds with various biological activities and future research should focus on their hydrosols.

Metabolic profiling of cancer cells has ever growing role in elucidation of molecular base of cancer development, progression and prognosis. In this study, we investigated the effect of a novel thieno[2, 3-b]pyridine derivative on the metabolic profile of two breast cancer lines with different hormonal status ; MDA-MB-231 (triple negative) and MCF-7 (ER, PR, HER2-). The thieno[2, 3-b]pyridines were initially discovered as potential inhibitors of phospholipase C (PLC) isoforms by virtual high throughput screen (vHTS). GC-MS coupled system was used for separation and identification of metabolites. The objective was to identify the compound effect on each metabolite and to find metabolites significantly different between treated and control cells in each line. Data processing, data normalization, statistical analysis and high-level functional interpretation was performed using MetaboAnlayst, a webbased platform for comprehensive analysis of quantitative metabolomics data. Test of statistical significance shows that results for MDA-MB-231 cells are more significant than for MCF-7 cells. PCA results show that drug treatment causes metabolic alternations in both cell lines. Quantitative enrichment analysis was employed to identify patterns of metabolite concentrations and to help elucidate possible biological mechanisms. The results of analysis reveal that treatment has a major impact in glucose/energy metabolism specifically in glycolysis/gluconeogenesis, pyruvate metabolism, Warburg effect, inositol metabolism for both lines. Metabolic profiling of cancer cells plays an increasingly important role for research on the mechanism of action of anticancer drug candidates.

Cells in every epithelium can be roughly divided in three compartments: stem cell (SC) compartment, transient amplifying cell (TA) compartment and mature or functional cell (FC) compartment. Maturation of stem cells is characterized epithelial stromal interaction and sequential maturational movement of stem cell’s progeny through those compartments. In this work we hypothesize that providing an artificial stroma, which murine breast cancer metastatic cells can infiltrate, will induce their differentiation. BALB/c female mice were injected with 106 isogenic 4T1 breast cancer cells labeled with GFP. After 20 days primary tumors were removed, and artificial ε-PCL implants were implanted on the contralateral side. After 10 more days mice were sacrificed and implants along with lung tissue were harvested. Mice were divided in four groups: tumor removal with sham implantation surgery (n=5), tumor removal with ε-PCL implant (n=5), tumor removal with VEGF enriched ε-PCL implant (n=7) and mice without tumor with VEGF enriched ε-PCL implant (n=3). Differentiational status of GFP+ cells was assessed by Ki67 and activated caspase 3 expression, thus dividing the population in SC like cells (Ki67+ aCasp3-), TA like cells (Ki67+ aCasp3+) and FC like cells (Ki67- aCasp3+/-) on flow cytometry. Lung metastatic load was reduced by 20% in mice with simple ε-PCL implant when compared to tumor bearing group with no implant (p

The Capparidaceae family consists of 16 genera and 480 species, which are mostly distributed in warm regions all around the world with Capparis (250) being the largest one. The genus Capparis is native to the Mediterranean basin, but is now widely distributed from Atlantic coasts (Morocco) to Black Sea lands and Caspian Sea. Very important group of bioactive components occurring in capers are glucosinolates (GSL). Three different species from this family were investigated. Leaves of Capparis micracantha DC. were collected in Vienna, Austria, while the fruits of Maerua baillonii Hadj-Moust. were from Madagascar. The whole plants of wild-growing Capparis spinosa L. (stem, leaf, flower, fruit), were harvested from four different locations in Croatia, Split, Mali Ston, Island of Brač, and Island of Hvar. GSLs were identified and quantified by their desulfo counterparts using LC-MS/MS analysis in different plant parts. Methyl GSL (glucocapparin) was predominant in all species ; C. micracantha (4.02 μmol/ g DW), C. spinosa (2.72-13.39 μmol/ g DW) and M. baillonii (12.90 μmol/ g DW). Tryptophan derived GSLs were the second most abundant GSLs: glucobrassicin in C. spinosa (0.26-8.44 μmol/ g DW), 4-hydroxyglucobrassicin in M. baillonii (1.03 μmol/ g DW). In addition, glucohirsutin and glucoarabin were detected as minor ones in C. spinosa. In order to investigate biological potential of GSL degradation products, the isolation of volatiles from plant material collected in Split and islands of Hvar and Brač was performed using conventional hydrodistillation (HD), and using modern microwave assisted distillation (MAD), and hydrodiffusion and gravity (MHG). The isolates rich in methyl isothiocyanate were tested for cytotoxic activity on breast cancer cell line MDA-MB-231 and human bladder cancer cell line T24 using MTT assay. The best cytotoxic activity against MDA-MB-231 cell line was observed after 72h incubation time for Brač MHG isolate (IC50=3.81 μg/mL), and against T24 cell line after 72h for Brač MAD isolate (IC50= 5.95 µg/mL), both samples can be considered as highly active.

