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Background: Laryngeal dystonia (LD) is a rare neurological movement disorder with an unknown cause affecting the intrinsic muscles of the larynx with a prevalence of 14-35 per 100 000. Vocal symptoms range from sporadic difficulty to sustained inability to phonate, with vocal tremors (voice breaks) or strained or choked speech. In severe cases, an affected subject may barely speak, which significantly impacts the quality of life and may cause psychiatric disturbances, including depression and anxiety. The diagnosis is challenging due to a lack of pathophysiological biomarkers. Alterations in the microstructural and functional integrity of the corticobulbar tract, descending from the primary motor cortex for representation of laryngeal musculature to the brain stem nuclei involved in voice/speech production might be implicated in the pathophysiology of LD [1, 2]. Neurophysiological studies indicate altered inhibitory mechanisms in LD, as with cervical dystonia and focal hand dystonia. More precisely, the cortical silent period (cSP), as a measure of intracortical inhibition in primary motor cortex (M1), has been reported to be shortened in laryngeal muscles in LD subjects [3]. Objective: To present two LD cases, abductor and adductor LD type, and the use of navigated TMS in mapping the laryngeal motor cortex by presenting results of MEP and cSP. Methods: Single magnetic pulse was applied over the M1 for laryngeal muscle representation during slight vocalization. Hook wire electrodes were used to record MEP and cSP from cricothyroid and vocal muscle. Results: In the subject with adductor LD, MEP was elicited from the left cricothyroid muscle (latency of 11.3 ms) with no cSP induced in the cricothyroid and vocal muscle at maximal stimulator output that subject could tolerate. In subject with abductor LD (left vocal muscle abduction), alterations in the duration of cSP were observed when comparing left and right cricothyroid muscles, and no cSP was elicited from the left vocal muscle. Conclusions: In LD, the cortical activation during phonation may not be efficiently or effectively associated with inhibitory processes, leading to muscular dysfunction. These cSP findings may give insight into the maladaptive cortical control during phonation in people with LD. Promising neuromodulatory techniques such as TMS might bring new light to the diagnosis and treatment of LD disorder. References [1] Simonyan, K. ; Tovar- Moll, F. ; Ostuni, J. ; Hallett, M. ; Kalasinsky, V.F. ; Lewin-Smith, M.R. ; Rushing, E.J. ; Vortmeyer, A.O. ; Ludlow, C.L. Focal white matter changes in spasmodic dysphonia: A combined diffusion tensor imaging and neuropathological study. Brain 2007, 131, 447–459. [2] Rogić Vidaković, M ; Gunjača, I ; Bukić, J. ; Košta, V. ; Šoda, J ; Konstantinović, I. ; Braco Bošković, B. ; Bilić, I. ; Režić Mužinić, N. Neurophysiological Basis and Treatment of Focal Laryngeal Dystonia: A Narrative Review and Two Case Reports Applying TMS over the Laryngeal Motor Cortex. J. Clin. Med. 2022, 15, 11(12), 3453. The Patho- [3] Chen, M. ; Summers, R. ; Goding, G.S. ; Samargia, S. ; Ludlow, C.L. ; Prudente, C.N. ; Kimberley, T.J. Evaluation of the Cortical Silent Period of the Laryngeal Motor Cortex in Healthy Individuals. Front. Neurosci. 2017, 11, 88.

The benefits of regular physical activity are well known, but its effect on the immune response is not cleared yet. PURPOSE: The aim of this study is to determine the possible influence of various intensity training on acute pro-inflammatory monocyte activation. METHODS: Ten male recreative runners were tested, using the spiroergometric ramp test, to determine their individual gas exchange threshold (GET). Afterward the subjects were tested three times with three different intensitys (intensity GET, intensity GET– 10% and intensity GET +10%) and performed a 20-minute training during which the blood samples were collected. Using flow cytometry method blood samples were used to analyse CD14++CD16- monocytes activity. RESULTS: Results have shown that physical activity affects on pro-inflammatory monocytes activation, and that there is a connection between intensity and monocytes activation. Our results also suggested that time of blood sampling and the number of tested subjects have an influence on results. CONCLUSION: Recreative running with intensity around aerobic threshold affects CD14++CD16- monocytes activation. But for a better understanding of intensity influence, different blood sampling times and bigger samples should be considered

Background: The primary pathological event in multiple sclerosis (MS) is demyelination with degeneration and loss of axons, which correlates with a permanent functional deficit. Monocytes, together with Th1 and Th17 cells, play a key role in CNS inflammation in MS. Gjelstrup et al. [1] showed a decreased expression of CD40 and CD192 markers in the total monocyte population in people with MS compared to the control group. Further, recent findings suggest an association between the pathophysiological mechanisms of MS (demyelination and loss of axons) and TMS neurophysiological measures [2-4]. Objective: To investigate CD40+ and CD192+ blood monocyte subpopulations in relapsing-remitting MS subjects undergoing TMS assessment of corticospinal tract integrity by recording MEPs from upper and lower extremity muscles. The CD40+ and CD192+ blood monocyte subpopulations were compared to healthy controls and MS subjects having prolonged latency of MEPs and subjects having neat MEP findings. Methods: Blood samples needed for flow cytometry were collected from 23 MS subjects and 10 healthy controls and incubated with anti-human-CD14 FITC antibodies, of phycoerythrin-conjugated antibodies reactive to human CD16, mouse antibodies reactive to human CD192 conjugated with BB700 and Alexa Flour 647 conjugated antibodies reactive to human CD40. Results: All MS subjects and MS subjects with prolonged MEP latency differed from the healthy controls in the percentage of CD192 in non- classical anti-inflammatory CD14+CD16++ monocytes, expression of CD40 in CD14++CD16-, percentage of total monocytes positive for CD40, and expression of CD192 in classical CD14++CD16-. MS subjects with prolonged MEP latency differed from MS subjects with neat MEP finding in the expression of CD40 in CD14++CD16- and the percentage of total monocytes positive for CD40.The percentage of CD192 in anti-inflammatory CD14+CD16++ monocytes correlated with the intensity of stimulation used to elicit MEP responses from lower extremity muscles. Conclusions: Distinct results were found concerning the percentage of CD192+ non-classical monocytes and expression of CD192 in classical monocytes when comparing MS subjects with altered MEP latency and those with neat MEP findings. Study results point to the subclinical relevance of CD192+ monocyte subpopulations in MS. The study represents the first attempt to apply TMS in evaluating corticospinal tract integrity with the immunological investigation of MS. References [1] Gjelstrup MC, Stilund M, Petersen T, i sur. Subsets of activated monocytes and markers of inflammation in incipient and progressed multiple sclerosis. Immunology & Cell Biology 2018 ; 96(2):160-174. doi: 10.1111/imcb.1025 [2] Chalah MA, Palm U, Ayache SS. Editorial: Corticospinal Excitability in Patients With Multiple Sclerosis. Frontiers in Neurology 2021 ; 11: 635612. doi: 10.3389/fneur.2020.635612 [3] Neva JL, Lakhani B, Brown KE, i sur. Multiple measures of corticospinal excitability are associated with clinical features of multiple sclerosis. Behavioral Brain Reserach 2016 ; 297:187-95. doi: 10.1016/j.bbr.2015.10.015 [4] Stampanoni Bassi M, Buttari F, Gilio L, i sur. Inflammation and Corticospinal Functioning in Multiple Sclerosis: A TMS Perspective. Front Neurol 2020 ; 11:566. doi: 10.3389/fneur.2020.00566

Background: Examining the integrity of the corticospinal tract by navigated TMS could help in understanding the neurophysiological correlates of multiple sclerosis (MS) symptoms. Objective: To investigate MEP measures of corticospinal tract integrity with navigated TMS and its correspondence with neurological (EDSS) and neuro- radiological (MRI) classifications in people with relapsing-remitting MS (pwMS). Methods: In a cross-sectional study, MEP measures of corticospinal excitability (RMT, MEP latency, MEP amplitude), clinical disease-related (EDSS), and MRI lesion data were collected in 23 pwMS receiving teriflunomide and compared to non- clinical samples. Results: PwMS subjects differed from non-clinical samples in MEP latency for upper and lower extremity muscles. PwMS with altered TMS finding (prolonged MEP latency or absent MEP), compared to pwMS with neat TMS finding, had higher EDSS score [Median = 3.5(range 0-4) vs. 0.5(0- 2.5)] and EDSS functional pyramidal score [Median = 3.0(0-3.5) vs. 0.5(0-2)]. RMT intensity for mapping representations for lower extremity muscles was predictive for EDSS functional pyramidal scores. Overall, TMS findings classified pwMS as the same as EDSS in 70-83% of cases and were similar or more successful than MRI, which corresponded to EDSS in 57- 65% of cases. PwMS with altered TMS findings differed from those with neat TMS finding on the total number of lesions in the brain corticospinal and in cervical corticospinal tract. Conclusions: The correspondence of TMS with MRI, and EDSS classifications implies the clinical utility of navigated TMS in assessing corticospinal tract integrity in pwMS.

Multiple sclerosis (MS) is currently an incurable autoimmune demyelinating and inflammatory disease affecting more than two million people worldwide. The neurophysiologic studies of the central nervous system (CNS) function comprising motor evoked potentials (MEP) elicited by transcranial magnetic stimulation (TMS) reported consistent and substantial impairments in CNS generally correlating with disability. Studies of CNS showed prolonged central motor conduction times, asymmetry of nerve conduction motor pathways, and prolonged latencies, while resting motor threshold, MEP amplitude, and cortical silent periods showed conflicting results. Studies of peripheral nervous system (PNS) function comprising electroneuronography (ENG) reported impairments of the PNS in MS that were less pronounced and inconsistent. However, neurophysiological investigations applying both TMS and ENG are not frequently used in routine diagnostic procedures to elucidate neurophysiological changes in CNS and PNS in patients with MS. We describe a case of a patient with MS examined with TMS and ENG. The ENG findings did not show clinically meaningful deviations in the PNS of upper and lower extremity muscles. Simultaneously, TMS proved pathological findings in MEPs of upper and lower extremity muscles.

