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Radiotherapy-induced toxicity is a major dose-limiting factor in anti-cancer treatment. Ionizing radiation leads to the formation of reactive oxygen and nitrogen species (ROS/RNS) that are associated with radiation-induced cell death. Investigations of biological effects of fullerenol have provided evidence for its ROS/RNS scavenger properties in vitro and radioprotective efficiency in vivo. Therefore we were interested to evaluate its radioprotective properties in vitro in the human erythroleukemia cell line. Pretreatment of irradiated cells by fullerenol exerted statistically significant effects on cell numbers and the response of antioxidative enzymes to X-ray irradiation-induced oxidative stress in cells. Our study provides evidence that the pre-treatment with fullerenol enhanced the enzymatic activity of superoxide dismulase and glutathione peroxidase in irradiated K562 cells. null

Cell culture K562 samples were treated with fullerenol (C6o(OH)24) at a concentration of 10 nmol/mL and thereafter irradiated with X-rays (24Gy). The activity of gamma-glutamyltransfrease (γ-GT), total superoxide-dismutase (SOD) and glutathion-peroxidase (GSH-Px) was determined 1, 24 and 48 hours after irradiation. Irradiation induces an increase in the activity of all the investigated enzymes. Fullerenol in the applied dose decreased the γ-GT activity 24 and 48 h after irradiation. The total SOD activity is increased in both pretreated groups except in the irradiated group at the 48th hour. Treatment with fullerenol before irradiation increased GSH-Px activity in irradiated groups and decreased it in the control groups. U uzorcima ćelijske kulture humane eritroleukemije (K562) ozračenim X-zracima doze 24 Gy i predtretiranim fulerenolom (C60(OH)24) koncentracije 10 nmol/mL određivana je aktivnost gama-glutamiltransferaze (γ-GT), ukupne superoksid-dismutaze (SOD) i glutation-peroksidaze (GSH-Px) 1, 24 i 48 sati nakon zračenja. Iradijacija povećava aktivnost sva tri ispitivana enzima. U 24-om i 48-om satu nakon zračenja zapaža se sniženje aktivnosti γ-GT u grupama predtretiranim fulerenolom. Fulerenol utiče na povišenje aktivnosti SOD u obe tretirane grupe, osim u grupi ozračenih uzoraka u 48-om satu gde je došlo do sniženja aktivnosti. Predtretman fulerenolom u zračenim grupama povećava, a u neozračenim eksperimentalnim grupama snižava aktivnost GSH-Px. null

Breast cancer is the most prevalent malignancy in women worldwide. It is estimated that every eight women in the world will be diagnosed with breast cancer at some moment of life. The modern medical approaches have reshaped treatment and diagnostic strategies in management of patients with breast neoplasms, significantly improving quality of life and therapy outcomes. Despite these advances, breast cancer is currently the second leading cause of all cancer-related mortality. Two of the breast cancer subtypes, hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) positive breast cancers, have specific entities on their surface successfully employed in designing targeted therapies. Such treatments enable distinction between malignant and normal tissue, resulting in selective induction of apoptosis in cancer cells. Therefore, development of targeted therapies strongly advanced the prognosis for patients with these breast cancer subtypes. The third type of breast cancers, triplenegative breast cancer (TNBC) represents its most aggressive form, characterized by substantial metastatic potential and higher recurrence rates. In addition, thus far there are no known cell surface receptors that could differentiate the TNBC from normal cells which could be efficiently employed for targeted therapy desing. Thus, this subset is treated with non-specific approaches alone, such as chemotherapy, radiotherapy and surgical procedures. Therefore, TNBC represents a subset of breast cancers with the greatest need for further elucidation of the pathological mechanisms underlying the malignant behavior and investigation of the novel treatment strategies that could exploit this knowledge. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that regulates the expression of genes involved in essential cellular processes including cell growth, survival, proliferation, differentiation, migration and immune response. Constitutive activation of STAT3 has been reported in a wide spectrum of malignant diseases, including both solid and hematological malignancies. The importance of STAT3 signaling in breast cancer is displayed by its abnormal activation in 70% of all breast cancers, accounting for essentially all triple-negative breast cancers. Therefore, targeting STAT3 holds a great potential in treatment of such malignancies. STAT3 is an intracellular protein that is physiologically activated in response to external stimuli, such as cytokines and growth factors. Upon activation, it shuttles from cytoplasm to nucleus, where it binds to its nine base pair consensus sequence in the promoter of its target genes to regulate their transcription. Since transcription factors like STAT3 have large and relatively flat molecular surfaces to allow protein-DNA and protein-protein interactions, it makes it challenging to directly target these proteins using small molecules. Given the difficulties in direct targeting, we considered whether metabolic changes driven by constitutive activation of STAT3 could provide vulnerability in breast cancer cells that can be exploited therapeutically. Recognizing that malignant transformation is associated with changes in a variety of cellular metabolites, we investigated how STAT3 affects lipid distribution in mammary epithelial cells, in both qualitative and quantitative manner. To evaluate the metabolic architecture resulting from persistent STAT3 activation, we inhibited or activated STAT3 in two complementary mammary epithelial cellular systems and performed mass spectrometry-based lipid profiling. We found that STAT3 prominently modulated several lipid classes, with most profound reduction of N-acyl taurine (NAT) and arachidonic acid (AA). To investigate if STAT3 activation was accompanied by these metabolic changes in patients, we performed in silico gene set enrichment analyses using publicly available microarray data of 129 breast cancer patients. We confirmed that STAT3 gene expression signature was