Black mustard (Brassica nigra L.) is a plant that belongs to Brassicaceae family and contains glucosinolates which in contact with the enzyme myrosinase usually decompose into isothiocyanates which give mustard its flavour [1]. The most researched degradation products of glucosinolates are isothiocyanates, which are generally known to be the most biologically active [2], but in the presence of epithiospecifier protein (ESP), epithionitriles and nitriles may be formed [3, 4]. Isothiocyanates and their metabolites help reduce the risk of developing various types of cancer such as lung, liver, esophagus, stomach, small and large intestine [5]. Degradation products of glucosinolates may exert their antitumor properties via few different mechanisms that include the immediate detoxification of carcinogens such as the prevention of the formation of carcinogen-induced DNA-adducts [6]. The aim of this study was: to determine the influence of the sample preparation method on the chemical composition of black mustard seeds volatile isolates obtained by microwave-assisted distillation (MAD), microwave hydrodiffusion and gravity (MHG), and Clevenger hydrodistillation. Also, the aim of the study was to determine the antiproliferative activity of volatile isolates against two tumor cell lines (breast cancer cell line MDA-MB-231, and bladder cancer cell line TCCSUP) by MTT assay and its proapoptotic effects determined by flow cytometry. GC-MS analysis showed that but-3-enyl isothiocyanate is a main component in Brassica nigra L. after Clevenger hydrodistillation (80.58%). After MAD and MHG, the main component in that two isolates is 4, 5-epithio-pentanenitrile (50.70%, and 59.93%). After 72 h, MHG volatile isolate from B. nigra showed high antiproliferative activity (9, 06 µg/mL) against human breast cancer cell line MDA-MB-231. The best antiproliferative activity of B. nigra MHG volatile isolate against human bladder cancer cell line TCCSUP was after 48 h and 72 h (58.95 and 27.21 µg/mL).

Introduction: Tropaeolum majus L. altum is a plant that belongs to Tropaeolaceae family and is known for its ornamental and medicinal properties [1]. It contains benzyl glucosinolate (glucotropaeolin) whose degradation product benzyl isothiocyanate shows various biological activities such as anticancer, antimicrobial and anti-inflammatory [2, 3, 4]. Objectives: The aim of this study was to investigate the anticancer activity of T. majus essential oil against human bladder cancer cell line TCCSUP and to determine if membrane alterations are a part of the anticancer mechanism of T. majus essential oil. Materials & methods: The essential oil of T. majus seeds was obtained by microwave-assisted distillation. Gas chromatograph equipped with mass spectrometer was used to identify volatile compounds. The half maximal inhibitory concentration (IC50) of T. majus essential oil was determined against TCCSUP cell line via the MTT assay. Atomic force and optical microscopy were used to detect morphology and membrane changes of cells treated with sub-IC50 concentration. Results: T. majus essential oil has shown anticancer activity against TCCSUP cell line due to high amount of benzyl isothiocyanate. Bright-field microscopy of sub-IC50 concentration treated cells reveals the change in cell morphology and possible membrane blebbing of some cells. Atomic force microscopy analysis suggests notable cell surface alterations/membrane bleb formation as a part of the anticancer mechanism. Conclusion: The T. majus essential oil exhibits anticancer activity against TCCSUP cancer cells by altering cell morphology after exposure with sub-IC50 concentration.

Hedge mustard (Sisymbrium officinale (L.) Scop.), a plant from Brassicaceae family, was collected in Split and in the Krka River area during May 2019. The aim of the study was to isolate and identify glucosinolates (GSLs) and their breakdown products. Different isolation methods have been applied in this work: hydrodistillation in the modified Clevenger apparatus, autolysis, and microwave-assisted distillation and microwave hydrodiffusion and gravity. GC-MS analysis of the volatile isolates indicated that the most abundant volatile product was isopropyl isothiocyanate (ITC), originated from glucoputranjivin degradation. GSLs were also identified and quantified by their desulfo counterparts using HPLC-DAD analysis in different plant parts (root, stem, leaf, flower). Three GSLs were identified in all plant parts: glucoputranjivin (0.68-16.00 µmol/g DW), 4-hydroxyglucobrassicin (0.04-2.73 µmol/g DW) and glucobrassicin (0.24- 6.40 µmol/g DW). Furthermore, glucosinalbin, 4- methoxyglucobrassicin and neoglucobrassicin were also identified in the roots. In order to investigate biological properties (cytotoxic and antiviral), isopropyl ITC was synthesized by one-pot synthesis by reaction of isopropyl amine with CS2 in aqueous K2CO3 solution affording the dithiocarbamate intermediate, which was further desulfurized by adding cyanuric acid at 0 °C to provide the corresponding ITC. After purification with petroleum ether, the ITC was confirmed by GC-MS and spectroscopic techniques.

