Rat C6 glioma model as a tool for discovering new therapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije.RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH.Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased.RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH.In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness..

Rat C6 glioma model as a tool for discovering new therapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije.RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH.Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased.RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH.In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness..

Etički aspekti upotrebe matičnih ćelija kao zdravstvenih tehnologija u personalizovanoj medicini

Objective: Stem cells could be defined as cell with an extraordinary capacity for self-renewal and the ability to differentiate into different cell types. The aim of this study is to examine students' opinion about the use of stem cells as health technologies in disease treatment and radical life extension. Method: Prospective cross-sectional study was conducted as a questionnaire, which was filled anonimly and voluntarily. Results: In this study 655 respondents have participated, with an average age 21.9±}1.8 years. High percentage distribution was concluded with most of the statements, with especially positive attitude towards the preservation of stem cells for their own treatment (84.2%), and treatment of their families (85.4%). Dominantly neutral attitude was expressed according to a statement about the reliability of stem cells and doubts about their implementation.(49.4%).There was no statistically significant difference in students' opinion between different faculties. Conclusion: It is important to evaluate students' opinion because it can obtain information on how the respondents agree with the potential implementation of health technology in the future and, accordingly, whether it is justified to continue further research. We should be more dedicated to informing students in the future, and this type of research should be implemented on a wider population to obtain the relevant results and perform more reliable conclusion.Uvod: Matične ćelije funkcionalno se mogu definisati kao ćelije koje poseduju izvanredni kapacitet samoobnavljanja i sposobnost diferenciranja u različite tipove ćelija. One nisu samo karakarakteristične za embrionalni stadijum razvića individue, već su prisutne i u adultnim tkivima, gde predstavljaju bazu za obnavljanje ćelija tokom života. Zbog toga su kao nove zdravstvene tehnologije tesno povezane sa personalizovanim pristupom lečenju, kao i sa radikalnim produženjem života. Cilj rada: Ispitati stavove studenata u vezi sa upotrebom matičnih ćelija kao zdravstvenih tehnologija u lečenju bolesti i radikalnog produženja života. Metod rada: Sprovedena je prospektivna studija preseka sa namenski kreiranim upitnikom, koji su ispitanici popunjavali anonimno i dobrovoljno. Rezultati: U istraživanju je učestvovalo 655 ispitanika (studenata), prosečne starosti 21,9±}1,8 godina. Utvrđena je visoka procentualna zastupljenost slaganja sa većinom datih izjava i sa naročito pozitivnim stavom prema čuvanju matičnih ćelija za sopstveno lečenje (84,2%), odnosno lečenje svoje porodice (85,4%). Dominantno neutralan stav ispitanika je iskazan prema izjavi o pouzdanosti matičnih ćelija i sumnje u vezi sa njenom primenom (49,4%). Nije utvrđeno postojanje statistički značajne razlike stavova studenata različitih fakulteta ni sa jednom izjavom. Zaključak: Ispitivanje stavova studenata je značajno jer se dobijaju informacije o tome koliko su ispitanici saglasni sa potencijalnom implementacijom zdravstvene tehnologije u budućnosti i shodno tome da li je opravdano nastaviti dalja istraživanja. Rezultati ovog istraživanja su pokazali da su studenti uglavnom saglasni sa čuvanjem matičnih ćelija kako u cilju sopstvenog tako i lečenja članova svoje porodice, ali i da su, nasuprot tome, skeptični po pitanju pouzdanosti u primeni ovih metoda lečenja. U budućnosti bi trebalo posvetiti veću pažnju informisanju studenata i ovaj tip istraživanja je potrebno sprovesti na široj populaciji radi dobijanja što relevantnijih rezultata i izvođenja pouzdanijeg zaključka

Enhanced electrochemical detection of gallic acid using modified glassy carbon electrodes with Zn/Ga-dopped cobalt ferrite