significantly enriched in patient’s samples with low expression of cysteine dioxygenase (CDO1) and cystathionine gamma lyase (CTH), the two enzymes required for taurine biosynthesis from homocysteine. On the contrary, STAT3 signature was highly enriched in patients’ samples with high expression of AA metalytic enzymes cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX), whereas enzyme that releases AA from plasma membrane, phospholipase A2 (PLA2G4A) did not correlate with STAT3 activity. These findings indicate that STAT3 activation results in reduction of cellular AA and taurine levels by modulating the expression of enzymes involved in their metabolism. As both of taurine and arachidonic acid are involved in plasma membrane remodeling, we examined utilizing these STAT3-driven metabolic alterations in identifying selective nano-size drug carriers that could exploit these lipid properties. We screened a library of 12 layer-by-layer (LbL) nanoparticles (NP) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-L-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to non-transformed cells. This was not a nonspecific consequence of malignant transformation, as nanoparticles coated with other surface chemistries did not display such an effect. Furthermore, the targeting property of PLE-NPs was lost when STAT3 was abrogated using phosphorylation or transcriptional inhibitors, ruxolitinib and pyrimethamine. As STAT3 is constitutively activated in almost all triple-negative breast cancers, we investigated utilizing PLENPs as a STAT3-targeting approach for this subset of breast cancers. The nanoparticles terminally layered with PLE showed potent binding to TNBC cells MDA-MB-231 and SUM159PT, and the heightened NP-cell binding was attenuated when STAT3 expression was diminished using two different small interfering RNAs (siRNAs). As our high-resolution microscopy data indicates that these nanoparticles predominantly bind to cell membrane and might not necessarily be internalized into the cytoplasm, we further examined the tumor penetrating properties of PLE-NPs in three-dimensional breast cell organoids. STAT3-transformed organoids grew at much higher cellular density and formed greater numbers of massive colonies, which could have been expected to impede penetrating ability of nanoparticles through such dense cellular structures. However, PLE-NPs displayed affinity towards STAT3-driven cells even in the three-dimensional breast cellular model and distributed in depth of the STAT3- transformed tumor organoids. Next, we examined the translational potential of these findings. As STAT3- transformed cells show greater resistance to cytotoxic agents, we evaluated if enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven breast cancer cells, including TNBC, at lower concentrations compared to cells lacking activated STAT3. On the contrary, these cells showed significantly greater resistance to apoptosis induced by either un-encapsulated cisplatin or cisplatin-loaded NPs lacking the targeting layer or coated with a non-targeting dextran sulfate (DXS). These findings further emphasize the therapeutic potential of targeting STAT3-driven cells using PLE-coated LbL nanoparticles. In addition, since radiation is commonly used in breast cancer treatment and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using uniquely designed LbL nanoparticles complemented with surface poly-L-glutamic acid. These nanoparticles hold great potential for the delivery of anticancer agents to breast malignant cells, including triple-negative breast cancer cells, with decreased toxicity in normal tissue. The combination treatment with radiotherapy may offer synergy with PLE-nanoparticles and enhance their targeting affinity. Taken together, the results of this dissertation indicate a potential novel treatment approach to triple-negative breast cancers, which currently lack targeted therapeutic options., Karcinom dojke je na globalnom nivou najučestalije maligno oboljenje kod žena. Prema procenama, svakoj osmoj ženi će u nekom momentu života biti dijagnostikovan karcinom dojke. Savremeni medicinski pristupi su unapredili metode lečenja i dijagnostike u zbrinjavanju pacijenata sa neoplazmom dojke, značajno poboljšavajući kvalitet života i rezultate terapije. Uprkos napretku, karcinom dojke je trenutno drugi vodeći uzrok smrtnosti uzrokovane malignitetom. Dva molekularna podtipa karcinoma dojke, hormon receptor (HR) i humani epidermalni faktor rasta 2 (HER2) pozitivni karcinomi dojke, poseduju specifične strukture na površini maligne ćelije koje su uspešno iskorišćene za dizajniranje ciljanih terapija. Date terapije omogućavaju diferencijaciju između malignog i normalnog tkiva, što rezultira selektivnom indukcijom apoptoze u malignim ćelijama. Stoga je razvoj ciljanih terapija značajno doprineo poboljšanju prognoze za pacijente sa navedenim podtipovima maligniteta dojke. Treći tip karcinoma dojke, trostruko-negativni karcinom dojke (TNBC) predstavlja njegov najagresivniji oblik, koji karakteriše izražen metastatski potencijal i visoka stopa recidiva. Pored toga, trenutno nisu poznati receptori na površini ćelijske membrane koji bi mogli razlikovati TNBC od normalnih ćelija i efikasno bili upotrebljeni za razvoj ciljanih terapija. Stoga se ovaj maligni oblik leči isključivo nespecifičnim pristupima, kao što su hemioterapija, radioterapija i hirurški zahvati. Usled svega navedenog, TNBC predstavlja tip karcinoma dojke sa najvećom potrebom za daljim rasvetljavanjem patoloških mehanizama na kojima zasniva maligno ponašanje kao i istraživanje novih strategija lečenja koje bi mogle iskoristiti dato znanje. Prenosilac signala i aktivator transkripcije 3 (STAT3) je onkogeni transkripcioni faktor koji reguliše ekspresiju gena uključenih u esencijalne ćelijske procese, uključujući ćelijski rast, preživljavanje, proliferaciju, diferencijaciju, migraciju i imunološki odgovor. Konstitutivna aktivacija STAT3 je zabeležena u širokom spektru malignih bolesti, uključujući solidne i hematološke malignitete. Značaj STAT3 signalizacije u karcinomu dojke se odlikuje njegovom patološkom aktivacijom u 70% svih karcinoma dojke, i praktično svim trostruko-negativnim karcinomima dojke. Stoga, terapije selektivno usmerene na STAT3 imaju veliki potencijal u lečenju takvih malignih oboljenja. STAT3 je intracelularni protein koji se fiziološki aktivira