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Interleukin 17 (IL-17) je središnji medijator upale s višestrukom ulogom u nastanku, napredovanju i rezistenciji na terapiju tumora različitih sijela. Pojedine studije govore u prilog antitumorske aktivnosti IL-17 dok je prema drugima nađeno da povećana izraženost IL-17 u tumorskom tkivu dojke doprinosi agresivnosti tumora i lošijoj prognozi. Uloga IL-17 dosad je većinom ispitivana na in vitro staničnim modelima i ljudskim ksenograftima. Nedostatak podataka o učincima IL-17 in vivo predstavlja nepremostivu prepreku u terapijskom moduliranju IL-17 posredovanih imunoloških reakcija radi uspješnijeg liječenja bolesnika oboljelih od raka. U ovom istraživanju analizirana je koncentracija IL-17 u serumu 118 žena oboljelih od ranog invazivnog raka dojke prije operacije i tijekom adjuvantnog liječenja i uspoređena s koncentracijom IL-17 u serumu kontrolne skupine zdravih žena. Analizirana je razina izraženosti IL-17 u različitim komponentama pridruženih intraoperativnih uzoraka bolesnica uključujući invazivnu i in situ komponentu, peritumorsku stromu i normalno tkivo dojke. Utvrđena je značajno viša serumska koncentracija IL-17 u serumu žena oboljelih od ranog raka dojke prije početka liječenja (P, Interleukin 17 (IL-17) has a key role in inflammatory responses. Increased levels of IL-17 have been reported in the serum of cancer patients. Some studies suggest antitumor activity of IL-17 and others speak in favor of its association with poorer prognosis. The lack of data on IL-17 behavior in vivo precludes the usage of IL-17 antibodies in cancer patients. This study included 118 patients with early invasive breast cancer. The serum concentration of IL-17 was measured before surgery and during adjuvant treatment and compared with healthy controls. The level of IL-17 expression in the corresponding tumor tissue samples was analyzed. Significantly higher serum concentrations of IL-17 were found in the serum of women with early breast cancer before treatment and during adjuvant treatment without correlation to tumor tissue IL-17 expression. There was a significant decrease in serum IL-17 levels in women with early breast cancer after surgery compared to preoperative values. Serum IL-17 levels were significantly negatively correlated with tumor estrogen receptor expression. The results suggest that the immune response in early breast cancer is mediated also by IL-17, particularly in triple-negative breast cancer. IL-17-mediated inflammatory response subsided after tumor removal, but IL-17 levels remained elevated compared to healthy controls.

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.

Trostruko negativni rak dojke (TNRD) javlja se u oko šestine svih bolesnica s rakom dojke, najagresivnijeg je ponašanja i najgore prognoze. To je heterogena bolest, sa specifičnim molekularnim karakteristikama i prirodnom dinamikom ranog recidiva i brze progresije. U nedostatku valjanog biobiljega, i time bilo kakvog ciljanog liječenja, danas je okosnica liječenja TNRD kemoterapija, pri čemu se uočava tzv. TNRD paradoks – bolja kemosenzitivnost i odgovor na terapiju, no lošije preživljenje. Danas se sve više nade polaže u pronalazak biobiljega koji će biti od prediktivnog i prognostičkog značaja u TNRD, kao što su to androgeni receptori (AR) i tumor – infiltrirajući limfociti (TIL). Provedena je retrospektivna kohortna studija na susljednom uzorku od 152 bolesnice dijagnosticirane i liječene od ranog TNRD u Klinici za tumore, KBC Sestre milosrdnice, u razdoblju od 1.1.2009. do 31.12.2012. godine. Analizirane su sociodemografske i kliničkopatološke varijable dostupne iz računalnog zapisa te je učinjena imunohistokemijska analiza AR te morfološka HE analiza TIL. Granična vrijednost za pozitivnost AR iznosila je ≥1%, a TIL su