Three strains of laboratory rats (Lewis, Wistar and Sprague–Dawley) commonly used for the study of movement disorders differ in gait traits. Sialic acid containing glycosphingolipids, called gangliosides, are abundant in the nervous system, where they affect numerous neurochemical events. Cerebellum plays the key role in the integration of body movements. This study investigated glycosphingolipid phenotypes with the hypothesis that they differ in cerebella of the three rat strains. In addition, forebrain and brain stem glycosphingolipid phenotypes were determined. Total glycosphingolipid fractions (neutral and acidic) were analysed by high performance thin- layer chromatography (HPTLC). Complex gangliosides were detected with HPTLC immunostaining by using cholera toxin B subunit after the neuraminidase pretreatment. The most significant differences were found in the cerebellum glycosphingolipid content. Lewis rats showed three fold higher monohexaosylceramides (galactosylceramide + sulfatide)/gangliosides ratio compared to Wistar rats. On the other hand, the cerebellum of Wistar rats contained an increased content of complex gangliosides GD1b, GT1b and GQ1b, fourfold, twofold and tenfold, respectively, compared to Lewis rats. This study shows that Wistar and Lewis rat strains have pronounced differences in glycosphingolipid content and the composition of cerebellum.

Horseradish (Armoracia rusticana P.Gaertn., B.Mey. & Scherb.) is a large-leaved, hardy perennial plant of the Brassicaceae family originating from the temperate Eastern Europe. The word chren is a common name for the horseradish in Slavic languages of Eastern Europe, and it is the most primitive word for the horseradish worldwide. The roots and the leaves were used for medicinal purposes and as a condiment since ancient times. Nowadays, it is primarily cultivated for the delicious, intense pungency of its roots whereas the leaves are used in a lesser extent for salad mixes. Agro-industrial and pharmaceutical sector encourage its usage as a functional food. Horseradish is rich in glucosinolates which degrade into bioactive volatile products, mainly isothiocyanates (ITCs) and nitriles. The roots and the leaves of horseradish were collected from the wild-growing population in Konavle (near Dubrovnik, Croatia) during the spring flowering phenological stage, March 2019. The isolation of volatiles was performed by the solvent free microwave assisted distillation and extraction using 500 W for 30 min and 15 min, respectively. Volatile isolates were analyzed by GC-MS and found to contain allyl ITC as the main glucosinolate degradation product originating form sinigrin, along with phenylethyl ITC originating from gluconasturtiin. Allyl ITC was tested for cytotoxic activity on human bladder carcinoma UM-UC-3 and glioblastoma LN-229 cell lines using MTT assay. Both cell lines were incubated for 4h, 24h, 48h and 72h. The best cytotoxic activity was observed against bladder cancer UM-UC-3 cell line after 24h incubation time (IC50=23.3 μg/mL), and against glioblastoma LN229 cell line after 48h (IC50= 36.5 µg/mL) ; both of which can be considered as moderately active