kao odgovor na spoljašnje stimuluse, poput citokina i faktora rasta. Kada je aktiviran, STAT3 se translocira iz citoplazme u jedro, gde se vezuje za svoju konsenzus sekvencu od devet baznih parova u promoteru ciljnih gena i reguliše njihovu transkripciju. Budući da faktori transkripcije poput STAT3 imaju velike i relativno ravne molekularne površine kako bi omogućili interakcije sa drugim proteinima i DNK, istovremeno takva molekulska struktura je poprilično problematična za direktno vezivanje malih molekula. S obzirom na poteškoće u direktnom ciljanom delovanju na STAT3, u okviru ove disertacije smo razmotrili mogućnost da metaboličke promene uzrokovane konstitutivnom aktivacijom STAT3 mogu predstavljati vulnerabilnost ćelija karcinoma dojke koja se može terapijski iskoristiti. Imajući u vidu da je maligna transformacija povezana sa promenama različitih ćelijskih metabolita, istražili smo kako STAT3 utiče na kvantitativnu i kvalitativnu distribuciju lipida u epitelnim ćelijama karcinoma dojke. Kako bismo ispitali efekte konstitutivne STAT3 aktivacije na metabolički profil ćelija karcinoma dojke, inhibirali smo ili aktivirali STAT3 u dva komplementarna ćelijska sistema i potom analizirali lipidni profil korišćenjem masene spektrometrije. Otkrili smo da STAT3 vrši modulaciju nekoliko klasa lipida, sa najizraženijom redukcijom ćelijskog nivoa N-acil taurina (NAT) i arahidonske kiseline (AA). Da bismo ispitali da li je aktivacija STAT3 praćena datim metaboličkim promenama kod pacijenata, izvršili smo in silico analize obogaćivanja gena pomoću javno dostupnih podataka RNK mikromatrica od 129 pacijenata obolelih od karcinoma dojke. Potvrdili smo da je STAT3 genska signatura značajno obogaćena u uzorcima pacijenta sa niskom ekspresijom cistein dioksigenaze (CDO1) i cistationin gama liaze (CTH), dva enzima potrebna za biosintezu taurina iz homocisteina. Suprotno tome, STAT3 genska signatura je značajno obogaćena u uzorcima pacijenata sa visokom ekspresijom metaboličkih enzima AA ciklooksigenaze 2 (COX-2) i 5-lipoksigenaze (5-LOX), dok ekspresija enzima koji oslobađa AA iz plazma membrane, fosfolipaze A2 (PLA2G4A), nije u korelaciji sa STAT3 aktivnošću. Ovi nalazi ukazuju na to da aktivacija STAT3 rezultira smanjenjem ćelijskog nivoa AA i taurina modulacijom ekspresije enzima koji su uključeni u njihov metabolizam. Kako su i taurin i arahidonska kiselina uključeni u modeliranje plazme membrane, ispitali smo korišćenje datih STAT3-uzrokovanih metaboličkih promena u identifikovanju selektivnih nanonosača lekova koji bi mogli da iskoriste date lipidne karakteristke. Analizirali smo ćelijsko vezivanje biblioteke od 12 višeslojnih (Layer-byLayer, LbL) nanočestica (NP) koje se razlikuju u površinskom sloju, s obzirom da omotač modulira njihovu interaktivnost sa ćelijskom membranom. Otkrili smo da se NP obložene poli-L-glutaminskom kiselinom (PLE) vezuju za ćelije karcinoma dojke sa aktiviranim STAT3 sa 50% većom efikasnošću nego za netransformisane ćelije. Navedena pojava ne predstavlja nespecifičnu posledicu maligne transformacije, jer nanočestice presvučene drugim strukturama nisu pokazale dati efekat. Pored toga, svojstvo ciljanog delovanja PLE-NP nestaje kada se inhibira fosforilacija ili transkripciona aktivnost STAT3 korišćenjem ruksolitiniba i pirimetamina. S obzirom da je STAT3 konstitutivno aktiviran u gotovo svim trostruko-negativnim karcinomima dojke, ispitali smo korišćenje PLE-NP kao seletivnog pristupa ovom tipu karcinoma dojke zasnovanog na ciljanom delovanju na STAT3. Nanočestice obložene sa PLE pokazale su povećano vezanje za TNBC ćelijske linije MDA-MB-231 i SUM159PT, koje se značajno smanjuje inhibicijom STAT3 ekspresije korišćenjem dve različite kratke interferirajuće RNK (siRNK). Kako naši mikroskopski podaci visoke rezolucije pokazuju da se date nanočestice pretežno vezuju za ćelijsku membranu i ne moraju nužno biti internalizovane u citoplazmu, dodatno smo ispitali svojstva penetracije PLENP u trodimenzionalne strukture organoida ćelija dojke. Rast STAT3-transformisanih organoida pokazuje izraženo veću ćelijsku gustinu uz formiranje većeg broja masivnih kolonija, te se mogla očekivati otežana penetracija nanočestica kroz tako guste ćelijske strukture. Međutim, PLE-NP su pokazale afinitet prema ćelijama sa aktiviranim STAT3 čak i u trodimenzionalnom modelu karcinoma dojke i distribuciju u dubini STAT3 transformisanih tumorskih organoida. Nakon datih otkrića, evaluirali smo translacioni potencijal ovih nalaza. Uzimajući u obzir da STAT3-transformisane ćelije pokazuju veću otpornost na citotoksične agense, ispitali smo da li bi poboljšana ciljana isporuka lekova putem PLE-NP-a pružila terapijsku prednost. Otkrili smo da PLE-NP ispunjene sa cisplatinom indukuju apoptozu ćelija karcinoma dojke sa aktiviranim STAT3, uključujući TNBC, u nižim koncentracijama u poređenju sa ćelijama kojima nedostaje STAT3 aktivnost, uključujući nemaligne ćelije dojke. Suprotno tome, STAT3- aktivirane ćelije su pokazale značajno veću apoptotsku otpornost na slobodan cisplatin kao i na nanočestice ispunjene cisplatinom koje nemaju ciljni sloj ili su obložene neciljajućim dekstran sulfatom (DXS). Ovi nalazi dodatno ističu terapijski potencijal selektivnog delovanja na maligne ćelije sa aktiviranim STAT3, korišćenjem PLE obloženih LbL nanočestica. Konačno, s obzirom da se terapija gama zraćenjem često koristi u lečenju maligniteta dojke i može delovati na ćelijsku distribuciju lipida, analizirali smo njen uticaj na ćelijsko vezivanje PLE-NP. Ozračivanje ćelija poboljšalo je selektivna svojstva PLE-NP ka STAT3-transformisanim ćelijama na dozno zavisan načina, sugerišući potencijalnu sinergiju između datih terapijskih modaliteta. Rezultati ove disertacije ukazuju da ćelijske lipidne promene uzrokovane aktiviranim STAT3 mogu biti terapijski iskorišćene LbL nanočesticama obloženim površinskom poli-L-glutaminskom kiselinom. Navedene nanočestice imaju veliki potencijal za isporuku lekova malignim ćelijama dojke, uključujući trostrukonegativne ćelije karcinoma dojke, uz smanjenu toksičnost ka normalnom tkivu. Kombinovani tretman sa radioterapijom može ponuditi sinergiju sa PLEnanočesticama i povećati njihov afinitet ciljanog delovanja. U zaključku, rezultati ove doktorske disertacije ukazuju na potencijalni novi pristup lečenju trostruko-negativnog karcinoma dojke, kome trenutno nedostaju ciljani terapijski modaliteti.