The aim of our study was to investigate the effect of white wine consumption on the expression of inflammatory markers/mediators (MMP-2, MMP-9, NF-ĸB and TGF-β) in the myocardial tissue following experimentally induced permanent myocardial ischemia. Male Sprague-Dawley rats (n=10) were randomized into two groups: drinking a combination of wine (Graševina) and water and those drinking water only for 28 days. After performing the coronary ligation, animals were left to survive for 24 hours and then sacrificed. Three representative zones for each group of animals: infarct/ischemic, peri-infarct/border zone and control/non-ischemic zones were analysed for the expression of immunoreactivity for the above markers. The threshold area % of signal density for each marker was measured and For MMP-9, a significantly smaller expression was found in all three zones of wine drinking animals group (p

Small subpopulation of cancer stem cells (characterized by CD44+CD24− phenotype) expresses GM3(NeuAc) and CD15s glycoconjugates. Acidic glycosphingolipid GM3(NeuAc), containing neuraminic acid substituted with acetyl residue, is known to inhibit tyrosine kinase associated with fibroblast growth factor receptor. CD15s glycoconjugate is the major ligand of endothelial (E) selectin, responsible for cancer cell metastasizing. We performed preliminary studies of viability, and GM3(NeuAc) and CD15s expression after treatment of MDA-MB-231 triple-negative breast cancer cells with a newly developed thienopyridine anticancer compound (1). The MTT assay was used for the cellular metabolic activity determination. Cells expressing highly GM3(NeuAc) and CD15s were analysed for their CD44+CD24- subpopulation percentage, and GM3(NeuAc) and CD15s median fluorescence intensity in CD44+CD24- gate using flow cytometry. The percentages of highly expressing GM3(NeuAc) and CD15s cancer stem cells were lower, and their GM3(NeuAc) median fluorescence intensity was higher after treatment with compound 1 in comparison to non-treated cells. There was no difference in CD15s expression after treatment with the same compound. Knowing the inhibitory role of GM3(NeuAc) in fibroblast growth factor signal transduction, these findings render compound 1 potentially useful for triple- negative breast cancer treatment.

Introduction: The aim of our study was to investigate the effect of white wine consumption on the expression of early inflammatory markers (MMP-2, MMP-9, NF-B) in the myocardial tissue following experimentally induced permanent myocardial ischemia. Materials and methods: Male Sprague-dawley rats (n=10) were randomized into two groups: drinking a combination of wine and water and those drinking water only for 28 days. After performing the coronary ligation, animals were left to survive for 24 hours and then sacrificed. Three representative zones for each group of animals: infarct/ischemic, periinfarct/ border zone and control/nonischemic zones were analysed for the expression of immunoreactivity for the above markers. The threshold area % of signal density for each marker was measured and compared. Results: For MMP-9, a significantly smaller expression was found in all three zones of wine drinking animals group (p

The aim of this study was to estimate effects of hyperbaric (HB) treatment by determination of CD15s and CD11b leukocyte proinflammatory markers expression. In addition, this study describes changes in CD77 and CD34 expression on rat endothelial cells in renal, pulmonary and cardiac tissue following exposure to hyperbaric pressure. Expression of CD11b and CD15s on leukocytes, as well as CD77 and CD34 expression on endothelial cells in cell suspensions of renal, pulmonary and cardiac tissue in rats after hyperbaric treatment and in control rats were determined by flow cytometry. Hyperbaric treatment significantly increased percentage of leukocytes expressing CD15s+CD11b- (from 1.71 ±1.11 to 23.42 ±2.85, P < 0.05). Hyperbaric treatment significantly decreased sum percentage of CD77+CD34- and CD77+CD34+ renal cells (from 16.35±5.5 to 4.48 ±1.28, P < 0.05). Hyperbaric treatment significantly increased percentage of CD34+ pulmonary cells (from 3.27±2.01 to 11.92 ±6.22, P < 0.05). Our study is the first reporting the hyperbaric environment influence on CD34+ heart cells in rats. The current findings of increased percentage of leukocytes expressing endothelial selectin ligand CD15s after hyperbaric treatment, point its role in endothelial damage prevention. We found out a significantly increase in percentage of CD34+ cardiac cells as well as CD34+ pulmonary cells in rats after HB treatment which could be a part of repair mechanism of injured endothelium caused by hyperoxia.

Introduction: After myocardial infarction (MI), CD44 is critical for healing and left ventricle remodelling. Monocyte CD44 antigen is hyaluronan ligand. Hyaluronan is responsible for extracellular matrix structural maintenance and inflammation regulation. CD11b+ monocytes have a role in slowing down angiogenesis and stimulation of inflammation after MI. The aim of this study was to estimate the effect of wine consumption at CD44 and CD11b monocyte expression after MI. In addition, CD15s glycoantigen, known as CD44 and CD11 branch, was determined. Materials and Methods: CD44, CD11b and CD15s positive monocytes in peripheral blood were measured by flow cytometery 24h after MI in male Sprague–Dawley rats (n=9) that consumed white wine for 4 weeks, and compared with control (C), water drinking (C6h and C24h after MI) and sham group. Results: Relative to C24h group, wine-consuming rats differed as follows: percentages of CD15s+CD11b+ monocyte and large monocyte subpopulations were decreased, % of CD44+ monocytes was increased ; expression of CD44 per one cell was two and five fold increased at CD11b+ monocytes and large monocytes, respectively ; CD44 was increased at CD15s+CD11b- monocytes ; CD11b was decreased at CD15s+CD44+ monocytes. In comparison to wine- consuming rats, C6h group showed additional significant difference in the increase of % of CD44+ monocytes and CD44 expression at CD11b+ and CD15s+CD11b- monocytes and CD11b+ large monocytes. Conclusions: Considering that CD44+ monocytes favour left ventricle remodelling and CD11b+ monocytes slow down angiogenesis and can promote injurious inflammation, our findings could indicate beneficial effects of wine pre-treatment on cardiac inflammatory response to MI.

Metastasis, tumor relapse and resistance to therapy remain the principal causes of death for breast cancer patients. The emerging paradigm posits that tumor progression is driven by a small subpopulation of cancer cancer stem cells that exhibit the ability to self-renew and the ability to regenerate the phenotypic heterogeneity of the parental tumor. Many cancer cellular functions have been discovered to be regulated by phospholipase C-gamma 2 (PLC) activation, suggesting that it represents an important therapeutic target for development of anticancer drugs. Here, we investigate the influence of a newly developed, small molecule PLC gamma inhibitor, with or without glucosylceramide synthase inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, D-PDMP) therapy, on the growth, survival and glycophenotype (CD15s and GM3) of breast cancer stem cells (CSCs: CD44+CD24-). MDA-MB-231 breast cancer cells were incubated with glucosylceramide synthase inhibitor (D-PDMP) and/or PLC gamma inhibitor. The viable cells were determined by the MTT assay. Flow cytometric analysis of cells positive to anti-CD44 and glycoantigens, and negative to CD24 was performed 48h after inhibitor treatment. Treatment of the MDA-MB-231 cells with the PLC gamma inhibitor decreased the number of total viable cells. Additional decrease was achieved after combined inhibitor treatment. Percentage of CSCs was significantly decreased only after PLC inhibitor alone treatment. CSC GM3 geometric mean fluorescence intensity (GMI) was increased after PLC inhibitor alone and combined inhibitor treatment. CSC CD15s GMI was slightly decreased after PLC inhibitor alone treatment but significantly after combined inhibitor treatment. PLC gamma inhibitor alone was the most effective against CSCs, but observed decrease of CSC CD15s GMI after combined inhibitor treatment indicates possible lower metastatic ability of dually treated cells.