analizirani potpuno, stromalni (sTIL) i intratumorski (iTIL), u centralnom tumoru (CT) i na invazivnoj margini (IM). Izračunate su korelacije svih navedenih parametara te petogodišnje preživljenje bez bolesti (DFS) i sveukupno preživljenje (OS). Bolesnice su bile medijana dobi 58 godina, većinom postmenopauzalne, najčešće stadija II bolesti, gradusa III i medijana Ki-67 57%. Liječene su operacijski i potom u gotovo 90% slučajeva adjuvantnom kemoterapijom, uglavnom antraciklinima i taksanima te adjuvantnom radioterapijom. U petogodišnjem praćenju povrat bolesti bilježi se u trećine bolesnica, najčešće u prvoj godini od dijagnoze, uglavnom u obliku udaljene diseminacije u pluća i kosti. Petogodišnji DFS iznosi 67.1%, a OS 73.7%. Uz povrat bolesti i smrt najznačajnije su bili povezani čimbenici poput mlađe dobi, većih tumora, zahvaćenih limfnih čvorova, odnosno višeg stadija bolesti te višeg proliferacijskog indeksa Ki-67. AR su bili izraženi (≥1%) u 31.1% bolesnica, a u njih 18.5% AR su >50%. Evaluacijom TIL prema navedenim odjeljcima, najgušće zastupljeni bili su sTIL na IM, s medijanom intenziteta od 30%, i čak 85.5% bolesnica s ≥10%, dok su najrjeđi i najmanjeg intenziteta bili iTIL u CT, s medijanom intenziteta od 1% i tek 23% bolesnica s ≥10% intenzitetom. Ukupno je četvrtina bolesnica imala TIL>50% u nekom od evaluiranih odjeljaka. Istovremeno izražene AR (≥1%) i prisutne TIL (>1%) imalo je 30% bolesnica, a izraženost AR nije bila statistički značajno povezana s prisutnošću TIL. Izraženost AR nije pokazala statistički značajan utjecaj na preživljenje bez bolesti, kao niti na ukupno preživljenje. Evaluacija TIL pokazala je statistički značajan povoljan utjecaj na DFS od sTIL na IM, ali ne i od ostalih odjeljaka. Sveukupno, pokazalo se da su bolesnice koje su imale TIL≥10% u sva četiri evaluirana odjeljka (sTIL i iTIL, u CT i na IM) imale statistički značajno bolji DFS, u odnosu na one s TIL, Triple-negative breast cancer (TNBC) occurs in about one-sixth of all BC patients, with the most aggressive behavior and the worst prognosis. It is a heterogeneous disease, with specific molecular characteristics and natural dynamics of early relapse and rapid progression. In the absence of a valid biomarker, and thus any targeted treatment, chemotherapy is nowadays still the backbone of treatment, with the so-called. TNBC paradox - better chemosensitivity and response to therapy, but worse survival. Today, more hope lies in finding new biomarkers that will be predictive and prognostic in TNBC, such as androgen receptors (AR) and tumor-infiltrating lymphocytes (TIL). Retrospective cohort study was conducted on a consecutive sample of 152 patients diagnosed and treated for early TNBC at the University Hospital for Tumors, Sestre milosrdnice University Hospital Center, from Jan 1st 2009 untill Dec 31st 2012. Sociodemographic and clinicopathological variables available from the hospital record were analyzed and IHC AR analysis and TIL morphological analysis by HE were performed. Limit for AR positivity was ≥1%, and TILs were fully analyzed, stromal (sTIL) and intratumoral (iTIL), in the central tumor (CT) and at the invasive margin (IM). Correlations of all parameters were calculated as well as 5y DFS and OS. Patients were at median age of 58, mostly postmenopausal, most commonly stage II disease, grade III, and a median Ki-67 of 57%. They were treated surgically and then in almost 90% of cases with adjuvant chemotherapy, mainly anthracyclines and taxanes and adjuvant radiotherapy. At 5y follow-up, disease recurrences are reported in one third of patients, most often in the first year after diagnosis, mainly in the form of distant metastases to the lungs and bones. The 5y DFS was 67.1% and the OS was 73.7%. Factors such as younger age, larger tumors, involved lymph nodes, or