Hirurško vađenje impaktiranih donjih umnjaka veoma je česta oralnohirurška intervencija, koja je, u postoperativnom toku, obično praćena neprijatnim tegobama (bol, edem i trizmus), a nekad i potencijalno ozbiljnim komplikacijama (alveolitis i postoperativna infekcija). Stoga se savetuje primena različitih medikamenata i postupaka kojima bi se pojava ovih tegoba i komplikacija predupredila ili ublažila. S tim u vezi, u stručnoj i naučnoj literaturi vodi se rasprava o svsishodnosti profilaktičke primene antibiotika. Interesantno je da se mogu naći brojni radovi u kojima se preporučuje profilaktička primena antibiotika prilikom hirurškog vađenja impaktiranih donjih umnjaka, ali i radovi kojima se obrazlaže neslaganje sa takvim pristupom, potkrepljujući sumnju u odnos koristi spram rizika od uzgrednih efekata antibiotika, tim pre što je rastuća rezistencija mikroorganizama na antibiotike potencijalno ozbiljna pretnja globalnom zdravlju. Po pitanju profilaktičke primene antibiotika u ovoj indikaciji još nije postignut konsenzus, a nijedan od dva suprostavljena stava nije verifikovan ubedljivim naučno potvrđenim dokazima. Cilj. Imajući sve napred rečeno u vidu, osnovni cilj istraživanja bio je da se utvrdi opravdanost profilaktičke primene antibiotika u cilju ublažavanja osnovnih tegoba i komplikacija nakon hirurškog vađenja impaktiranih donjih umnjaka i da se, istovremeno, ispita efikasnost novijih vrsta antibiotika (moksifloksacin i cefiksim) primenjenih na taj način. Pri tom, izvršena su i laboratorijska (mikrobiološka) istraživanja senzitivnosti izolovanih mikroorganizama na antibiotike korišćene u istraživanju, koja bi mogla korisno da verifikuju klinički dobijene rezultate. Imajući to u vidu, na osnovu naučno verifikovanih rezultata, mogu se očekivati dve velike koristi od planiranog istraživanja: (1) potvrda ili negiranje opravdanosti profilaktičke primene antibiotika radi suzbijanja bilo koje od tegoba ili komplikacija posle hirurškog vađenja impaktiranih donjih umnjaka; (2) potvrda efikasnosti fluorohinolona (moksifloksacin) i cefalosporina (cefiksim) u suzbijanju dentogenih infekcija. Materijal i metode. Kliničko istraživanje sprovedeno je u Službi oralne hirurgije sa operacionim blokom i stacionarom Klinike za stomatologiju Vojvodine, po dizajnu dvostruko-slepe prospektivne kliničke studije, koje je obuhvatilo 177 ispitanika. Mikrobiološka istraživanja, sprovedena su u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine, na uzorcima brisa iz operativne rane pacijenata s postoperativnom infekcijom ili eksudata (gnoja) dobijenog iz perikoronarnog prostora pacijenata sa perikoronitisom. Pri tom, antibiogramom je utvrđivana senzitivnost izolovanih mikroorganizama na moksifloksacin, cefiksim i druge antibiotike koji se često koriste u oralnoj hirurgiji (penicilin i njegovi derivati, eitromicin, klindamicin i etraciklin). U studiju su uključeni samo ispitanici dobrog opšteg zdravstvenog stanja (ispitanici klasifikovani kao ASA I i ASA II), a u studiji nisu mogli da budu uključeni ispitanici kod kojih postoji sumnja na alergiju na antibiotike uopšte, a posebno na antibiotike koji su korišćeni u istraživanju, te maloletna lica, trudnice i dojilje. Zbog nepridržavanja datih uputstava u postoperativnom periodu ili neodazivanja na zakazane kontrolne preglede iz studije je isključeno 20 ispitanika. Istraživačke grupe formirane su nakon završenog istraživanja i otkrivanja odgovarajućeg šifarnika. Nakon završenog istraživanja, po otvaranju šifarnika, ustanovljeno je da je prvu studijsku grupu (M) činilo 52 ispitanika, kojima je u postoperativnom periodu ordiniran moksifloksacin. Drugu studijsku grupu (C) činilo je 53 ispitanika, kojima je u postoperativnom periodu ordiniran cefiksim. Treću, kontrolnu grupu (P) činilo je 52 ispitanika, kojima u postoperativnom periodu nije ordiniran antibiotik, već placebo tablete (koje su bile, izgledom, identične korišćenim antibioticima, ali su sadržavale samo indiferentne supstancije). Zavisno od preoperativno postojeće infekcije oko krune delimično izraslog umnjaka, tj. perikoronitisa, ispitanici sve tri formirane grupe, u svakoj od njih, svrstani su u dve podgrupe: podgrupu a (bez znakova perikoronitisa) i podgrupu b (sa prisutnim perikoronitisom). Na osnovu ovog kriterijuma, u grupi M, podgrupu a činilo je 39 ispitanika, a podrgupu b 13 ispitanika; u grupi C, podgrupu a činilo je 39 ispitanika, a podgrupu b 14 ispitanika; u kontrolnoj grupi (P), podgrupu a činilo je 39 ispitanika, a podgrupu b 13 ispitanika. Procena efekata primenjenih lekova vršenaje na osnovu postoperativnih tegoba ili komplikacija. Procena postoperativne analgezije vršena je pomoću vizuelno analogne skale (VAS) i verbalne skale rangiranja (VRS). Kod svakog ispitanika bio je registrovan momenat nastanka postoperativnog bola i njegov intenzitet, a registrovana je i potreba za dodatnim analgeticima. Postoperativni koeficijent edema dobijen je milimetarskim premeravanjem preoperativnog i postoperativnog odstojanja između pojedinih referentnih tačaka: spoljašnjeg ugla oka i ugla donje vilice; ugla usana i tragusa; vrha brade i tragusa. Procena stepena postoperativnog trizmusa vršena je merenjem razdaljine između mezijalno-incizalnih uglova gornjih i donjih centralnih sekutića pri maksimalno otvorenim ustima. U postoperativnom periodu registrovana je i pojava alveolitisa, kao i znaci postoperativne infekcije. Statistička analiza dobijenih podataka izvršena je primenom parametarskih (ANOVA i ANOVA ponovljenih merenja) i neparametarskih testova (Kruskal-Wallis test, Friedman test, Wilcoxon Signed Ranks test, McNemar test i Pearson χ2 test). Rezultati. Opisana prospektivna, dvostruko-slepa klinička studija, kao i izvršeno mikro-biološko istraživanje, pružili su sledeće rezultate: (1) kod ispitanika kod kojih su profilaktički primenjivani moksifloksacin ili cefiksim, utvrđena je statistički značajno manja učestalost, kao i manji intenzitet i kraće trajanje postoperativnih tegoba (bol, edem i trizmus) u odnosu na ispitanike kod kojih je primenjen placebo; (2) ispitanici kod kojih je profilaktički, u sklopu hirurškog vađenja impaktiranih donjih umnjaka, primenjivan moksifloksacin, imali su statistički značajno manju učestalost alveolitisa u odnosu na ispitanike kod kojih je primenjen cefiksim, kao i u odnosu na ispitanike kod kojih je primenjen placebo; (3) ispitanici kod kojih su profilaktički primenjivani moksifloksacin ili cefiksim, imali su statistički značajno manju učestalost postoperativne infekcije u odnosu na ispitanike kod kojih je primenjen placebo, pogotovu ako je inflamacija bila i preoperativno prisutna oko krune umnjaka. Pri tom, kod ispitanika koji su primili moksifloksacin, postoperativna infekcija nije zabeležena ni u jednom slučaju; (4) mikroorganizmi izolovani iz brisa perikoronoranog prostora kod ispitanika sa perikoronitisom ili iz brisa operativne rane u slučaju pojave postoperativne infekcije, na osnovu antibiograma, bili su osetljivi na moksifloksacin i na cefiksim, što ukazuje na efikasnost ovih antibiotika na prozrokovače oralnih infekcija, dok je zapažen povećan procenat rezistencije mikroorganizama na standardno primenjivane antibiotike, pogotovu na penicilin i njegove derivate. Zaključak. Na osnovu svih rezultata istraživanja, zaključeno je da su potvrđene hipoteze o efikasnosti moksifloksacina i cefiksima u profilaksi postoperativne infekcije nakon hirurškog vađenja impaktiranih donjih umnjaka i da su mikroorganizmi oralne flore, generalno, senzitivni na pomenute antibiotike. Međutim, hipoteze da moksifloksacin i cefiksim neće ispoljiti efekat na profilaksu inflamacijskih postoperativnih tegoba (bol, edem i trizmus), kao i alveolitisa, nisu potvrđene i mogu se odbaciti. Iz svih navedenih rezultata, može se zaključiti da je profilaktička primena moksifloksacina ili cefiksima u sklopu hirurškog vađenja impaktiranih donjih umnjaka svrsishodna u smislu ublažavanja i suzbijanja postoperativnih inflamacijskih tegoba i komplikacija., Surgical removal of impacted