Lunaria annua L. (annual honesty) belongs to Brassicaceae family, which is highly characterized by glucosinolates (GSL). It is native to the Balkans and southwest Asia. Unmodified L. annua oil has been used on a small scale as an industrial lubricant and as a source of nervonic acid used in pharmaceutical and nutraceutical applications. The goal of the present study was the isolation of the volatiles by conventional methods which include hydrodistillation using a Clevenger type apparatus, CH2Cl2 extraction after autolysis as well as by modern technique using microwave (MW) distillation and extraction. Isothiocyanates (ITCs), the main volatiles identified by GC-MS, originated from GSL degradation, i.e. isopropyl ITC from glucoputranjivin (1), 5-(methylsulfinyl)pentyl ITC from glucoalyssin (2) and 6- (methylsulfinyl)hexyl ITC from glucohesperin (3). Two other ITCs, namely pent-4-enyl ITC and hex-5-enyl ITC were also detected indicating the presence of two more corresponding GSLs. However, LC-PDA-MS/MS analysis of desulfoGSLs confirmed the presence of only 1, 2, and 3. Thus, it can be suggested that pent-4-enyl ITC and hex-5-enyl ITC are formed due to the thermal instability of 5-(methylsulfinyl)pentyl ITC and 6-(methylsulfinyl)hexyl ITC, respectively during GC-MS analysis. Hydrodistillate, CH2Cl2 extract after autolysis as well as MW extract were evaluated for their cytotoxic activity against two tumor cell lines. The activities were observed after 4, 24, 48, and 72 h and expressed as IC50. Hydrodistillate, CH2Cl2 extract and MW extract showed the best cytotoxic activity on human lung cancer cell line A549 during incubation time of 72 h (IC50 152.8, 18.79, and 33.46 μg/mL, respectively), while the best effect on breast cancer cell line MDA-MB-231 was after 48 h (IC50 67.44, 6.0 and 11.8 μg/mL, respectively). Following the Al-Gendy, et al. criteria, who categorized the activities against the tested cell lines based on IC50 values, CH2Cl2 extract and MW extract may be considered as highly active, while hydrodistillate as moderately active.

Records of herbal extracts date back two millennia when essential oils were first obtained by distillation. Only a few minor modifications were made during the period of industrialization and the advancement of science. A recent patent describes a new method for extracting natural products without added any solvent or water by using microwave energy. The solvent free microwave extraction apparatus is an original combination of microwave heating and dry distillation at atmospheric pressure. Based on a relatively simple principle, this method involves placing plant material in a microwave reactor. The internal heating of the in situ water within the plant material distends the plant cells and leads to rupture of the glands and oleiferous receptacles. Brassica is a genus which comprises over 38 species, many of them well-known ones such as broccoli, cabbage, cauliflower, kale, turnip, rapeseed, etc. The distinctive taste, smell as well as pharmacological benefits are mostly due to isothiocyanates and nitriles that derive from glucosinolate degradation [1]. In this work, the isolation of volatiles was performed from commercially obtained samples of three different varieties of Brassica oleracea (broccoli, cabbage, kohlrabi) by microwave assisted distillation (for 35 min) and extraction (15 min) using 500 W. Volatile isolates were analysed by GC-MS technique and evaluated against two tumour cell lines: A549 (human lung cancer cells) and MDA-MB-231 (human breast adenocarcinoma) using MTT assay.

White (Sinapis alba L.), brown (Brassica juncea L.) and black (Brassica nigra L.) mustard belong to Brassicaceae family which is known as the mustards, the crucifers or the cabbage family that contains sulfur compounds (glucosinolates). Isothiocyanates (ITCs) are degradation products of glucosinolates which have various biological activities (anticancer, antioxidant, antimicrobial, antifungal). The aim of this research was the extraction of volatiles rich in ITCs from selected mustard seeds using conventional extraction with organic solvent after autolysis and using microwave extraction and the evaluation of their effects on human cancer cells proliferation. GC/MS analysis showed that fractions from white and brown mustard seeds contain allyl isothiocyanate as a dominant compound. The highest antiproliferative activity against both cancer cells showed brown mustard volatile fraction after microwave extraction (70% and 50% after 72 h and 48 h at the concentration of 100 ug/mL against breast and bladder cancer cells). The use of microwave significantly decreases extraction period and it is appropriate manner to obtain volatile fraction of brown mustard seeds rich with allyl isothiocyanate that shows high antiproliferative activity against human breast and urinary bladder cancer cells.

BACKGROUND: Ganglioside GM3 is found in the plasma membrane, where its accumulation attenuates insulin receptor signaling. Considering the role of skeletal muscles in insulin-stimulated glucose uptake, the aim of the present study was to determine the expression of GM3 and its precursors in skeletal muscles of rat models of type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively). METHODS: Diabetes was induced in male Sprague-Dawley rats by streptozotocin injection (55 mg/kg, i.p., for T1DM induction ; 35 mg/kg, i.p., for T2DM induction), followed by feeding of rats with either a normal pellet diet (T1DM) or a high-fat diet (T2DM). Rats were killed 2 weeks after diabetes induction and samples of skeletal muscle were collected. Frozen quadriceps muscle sections were stained with a primary antibody against GM3 (Neu5Ac) and visualized using a secondary antibody coupled with Texas Red. The muscle content of ganglioside GM3 and its precursors was analyzed by high-performance thin- layer chromatography (HPTLC) followed by GM3 immunostaining. RESULTS: Muscle GM3 content was significantly higher in T2DM compared with control rats (P < 0.001). Furthermore, levels of the GM3 precursors ceramide, glucosylceramide, and lactosylceramide were significantly higher in T2DM compared with control rats (P < 0.05), whereas ceramide content was significantly lower in T1DM rats (P < 0.05). The intensity of the GM3 band on HPTLC was significantly higher in T2DM rats (P < 0.001) and significantly lower in T1DM rats (P < 0.05) compared with control. CONCLUSIONS: The expression patterns of GM3 ganglioside and its precursors in diabetic rats suggest that the role of glycosphingolipid metabolism may differ between T2DM and T1DM.