a higher stage of disease, and a higher Ki 67 were most significantly associated with disease recurrence and death. AR were expressed (≥1%) in 31.1% of patients, and in 18.5% of them AR were> 50%. By evaluating TIL according to the above mentioned technique, sTIL on IM were most frequently represented, with a median intensity of 30%, and as many as 85.5% of patients with ≥10%, while the rarest and of lowest intensity were iTIL in CT, with a median intensity of 1% and 23% of patients with ≥10%. In total, quarter of patients had TIL>50% in any of the evaluated compartments. Around 30% of patients had expressed AR (≥1%) and present TIL(>1%) at the same time, and AR expression was not statistically significantly associated with the presence of TIL. Expression of AR showed no statistically significant effect on DFS, or OS. Evaluation of TIL showed a statistically significant favorable effect on DFS of sTIL on IM, but not of the other compartments. Overall, patients with TIL≥10% in all four evaluated compartments (sTIL and iTIL, in CT and IM) were shown to have statistically significantly better DFS, compared to those with TIL

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.

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: Cilj ovog istraživanja bio je utvrditi antitumorske učinke tretmana matičnih stanica karcinoma dojke novosintetiziranim inhibitorom fosfolipaze, spojem tieno [2,3-b] piridinom, preciznije, njegovo djelovanje na metastatski biljeg CD15s na MDA-MB-231 staničnoj liniji raka dojke. Materijali i metode: Trostruko negativna stanična linija raka dojke MDA-MB-231 tretirana je novosintetiziranim tieno-piridinskim spojem (3-amino-5-okso-N-naftil-5,6,7,8-tetrahidrotieno [2,3-b] kinolin-2-karboksamid) koncentracije 2 µM, tijekom 48h, kako bi se utvrdio postotak subpopulacije matičnih stanica raka pozitivnih na biljeg CD15s i na prosječni izražaj CD15s biljega po jednoj matičnoj stanici raka nakon tretmana. Nakon imunobojenja, pozitivne stanice detektirane su metodom protočne citometrije. Rezultati: Tretman novosintetiziranim inhibitorom PLC značajno je smanjio postotak subpopulacije matičnih stanica pozitivnih na biljeg CD15s, ali nije imao utjecaja na prosječni izražaj CD15s biljega po jednoj matičnoj stanici. Zaključci: Zbog utvrđenog smanjenja postotka subpopulacije matičnih stanica trostruko negativnog raka dojke koje na svojoj površini eksprimiraju metastatski biljeg CD15s, novosintetizirani inhibitor fosfolipaze C iz skupine tieno [2,3-b] piridina potencijalni je lijek za trostruko negativni karcinom dojke., The aim of this study: The aim of this study was to determine the effects of treating of breast cancer stem cells with the newly synthesized thieno[2,3-b]pyridine anticancer agent, more precisely, focusing on its effect on metastatic marker, CD15 glyconjugate, expressed on the surface of cacer stem cells. Methods: The MDA-MB-231 triple-negative breast cancer cell line was treated during 48h with a 2 µM newly developed thienopyridine anticancer compound (3-amino-5-oxo-N-naphtyl-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide) to determine its effect on percentage of subpopulation of breast cancer stem cells that express CD15s and on the mean expression of CD15s on an individual breast cancer stem cells. Flow cytometric analysis was performed after immunostaining. Results: PLC inhibitor has significally decreased the percent of breast cancer stem cells positive on CD15s, but it was without effect upon mean expression of CD15s on an individual breast cancer stem cell. Conclusion: Due to its decreasing effect upon the percentage of triple negative breast cancer stem cells that are positive for the metastatic CD15s glycoconjugate, the investigated PLC inhibitor can be a potential drug for triple-negative breast cancer treatment.