mandibular wisdom teeth is a frequent intervention, usually accompanied by unpleasant sequelae (pain, oedema and trismus) in the postoperative period, sometimes even with possibly serious complications (dry socket and postoperative infection). It is therefore advisable to use various medications and procedures to prevent or alleviate the occurrence of these sequelae and complications. Accordingly, there is a debate in the literature on the expedience of prophylactic use of antibiotics. It is interesting that numerous articles recommending prophylactic antibiotic use to patients undergoing the impacted mandibular third molar surgerymay be found, as well as those providing arguments for the disagreement with such an approach, considering that its benefits do not outweigh the risks of adverse side effects, especially due to growing resistance of microorganisms towards antibiotics, which is a possible serious threat to global health. Concerning the prophylactic use of antibiotics for this indication, consensus has not yet been reached, and neither of the views has been verified by convincing scientific evidence. Aim. Having the aforementioned dilemma in mind, the main goal of the study was to determine the validity of prophylactic use of antibiotics as to alleviate customary sequelae and possible complications after surgical removal of impacted mandibular third molars and, at the same time, the effectiveness of newer antibiotics (moxifloxacin and cefixime) when used for this purpose. Moreover, microbiological investigations of sensitivity of the isolated microorganisms to antibiotics used in this research were performed, which could verify the clinically obtained results.With this in mind, two major contributions, based on scientifically verified results, could be expected from the research: (1) confirmation or refuting the validity of prophylactic antibiotic use to controlsequelae or complications that could follow the impacted mandibular third molar surgery; and (2) confirmation of the efficacy of fluoroquinolones (moxifloxacin) and cephalosporins (cefixime) in controlling odontogenic infections. Materials and methods. Clinical research was conducted at the Oral Surgery Department of the Dentistry Clinic of Vojvodina, adopting the double-blind prospective clinical study design, which included 177 subjects. Microbiological studies were performed at the Center for Microbiology of the Institute of Public Health of Vojvodina, on swab samples obtained from the surgical wounds of patients with postoperative infection or the exudate (pus) obtained from the peri-coronal space of patients with pericoronitis. The sensitivity of isolated microorganisms to moxifloxacin, cefixime and other antibiotics commonly used in oral surgery (penicillin and its derivatives, erythromycin, clindamycin and tetracycline) was tested microbiologically. Only subjects in good general health (subjects classified as ASA I and ASA II) were included in the study, while excluding those with suspected allergy to antibiotics in general, and in particular to the antibiotics used in the research, as well as under-age persons and pregnant and lactating women. Due to non-compliance with the instructions given for the postoperative period or failure to attend the scheduled check ups, 20 subjects were later on excluded from the study. Study groups were formed upon the study completion, once the treatment codebook was opened. Following the completion of the study, after opening the codebook, it was established that the first study group (M) consisted of 52 subjects, to whom moxifloxacin was administered in the postoperative period. The second study group (C) consisted of 53 subjects, to whom cefixime was administered in the postoperative period. The third, control group (P) consisted of 52 subjects who, instead ofreceiving an antibiotic in the postoperative period, were given placebo tablets (which were identical in appearance to the studied antibiotics, but contained only indifferent substances). Depending on the presence of preoperative infection around the crown of the partially erupted third molar, i.e. pericoronitis, subjects in all three groups were further separated into two subgroups: subgroup a (without signs of previous pericoronitis) and subgroup b (with pericoronitis present). Based on this criterion, in group M, subgroup a consisted of 39 participants, and subgroup b had 13 participants; in group C, subgroup a consisted of 39 participants, and subgroup b had 14 participants; and in the control group (P), subgroup a comprised of 39 participants and subgroup b of 13 participants. Evaluation of the effects of the applied medication was performed on the basis of postoperative sequelae or complications. Postoperative analgesia was assessed using a visual analogue scale (VAS) and a verbal rating scale (VRS). For each subject, the timing of postoperative pain emergence and its intensity was noted, and the need for additional analgesics was registered as well.The postoperative oedema coefficient was obtained by measuring the preoperative and postoperative distance between specific reference points in millimetres. The reference points were: the outer corner of the eye and the angle of the lower jaw; lipcorner and tragus; and chin tip and tragus.The degree of postoperative trismus was assessed by measuring the distance between mesial-incisal angles of the upper and lower central incisors at the maximum mouth opening ability. In the postoperative period, emergence of dry socket was registered, along with signs of postoperative infection as well. Statistical analysis of the obtained data was performed via parametric (ANOVA and repeated measures ANOVA) and nonparametric (Kruskal-Wallis test, Friedman test, Wilcoxon Signed Ranks test, McNemar test and Pearson χ2 test) tests. Results. The described prospective, double-blind clinical study, as well as the performed microbiological study, yielded the following results: (1) in subjects who were prophylactically administered moxifloxacin or cefixime, a lower incidence, as well as a lower intensity and shorter duration of postoperative sequelae (pain, oedema and trismus) was noticed, which was statistically significant in relation to subjects who received placebo; (2) subjects who prophylactically received moxifloxacin did not manifested dry socket at all, which statistically significantly differed from subjects who were administered cefixime or placebo; (3) subjects who prophylactically received moxifloxacin or cefixime did not have postoperative infection at all, unlike those who received placebo, especially if inflammation around the crown of the third molarwas present preoperatively; (4) microorganisms isolated from swabs taken from peri-coronal space in subjects with pericoronitis or from surgical wound in case of postoperative infection, based on bacterial sensitivity test, were sensitive to moxifloxacin and cefixime, indicating the efficacy of these antibiotics against bacteria causing oral infections; however, an increased rate of resistance of oral bacteria was noticed to commonly prescribed antibiotics, particularly to penicillin and its derivatives. Conclusion. Based on all the study results, it was concluded that the hypotheses that moxifloxacin and cefixime effectively prevent infection after surgical removal of impacted mandibular third molars and that the microorganisms of the oral flora are generally sensitive to these antibiotics were confirmed and supported. However, the hypotheses that moxifloxacin and cefixime will not affect the prophylaxis of inflammatory postoperative sequelae (pain, oedema and trismus), as well as dry socket, were not confirmed by the findings and they were rejected. Based on all aforementioned results, it can be concluded that prophylactic use of moxifloxacin or cefixime in patients undergoing the impacted mandibular third molar surgery is appropriate for alleviating and suppressing postoperative inflammatory sequelae and complications.