Volatile sulfur compounds make up a large group of different species found in a 43 plant families and 173 plant genera, including the most important family, Brassicaceae. Glucosinolates, β-thioglucoside-N-hydroxysulfates with variable side chains are secondary metabolites identified in over 16 different plant families. These compounds are found particularly in the Brassicaceae family. Bunias erucago L. (Corn Rocket, Crested wartycabbage) is wild-growing plant in Southern Europe. Leaves, young stems as well as root are edible parts eaten raw or cooked. “Pazija”, (a Turkish name for beet (Beta)] or “pakoleć” is traditional food prepared as mixture of wild vegetables boiled in a mix where the dominant species is B. erucago [1]. Samples of B. erucago are collected from two different locations: Marjan hill (Split) and island of Brač (Škrip). The glucosinolate identification is carried out indirectly by their degradation volatile products obtained by hydrodistillation and organic solvent extraction, mostly isothiocyanates and nitriles using GC-MS technique. Analysis of B. erucago from island Brač had shown the presence of glucotropaeoline and glucodehydroerucin while in the plant collected on Marjan hill five types of glucosinolates had been identified: gluconapin, glucotropaeolin, heptyl glucosinolate, glucodehydroerucin and glucoerucin. Volatile sulfur isolates were also tested for their antioxidative, anticancer, and cholinesterase inhibitory activities.

This study focuses on in-vitro research regarding rotating magnetic field (RMF) exposure as a new treatment possibility for glioblastoma multiforme (GBM) - highly destructive malignant brain tumor in humans. In this study we demonstrated that exposure of LN229 GBM cells to 0.4T 10 Hz RMF resulted in ambiguous data regarding cell viability. Some exposure protocols (12h/day for 2 days) resulted in statistically significant reduction of tumor cell viability after exposure to aforementioned RMF while others (3h/day for 3 days) resulted in increased cell viability. Larger, comparative study using multiple cell lines, different exposure parameters and protocols, should be performed before a final conclusion about the link between viability of human glioblastoma cells and rotating magnetic field exposure can be made.

The anticancer activity of volatile isolates from Allium commutatum Guss., Allium sativum L., and Lepidium graminifolium L., analysed by GC-MS, as well as the pure sulfur volatiles, were evaluated against two human tumor cell lines: glioblastoma cell line LN-229 and bladder cancer cell line UM-UC-3, using MTT assay. The major sulfur volatiles found in A. commutatum hydrodistillate from flower and A. sativum bulb originated from S-alk(en)yl cysteine sulfoxide degradation. The most abundant sulfur volatile in A. commutatum isolate was dipropyl trisulfide, and in A. sativum diallyl disulfide. A. commutatum distillate showed very weak cytotoxic effect on both cancer cell lines except at incubation time of 72h on LN229 cell line (IC50 6.364 µg/mL), while dipropyl trisulfide showed much stronger cytotoxic effect: with IC50 12.34 and 10.35 µg/mL, and IC50 22.19 and 8.434 µg/mL for UM-UC-3 and LN229 cell line, respectively during incubation time of 48 and 72h. Both A. sativum extract and distillate showed strong time and concentration-dependent cytotoxic activity on both cancer cell lines, with the best results at incubation time of 48 and 72h. A. sativum extract had IC50 14.49 and 12.48 µg/mL, and 40.84 and 10.41 µg/mL for UM-UC-3, and for LN229 cell line respectively, during incubation time of 48 and 72h. A. sativum distillate showed very similar results: IC50 20.86 and 14.13 µg/mL, and 17.41 and 12.01 µg/mL for UM-UC-3, and for LN229 cell line, respectively . As expected, an active compound from A. sativum diallyl disulfide showed very strong anticancerogenic potential with IC50 22.3 and 19.07 µg/mL, and 44.69 and 8.85 µg/mL for UM-UC-3, and LN229 cell line, respectively. L. graminifolium extract and distillate sulfur volatiles originated from glucosinolates degradation i.e. 3-methoxybenzyl isothiocyanate, and benzyl isothiocyanate. They didn’t show strong cytotoxic activity on UM-UC-2 cell line. while there was effect on LN229 cell line of distillate at incubation time of 48 h (IC50 53.92 µg/mL) and of extract at incubation time of 48 and 72h (IC50 30.71 and 54.37 µg/mL, respectively). On the contrary, benzyl isothiocyanate showed much stronger cytotoxic effect: with IC50 13.16 and 12.3 µg/mL, and IC50 6.48 and 12.29 µg/mL for UM-UC-3, and LN229 cell line, respectively during incubation time of 48 and 72h.