Mikrodomene endotelnih plazma membrana, takozvane lipidne splavi/kaveole, bogate su neutralnim glikosfingolipidima, globotriozilceramidom Gb3Cer ili CD77. Nekoliko membranskih Ca2+ kanala i pumpi nađeni su u lipidnim splavima/kaveolama. Povećani unos Ca2+ mogao bi uzrokovati endotelni proupalni fenotip. Zbog toga je cilj ove studije odrediti utjecaj hiperkalcijemije u štakora određivanjem izražaja CD77 na CD34+ endotelnim stanicama srca, bubrega i vene kave. Potencijalni proupalni fenotip uzrokovan kalcijem određen je izražajem CD11b i CD15s na leukocitima. Da bi se uzrokovala hiperkalcijemija, Spraque-Dawley mužjacima štakora davana je 1,5% otopina CaCl2 tijekom 14 dana. Izražaj CD77 na CD34+ endotelnim stanicama stanične suspenzije srca, bubrega i vene kave, te izražaj leukocitnih CD11b i CD15s u hiperkalcijemijskoj i kontrolnoj skupini štakora određeni su protočnim citometrom. Koncentracija ioniziranog kalcija u plazmi je bila 1,37 ± 0,01 mM u hiperkalcijemijskoj naspram 1,19 ± 0,03 mM u kontrolnoj skupini štakora. Skupina s hiperkalcijemičnim štakorima pokazala je statistički značajno niži postotak CD34+CD77 stanica u bubregu i veni kavi uz istodobno povećanje postotka leukocita koji imaju izražene proupalne biljege CD11b i CD15s. Pretpostavlja se da su plazma membrane CD77 endotelnih stanica siromašnije u lipidnim mikrodomenama, a time slabije u kontroli Ca2+ izljeva. Postotak CD11b+CD15s+ leukocita mogao bi biti mjera proupalnog učinka kod blage hiperkalcijemije., Endothelial plasma membrane lipid microdomains, so called lipid rafts/caveolae, are rich in neutral glycosphingolipid, globotriaosylceramide, Gb3Cer or CD77. Several plasma membrane Ca2+ channels and pumps are located in lipid rafts/caveolae. Increased Ca2+ influx could cause the development of an endothelial proinflammatory phenotype. Therefore, the aim of this study was to estimate effects of hypercalcemia in rats by determination of CD77 expression on CD34+ endothelial cells in heart, kidney and vena cava. In addition, potential proinflammatory calcium effect was estimated by CD11b and CD15s expression on leukocytes. To achieve hypercalcemia, Sprague-Dawley male rats were drinking CaCl2 solution with a concentration of 1.5% elemental calcium during 14 days. CD77 expression on CD34+ endothelial cells in cell suspensions of heart, kidney and vena cava, as well as leukocyte expression of CD11b and CD15s in hypercalcemic and control rats were determined by flow cytometry. Ionized calcium concentration in plasma was 1.37  0.01 mM in hypercalcemic vs. 1.19  0.03 mM in control rats. Hypercalcemic group showed statistically significantly decreased proportion of endothelial CD34+CD77- cells in the kidney and vena cava in parallel with increase of percentage of leucocytes expressiong proinflammatory markers CD11b and CD15s. In conclusion, it is tempting to speculate that plasma membranes of glycosphingolipid CD77- endothelial cells are poorer in caveolae lipid microdomains and therefore weaker in controlling of Ca2+ influx. The percentage of CD11b+CD15s+ leukocytes could be a measure of proinflammatory effects of mild hypercalcemia.

INTRODUCTION: Prostate cancer remains the second most common cause of cancer related death among men, highlighting the need for new therapies. Many cancer cellular functions have been discovered to be regulated by phospholipase C (PLC) gamma activation, suggesting that it represents an important therapeutic target for development of anticancer drugs. Here, we investigate the influence of a newly developed, small molecule PLC gamma inhibitor, with or without taxane therapy, on the growth and survival of sub-populations of a prostate cancer cell line. MATERIALS AND METHODS: Cells were incubated 48h with Paclitaxel (5 nM) and PLC gamma inhibitor (1 microM) alone or in their combination.The viable cells were determined by the MTT assay. Flow cytometric analysis of cells positive to anti- CD44, anti-CD54, and propidium iodide staining was performed to characterise apoptotic Du145 sub- populations 48h after inhibitor treatment. RESULTS: Treatment of the DU145 prostate cancer cell line with the PLC gamma inhibitor resulted in cell cycle arrest with minimal increase in apoptosis. Sub-populations of prostate cancer cell lines have unique phenotypes (with CD44+ cells being more proliferative and CD54+ cells serving as better CD8+ T cell targets). We examined the effects of the PLC gamma inhibitor on these subpopulations and found that exposure decreased the percentage of both CD44+ (p=0.00007) and CD54+ (p=0.009) sub-populations. In contrast, treatment with Paclitaxel only effected CD44+ cells (p=0.0002). Combination treatment of the PLC gamma inhibitor and Paclitaxel however had synergistic effects on both CD44+ and CD54+ DU145 cells (p=0.005 and p=0.0002, respectively). CONCLUSIONS: These results suggest that a combination of PLC gamma inhibitor and Paclitaxel could be a novel strategy for the treatment of prostate cancer.

Background: The kidney expresses relatively high levels of glycosphingolipid globotriaosylceramide (Gb3Cer or CD77) compared with other organs. Glycosphingolipids are found in membrane lipid rafts where they influence function of receptors and transporters embedded in these domains. In addition, CD77 serves se E. coli verotoxin receptor. Renal glucose reabsorption in diabetes mellitus (DM) is altered, and glycosuria provides a favorable environment for bacteria to reside and grow in the urinary tract, often leading to urinary tract infection. Therefore, the aim of our study was to determine CD77 expression on renal endothelial and non-endothelial cells, in rat models of DM type 1 (T1DM) and type 2 (T2DM). Methods: Diabetes was induced with streptozotocin (55 mg kg−1 for T1DM and 35 mg kg−1 for T2DM) injection to rats which were fed with normal pellet diet (T1DM) or high-fat diet (T2DM). The endothelial marker CD34 and glycosphingolipid CD77 were determined in renal cell suspensions by flow cytometry. Results: Rats of T2DM model showed twice lower proportion of renal CD77 positive non-endothelial cells in comparison to corresponding control rats (P = 0.016). Conclusions: Our study showed that the CD77 positive non-endothelial renal cells could specifically mirror diabetic nephropathy. The reason of increased susceptibility to bacteria other than E. coli in diabetic patients could lay in changed membrane glycosphingolipid composition.

Globotetraosylceramide (Gb4Cer, GalNAc alpha1-3Gal alpha1-4Gal alpha1-4 Glc alpha1-1Cer) has been identified among the major renal neutral glycosphingolipids. Gb4Cer molecules are found in membrane lipid rafts where they can influence the function of receptors and transporters embedded in these domains. Apical membranes of the kidney proximal tubule epithelial cells contain lipid rafts that support absorptive strategy for nutrient reabsorption. Considering diabetic nephropathy and changed glycosphingolipid metabolism in diabetes, the aim of our study was to determine renal Gb4Cer expression in rat model of diabetes type 1. Diabetes was induced with streptozotocin (55 mg kg−1) injection two weeks before Gb4Cer analysis in the kidney of Sprague-Dawley rats weighing 140-160 g (8 males and 5 females) and compared to respective control animals (3 males and 3 females). Gb4Cer fractions in the tissues of diabetic and control rats were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific anti-Gb4Cer polyclonal antibody. Diabetic male rats showed increased expression of Gb4Cer. In addition, two bands of Gb4Cer derivative (corresponding to sulphated Gb4Cer), chromatographed bellow Gb4Cer, were also increased in diabetic male rats compared to control. In contrast to control male rats, control female rats had lower Gb4Cer and only one Gb4Cer derivative band. Diabetic female rats showed lower expression of Gb4Cer and Gb4Cer derivative compared to control female rats. Results of this study are significant in the view of the risk to develop end-stage renal disease that is doubled in men compared with women when age at diabetes type 1 onset is ≥15 years. Knowing that estrogen treatment decreases the content of Gb4Cer in rat kidney, our results point at the role of Gb4Cer in nephropathy development particularly in males.

Cilj istraživanja: Cilj ovog istraživanja je utvrditi kako trening različitog intenziteta (ispod ili iznad bazalnog metabolizma) utječe na monocitne subpopulacije na uzorku sportaša rekreativaca. Materijali i metode: Uzorci krvi svih sudionika uzeti su za analizu 30 minuta prije i nakon treninga. Uzorak je tretiran reagensom za blokiranje Fc receptora te je inkubirana 20 minuta u mraku na 25°C s 4 μL anti-humanih CD14s PerCP-Cy5.5 konjugiranih antitijela i 5 μL fikoeritrinom konjugiranih antitijela reaktivnih na ljudski CD16. Nakon lize crvenih krvnih stanica otopinom za lizu, stanice su analizirane na protočnom citometru. Mjereni su također i uzorci neobojanih stanica i obrađeni su kao negativne kontrole. Podaci prikupljeni citometrom analizirani su pomoću softvera FlowLogic. Rezultati: Kao rezultat istraživanja dobili smo podatak da tjelesna aktivnost ima utjecaj na aktivaciju protuupalne te upalne monocitne skupine. Povećanjem intenziteta tjelesne aktivnosti opažen je porast broja monocitnih skupina. Također rezultati govore da vrijeme uzorkovanja krvi i broj ispitanika utječu na rezultate. Zaključak: Trening različitog intenziteta kod sportaša rekreativaca (ispod ili iznad bazalnog metabolizma) utjecao je na porast broja upalnih i protuupalnih monocita, ali je za bolje razumijevanje utjecaja intenziteta potrebno uzeti u obzir vrijeme uzorkovanja krvi i veći uzorak., The aim of this study: The aim of this research is to determine how training at different intensities (below or above basal metabolism) affects monocyte subpopulations in a sample of recreational athletes. Methods: Blood samples of all participants were taken for analysis 30 minutes before and after training. The sample was treated with Fc receptor blocking reagent and incubated for 20 minutes in the dark at 25°C with 4 μL of anti-human CD14s PerCP-Cy5.5 conjugated antibodies and 5 μL of phycoerythrin-conjugated antibodies reactive to human CD16. After the lysis of red blood cells with lysis solution, the cells were analyzed on a flow cytometer. Samples of unstained cells were also measured and processed as negative controls. Data collected by cytometer were analyzed using FlowLogic software. Results: As a result of the research, we obtained an information that physical activity has an impact on the activation of anti-inflammatory and inflammatory monocyte groups. By increasing the intensity of physical activity, an increase in the number of monocyte groups was observed. The results also tell that the time of blood sampling and the number of subjects have an influence on results. Conclusion: Training of different intensity in recreational athletes (below or above basal metabolism) affects the increase in the number of inflammatory and anti-inflammatory monocytes, but for a better understanding of intensity influence, it is necessary to take into account the time of blood sampling and a larger sample.

