Title Utjecaj tvari s imunomodulatornim djelovanjem na galektin-3 Title (english) Effects of immunomodulatory drugs on galectin-3 Author Sanja Dabelić Mentor Jerka Dumić (mentor)Committee member Ivan Bašić (predsjednik povjerenstva) Committee member Jerka Dumić (član povjerenstva)Committee member Dražena Papeš (član povjerenstva)Granter University of Zagreb Defense date and country 2004, Croatia Scientific / art field, NATURAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Galektin-3, lektin koji specifično prepoznaje -galaktozidne strukture, djeluje kao jak
proinflamatorni signal koji modulira staničnu proliferaciju i adheziju, kemotaksiju,
fagocitozu te sintezu upalnih medijatora. Mnoge tvari s imunomodulatornim djelovanjem
djeluju na signalne puteve u kojima sudjeluju i transkripcijski faktori čija su vezna mjesta
prisutna u promotorskoj regiji gena za galektin-3 (LGALS3). Zbog česte uporabe ovih tvari u
terapijske svrhe te važnosti galektina-3 u fiziologiji stanica monocitno-makrofagne loze, u
ovom je radu ispitan utjecaj nesteroidnih (aspirina i indometacina) i steroidnih
(hidrokortizona i deksametazona) tvari s imunomodulatornim djelovanjem primijenjenih u
različitim terapijskim rasponima na ekspresiju LGALS3 i galektina-3 u nediferenciranim i
diferenciranim stanicama monocitne stanične linije THP-1 tijekom 72-satnog kultiviranja.
Niti jedna od ispitanih tvari u primijenjenim koncentracijama tijekom 72 sata kultiviranja
nije citotoksično djelovala na stanice. Količina mRNA za galektin-3 određena je primjenom
metode RT-PCR praćene analizom na uređaju AbiPrism 310 Genetic Analyser, a količina
galektina-3 u staničnim homogenatima Western-imunoblot metodom. Rezultati su pokazali
da ispitivane tvari mijenjaju ekspresiju LGALS3 i galektina-3 te da njihovi učinci ovise o
diferenciranosti stanica, koncentraciji primijenjene tvari kao i vremenu izlaganja. U
nediferenciranim stanicama sve ispitane tvari svih primijenjenih koncentracija inhibiraju
ekspresiju i LGALS3 i galektina-3, a inhibitorni je učinak u korelaciji s vremenom izlaganja.
Tijekom 48-satne preobrazbe nediferenciranih stanica u diferencirane, dolazi do snažne
indukcije ekspresije i LGALS3 (3 puta) i galektina-3 (3,5 puta). U diferenciranim stanicama
sve ispitivane tvari u početku inhibiraju ekspresiju LGALS3 nakon čega dolazi do uspostave
konstitutivne razine mRNA, dok na proteinskoj razini dugotrajnije izlaganje ispitivanim
tvarima potiče ekspresiju galektina-3. U oba slučaja, intenzitet promjena kao i njihov
vremenski slijed ovise o vrsti i koncentraciji primijenjene tvari. Na temelju dobivenih
rezultata moguće je zaključiti da primijenjene tvari s imunomodulatornim djelovanjem
utječu na različite signalne puteve kao i mehanizme regulacije ekspresije galektina-3 na
genskoj i proteinskoj razini, a da promjene ovise o vrsti i koncentraciji tvari te vremenu
izloženosti, kao i o diferencijacijskom stupnju stanica. Dobiveni rezultati predstavljaju važan
korak u razumijevanju djelovanja tvari s imunomodulatornim djelovanjem na galektin-3 u
stanicama monocitno-makrofagne loze, a time i na njihove brojne fiziološke funkcije.
Abstract (english) Galectin-3, a β-galactoside binding lectin, acts as a strong pro-inflammatory signal that
modulates cell proliferation and adhesion, chemotaxis, phagocytosis and synthesis of
inflammatory mediators. Many immunomodulatory drugs affect signaling pathways that
comprise transcriptional factors which binding sites are present in the promoter region of
galectin-3 gene (LGALS3). Because of the frequent therapeutic application of
immunomodulatory drugs and the importance of galectin-3 in the physiology of monocytes
and macrophages, we investigated effects of non-steroidal (aspirin and indomethacin) and
steroidal (hydrocortisone and dexamethasone) immunomodulatory drugs, applied in
different therapeutic ranges, on the expression of LGALS3 and galectin-3 in nondifferentiated
and differentiated cells of monocytic THP-1 cell-line during 72 hours of
cultivation. None of the studied drugs in applied concentrations during 72 hours of
cultivation had cytotoxic effect on the cells.
The targeted mRNA level was evaluated using relative RT-PCR technique and analysis on
the AbiPrism 310 Genetic Analyser. Galectin-3 expression in cell homogenates was
determined by western-immunoblot analysis. The results showed that the chosen
immunomodulatory drugs affect LGALS3 and galectin-3 expression and that their effects
depend on cell differentiation level, concentration of the applied drug, as well as time of
exposure. In undifferentiated cells all of the studied drugs (in all applied concentrations)
inhibit expression of LGALS3 and galectin-3, and inhibitory effect correlates with time of
exposure. Differentiation of monocytic THP-1 cells into macrophages during 48-hours
strongly induces expression of LGALS3 (3 times) and galectin-3 (3,5 times). In differentiated
cells, all studied drugs inhibited LGALS3 expression at the beginning, but during further
incubation constitutive level of galectin-3 mRNA was reestablished. On the protein level,
prolonged exposure to all applied drugs induce galectin-3 expression. Intensity and timecourse
of both, mRNA and protein level changes depend on drug type and on applied
concentration. These findings indicate that the applied immunomodulatory drugs affect
different signaling pathways and mechanisms of regulation of galectin-3, on both mRNA
and protein level. The changes depend on drug type and concentration, time of exposure as
well as cell differentiation level. The results obtained in this study represent important step
in the understanding of the effects of immunomodulatory drugs on galectin-3 in
monocytes/macrophages, hence on their numerous physiological roles.
Keywords
aspirin
deksametazon
ekspresija
galektin-3
hidrokortizon
indometacin
LGALS3
Keywords (english)
aspirin
dexamethasone
expression
galectin-3
hydrocortisone
indomethacin
LGALS3
Language croatian URN:NBN urn:nbn:hr:163:513894 Promotion 2004 Study programme Title: Biology Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2017-11-24 11:45:13