Antimicrobial peptides (AMPs) are endogenous antibiotics present in all organisms acting directly towards pathogens and also showing immunomodulatory properties. Skin secretions of many tested anuran species have been shown to contain a variety of bioactive peptides that often exhibit antibacterial activity, also against multidrug resistant isolates. We have selectively amplified transcripts likely to encode for AMPs, thus providing their sequences. After RNA extraction from frog skin tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose, forward degenerate primers were designed based on highly conserved signal peptide regions from Ranidae, Hylidae and Bombinatoridae together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions themselves were derived from sequences already available in DADP database and transcriptome data in SRA database. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads which were then assembled into contigs representing nearly full-length AMP-encoding transcripts. Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides from 5 different specimens belonging to 5 different frog species, mostly from Ranidae species. Based on appropriate biophysical properties (e.g. charge, hydrophobicity, amphipathicity) six of the most promising candidates were chosen for chemical synthesis and extensive characterization. All peptides were tested against a panel of Gram-negative and Gram-positive bacteria, as well on tumour cell lines. One peptide, identified in Rana arvalis, proved to be active against both reference ATCC strains and cancer cells, while non-toxic for human MEC-1 cells in a range of concentrations several folds higher then minimal inhibitory concentration (MIC) and IC50 values. Furthermore, the peptide was found to disrupt the bacterial membrane even at sub-MIC concentrations, as observed by flow cytometry and visualised by atomic force microscopy (AFM).

Skin secretions of many tested anuran species have been shown to contain a variety of antimicrobial peptides (AMPs) acting directly towards pathogens, including multidrug resistant isolates, and also showing immunomodulatory properties [1]. We have selectively amplified transcripts likely to encode for AMPs, thus providing their sequences. After RNA extraction from frog skin tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose, forward degenerate primers were designed based on highly conserved signal peptide regions [2], together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions were derived from sequences deposited in DADP database [3] and transcriptome data in SRA database [4]. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads. Those were then assembled into contigs representing nearly full-length AMP-encoding transcripts. Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides from 5 different specimens belonging to 5 different frog species, mostly from Ranidae species. Based on appropriate biophysical properties (e.g. charge, hydrophobicity, amphipathicity) six of the most promising candidates were chosen for chemical synthesis and extensive characterization. All peptides were tested against a panel of Gram-negative and Gram-positive bacteria, as well on tumour cell lines. One peptide, identified in Rana arvalis, proved to be active against both reference ATCC strains and epithelial cancer cell lines, while less toxic for circulating MEC-1 cells in a range of concentrations several fold higher then minimal inhibitory concentration (MIC) and IC50 values. Furthermore, the peptide was found to disrupt the bacterial membrane even at sub-MIC concentrations, as observed by flow cytometry and visualised by atomic force microscopy (AFM). [1] A. Nijnik and R. Hancock, Emerg. Health Threats J. 2, (2009). [2] V. Tessera, F. Guida, D. Juretić, and A. Tossi, FEBS J. 279, 724 (2012). [3] M. Novković, J. Simunić, V. Bojović, A. Tossi, and D. Juretić, Bioinformatics 28, 1406 (2012). [4] Y. Kodama, M. Shumway, and R. Leinonen, Nucleic Acids Res. 40, D54 (2012).