Objectives: The purpose of this study was to determine the effects of treating bladder cancer cells with the newly synthesized thieno[2,3-b]pyridine anticancer agents, by focusing on its cytotoxic effects on the investigated human cell line T24. Methods: The T24 bladder cancer cell line was treated with a newly developed thieno[2,3-b]pyridine anticancer compounds: Inhibitor 5 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, Inhibitor 6 3-amino-N-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, Inhibitor 8 (E)-3-amino-5-(3-(3-bromophenyl)acryloyl)-N-(3-chloro-2-methylphenyl)-6-methylthieno[2,3-b]pyridine-2-carboxamide and Inhibitor 9 (E)-3-amino-5-(3-(3-bromophenyl)-1-hydroxyallyl)-N-(3-chloro-2-methylphenyl)-6-methylthieno[2,3-b]pyridine-2-carboxamide to determine its cytotoxic effect on T24 cells. The 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to analyze the cellular metabolic activity and determine the cytotoxic effect. Results: Inhibitors 5, 6, 8 and 9 all showed significant cytotoxic effect on the bladder cancer cells in a dose- and time-dependent manner. The most effective was Inhibitor 8 with an IC50 value of 0.4041 μg/mL after 72 hours. Conclusion: Due to their cytotoxic effect on bladder cancer cells, Inhibitors 5, 6, 8 and 9 deserve further attention as a potential treatment for transitional cell cancer., Ciljevi: Svrha ove studije bila je utvrditi učinke tretiranja stanica raka mokraćnog mjehura novosintetiziranim tieno[2,3-b]piridin antitumorskim spojevima, fokusirajući se na njihove citotoksične učinke na ispitivanu humanu staničnu liniju T24. Materijali i metode: T24 stanična linija raka mokraćnog mjehura tretirana je novosintetiziranim tieno[2,3-b]piridin antitumorskim spojevima: Inhibitor 5 - 3-amino-N-(3-kloro-2-metilfenil)-5-okso-5,6,7,8-tetrahidrotieno[2,3-b]kinolin-2-karboksamid, Inhibitor 6 - 3-amino-N-(naftalen-1-il)-5-okso-5,6,7,8-tetrahidrotieno[2,3 -b]kinolin-2-karboksamid, Inhibitor 8 - (E)-3-amino-5-(3-(3-bromofenil)akriloil)-N-(3-klor-2-metilfenil)-6-metiltieno[2, 3-b]piridin-2-karboksamid i Inhibitor 9 - (E)-3-amino-5-(3-(3-bromofenil)-1-hidroksialil)-N-(3-klor-2-metilfenil)-6- metiltieno[2,3-b]piridin-2-karboksamid, kako bi se odredio njihov citotoksični učinak na T24 stanice. Proveden je test 3-(4,5-dimetiltiazolil-2)-2,5-difeniltetrazolij bromida (MTT) kako bi se analizirala stanična metabolička aktivnost i odredio citotoksični učinak. Rezultati: Svi inhibitori 5, 6, 8 i 9 pokazali su značajan citotoksični učinak na stanice raka mokraćnog mjehura i taj učinak je ovisan o dozi i vremenu inkubacije. Najučinkovitiji je bio Inhibitor 8 s IC50 vrijednošću od 0,4041 μg/mL nakon 72 sata. Zaključci: Zbog svog citotoksičnog učinka na stanice raka mokraćnog mjehura, Inhibitori 5, 6, 8 i 9 zaslužuju daljnju pozornost kao potencijalni tretman za rak prijelaznih stanica.

Cilj: Cilj ovog istraživanja jest dokazati djelovanje sintetski dobivenog inhibitora iz skupine tieno[2,3-b]piridina na apoptozu stanica staničnih linija SK-OV-3 i OVCAR-3 humanog karcinoma jajnika. Pretpostavljeno je da će se nakon tretiranja tumorskih staničnih linija inhibitorom postotak stanica u ranoj, kasnoj te ranoj i kasnoj apoptozi ukupno, povećati. Materijali i metode: Pomoću MTT metode određena je optimalna koncentracija inhibitora kojom su tretirane stanice staničnih linija SK-OV-3 i OVCAR-3 humanog karcinoma jajnika tijekom 48 sati. Dodatkom aneksina V i propidij jodida stanice su analizirane protočnim citometrom kojim se dobije postotak stanica u ranoj i kasnoj apoptozi kod tretiranih i netretiranih stanica raka, a dobije se i grafički prikaz stanica. Rezultati: Prikazani su kao postotak rane i kasne apoptoze i preživjelih stanica. Djelovanjem inhibitora ostvaren je statistički značajan porast apoptotskih stanica stanične linije SK-0V-3 u ranoj, kasnoj te ranoj i kasnoj apoptozi ukupno u odnosu na netretirane stanice. Slični rezultati zabilježeni su i za staničnu liniju OVCAR-3, gdje se također bilježi statistički značajan porast apoptotskih stanica u odnosu na netretirane stanice. Zaključci: Novo-sintetizirani tieno[2,3-b]piridinski spoj doveo je do statistički značajnog porasta postotka stanica u ranoj, kasnoj te ranoj i kasnoj apoptozi ukupno u odnosu na netretirane stanice. Ovakvi rezultati nisu zanemarivi, a spoj je potencijalno važan za razmatranje i daljnje istraživanje u području liječenja karcinoma., Objectives: The aim of this study was to demonstrate the effect of a synthetically derived inhibitor from the thieno[2,3-b]pyridine group on cell apoptosis of SK-OV-3 and OVCAR-3 cell lines of human ovarian cancer. It is hypothesized that after the treatment of tumor cell lines with an inhibitor, the percentage of cells in early, late, and early and late apoptosis together will increase. Materials and methods: Using MTT method, optimal concentration of inhibitor was determined with whom ovarian cancer cells of SK-OV-3 and OVCAR-3 cell lines were treated for 48 h. After adding annexin V and propidium iodide, the cells were analyzed by flow cytometer to obtain the percentage of cells in early, late and early and late apoptosis in total in treated and untreated cancer cells, and a graphical representation of the cells is also obtained. Results: They are represented as the percentage of early and late apoptosis and surviving cells. The effect of inhibitor resulted in a statistically significant increase in apoptotic cells of the SK-OV-3 cell line in early, late and early and late apoptosis in total compared to untreated cells. Similar results were recorded for the OVCAR-3 cell line, where a statistically significant increase in apoptotic cells was also recorded compared to untreated cells. Conclusions: The treatment of ovarian cancer cells with newly synthesized thieno[2,3-b]pyridine led to a statistically significant increase in the percentage of cells in early, late and early and late apoptosis in total compared to untreated cells. These results are not negligible, and the compound is potentially important for the further investigation in the cancer therapy field.

Cilj istraživanja: Cilj je istraživanja bio ispitati potencijalno citotoksično djelovanje fenolnih spojeva izoliranih iz pupa masline sorti Leccino i Frantoio na dvije stanične linije humanih karcinoma dojke: MDA-MB-231 i MCF-7. Broj karcinomskih stanica nakon izlaganja uzorcima pretpostavljeno se trebao smanjiti u odnosu na kontrolnu skupinu. Materijali i metode: Ispitivanje citotoksičnosti fenolnih spojeva na dvjema staničnim linijama karcinoma dojke rađeno je MTT testom nakon 4, 24, 48 i 72 sata inkubacije. Usporedbom apsorbancije nastalog formazana kod stanica tretiranih spojevima dobivenih iz uzoraka masline te apsorbancije kontrolne skupine, odnosno netretiranih stanica, dobiven je postotak preživljenja karcinomskih stanica. Rezultati: Rezultati izlaganja stanica karcinoma dojke (stanične linije MDA-MB-231 i MCF- 7) ekstraktima masline E3 (sorta Leccino) i E4 (sorta Frantoio) prikazani su grafički u odnosu postotka metabolički aktivnih stanica i koncentracije spoja. Najznačajniji učinak na staničnu liniju MDA-MB-231 postignut je nakon inkubacije od 48 sati pri koncentraciji od 200 μg/mL za obje sorte, a na staničnu liniju MCF-7 nakon inkubacije od 72 sata pri koncentraciji od 100 μg/mL za sortu E3 i koncentraciji od 200 μg/mL za sortu E4. Zaključak: Ekstrakti pupa masline pokazuju određen citotoksični utjecaj na stanične linije humanih karcinoma dojke MDA-MB-231 i MCF-7, koji ovisi o vremenu izlaganja i koncentraciji, te u određenoj mjeri smanjuju preživljenje stanica karcinoma. Učinak ekstrakata nije uvijek razmjeran povećanju koncentracije. Ekstraktima sorti maslina korištenih u našem istraživanju nije dostignuta IC50 vrijednost., Objective: The aim of the research was to examine the potential cytotoxic effects of phenolic compounds isolated from Leccino and Frantoio olive buds on two cell lines of human breast cancer: MDA-MB-231 and MCF-7. The number of carcinoma cells was assumed to be reduced after exposure to phenolic compounds in comparison with the control group. Materials and methods: Cytotoxic effects of phenolic compounds on breast cancer cell lines were determinged with MTT assay, after the periods of 4, 24, 48 and 72 hours of incubation. The comparison between the absorbance of created formazan in cells treated with samples obtained from the olive buds and the absorbance of the control group (cells untreated with olive bud samples) established the survival rates of carcinoma cells. Results: The results of breast cancer cells (cell lines MDA-MB-231 and MCF-7) exposure to olive extracts E3 (Leccino sort) and E4 (Frantoio sort) are shown graphically as relation of the percentage of metabolically active cells to the concentration of the compound. The most significant effect on the MDA-MB-231 cell line was achieved after incubation of 48 hours at a concentration of 200 μg/mL for both sorts, and on the MCF-7 cell line after incubation of 72 hours at a concentration of 100 μg/mL for the E3 sort and concentration of 200 μg/mL for the E4 sort. Conclusion: Olive bud extracts show a certain cytotoxic effect on human breast cancer cell lines MDA-MB-231 and MCF-7, which depends on exposure time and concentration, and to a certain extent reduce the survival of said cancer cells. The effect of extracts is not always proportional to the increase in concentration. The extracts of the cultivars used in our research did not reach the IC50 value.