Zahvaljujući amfifilnoj strukturi i mogućnosti građenja konjugata, žučne kiseline - endogeno sintetisani produkti katabolizma holesterola su prepoznate kao potencijalni nosači lekova i promoteri transporta kroz biološke membrane. Otkriće da aktivacijom specifičnih nuklearnih receptora regulišu ekspresiju gena uključenih u plejadu signalnih puteva uključenih u metabolizam, proliferaciju i diferencijaciju ćelija i onkogenezu, proširilo je ulogu žučnih kiselina u odnosu na inicijalno opisanu ulogu intestinalnih emulgatora. Žučne kiseline se danas ne smatraju samo pasivnim nosačima lekova i promoterima transporta kroz biološke membrane već i molekulima sa farmakodinamskom funkcijom, koji regulišu različite aspekte integrativnog ćelijskog metabolizma. Doksorubicin je jedan od najčešće korišćenih antineoplastičkih agenasa i sastavna je komponenta mnogih hemoterapijskih protokola u lečenju solidnih i hematoloških maligniteta. Međutim, hepatotoksični i kardiotoksični efekti značajno ograničavaju upotrebu ovog, inače veoma korisnog antitumorskog agensa. Pojava odložene dozno-zavisne kardiotoksičnosti predstavlja značajan zdravstveni problem onkoloških pacijenata sa uspešno lečenim malignitetom, naročito pacijenata lečenih u pedijatrijskom uzrastu. Budući da je razvoj novih lekova veoma dug i skup proces sa neizvesnim ishodom, poboljšanje farmakodinamskih i farmakokinetskih svojstava već postojećih antitumorskih agenasa sa dokazanom efikasnošću, uz smanjenje toksičnih efekata, predstavlja racionalan istraživački pristup u savremenoj medicini. Osnovni cilj ovog rada je ispitivanje uticaja žučnih kiselina ursodeoksiholne, henodeoksiholne i 12-okso-henodeoksiholne kiseline (12-monoketoholne kiseline) na citotoksičnu aktivnost doksorubicina prema MCF-7 ćelijskoj liniji humanog adenokarcinoma dojke i ispitivanje molekularnih mehanizama odgovornih za farmakodinamske efekte. Takođe su navedene žučne kiseline ispitane kao promoteri transporta koji utiči na prodor i kumulaciju doksorubicina u malignim ćelijama. U ovom radu je ispitan uticaj koadministracije navedenih žučnih kiselina sa doksorubicinom na odložene toksodinamske efekte (hepatotoksičnost i kardiotoksičnost) kod pacova, ali i efekti pretretmana žučnim kiselinama na koncentracije doksorubicina u krvi, bilijarnu ekskreciju leka kao i kumulaciju u jetri i miokardu eksperimentalnih životinja. Žučne kiseline su u netoksičnim koncentracijama potencirale in vitro citotoksične efekte doksorubicina na MCF-7 ćelijskoj liniji pri čemu je henodeoksiholna ispoljila sinergistički efekt, dok su ursodeoksiholna u 12-monoketoholna ispoljile aditivni citotoksični efekt sa doksorubicinom. Ispitivanjem molekularnih mehanizama citotoksičnih efekata utvrđeno je da su žučne kiseline u različitom stepenu potencirale apoptozu ćelija mitohondrijalnim putem uticajem na ekspresiju pro- i antiapoptotskih proteina na transkripcionom nivou i povećale stres endoplazmatskog retikuluma, ali i dovele do alteracija ekspresije gena koji kodiraju sintezu antioksidativnih enzima, transmembranskih efluks proteina i enzima uključenih u metaboličku inaktivaciju leka. Žučne kiseline u netoksičnim koncentracijama su takođe značajno povećale prodor i kumulaciju doksorubicina u MCF-7 ćelijskoj liniji. U in vivo sistemu, koadministracija žučnih kiselina nije rezultovala u poboljšanju odloženih toksodinamskih efekata visokih doza doksorubicina na biohemijskom i molekularnom nivou. Međutim, nakon pretretmana žučnim kiselinama, vrednosti koncetracija doksorubicina u serumu su bile povišene nakon pretretmana urso- i henodeoksiholnom kiselinom i snižene nakon pretretmana 12-monoketoholnom kiselinom uz povećanje bilijarne sekrecije doksorubicina. Pored promena u farmakokinetskom profilu doksorubicina, pretretman žučnim kiselinama je blago redukovao prodor i kumulaciju doksorubicina u hepatocite i kardiomiocite. Na osnovu rezultata ove studije može se zaključiti da primena ispitivanih žučnih kiselina sa doksorubicinom povećava prodor i poboljšava farmakodinamski profil doksorubicina in vitro, na ćelijskom modelu humanog adenokarcinoma dojke. Poboljšanje selektivnog preuzimanja i prodora doksorubicina u maligne ćelije koje nije praćeno povećanom kumulacijom u normalnim tkivima, kao i poboljšanje antitumorskog dejstva doksorubicina sa mogućim smanjenjem doze uz smanjenje pojave dozno-zavisnih neželjenih dejstava doksorubicina čini žučne kiseline molekulima kandidatima za dalja ispitivanja u cilju razvoja novih, poboljšanih antitumorskih terapijskih strategija., Due to the amphiphilic structure and the significant conjugation potential, bile acids - endogenously synthesized products of cholesterol catabolism have been recognized as potential drug carriers and promoters of transport through biological membranes. The discovery that by activating specific nuclear receptors bile acids regulate the expression of genes involved in various signaling pathways including metabolism, cell proliferation and differentiation as well as carcinogenesis, expanded initially ascribed role of intestinal emulsifiers to the various fields. Bile acids are now considered not to act only as passive carriers of drugs and promoters of transport through biological membranes, but also as the molecules with pharmacodynamic activity, regulating various aspects of integrative cellular metabolism. Doxorubicin is one of the most commonly prescribed antineoplastic agents as an integral component of many chemotherapy protocols in the treatment of both solid and hematologic malignancies. However, hepatotoxic and cardiotoxic effects significantly limit the use of this, otherwise, very useful anti-tumor agent. The development of dose-dependent cardiotoxic side effects represents particular health issue in successfully treated oncological patients, especially among survivors of pediatric malignancies. Since the development of new drugs is very long and expensive process with an uncertain outcome, improving the pharmacodynamic and pharmacokinetic properties of the existing agents with proven efficacy, while reducing toxic side effects, represents a rational approach to research in modern medicine. The main objective of this work is to examine the role of bile acids: ursodeoxycholic, chenodeoxycholic and 12-oxo-chenodeoxycholic acid (12-monoketocholic acid) on the cytotoxic activity of doxorubicin in the MCF-7 human breast adenocarcinoma cell line, and to get insight on molecular mechanisms responsible for underlying pharmacodynamic effects. The capacity of bile acids to promote the transport and accumulation of doxorubicin in malignant cells was also evaluated. In addition, the effect of co-administration of the bile acids with doxorubicin on delayed toxodynamic effects (hepatotoxicity and cardiotoxicity) in rats, as well as the effects of bile acid pretreatment on the doxorubicin serum concentration and pharmacokinetic profile, biliary excretion of the drug as well as accumulation in the liver and myocardial cells of experimental animals were examined. Bile acids applied in non-toxic concentrations potentiated in vitro cytotoxic effects of doxorubicin in MCF-7 cell line. Chenodeoxycholic acid exhibited a synergistic effect, whereas ursodeoxycholic and 12-monoketocholic acid exhibited an additive cytotoxic effect with doxorubicin. By examining the underlying molecular mechanisms of cytotoxic effects, bile acids have been found to potentiate apoptosis of cells by mitochondrial-dependent pathway by modifying the expression of pro- and anti-apoptotic proteins at the transcriptional level and to increase endoplasmic reticulum stress, but also have altered the expression of genes encoding the synthesis of antioxidant enzymes, transmembrane efflux proteins and enzymes involved in metabolic inactivation of the drug. Non-toxic concentrations of bile acids also significantly increased the penetration and accumulation of doxorubicin in MCF-7 cell line. In the in vivo system, the co-administration of bile acid did not improved delayed toxodynamic effects of high dose of doxorubicin both at the biochemical and molecular levels. However, pretreatment with bile acids resulted in alterations of serum doxorubicin concentrations. Chenodeoxycholic and ursodeoxycholic acid elevated whereas 12-monoketocholic acid decreased serum doxorubicin concentrations. In addition to changing pharmacokinetic profile of doxorubicin on bile acid species-dependent manner, all bile acids have also increased excretion of drug by the biliary route, and slightly reduced penetration and accumulation of doxorubicin in hepatocytes and cardiomyocytes. Based on the results of this study, the administration of the examined bile acids with doxorubicin increases the penetration and improves the pharmacodynamic profile of doxorubicin in vitro on the cell model of human breast adenocarcinoma. The improvement of selective uptake and penetration of doxorubicin into malignant cells that is not accompanied by increased accumulation in normal tissues, as well as the improvement in the anti-tumor effects of doxorubicin with a possibility to reduce the dose and thereby the occurrence of dose-dependent undesirable effects of doxorubicin, render bile acids as the potential candidate molecules in developing novel antitumor therapeutic strategies.