Sesquiterpenes are C15-terpenoids built from three isoprene units. These compounds are commonly found in higher plants, marine organisms and fungi. Naturally, they occur as hydrocarbons or in oxygenated forms including lactones, alcohols, acids, aldehydes, and ketones. Over the last decade, sesquiterpenes have attracted significant attention because of the roles they play in biological systems and their diverse biological activities, such as anti-inflammatory, antibacterial, antioxidant and anti-carcinogenic. The antiproliferative activity of three sesquiterpenes (thuyopsene, nerolidol and farnesol) was evaluated on three lines of human bladder cancer (T24, UM-UC-3, TCCSUP, ) using MTT assay. Thuyopsene is one of the major sesquiterpene constituents of cedarwood, while farnesol and nerolidol can be found in the essential oils of many types of plants and flowers. The tested compounds showed different antiproliferative activity on the cancer cell lines. Farnesol showed the highest impact on T24 cells growth (52% growth inhibition) at concentration of 56 μM, while thuyopsene showed the strongest growth inhibition of 50% and 27% on UM-UC-3 and TCCSUP cell line at concentration of 171 and 122 μM, respectively. Generally, nerolidol showed the lowest antiproliferative potential on the cancer cells. In order to determine type of cell death induced by treatment, Annexin-V-FITC assay was used for the detection of apoptosis by flow cytometry. The results obtained for these three sesquiterpenes open a perspective for their use as antiproliferative agents.

Karcinom je složena bolest u kojoj stanice više ne odgovaraju na signale unutar tkiva u kojem reguliraju staničnu diferencijaciju, preživljavanje, proliferaciju i smrt. Kao rezultat toga, ove stanice se nakupljaju unutar tkiva, dovodeći do lokalnog oštećenja i upale. Postoji više od 200 različitih vrsta karcinoma. [1] Kupusnjače ili krstašice (Brasicaceae, prethodno Cruciferae), porodica su cvjetnica (red Brassicales) i sadrže 338 rodova i oko 3700 vrsta.[2] Izotiocijanati su biološki aktivni produkti hidrolize glukozinolata. Kupusnjače sadrže razne glukozinolate, od kojih svaki hodrolizom formira različite izotiocijanate.[3] In vitro je ispitivano citotoksično djelovanje izotiocijanata izoliranih iz porodice kupusnjača na dvije stanične linije: T24 (karcinom mokraćnog mjehura) i A1235 (glioblastom). Stanična živost i proliferacija određena je mjerenjem staničnog metabolizma koristeći MTT metodu. MTT testom se određuje postotak metabolički aktivnih stanica nakon izlaganja jednom od ekstrakata biljaka, jer mrtve, odnosno metabolički neaktivne stanice ne vežu MTT. Omjer apsorbancije stanica tretiranih vodenim otopinama izotiocijanata te apsorbancije onih koje nisu tretirane pokazatelj nam je citotoksične aktivnosti korištenih izotiocijanata. Najbolji citotoksični učinak na staničnu liniju T24 pokazao je izopropil izotiocijanat pri koncentraciji od 5mM nakon 72h inkubacije. Najbolji citotoksični učinak na staničnu liniju A1235 pokazao je benzil izotiocijanat pri koncentraciji od 20μM nakon 24h inkubacije. In vitro izlaganje stanica karcinoma mokraćnog mjehura (T24) i glioblastoma (A1235) izotiocijanatima izoliranim iz biljne porodice kupusnjača (Brasicaceae) dovodi do smanjenja preživljenja tih stanica. Ispitivani izotiocijanati pokazuju citotoksični učinak ovisan o vremenu inkubacije i koncentraciji. Djelovanje izotiocijanata nije uvijek razmjerno povećanju koncentracije i vremenu inkubacije, te u pojedinim slučajevima dolazi do oporavka stanica. Citotoksični učinak izotiocijanata, što je ujedno i hipoteza našeg ispitivanja, je potvrđen, a idući korak je potvrđivanje tih učinaka in vivo ispitivanjem na modelima karcinoma mokraćnog mjehura i glioblastoma kod životinja. „The research was funded by HRZZ project BioSme (IP-2016-06-1316)” LITERATURA: [1] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4 ; 144(5):646-74. doi:10.1016/j.cell.2011.02.013. [2] The Editors of Encyclopædia Britannica. Brassicaceae. Encyclopædia Britannica. July 17, 2015. URL:https://www.britannica.com/plant/Brassicace ae. (datum pristupa: 30.11.2016.) [3] http://lpi.oregonstate.edu/mic/dietary- factors/phytochemicals/isothiocyanates (datum pristupa:10.4.2017.)