In this study, solvothermally synthesized nanoparticles of cobalt ferrite (CoFe2O4, CFO) and Zn/Ga-dopped cobalt ferrite (Co0.5Zn0.5Fe2O4, CFO_Zn and CoFe1.5Ga0.5O4, CFO_Ga, respectively) with dihydrocaffeic acid (DHCA) as a surfactant were examined for electrochemical detection of gallic acid. Based on X-ray diffraction analysis, diffraction maxima of all samples correspond to the cubic spinel phase, while transmission electron microscopy showed that the nanoparticles are non-agglomerated, sphere-like with an average size of 5±1 nm. The presence of DHCA on the surface of nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy (FTIR). For the purpose of electrochemical detection of gallic acid the modified glassy carbon electrode (m-GCE) was prepared from CFO, CFO_Zn and CFO_Ga nanoparticles. Initial tests have shown instability/disintegration of m-GCE in aqueous solutions, because of which samples were annealed at 450 °C in air. Cyclic voltammograms of all annealed samples showed good reversibility in the system [Fe(CN)6] 3-/4- . The most efficient electron transfer was achieved when the mass ratio between sample and Nafion® was 85:15, respectively. The best sensitivity to gallic acid (10-4 M, in Britton-Robinson buffer) was observed for CFO_Zn sample. Results are showing that by selective functionalization and surface modification of nanoparticles, it is possible to optimize the electrochemical properties suitable for sensing, which is the basis of this method and the goal of further research.Twenty-First Young Researchers’ Conference - Materials Science and Engineering: Program and the Book of Abstracts; November 29 – December 1, 2023, Belgrade, Serbi

Decreased TSPAN14 Expression Contributes to NSCLC Progression

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression

Supplementary data for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Stojković, S.; Mandić, B.; Tešević, V.; Vajs, V.; Isaković, A.; Pešić, M. Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus Glutinosa) and Their Interaction with Anticancer Drugs. Planta Medica 2014, 80 (13), 1088–1096. https://doi.org/10.1055/s-0034-1382993

Supplementary material for: [https://doi.org/10.1055/s-0034-1382993]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1845

A SUGGESTION FOR PROACTIVE CARDIOLOGIC APPROACH TO CUSHING’S SYNDROME OR DISEASE

Numerous studies and reviews agree about the increased cardiovascular risk in Cushing’s syn-drome. Therefore, the aim of the paper is to suggest a few common diagnostic and therapeutic cardiologic preferences for the majority of Cushing’s syndrome/Cushing’s disease (CS/CD) pa-tients which are not yet routine but have the rationale to become standard procedures. This may serve as an initial working document, to be improved by the experts in the field. A narrative review is used to present synthesis and deduction of several approaches in cardiology regarding the actu-al topic. Results are systematized as the risk factors or co-morbidities list (prevalent in CS/CD) coupled with current and adapted cardiologic suggestions for practice

Surface, structural and electric properties of ion beam irradiated graphene oxide papers

In this paper, the physicochemical properties of nanocomposites of graphene oxide (GO) and cobalt ferrite nanoparticles CFO NPs (pristine and doped with zinc and gallium, CFO_Zn, CFO_Ga) were investigated. CFO, CFO_Zn and CFO_Ga NPs were synthesized using the solvothermal method with oleic acid (OA) as a surfactant. X-ray diffraction (XRD) confirmed the presence of pure ferrite phase while transmission electron microscopy (TEM) revealed spherical and non-agglomerated NPs with average size of 5±1 nm. Fourier transform infrared spectroscopy (FTIR) has confirmed bridging bidentate interaction between the oleic acid and the metal atoms on the surface of the NPs, while Raman spectroscopy results confirmed the incorporation of zinc and gallium in the spinel structure. Prior to preparation of nanocomposite with GO, an OA coating has been exchanged with dihydrocaffeic acid (DHCA). DHCA binds to surface of NPs through both covalent bridging bidentate interaction and catechol groups, as determined by FTIR. Nanocomposite has been obtained by adding CFO(_Zn/Ga) NPs to a GO suspension with nominal fractions of 5 to 15 wt.%, followed by homogenization and hydrothermal treatment (T=120 °C, t=3 h). TEM results revealed that the shape and size of NPs remain unchanged in nanocomposite, while FTIR results have shown that hydrogen bond is established between the functional groups of GO and DHCA. Electrochemical properties of individual components and nanocomposite were examined by cyclic voltammetry. The best electrochemical properties, in terms of the highest specific capacity, were shown by the composite which contains 15 wt.% CFO NPs.Nineteenth Young Researchers' Conference Materials Science and Engineering, December 1-3, 2021, Belgrade, Serbi

Chemical Profiling and Assessment of Antineurodegenerative and Antioxidant Properties of Veronica teucrium L. and Veronica jacquinii Baumg.