Žučne kiseline deluju kao signalni molekuli u organizmu i uključene su u regulaciju brojnih metaboličkih, inflamatornih i imunomodulatornih procesa. Ova endogena jedinjenja ostvaruju svoje efekte najvećim delom putem nuklearnih receptora. Farnezoid X receptor (FXR) je glavni regulator homeostaze žučnih kiselina, a pokazano je da je značajno uključen i u procese inflamacije i kancerogeneze, prevashodno u jetri i intestinalnom traktu. Aktivacija FXR receptora predstavlja značajnu farmakološku strategiju za terapiju holestatskih bolesti jetre, inflamatorne bolesti creva i karcinoma kolona. Definisana je uloga žučnih kiselina u signalnim putevima koji regulišu ćelijski ciklus i doprinose razvoju ili regresiji maligniteta, ali je malo poznat uticaj ovih jedinjenja na epigenetske mehanizme regulacije ključnih ćelijskih procesa. Imajući u vidu da su efekti žučnih kiselina determinisani njihovom polarnošću, cilj istraživanja je bio da se ispita uticaj sintetski dobijenog keto derivata holne kiseline, 12-monoketoholne kiseline (MKH), u komparaciji sa prirodnim žučnim kiselinama, hidrofobnom henodeoksiholnom kiselinom (HDH) i hidrofilnom ursodeoksiholnom kiselinom (UDH), na ćelijske procese apoptoze, oksidativnog stresa i inflamacije, koji su od značaja za hemoprevenciju i terapiju karcinoma kolona, u in vitro i in vivo sistemima. Cilj istraživanja je takođe obuhvatao i ispitivanje uloge odabranih žučnih kiselina u epigenetskoj regulaciji ovih procesa u ćelijama karcinoma kolona. Na in vivo modelu intrahepatične holestaze kod eksperimentalnih životinja, pokazano je da UDH i MKH ispoljavaju antiapoptotski, antioksidativni i antiinflamatorni efekat u jetri i intestinumu. Utvrđeno je da UDH i MKH sprečavaju mitohondrijalni put aktivacije apoptoze u jetri, dok UDH ispoljava antiapoptotski efekat i u intestinumu eksperimentalnih životinja sa holestazom. Ove dve žučne kiseline su u značajnoj meri modulirale ekspresiju gena uključenih u antioksidativnu zaštitu, kao i aktivnost antioksidativnih enzima, u jetri i intestinumu eksperimentalnih životinja sa holestazom, ka nivoima ekspresije i aktivnosti kod zdravih, netretiranih životinja. Dok su UDH i MKH u dozi od 4 mg/kg ispoljile antiinflamatorno dejstvo u jetri i intestinumu smanjenjem ekspresije gena za proinflamatorni transkripcioni faktor NF-κB, primena HDH i MKH u dozi od 20 mg/kg je imala suprotan efekat. Na modelu HT-29 ćelijske linije adenokarcinoma kolona, utvrđeno je da polusintetska žučna kiselina MKH ispoljava značajno manju citotoksičnost u odnosu na HDH i nešto veću citotoksičnost u odnosu na UDH. Epigenetski lek vorinostat je ispoljio sinergističko citotoksično dejstvo sa sve tri ispitivane žučne kiseline. Vorinostat je ostvario proapoptotski i antiproliferativni efekat u HT-29 ćelijama, koji je bio najizraženiji u kombinaciji sa MKH, s obzirom da je došlo do značajnog povećanja odnosa ekspresije BAX i BCL2 gena i smanjenja ekspresije gena za marker proliferacije ciklin D1. Vorinostat je, takođe, značajno smanjio antioksidativni kapacitet HT-29 ćelija smanjenjem ekspresije NRF2 gena i sledstvenim smanjenjem ekspresije gena za antioksidativne enzime. HDH je dodatno smanjila, a MKH poboljšala antioksidativni kapacitet HT-29 ćelija modulacijom ekspresije NRF2 gena. U in vitro i in vivo sistemu u okviru ove doktorske disertacije je pokazano da, pored HDH kao poznatog endogenog agoniste FXR receptora, MKH takođe povećava ekspresiju gena za FXR i njegovog ciljnog gena za transkripcioni korepresor SHP, što ukazuje da ova polusintetska žučna kiselina može da aktivira FXR. Osim toga, utvrđeno je da žučne kiseline ispoljavaju različite efekte prema ekspresiji gena za histon deacetilaze HDAC1 i HDAC2 u jetri i intestinumu eksperimentalnih životinja, kao i u HT-29 ćelijama karcinoma kolona, a jedino je UDH značajno smanjila ekspresiju gena za oba ispitivana enzima uključena u epigenetsku regulaciju ćelijskih procesa, i u isptivanim tkivima i HT-29 ćelijama. Rezultati našeg rada ukazuju da bi se UDH i MKH mogle koristiti u hemoprevenciji karcinoma kolona u niskim dozama, s obzirom na utvrđene efekte u modulaciji ekspresije gena uključenih u procese apoptoze, oksidativnog stresa i inflamacije. Takođe, s obzirom na ostvaren sinergistički efekat žučnih kiselina sa epigenetskim antitumorskim agensom vorinostatom, otvara se mogućnost kombinovane farmakološke strategije u terapiji solidnih tumora, koji u najvećem procentu pokazuju rezistenciju prema samom vorinostatu., Bile acids act as signaling molecules in the organism and they are involved in the regulation of numerous metabolic, inflammatory and immunomodulatory processes. These endogenous compounds exert their effects mostly by binding and activation of nuclear receptors. Farnesoid X receptor (FXR) is the main regulator of bile acid homeostasis, and has been shown to be significantly involved in processes of inflammation and carcinogenesis, primarily in the liver and intestinal tract. Activation of FXR receptor represents a significant pharmacological strategy for the treatment of cholestatic liver disease, inflammatory bowel disease, and colon carcinoma. The role of bile acids in signaling pathways regulating the cell cycle and contributing to the development or regression of malignancies is well determined, but the effects of these compounds on epigenetic mechanisms of key cellular processes regulation is yet to be elucidated. Given that the effects of bile acids are mostly determined by their polarity, the aim of our study was to investigate in vitro and in vivo effects of semi-synthetic keto derivative of cholic acid, 12-monoketocholic acid (MKC), in comparison to natural bile acids, hydrophobic chenodeoxycholic acid (CDC) and hydrophilic ursodeoxycholic acid (UDC), on processes of apoptosis, oxidative stress and inflammation, which are significant for both chemoprevention and therapy of colon cancer. Besides, the aim of our study was to examine the role of selected bile acids in the epigenetic regulation of these processes in colon cancer cells. In in vivo model of intrahepatic cholestasis in experimental animals, it has been demonstrated