Cilj istraživanja: Cilj ovog istraživanja bio je otkriti hoće li primjena novosintetiziranog inhibitora fosfolipaze C (PLC) iz skupine tieno [2,3-b] piridina smanjiti postotak matičnih stanica (CSC, od engl. cancer stem cells) trostruko negativnog tumora dojke na ispitivanoj MDA-MB-231 staničnoj kulturi. Dodatni cilj je bio, u jednakim uvjetima pokusa, usporediti učinak inhibitora s učincima postojećeg lijeka paklitaksela kao i kombinacije inhibitora i paklitaksela. Materijali i metode: Novosintetizirani inhibitor fosfolipaze C, 3-amino-5-okso-N-naftil-5,6,7,8-tetrahidrotieno [2,3-b] kinolin-2-karboksamid, primijenjen je na MDA-MB-231 staničnoj liniji trostruko negativnog karcinoma dojke. Nakon tretmana stanica 2 M inhibitorom PLC i/ili 40 nM koncentracijom paklitaksela tijekom 48 sati, fluorescencije uzoraka imunobojenih protutijelima na CD44 i CD24 izmjerene su protočnom citometrijom. Rezultati: Inhibitor je blago, ali statistički značajno smanjio CSC (s 99,09±0,53 na 96,78±1,03). Paklitaksel je izazvao statistički neznačajno povećanje postotka CSC. Kombinirani tretman inhibitorom i paklitakselom nije utjecao na postotak CSC. Zaključci: Novosintetizirani tieno [2,3-b] piridin smanjuje postotak matičnih stanica u MDA-MB-231 staničnoj liniji trostruko negativnog raka dojke što bi moglo predstavljati novi terapijski pristup TNBC. Kombinirani tretman inhibitorom PLC i paklitakselom nije utjecao na postotak CSC, moguće je da se suprotni učinci paklitaksela i inhibitora međusobno poništavaju., The aim of this study: The aim of this study was to determine whether the newly synthesized phospholipase C inhibitor from the group of thieno[2,3-b]pyridine inhibitors reduces the percentage of triple negative breast cancer stem cells (CSC) on the investigated human cell line MDA-MB-231. An additional aim was, under the same experimental conditions, to compare the effect of the inhibitor with the effects of the existing paclitaxel drug as well as the combination of the inhibitor and paclitaxel. Methods:The newly synthesized phospholipase C inhibitor, 3-amino-5-oxo-N-naphtyl-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, was administered to MDA-MB-231 triple-negative breast cancer cell line. After cell treatment with 2 M phospholipase C inhibitor and / or with 40 nM concentration of paclitaxel for 48 hours, fluorescences of immunostained samples with antibodies to CD44 and CD24 were measured by flow cytometry. Results: The inhibitor slightly but statistically significantly reduced the percentage of CSC (from 99,09±0,53 to 96,78±1,03). Paclitaxel caused a statistically insignificant increase in the percentage of CSCs. The combined treatment with inhibitor and paclitaxel did not affect the percentage of CSC. Conclusion: The newly synthesized thieno [2,3-b] pyridine reduces the percentage of stem cells in the MDA-MB-231 cell line of triple-negative breast cancer which could represent a new therapeutic approach to TNBC. The combined treatment with PLC inhibitor and paclitaxel did not affect the percentage of CSC. Therefore, it is possible that the opposite effects of paclitaxel and inhibitors cancel each other out.