Cilj istraživanja: Cilj istraživanja bio je odrediti postotke monocitnih subpopulacija kod pacijenata s Crohnovom bolešću koji su liječeni biološkom ili nebiološkom terapijom. Materijali i metode: Uzorci krvi, uzeti svim sudionicima istraživanja u jutarnjem razdoblju nakon najmanje 10-satnog posta, korišteni su za biokemijsku analizu i protočnu citometriju. Krv je uzeta iz antekubitalne vene koristeći polietilenski kateter. Sto mikrolitara pune krvi tretirano je reagensom za blokiranje Fc receptora (Miltenyi Biotec GmbH, Bergisch Gladbach, Njemačka) što je onemogućilo nespecifično vezanje protutijela. Uz to, dodano je 4 μL anti-humanog CD14s PerCP-Cy5.5- konjugiranog protutijela (eBioscience, SAD), 4 μL protutijela konjugiranog fikoeritrinom reaktivnog na humani CD16 (eBioscience, SAD) i 10 μL mišjeg protutijela reaktivnog na humani CD44 konjugiranog s FITC (BD Pharmingen, San Diego, CA). Zatim su uzorci inkubirani u trajanju od 20 minuta na 25 °C u mraku. Nakon liziranja eritrocita dodatkom otopine za lizu (Miltenyi Biotec GmbH, Bergisch Gladbach, Njemačka), monociti su analizirani protočnim citometrom (BD Accuri C6, BD Biosciences, Belgija). Rezultati: Rezultati koje smo dobili pokazali su dvostruko veći postotak CD14⁺⁺CD16⁺ monocita u umjereno aktivnoj Crohnovoj bolesti u odnosu na blagu i tešku (p = 0,002). Isto tako, primjena nebiološke terapije smanjila je za 2,3 puta postotak neklasičnih CD14⁺CD16⁺⁺ monocita, dok su pacijenti koji su primali biološku terapiju imali sličan postotak kao kontrolna skupina. Zaključci: Biološka terapija je povoljnija od nebiološke jer ne snižava postotak neklasičnih protuupalnih CD14⁺CD16⁺⁺ monocita., The aim of this study: The aim of the study was to determine the percentages of monocyte subpopulations in patients with Crohn's disease treated with biological or non-biological therapy. Methods: Blood samples, taken from all research participants in the morning period after at least a 10- hour fast, were used for biochemical analysis and flow cytometry. Blood was taken from the antecubital vein using a polyethylene catheter. 100 microliters of whole blood were treated with Fc receptor blocking reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) to prevent nonspecific binding. In addition, 4 μL of anti-human CD14s PerCP-Cy5.5-conjugated antibodies (eBioscience, USA), 4 μL of phycoerythrin-conjugated antibodies reactive to human CD16 (eBioscience, USA), and 10 μL of mouse antibodies reactive to human CD44 conjugated with FITC (BD Pharmingen, San Diego, CA). The samples were incubated for 20 minutes at 25 °C in the dark. After lysing erythrocytes with additional lysis solution (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), monocytes were analyzed with a flow cytometer (BD Accuri C6, BD Biosciences, Belgium). Results: The results we obtained showed a twice higher percentage of CD14⁺⁺CD16⁺ monocytes in moderately active Crohn's disease compared to mild and severe (p = 0.002). Likewise, the use of non-biological therapy decreased by 2.3 times the percentage of non-classical CD14⁺CD16⁺⁺ monocytes, while patients who received biological therapy had a similar percentage as the control group. Conclusion: Biological therapy is more favorable than non-biological therapy because it does not lower the percentage of non-classical anti-inflammatory CD14⁺CD16⁺⁺ monocytes.

Cilj istraživanja: Cilj je istraživanja usporedba upalnog odgovora kod različito liječenih pacijenata oboljelih od ulceroznog kolitisa. Materijali i metode: Uzorci krvi svih sudionika uzeti su za analizu. Uzorak je tretiran Fc-receptor blokirajućim reagensom te je uzorak inkubiran 20 minuta bez izvora svjetlosti pri sobnoj temperaturi uz dodatak anti-humanog CD14s PerCP-Cy5.5-konjugiranog protutijela i protutijela konjugiranog fikoeritrinom, reaktivnog na humani CD16 te dodatak mišjih antitijela reaktivnim na humani CD44, koji je konjugiran s fluorescein izotiocijanatom. Nakon lize stanica, stanice su analizirane pomoću protočne citometrije. Podatci su obrađeni korištenjem FlowLogic Software-a. Rezultati: Pacijenti oboljeli od ulceroznog kolitisa liječeni biološkom terapijom imali su značajno smanjenje medijana intenziteta fluorescencije CD44 biljega na CD14+CD16++ subpopulaciji monocita. Medijan intenziteta fluorescencije CD44 biljega na CD44+CD14+ limfocitima gotovo je dvostruko veći kod pacijenata liječenih biološkom terapijom u usporedbi s pacijentima liječenih nebiološkom terapijom. Postotci CD44+CD14+ limfocita nisu se pokazali različitima između navedenih skupina. Zaključak: Opisana je različita ekspresija CD44 biljega na neklasičnim monocitima pacijenata, s obzirom na terapiju koju pacijent prima. Osim neklasičnih monocita, zahvaćeni su i CD44+CD14+ limfociti – uočena je različita ekspresija CD44 biljega ovisno o terapiji., The aim of research: The aim of this research was comparison of inflammatory response between differently treated patients with ulcerative colitis. Materials and methods: Blood samples of all participants were taken for analysis. The sample was treated with Fc receptor blocking solution, then incubated for 20 minutes without a light source at room temperature with the addition of anti-human CD14s PerCP-Cy5.5-conjugated antibodies and phycoerythrin-conjugated antibodies reactive to human CD16 and with murine antibodies reactive to human CD44, conjugated with fluorescein isothiocyanate. After cell lysis, cells were analyzed by flow cytometry. Data were processed using FlowLogic Software. The results: Ulcerative colitis patients treated with biological therapy had a significant decrease in the median fluorescence intensity of the CD44 marker on the CD14+CD16++ subpopulation of monocytes. The median fluorescence intensity of the CD44 marker on CD44+CD14+ lymphocytes is almost twice as high in patients treated with biological therapy compared to patients treated with non-biological therapy. The percentages of CD44+CD14+ lymphocytes did not differ between the mentioned groups. Conclusions: Different expression of the CD44 marker on non-classical monocytes of patients was described, with regard to the therapy the patient received. In addition to non-classical monocytes, CD44+CD14+ lymphocytes are also affected – different expression of the CD44 marker was observed depending on the therapy.

Cilj istraživanja: Cilj istraživanja bio je dokazati apoptotsko djelovanje tieno[2,3-b]piridinskog derivata (E)-3-amino-5-(3-(3-bromfenil)akriloil)-N-(3-kloro-2-metilfenil)-6-metiltieno[2,3-b]piridin-2-karboksamida (inhibitor 8) na stanice humanog karcinoma grlića maternice staničnih linija HeLa i SiHa. Materijali i metode: MTT testom provedeno je ispitivanje citotoksičnosti na staničnim linijama HeLa i SiHa te je određena potrebna koncentracija inhibitora 8 kojom je ispitivano njegovo apoptotsko djelovanje. Apoptotski učinak inhibitora 8 na stanične linije HeLa i SiHa praćen je protočnom citometrijom. Rezultati: Prikazani su grafički kao postotak rane i kasne apoptoze i preživjelih stanica. Inhibitor 8 je inducirao ranu apoptozu kod obje ispitivane stanične linije. Nije pokazao značajan učinak na HeLa stanice u kasnoj apoptozi, dok je na SiHa stanice u ranoj apoptozi pokazan najznačajniji učinak. Zaključci: In vitro izlaganje staničnih linija humanog karcinoma grlića maternice, HeLa i SiHa, tieno[2,3-b]piridinskom derivatu dovodi do povećanja postotka stanica u fazi apoptoze. Tretiranje karcinomskih stanica tieno[2,3-b]piridinskim derivatom mogao bi predstavljati novi terapijski pristup liječenja. Potrebna su daljnja in vitro i in vivo ispitivanja na životinjskim modelima kako bi se potvrdilo djelovanje., Objectives: The aim of the research was to prove the apoptotic action of the thieno[2,3-b]pyridine derivative (E)-3-amino-5-(3-(3-bromophenyl)acryloyl)-N-(3-chloro-2- methylphenyl)-6-methylthieno[2,3-b]pyridine-2-carboxamide (inhibitor 8) on human cervical carcinoma cells of HeLa and SiHa cell lines. Materials and methods: The MTT test was used to test cytotoxicity on HeLa and SiHa cell lines, and the required concentration of inhibitor 8 was determined to test its apoptotic effect. The apoptotic effect of inhibitor 8 on HeLa and SiHa cell lines was monitored by flow cytometry. Results: They are shown graphically as a percentage of early and late apoptosis and surviving cells. It can be concluded that inhibitor 8 is effective on both tested cell lines in early apoptosis and in early and late apoptosis overall. It did not show a significant effect on HeLa cells in late apoptosis, while the most significant effect was shown on SiHa cells in early apoptosis. Conclusions: In vitro exposure of human cervical cancer cell lines, HeLa and SiHa, to a thieno[2,3-b]pyridine derivative leads to an increase in the percentage of cells in the apoptotic phase. Treatment of cancer cells with a thieno[2,3-b]pyridine derivative could represent a new therapeutic approach. Further in vitro and in vivo studies with animal models are required to confirm the effect.