that UDC and MKC exhibit antiapoptotic, antioxidant, and antiinflammatory effects in the liver and intestine. It was shown that UDC and MKC prevent the mitochondrial pathway of apoptosis activation in the liver, while UDC exhibits an antiapoptotic effect in the intestine of experimental animals with cholestasis as well. These two bile acids significantly modulated the expression of genes involved in antioxidant protection, as well as the activity of antioxidant enzymes, in the liver and intestine of experimental animals with cholestasis, towards levels of expression and activity in healthy, untreated animals. While UDC and MKC at a low dose of 4 mg/kg exhibited an antiinflammatory effect in the liver and intestine by reducing the expression of the gene encoding the proinflammatory transcription factor NF-κB, the application of CDC and MKC at a high dose of 20 mg/kg exerted the opposite effect. In HT-29 human adenocarcinoma cell line, it has been demonstrated that semi-synthetic bile acid MKC exhibits significantly lower cytotoxicity than CDC and slightly higher cytotoxicity than UDC. The epigenetic drug vorinostat has exhibited a synergistic cytotoxic effect with all three investigated bile acids. Vorinostat exerted proapoptotic and antiproliferative effects in HT-29 cells, which were most pronounced in combination with MKC, as there was a significant increase in the ratio of BAX and BCL2 genes expression and a decrease of the proliferation marker cyclin D1 gene expression. Vorinostat also significantly reduced the antioxidant capacity of HT-29 cells by reducing the expression of NRF2 gene and consequently decreasing the expression of genes encoding antioxidant enzymes. CDC further reduced, while MKC improved the antioxidant capacity of HT-29 cells by modulating the expression of NRF2 gene. In both in vitro and in vivo systems, it was demonstrated that, in addition to CDC as a known endogenous FXR agonist, MKC also increased the expression of the gene encoding FXR, and FXR target gene encoding transcriptional co-repressor SHP as well, indicating that this semi-synthetic bile acid can also activate FXR. Besides, bile acids have been shown to exert distinct effects on the expression of the histone deacetylases HDAC1 and HDAC2 gene in the liver and intestine of experimental animals, and in HT-29 colon cancer cells. Only UDC significantly reduced the expression of the genes for both studied enzymes involved in the epigenetic regulation of cell processes, in both tissues and HT-29 cells. The results of our work indicate that UDC and MKC could be used in chemoprevention of colon cancer at low doses, considering determined effects in the modulation of expression of the genes involved in processes of apoptosis, oxidative stress and inflammation. Furthermore, synergistic effects of bile acids with the epigenetic antitumor agent vorinostat open up the possibility of a combined pharmacological strategy in the treatment of solid tumors, which are at the high percentage resistant to the effects of vorinostat alone.

Ugljenične nanocevi (UNC) imaju sve veću primenu u elektronici, kompjuterskoj i optičkoj industriji, kao i u biomedicini. Dok proizvodnja jednoslojnih UNC nanocevi beleži sve veći rast poslednjih godina, rizik koji nosi izlaganje ovom nanomaterijalu ostaje nerazjašnjen. Oskudni i često kontradiktorni podaci o toksičnim efektima jednoslojnih UNC ukazuju na potrebu za daljim ispitivanjima. U našim istraživanjima ispitivane su promene u ćelijskom odgovoru kao i morfološke promene nakon delovanja jednoslojnih ugljeničnih nanocevi na ćelijskoj liniji humanih fetalnih fibroblasta pluća MRC-5 i ćelijskoj liniji humanog adenokarcinoma pluća A549. U ovoj studiji korišćene su jednoslojne ugljenične nanocevi koje su sadržale rezidualne nečistoće poput gvožđa. Citotoksičnost jednoslojnih UNC (engl. single-walled carbon nanotubes – SWCNT) je ispitivana kolorimetrijskim MTT testom. Tokom 24 i 48h niske koncentracije jednoslojnih ugljeničnih nanocevi (ćelija nakon tretmana sa jednoslojnim ugljeničnim nanocevima pojedinačno i u kombinaciji sa antioksidantima ukazala je na kompleksnost i raznolikost biološkog odgovora ispitivanih ćelija. U našem istraživanju ispitivana je i promena aktivnosti enzima antioksidativne zaštite i količine glutationa u ćeliji. Primena jednoslojnih ugljeničnih nanocevi u MRC-5 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD i GR, povećava specifičnu aktivnost GPx i ne utiče na promenu specifične aktivnosti GST i količine glutationa u ćeliji Primena jednoslojnih ugljeničnih nanocevi u A549 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD, ne utiče na promenu specifične aktivnost enzima GR, GST i GPx, i dovodi do povećanja količine glutationa u ćeliji. Ćelijska vijabilnost, morfološke promene, redoks homeostaza i ekspresija ispitivanih gena bile su promenjene nakon tretmana sa jednoslojnim ugljeničnim nanocevima. Iako su dobijeni rezultati značajni za procenu toksičnosti ugljeničnih nanocevi, neophodna su dalja istraživanja koja treba da doprinesu boljem razumevanju toksičnih efekata ugljeničnih nanocevi., Carbon nanotubes are being actively introduced in electronics, computer science, and optics as well as for various biomedical applications. While production of single-walled carbon nanotubes (SWCNT) has escalated in recent years, the knowledge on risk associated with exposure remains unclear. Contradictory data on the toxic effects of single-walled carbon nanotubes highlights the urgent need for further studies. In this study we investigated the alterations in cellular response along with morphological changes induced by single-walled carbon nanotubes in human lung fibroblast cell line MRC-5 and adenocarcinoma human alveolar basal epithelial cells A549. In this study we used SWCNT containing large amounts of residual metallic impurities such is iron, and the iron concentration increased in dose dependent manner in cells exposed to SWCNT. Cytotoxicity was evaluated by MTT assay and SWCNT showed little cytotoxic effect on the proliferation and viability of two cell lines tested at the concentrations used (