Cilj istraživanja. Karcinom dojke najčešći je maligni tumor u žena i važan javnozdravstveni problem diljem svijeta. Trostruko negativni karcinomi (TNBC) čine 10 do 20 % svih karcinoma dojke, karakterizirani su negativnim statusom receptora za steroidne hormone i HER-2-neu receptor, a povezuju se s agresivnijim kliničkim tijekom. Cilj ovog istraživanja bio je utvrditi imunohistokemijski izražaj E-kadherina u trostruko negativnim karcinomima dojke, te njegovu povezanost s kliničko-patološkim pokazateljima, kao što su dob, veličina tumora, klinički stadij, histološki tip, broj mitoza, bazalna morfologija, bazalni imunofenotip, stupanj diferencijacije, vaskularna invazija i zahvaćanje limfnih čvorova. Materijali i metode. U studiju su uključene 83 žene oboljele od trostruko negativnog karcinoma dojke operirane u Klinici za kirurgiju KBC Split od 2003. do 2010. godine. Uzorci tumora bojani su imunohistokemijski primjenom protutijela na CK5/6, CK14, EGFR, Ki-67 i E-kadherin. Bazalni imunofenotip definiran je pozitivnošću na jedan ili više bazalnih markera: CK5/6, CK14 i/ili EGFR. Imunohistokemijski izražaj E-kadherina određivan je semi-kvantitativno, uključujući postotak pozitivno obojenih stanica, cjelovitost i intenzitet membranskog bojenja. Za usporedbu varijabli u odnosu na imunohistokemijski izražaj E-kadherina korišten je χ2 test. Rezultati. Pozitivan imunohistokemijski izražaj E-kadherina zabilježen je kod 69 (87,3%) trostruko negativnih tumora. Većina tumora, 69 (83,1%) bila je visokog histološkog gradusa. uz visoke vrijednosti mitotskog (medijan 25,9/10 VPVP, raspon 2-110) i Ki-67 proliferacijskog indeksa (medijan 53.7%, raspon 3-95). U skupini tumora s negativnim izražajem E-kadherina bilo je značajno više tumora koje svrstavamo u posebne histološke tipove (p=0,001), te značajno više tumora s pozitivnom bazalnom morfologijom (p=0,024). Također, zabilježena je statistički značajna povezanost imunohistokemijskog izražaja E-kadherina s mitotskim i proliferacijskim indeksom (p=0,042; p=0,023), te kliničkim stadijem bolesti (p=0,027). Zaključak. Povezanost smanjene razine ekspresije E-kadherina sa lošijim kliničko-patološkim karakteristikama tumora upućuje da bi E-kadherin mogao poslužiti kao jedan od markera za izdvajanje trostuko negativnih tumora s agresivnijim kliničkim tijekom., Objectives: Breast carcinoma is the most common malignant tumor in female population and a very important public health issue around the world. Triple negative breast carcinomas (TNBC) are a subgroup of breast carcinomas, characterised by negative steroid hormone receptors and HER-2-neu receptor, associated with aggressive clinical behaviuor. Aim of this research was to establish immunohistochemical expression of E-cadherin in triple negative breast carcinomas and to identify its connection with clinical-pathological indicators, like age, tumor size, clinical stage, histological type, mitotic count, basal morphology, basal immunophenotype, grade , vascular invasion and lymph node metastasis. Patients and methods: Study enrolled 83 patients that where operated at the Department of surgery, University Hospital Split during the period from 2003. to 2010. Tumor samples where analyzed immunohistochemically by using antibodies against CK5/6, CK14, EGFR, Ki-67 and E-cadherin. Basal immunophenotype was defined with positivity for one or more basal markers CK5/6, CK14 and/or EGFR. For comparison of variables with immunohistochemical expression of E-cadherin χ2 test was used. Immunohistochemical expression of E-cadherin was determined semi-quantitatively, with positively stained cells ratio, integrity and intensity of membrane staining. Results: Positive immunohistochemical expression of E-cadherin was recorded in 69 (87,3%) of triple negative carcinomas. Most of the tumors 69 (83.1%) were histologically high grade, with high mitotic index (median value of 25,9 / 10 VPVP, scale from 2 to 110) and Ki-67 proliferation index (median value of 53.7%, scale from 3 to 95 ). Triple negative tumors with negative expression of E-cadherin included significant number of tumors classified as a specific histological types (p=0.001) and tumors with a positive basal morphology (p=0.024). Immunohistochemical expression of E-cadherin was significantely related to mitotic and proliferative index (p=0.042; p=0.023) and clinical stage (p=0.027).