Cilj istraživanja bio je utvrditi učinak primjene inhibitora fosfolipaze C iz skupine tieno[2,3-b] piridina na postotak epitelno mezenhimalnih CD44+CD24+, epitelnih CD44-CD24+ i luminalno epitelnih CD44-CD24- stanica pozitivnih na glikokonjugat GM3(NeuAc) te prosječni izražaj GM3(NeuAc) po jednoj stanici za svaku od navedenih subpopulacija trostruko negativnog karcinoma dojke.Stanice MDA-MB-231 linije kultivirane su tijekom 48h na četiri načina: bez dodatka inhibitora i/ ili paklitaksela, te uz dodatak samog inhibitora, samog paklitaksela ili njihove kombinacije. Metodom protočne citometrije izmjerena je fluorescencija suspenzija stanica imunobojenih protutijelima na CD44,CD24 i GM3(NeuAc) te analiziran postotak pojedine stanične subpopulacije kao i izražaj GM3(NeuAc).Inhibitor fosfolipaze je doveo do porasta postotka i izražaja GM3(NeuAc) u CD44+CD24+ te postotka u CD44-CD24- staničnoj subpopulaciji. Samo kod CD44+CD24+ subpopulacije, zapažen je povećani prosječni izražaj GM3(NeuAc) po jednoj stanici. Kombinirani tretman s paklitakselom još je učinkovitije povećao postotak GM3(NeuAc) na svim subpopulacijama.GM3(NeuAc) glikokonjugat pripada klasi kiselih glikosfingolipida te je opisan kao inhibitor prijenosa signala putem receptora za faktor rasta fibroblasta. Budući da inhibitor fosfolipaze iz ovog istraživanja dovodi do porasta postotka i izražaja GM3(NeuAc) u određenim subpopulacijama, zaslužuje daljnja in vitro i in vivo istraživanja kao mogući antitumorski agens., The aim of the study was to determine the effect of phospholipase C inhibitors from the thieno [2,3-b] pyridine group on the percentage of epithelial mesenchymal CD44+CD24+, epithelial CD44-CD24+ and luminal epithelial CD44-CD24-cells positive for GM3(NeuAc) glycoconjugate and the average expression of GM3(NeuAc) per cell for each of these subpopulations of triple-negative breast cancer.Cells of the MDA-MB-231 line were cultured for 48 h in four ways: without the addition of inhibitors and/or paclitaxel, and with the addition of the inhibitor alone, paclitaxel alone, or a combination with both of these. Flow fluorescence of antibody-stained cell suspensions to CD44, CD24 and GM3(NeuAc) was measured by flow cytometry and the percentage of each cell subpopulation as well as the expression of GM3 (NeuAc) were analyzed.The phospholipase inhibitor led to an increase in the percentage and expression of GM3(NeuAc) in CD44+CD24+ and the percentage in the CD44-CD24-cell subpopulation. Only in the CD44+CD24+ subpopulation, an increased average expression of GM3(NeuAc) per cell was observed. Combination treatment with paclitaxel increased the percentage of GM3(NeuAc) even more effectively in all subpopulations.GM3(NeuAc) glycoconjugate belongs to the class of acidic glycosphingolipids and has been described as an inhibitor of signal transduction via fibroblast growth factor receptors. Because the phospholipase inhibitor from this study leads to an increase in the percentage and expression of GM3(NeuAc) in certain subpopulations, it deserves further in vitro and in vivo studies as a possible antitumor agent.

Cilj istraživanja je dokazati djelovanje inhibitora fosfolipaze C, samog ili u kombinaciji s paklitakselom, na snižavanje postotka epitelnih CD44-CD24+ i luminalno epitelnih CD44- CD24- stanica pozitivnih na CD15s te na snižavanje prosječnog izražaja glikokonjugata CD15s po jednoj CD44-CD24+ i CD44-CD24- stanici.Na MDA-MB-231 staničnoj liniji trostruko negativnog karcinoma dojke, primijenjen je novosintetizirani inhibitor fosfolipaze C iz skupine tieno[2,3-b]piridina. Stanice su tretirane inhibitorom fosfolipaze C koncentracije 2 µM i/ili paklitakselom tijekom 48 sati,nakon čega je izmjerena fluorescencija uzoraka imunobojenih protutijelima na CD44, CD24 i CD15s pomoću protočnog citometra.Rezultati su prikazani statističkim dijagramima u kojima su prikazani postotci luminalnih i epitelnih stanica MDA-MB-231 stanične linije pozitivnih na CD15s te prosječni izražaj CD15s po pojedinoj stanici navedenih populacija stanica po skupinama ovisno o korištenju samog inhibitora, paklitaksela ili njihove kombinacije. Paklitaksel je snizio izražaj CD15s na epitelnim stanicama no bez statističke razlike. Inhibitor 6 je povećao, a paklitaksel je snizio izražaj CD15s na luminalnim stanicama, no bez statističke značajnosti. Inhibitor 6 i paklitaksel su smanjili prosječni izražaj biljega CD15s po epitelnoj stanici uz statističku značajnost, p = 0.011. Paklitaksel je smanjio prosječni izražaj biljega CD15s po luminalnoj stanici no bez statističke značajnosti. Tretman MDA-MB-231 stanične linije 2 µM koncentracijom inhibitora fosfolipaze C iz skupine tieno[2,3-b]piridina statistički značajno smanjuje postotak epitelnih CD44-CD24+ stanica na izražaj CD15s po jednoj matičnoj stanici raka dojke, no ne mijenja značajno postotak CD44-CD24+ subpopulacije matičnih stanica pozitivnih na biljeg CD15s. The aim of the research was to demonstrate the effect of phospholipase C inhibitor, alone or in combination with paclitaxel, on reducing the percentage of epithelial CD44-CD24+ and luminal epithelial CD44-CD24- cells positive for CD15s and on lowering the average expression of CD15s glycoconjugate per CD44-CD24+ and CD44-CD24+ cell. A newly synthesized phospholipase C inhibitor from the thieno [2,3-b] pyridine group was used on MDA-MB-231 triple-negative breast cancer cell line. Cells were treated with a 2 µM phospholipase C inhibitor and / or paclitaxel for 48 hours, after which the fluorescence of antibody-stained samples on CD44, CD24 and CD15s was measured using a flow cytometer. The results are presented in statistical diagrams showing the percentages of luminal and epithelial cells of the MDA-MB-231 cell line positive for CD15s and the average expression of CD15s per cell of these population cells by groups depending on the use of the inhibitor, paclitaxel or a combination thereof. Paclitaxel decreased CD15s expression on epithelial cells without statistical differences. Inhibitor 6 increased, and paclitaxel decreased CD15s expression in luminal cells, with no statistical significance. Inhibitor 6 and paclitaxel decreased the mean expression of the CD15s marker per epithelial cell with statistical significance, p = 0.011. Paclitaxel decreased the mean expression of the CD15s marker per luminal cell without statistical significance. Treatment of MDA-MB-231 cell line with 2 µM concentration of thieno [2,3-b] pyridine phospholipase C inhibitor statistically significantly reduced the percentage of epithelial CD44-CD24 + cells per CD15s expression per breast cancer stem cell, but did not significantly change the percentage of CD44 -CD24 + subpopulations of CD15s marker-positive stem cells.

Cilj istraživanja je bio dokazati citotoksični i apoptotički učinak izotiocijanata (ITC): 2-metoksifenila, 4-(metilsulfanil)fenila i 4-nitrofenila na staničnim linijama trostruko-negativnog karcinoma dojke MDA-MB-231 i karcinoma mokraćnog mjehura T24. Pretpostavka je da će se nakon tretiranja stanica spojevima smanjiti postotak metaboličkih aktivnih stanica te da navedeni spojevi uzrokuju programiranu staničnu smrt ispitanih stanica. Ispitivanje citotoksičnosti na staničnim linijama karcinoma dojke MDA-MB-231 i karcinoma mokraćnog mjehura T24 provedeno je MTT testom. Metabolička aktivnost stanica se određivala unutar 4, 24, 48 i 72 h inkubacije. Rezultati za test citotoksičnosti su prikazani grafovima na kojima su prikazan postotak metabolički aktivnih stanica u ovisnosti o koncentraciji spoja i vremenu izlaganja. Svi spojevi pokazuju najveću citotoksičnu aktivnost u koncentracijama 50 µg/mL i 100 µg/mL i u vremenu inkubacije od 72 h, a najbolju citotoksičnost pokazuje spoj 4-(metilsulfanil)fenil izotiocijanat. Bolji citotoksični učinak izotiocijanata je dokazan na staničnoj liniji T24. Nakon toga je ispitan učinak na apoptozu metodom protočne citometrije korištenjem Annexin-V/PI kita. Tretman stanica koncentracijama jednakim IC50 vrijednostima za 48 h inkubacije dokazao je da je mehanizam citotoksične aktivnosti za sva tri uzorka izotiocijanata bio poticanje apoptoze. 2-metoksifenil ITC pokazuje najbolji učinak na poticanje apoptoze na staničnoj liniji MDA-MB-231. 4-nitrofenil ITC pokazuje najbolji učinak na poticanje apoptoze na staničnoj liniji T24., The aim of the study was to demonstrate the cytotoxic and apoptotic effect of isothiocyanates (ITC): 2-methoxyphenyl, 4- (methylsulfanyl) phenyl and 4-nitrophenyl on MDA-MB-231 triple-negative breast cancer cell lines and T24 bladder cancer cell line. It is assumed that after treatment of cells with compounds, the percentage of metabolically active cells will decrease and that these compounds cause programmed cell death of the tested cells. Cytotoxicity testing on MDA-MB-231 breast cancer cell lines and T24 bladder cancer was performed by MTT assay. Metabolic activity of the cells was determined within 4, 24, 48 and 72 h of incubation. The results for the cytotoxicity test are shown by graphs showing metabolically active cells as a function of compound concentration and exposure time. All compounds showed the highest cytotoxic activity at concentrations of 50 µg/mL and 100 µg/mL and at an incubation time of 72 h, and the best cytotoxicity was shown by the compound 4-(methylsulfanyl) phenyl ITC. A better cytotoxic effect of ITC was demonstrated on the T24 cell line. The effect on apoptosis was then examined by flow cytometry using the Annexin-V / PI kit. Treatment of cells with concentrations equal to IC50 values for 48 h of incubation proved that the mechanism of cytotoxic activity for all three isothiocyanate samples was the induction of apoptosis. 2-methoxyphenyl ITC shows the best effect on inducing apoptosis on the MDA-MB-231 cell line. 4-nitrophenyl ITC shows the best effect on inducing apoptosis on the T24 cell line.