The Centaurea genus species herbal remedies have been used in ethnomedicines in Mediterranean area for treatment of various diseases as they possess a valuable source of bioactive compounds. Sesquiterpene lactones, phenols, flavonoids and lignans are usually regarded as a group of compounds responsible for diverse biological activity of Centaurea plant extracts The chemical constituents were obtained and tentatively identified using high performance liquid chromatography HPLC-DAD-ESI-TOF-MS in negative ionisation mode. The aqueous extracts of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastomma (A1235) cancer cell lines using the MTT assay. Cells were exposed subsequently to both extracts in order to determine type of cell death induced by treatment. The main compounds of herba extract were sesquiterpene lactones: solstitialin A-3, 13 diacetate and epoxyrepdiolide ; quinic acid as organic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, malic acid ; sesquiterpene lactone cynaropicrin ; phenolic compounds: chlorogenic acid and phenylpropanoid syringin. Although both extracts showed significant dose- and time-dependent cytotoxicity against both cancer cell lines, the flower extract exhibited slightly better activity. The majority of the cells died by treatment induced apoptosis. Flower extract (26.25%) compared to a leaf extract (22.15%) showed slightly higher percentage of an apoptosis in T24 cells compared to non-treated cells (3.27 %). Centaurea ragusina L., an endemic Croatian plant species, revealed a good anti- proliferative activity of aqueous extracts on human bladder (T24) and human glioblastomma (A1235) cancer cell lines. Centaurea ragusina aqueous extract represents a valuable source of sesquiterpene lactones and flavonoids with anticancer properties reported in numerous studies.

Apoptoza ili programirana stanična smrt je normalni fiziološki oblik smrti stanice. Proteini koji imaju važnu ulogu u procesu apotoze su: Bcl-2, Smac/Diablo, IAP, kaspaza-3 (ced-3), kaspaza-4 (ced-4), kaspaza-8 (ced-8), kaspaza-9 (ced-9), TNF, Fas, i brojni drugi, koji nizom signalnih puteva aktiviraju nizvodne puteve koji će dovesti do stanične smrti [1]. Oleuropein spada u fitokemijsku skupinu iridoida, točnije sekoiridoida. Iridoidi su derivati ciklopentanopirana, a kod sekoiridoida dolazi do otvaranje ciklopentanskog prstena. U biljkama nastaju biosintetskim putem mevalonske kiseline. Osim oleuropeina, u listu masline prisutni su i njegovi razni derivati, kaodemetiloleuropein, metilni ester oleozida, ligstrozid, dimetilni ester oleozida, oleurozid i drugi [2]. Stupanj apoptoze je praćen protočnom citometrijom. Stanice koje su u fazi rane apoptoze obojane su aneksinom-V obilježenim zelenim fluorokromom, dok su stanice u kasnoj apoptozi obojane propidij jodidom koji fluorescira crveno. Postotak apoptotičkih stanica je izraženkao broj fluorescirajućih stanica u ukupnom broju stanica, čija je vrijednost postavljena kao 100%. Iz rezultata je vidljivo da oleuropein i ekstrakt lista divlje masline djeluju na apoptozu stanične linije A1235. Pri višoj koncetraciji oleuropeina postotak ulaska stanice u apoptozu veći, te je time smanjen postotak preživjelih stanica. Kod ekstrakta lista divlje masline postotak ulaska stanica u apoptozu je povezan s koncentracijom. U odnosu na stanice tretirane oleuropeinom, veći je broj preživjelih A1235 stanica tretiranih ekstraktom lista divlje masline. Dokazanom učinku ekstrakta lista divlje masline vjerojatno doprinose i druge sastavnice poput(iridoidi, triterpeni, flavonoidi, kalkoni, fenolne kiseline, kumarini) koji upravo zbog fitokemijskih karakteristika pridonose ulasku stanice u apoptozu. Kojim mehanizmom oleuropein i ekstrakt lista divlje masline potiču apoptozu možemo samo prepostaviti. U dosadašnjim istraživanjima spominju se inhibicija putem TNF-α, NF-κB, COX-2 i indukcije miRNA [3]. LITERATURA: [1] E. Vrdoljak, M. Šamija, Z. Kusić, M. Petković, D., Gugić, Z.Krajina, Biologija raka: Genska osnova raka, u: Klinička onkologija, A. Raič (ur.), Medicinska naklada, Zagreb, 2013, 1–2. [2] European Medicines Agency. Assessment Report on Olea Europaea L., folium, EMA/HMPC/ 430506/2009, European Medicines Agency, London, 2011. [3] S. Lamy, A. Ben Saad, A. Zgheib, B. Annabi, Olive oil compounds inhibit the paracrine regulation of TNF-alpha-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression. J. Nutr. Biochem. (27) 2016, 136–145.