Neuroprotective potential of V. teucrium and V. jacquinii methanol extracts was analyzed. Chemical analysis of investigated extracts showed the presence of phenolic acid derivatives, flavonoids and one secoiridoid. The detected flavonoids derived from flavones (luteolin and isoscutellarein in V. jacquinii; apigenin, isoscutellarein and luteolin in V. teucrium) and flavonol (quercetin in V. jacquinii). Acteoside was the dominant compound in V. jacquinii, while plantamajoside and isoscutellarein 7-O-(6‴-O-acetyl)-ß-allosyl (1‴?2‴)-ß-glucoside were the major phenolics in V. teucrium. Additionally, the antineurodegenerative activity was tested at concentrations of 25, 50, and 100 µg/ml using acetylcholinesterase (AChE) and tyrosinase (TYR) assays. The inhibition of both enzymes was achieved with the investigated extracts, ranging from 22.78 to 35.40% for AChE and from 9.57 to 16.38% for TYR. There was no statistical difference between the activities of the analyzed extracts. Our data indicate that V. teucrium and V. jacquinii may have beneficial effects against Alzheimer's and Parkinson's disease.The authors are grateful to Fundac ~ao para a Ci^encia e a Tecnologia (FCT, Portugal) for financial support to CIMO (strategic project PEst-OE/AGR/UI0690/2011). J. C. M. Barreira and M. I. Dias thank FCT, POPH-QREN, and FSE for their grants (SFRH/BPD/72802/2010 and SFRH/BD/84485/2012, respectively). The GIP-USAL is financially supported by the Spanish Government through the project AGL2015-64522-C2-2-R. The authors are also grateful to the Serbian Ministry of Education, Science and Technological Development for financial support (Grant Numbers 173032, 173029, and 46013).info:eu-repo/semantics/publishedVersio

Electrochemical properties of composites of graphene oxide and cobalt ferrite doped with zink and gallium

The possibility of surface functionalization and formation of stable aqueous suspensions make graphene oxide (GO) suitable as base for composite materials, especially with nanoparticles. Cobalt ferrite (CFO) has attracted attention due to its unique properties such as large magnetic anisotropy, high coercivity, moderate saturation magnetization, excellent chemical stability, mechanical hardness, etc. This study presents the electrochemical properties of GO and CFO composites, as well as CFO doped with zinc (CFO_Zn) and gallium (CFO_Ga). Magnetic nanoparticles were synthesized using the solvothermal method, after which the oleic acid was exchanged with dihydrocaffeic acid to obtain the hydrophilic material. GO was synthesized using a modified Hummer's method. Composites of GO and magnetic nanoparticles were synthesized by the hydrothermal method (T = 120 °C, t = 3 h), with nominal fractions of 5 and 15 wt.% of magnetic nanoparticles. X-ray structural diffraction, as well as FTIR analysis, confirmed the complete oxidation of graphene layers. SEM and TEM images showed deposition of magnetic nanoparticles on GO layer with the different distribution density between 5 wt.% and 15 wt.% composites. Also, shape and size of magnetic nanoparticles remained unchanged. Based on FTIR analysis of hydrothermally treated GO and composites a partial reduction of epoxy groups was found along with hydrogen bond established between the components of the composite. The electrochemical charge storage of composites is mainly of pseudocapacitive nature, which originates from the oxidoreduction reactions of H+ ions from electrolytes and surface functional groups of GO. The best electrochemical properties, in terms of the highest specific capacity, were shown by the composite which contains 15 wt.% CFO, which value is 36.86 F g-1 at a polarization rate of 5 mV s-