Cilj: Cilj ovog istraživanja jest dokazati djelovanje sintetski dobivenog inhibitora iz skupine tieno[2,3-b]piridina na apoptozu stanica staničnih linija MCF7 karcinoma dojke i MDA-MB-231 trostruko negativnog karcinoma dojke. Pretpostavka je da će se nakon tretiranja tumorskih staničnih linija inhibitorom postotak stanica u ranoj, kasnoj te ranoj i kasnoj apoptozi ukupno, povećati. Materijali i metode: Pomoću MTT-a došli smo do potrebne koncentracije inhibitora kojom smo tretirali stanice raka dojke staničnih linija MCF7 i MDA-MB-231 tijekom 48 sati. Nakon dodatka Annexina V i propidij jodida stanice su analizirane protočnim citometrom kojim smo dobili postotak stanica u ranoj i kasnoj apoptozi kod tretiranih i netretiranih stanica raka. Rezultati: Prikazani su kao postotak rane i kasne apoptoze i preživjelih stanica. Djelovanje inhibitora na stanice stanične linije MCF7 pokazalo je smanjenje broja stanica u ranoj apoptozi u odnosu na kontrolu koje nadomješta statistički značajan porast stanica u kasnoj te ukupno ranoj i kasnoj apoptozi u odnosu na netretirane stanice. U ovom istraživanju inhibitor je pokazao blagi, ali statistički značajan porast postotka stanica MDA-MB-231, stanične linije trostruko negativnog raka dojke u ranoj apoptozi. Također inhibitor je pokazao značajni porast stanica u kasnoj te ukupno ranoj i kasnoj apoptozi proučavane stanične llinije. Zaključci: Novosintetizirani tieno[2,3-b]piridin doveo je do značajnog porasta postotka stanica karcinoma dojke koje ulaze u apoptozu. Djelovanje koje je pokazao taj inhibitor moglo bi biti značajno u liječenju trostruko negativnog raka dojke., Objectives: The aim of this study was to demonstrate the effect of a synthetically derived inhibitor from the thieno[2,3-b]pyridine group on cell apoptosis of MCF7 breast cancer cell line and MDA-MB-231 triple-negative breast cancer cell line. It is hypothesized that after the treatment of tumor cell lines with an inhibitor, the percentage of cells in early, late, and early and late apoptosis will increase. Materials and methods: Using MTT, we obtained required concentration of inhibitor with whom we treated breast cancer cells of the MCF7 and MDA-MB-231 cell lines for 48 hours. After the addition of Annexin V and propidium iodide, the cells were analyzed by flow cytometer to obtain the percentage of cells in early and late apoptosis in treated and untreated cancer cells. Results: They are presented as the percentage of early and late apoptosis and surviving cells. The effect of inhibitor on MCF7 cell line showed a decrease in the number of cells in early apoptosis compared to the control, which is compensated by the statistically significant increase in percentage of the cells in late and total early and late apoptosis compared to untreated cells. In this study, the inhibitor showed a mild, but statistically significant increase in the percentage of MDA-MB-231 cells, a triple-negative breast cancer cell line, in early apoptosis. The inhibitor also showed a significant increase in cells in late and total early and late apoptosis in observed cell lines. Conclusions: Newly synthesized thieno[2,3-b]pyridine led to a significant increase in the percentage of breast cancer cells which enter into apoptosis. The action shown by this inhibitor could be significant in the treatment of triple-negative breast cancer.

Objectives: The goal of this study was to determine GD3 expression after treatment with the novel thieno[2,3-b]pyridine derivative, by focusing on CD44+/CD24- CSCs, CD44- epithelial cells, as well as CD44+/ CD24+ hybrid cells. Methods: The MDA-MB-231 cell line was treated with 3-amino-N-(3-chloro-2- methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (compound 1). Cells were plated onto six-well plates and treated with 2 M compound 1 for 48 h. Following this, cells were stained with anti-GD3, anti-CD44 and anti-CD24 antibodies, and then analyzed via flow cytometry. The acquisition of data of triple-stained samples was done by a BD Accuri C6 cytometer (BD Biosciences) and analyzed with the FlowLogic Software. Results: Treatment of MDA-MB-231 cells with compound 1 has led to a reduction of the expression of GD3 in CD44+/CD24- CSCs, as well as CD44- epithelial cells. GD3 expression in CD44+/CD24+ hybrid cells was not significantly reduced. Conclusion: Due to its ability to reduce GD3 expression in different breast cancer subpopulations, compound 1 has a potential to be used in the treatment of triple-negative breast cancer., Ciljevi: Cilj ove studije bio je utvrditi ekspresiju GD3 nakon tretmana novim derivatom tieno[2,3-b]piridina, posebice na CD44+/CD24- matičnim stanicama raka (CSC), CD44- epitelnim stanicama, kao i CD44+/CD24+ hibridnim stanicama. Metode: Stanična linija MDA-MB-231 tretirana je s 3-amino-N-(3-kloro-2-metilfenil)-5- okso-5,6,7,8-tetrahidrotienom[2,3-b]kinolin-2-karboksamid (spoj 1). Stanice su nasađene na ploče sa šest jažica i tretirane s 2 M spojem 1 tijekom 48 sati. Nakon toga, stanice su obojane s protu-GD3, protu-CD44 i protu-CD24 protutijelima, a zatim analizirane protočnom citometrijom. Prikupljanje podataka trostruko obojenih stanica rađeno je citometrom BD Accuri C6 (BD Biosciences) i analizirano softverom FlowLogic. Rezultati: Tretman MDA-MB-231 stanica spojem 1 doveo je do smanjenja ekspresije GD3 na CD44+/CD24- CSC, kao i CD44- epitelnim stanicama. Ekspresija GD3 na hibridnim stanicama CD44+/CD24+ nije bila značajno smanjena. Zaključak: Zbog svoje sposobnosti smanjenja ekspresije GD3 u različitim subpopulacijama raka dojke, spoj 1 ima potencijal za upotrebu u liječenju trostruko-negativnog raka dojke.

CILJEVI ISTRAŽIVANJA: Ciljevi ovog istraživanja bili su odrediti izražaj CD15s na epitelnim (CD44-CD24+ i CD44-CD24-) i mezenhimalnim (CD44+CD24- tj. CSC) stanicama MDA-MB-231 stanične linije trostruko negativnog karcinoma dojke te odrediti izražaj CD44 kod CSC CD15s+ i CSC CD15s- stanica nakon primjene tienopiridinskog derivata. MATERIJALI I METODE: Novosintetizirani tienopiridinski derivat, 3-amino-N- (3-kloro-2-metilfenil)- 5 -okso -5,6,7,8-tetrahidrotieno [2,3-b] kinolin- 2-karboksamid, primijenjen je u 2 μM koncentraciji u trajanju od 48 sati na MDA-MB-231 staničnoj liniji. Potom su stanice imunobojene s anti-CD15s odnosno sekundarnim antitijelom na anti-CD15s konjugiranim s eFluor 660 fluorokromom, s anti-CD44-FITC i anti-CD24-PE te je fluorescencija pojedinih kromogena konačno mjerena protočnim citometrom. REZULTATI: U izražaju CD15s u CSC subpopulaciji nije bilo značajne razlike prije i nakon tretmana tienopiridinskim derivatom. Za razliku od CSC subpopulacije, ovaj je tretman rezultirao gotovo dvostrukim smanjenjem izražaja CD15s u subpopulaciji CD44- (P=0,004). Tretman nije utjecao na ekspresiju CD44 u CD15s+ CSC, ali je smanjio izražaj CD44 (P=0,008) u CD15s-CSC. ZAKLJUČAK: Nalazi sniženog izražaja CD15s kod epitelnih stanica i CD44 kod CD15- mezenhimalnih stanica ukazuju na potencijal novosintetiziranog tienopiridinskog derivata kao terapijskog sredstva za trostruko negativni rak dojke., Objectives: The aims of this study were to analyse the expression of CD15s in epithelial (CD44-CD24+) and mesenchymal (CD44+CD24-, CSC) cells in MDA-MB-231 triple-negative breast cancer cell line and to measure expression of CD44 in CSC CD15s+ and CSC CD15s- cells after being treated with thieno[2,3-b]pyridine derivative. Materials and methods: Newly synthesized thieno[2,3-b]pyridine derivative of 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide was used in 2 μM concentration for 48 hours on MDA-MB-231 cell line. Thereafter, the cells were stained with anti-CD15s secondary antibody conjugated with eFluor 660 on CD15s fluorochrome, anti-CD44-FITC, anti-CD24-PE, and then chromogen fluorescence was analyzed by flow cytometry. Results: There was no difference in expression of CD15s in CSC subpopulation before and after treatment with thieno[2,3-b]pyridine derivative. Unlike CSC subpopulation, this treatment has resulted in almost two times reduction of CD15 expression in CD44- subpopulation (P=0.004). The treatment didn’t have any influence on the expression of CD44 in CD15s+CSC but it had reduced expression of CD44 (P=0.008) in CD15-CSC. Conclusion: The finding of reduced expression of CD15s in epithelial cells and CD44- within mesenchymal cells show the potential of newly synthesized thieno[2,3-b]pyridine derivative as a potential treatment option for triple-negative breast cancer.

Cilj istraživanja bio je ispitati citotoksični učinak uzoraka dobivenih iz Sisymbrium spp. na tri stanične linije humanih karcinoma: A549, MDA-MB-231 i T24. Korišteni su destilati i ekstrakti iz biljnih vrsta Sisymbrium officinale (L.) i Sisymbrium orientale te izopropil izotiocijanat. Sve tri stanične linije tretirane su koncentracijama od 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL i 100 µg/mL. Citotoksičnost se određivala nakon 4, 24, 48 i 72 h korištenjem MTT metode, ali su prikazani samo najznačajniji rezultati. Omjer apsorbancije stanica tretiranih uzorcima dobivenih iz Sisymbrium spp. i izopropil izotiocijanatom te apsorbancije onih koje nisu tretirane pokazatelj je citotoksične aktivnosti korištenih uzoraka i izopropil izotiocijanata. Najznačajniji citotoksični učinak na staničnu liniju A549 vidljiv je nakon inkubacije od 72h, ali nema značajne razlike u djelovanju uzoraka dobivenih različitim metodama izolacije. Najznačajniji citotoksični učinak na staničnu liniju MDA-MB-231 postignut je djelovanjem uzorka Sisymbrium orientale dobivenog autolizom komuščice pri koncentraciji od 100 µg/mL gdje se nakon inkubacije od 72h broj metabolički aktivnih stanica smanjio na 38%. Na staničnu liniju T24 najznačajniji citotoksični učinak pokazuje također uzorak dobiven autolizom komuščice nakon inkubacije od 72h pri koncentraciji 100 µg/mL, ali je učinak slabiji nego kod MDA-MB-231 linije. Ispitivani uzorci i izopropil izotiocijanat pokazuju citotoksični učinak ovisan o vremenu inkubacije i koncentraciji. Djelovanje nije uvijek razmjerno povećanju koncentracije i vremenu inkubacije, te u pojedinim slučajevima dolazi do oporavka stanica. Izopropil izotiocijanat ima slabi citotoksični učinak na stanične linije karcinoma dojke (MDA-MB-231) i karcinoma mokraćnog mjehura (T24) i to samo pri najvećoj koncentraciji od 100 µg/mL nakon 72h inkubacije., The aim of this research was to examine the cytotoxic effect of samples obtained from Sisymbrium spp. and isopropyl isothiocyanate on three human cancer cell lines: A549, MDA-MB-231 and T24. Distillates and extracts from the plant species Sisymbrium officinale (L.), Sisymbrium orientale and isopropyl isothiocyanate were used. Three cell lines were treated with concentrations of 1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL and 100 μg/mL. Cytotoxicity was determined after 4, 24, 48 and 72 h using the MTT method, but only the most significant results were presented. The ratio of the absorbance of cells treated with samples obtained from Sisymbrium spp. and isopropyl isothiocyanate and the absorbance of those that are not treated is an indicator of the cytotoxic activity of the samples and isopropyl isothiocynate we used. The most significant cytotoxic effect on the A549 cell line was achieved after 72 h of incubation, but there was no significant difference in the action of the samples obtained by different isolation methods. The most significant cytotoxic effect is achieved after treatment of MDA-MB-231 cell line with Sisymbrium orientale sample obtained by silique autolysis at a concentration of 100 µg/mL after 72 hours of incubation. Number of metabolically active cells decreased to 38%. Sisymbrium orientale sample obtained by silique autolysis after 72 h of incubation at a concentration of 100 μg/mL showed the most significant cytotoxic effect on the T24 cell line. as well, but slightly weaker. Samples obtained from Sisymbrium spp. and isopropyl isothiocynate showed a cytotoxic effect dependent on the incubation time and concentration. The effectiveness of samples obtained from Sisymbrium spp. and isopropyl isothiocynate was not always correspondent to the increase of concentration and incubation time and, in some cases, cell recovery occurred. Isopropyl isothiocyanate has a weak cytotoxic effect on breast cancer cell lines (MDA-MB-231) and bladder cancer (T24) only at a maximum concentration of 100 µg/mL after 72 h of incubation.

Uvod: Karcinom prostate jedan je od najčešćih karcinoma u muškoj populaciji, stoga su zbog njegovog učestalog pojavljivanja predstavljena brojna istraživanja koja nastoje poboljšati, skratiti i omogućiti što kvalitetnije liječenje pacijenta. Progresija tumora može biti potaknuta od strane matičnih stanica karcinoma. Tijekom ovog istraživanja procijenjen je metabolizam stanica pomoću MTT testa nakon primjene novosintetiziranog spoja samostalno ili u kombinaciji s paclitaxel-om. Također im je određena rana i kasna apoptoza pomoću Annexin-V i PI bojenja. Upravo se razaranje matičnih stanica karcinoma prostate smatra obećavajućim ciljem liječenja karcinoma prostate kako bi se poboljšao ishod u pacijenata s naprednim stadijem bolesti. Cilj: Cilj ovog rada bio je utvrditi postotak vijabilnosti stanica karcinoma prostate Du-145 nakon tretmana s novosintetiziranim spojem (3-Amino-5okso-N-naftil-5,6,7,8-tetrahidrotieno[2,3-b]kinolin-2-karboksamid) - inhibitorom fosfolipaze C. Materijali i metode: Stanice Du-145 su inkubirane s novosintetiziranim spojem ili u kombinaciji s paclitaxel-om. Stanice su uzgajane u specifičnim uvjetima, a vijabilnost stanica određena je pomoću MTT testa. Vrsta stanične smrti procijenjena je nakon 48 sati pomoću Annexin-V-FITC testa i propidij jodida. Podaci su prikupljeni pomoću BD Accuri C6 citometra i analizirani korištenjem FlowLogic Softvera. Rezultati: Rezultati koji su dobiveni nakon tretmana s novosintetiziranim spojem ovisili su o koncentracijama i vremenskom razdoblju tretmana. Nakon 72h, niže koncentracije novosintetiziranog spoja, u iznosu od 1 μM pokazivale su značajnu citotoksičnost u odnosu na netretirane stanice. Također, rezultati su pokazali značajno smanjenje preživjelih stanica nakon korištenja kombinacije novosintetiziranog spoja (0,5 μM) i paclitaxel-a (25 μM) nakon 48 i 72 sata. 25 Zaključak: Ustanovljeno je kako novosintetizirani spoj, 3-amino-5-okso-N-naftil-5,6,7,8-tetrahidrotieno[2,3-b] kinolin-2-karboksamid, citotoksičan za stanice karcinoma prostate. Također vrlo važno je spomenuti kako su prilikom korištenja novositetiziranog spoja presudne koncentracije tog istog spoja te vremenski period u kojem je spoj korišten. Introduction: Prostate cancer is one of the most common cancer in the male population, therefore, due to its frequent occurrence, a number of researches are being carried out that seek to improve, shorten and enable the best quality of treatment for the patient. Tumor progression may be triggered by cancer stem cells (CSCs). During this study, cell metabolism was evaluated using the MTT assay after application of the newly synthesized compound alone or in combination with paclitaxel. Early and late apoptosis was also determined by using Annexin-V and PI staining. Precisely, this destruction of prostate cancer steam cells is considered as promising goal of prostate cancer treatment to improve outcome in patients with advanced stage of disease. Aim: The aim of this paper was to determine the cell viability of prostate cancer cells Du-145 after treatment with the newly synthesized compound (3-Amino-5-oxo-N-naphthyl-5,6,7,8-tetrahydrothieno [2,3-b] quinoline-2-carboxamide) inhibitor of phospholipase C. Materials and methods: Du-145 cells were incubated with either only newly synthesized compound or in combination with paclitaxel. The cells were cultured under specific conditions, and cell viability was determined by the MTT test. Cell death was evaluated after 48 hours using Annexin-V-FITC test and propidium iodide. The data were collected using the BD Accuri C6 cytometer and analyzed using FlowLogic Software. The results: The results obtained after treatment with the newly synthesized compound depended on the concentrations and the time of treatment. After 72 hours, lower concentration of the novosynthetic compound, in the amount of 1 μM showed significant cytotoxicity in comparison to untreated cells. Also, the results showed a significant reduction in surviving cells after using the combination of newly synthesized compound (0,5 μM) and paclitaxel (25 μM) after 48 and 72 hours. 27 Conclusion: It has been found that newly synthesized compound, 3-amino-5-oxo-N-naphthyl-5,6,7,8-tetrahydrothieno [2,3-b] quinoline-2-carboxamide is cytotoxic to prostate cancer cells. Also, it is very important to mention while using the newly synthesized compound, the critical is concentration of the same compound and the time period in which the compound is used.