Title Biološki učinci novih n-metoksimetiliranih derivata pirimidina na tumorske stanične linije čovjeka u uvjetima in vitro Title (english) Biological effects of novel n-methoxymethylated pyrimidine derivatives in human tumor cell lines Author Lana Sappe Mentor Mirela Sedić (mentor)Committee member Karlo Wittine (predsjednik povjerenstva)Committee member Kristina Sepčić (član povjerenstva) Committee member Igor Jurak (član povjerenstva)Committee member Mirela Sedić (član povjerenstva)Granter University of Rijeka Defense date and country 2020-05-25, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract U doktorskoj disertaciji istražili smo biološku aktivnost novih N-metoksimetiliranih (MOM) C-6 acikličkih derivata pirimidina, u uvjetima in vitro i in silico. Od svih spojeva, najsnažniji anti-proliferativni učinak na adherentne tumorske stanične linije je imao spoj 14b (6-(3-benziloksi-2-(benziloksimetil)propil-N,N-1,3-bis(metoksimetil)-5-metilpirimidin-2,4,dion), koji je ujedno pokazao i značajno jači citostatski učinak na leukemijskim stanicama K562 u usporedbi s klinički odobrenim lijekom Tasignom u uvjetima in vitro. In silico analiza pokazala je kako spoj 14b vjerojatno djeluje kao inhibitor RNA- i DNA- polimeraza, te kao inhibitor stanične adhezije. Daljnja in vitro istraživanja su potvrdila inhibitorni učinak spoja 14b na adheziju tumorskih stanica na podlozi sa fibronektinom, naročito kod metastatskih stanica kolorektalnog karcinoma SW620. Inhibitorni učinak spoja 14b na procese koje regulira DNA- polimeraza djelomično je bio potvrđen i analizom staničnog ciklusa koja je pokazala kako spoj 14b smanjuje postotak stanica SW620 u fazi staničnog ciklusa G1 te nakupljanje stanica u fazama staničnog ciklusa S i G2/M. Spoj 14b aktivira unutarnji (mitohondrijski) put apoptoze u stanicama SW620. Analiza staničnog metabolizma pokazala je i učinak spoja 14b na funkciju mitohondrija i glikolitičku aktivnost stanica u ovisnosti o koncentraciji, te je pri najnižoj koncentraciji od 0,1 μM uočena povećana metabolička aktivnost što upućuje na proliferativni učinak, dok je pri višoj koncentraciji od 1 μM, spoj 14b pokazao toksičan učinak na mitohondrije. Anti-proliferativna, pro-apoptotska i anti-adhezivna svojstva spoja 14b mogu se pripisati inhibiciji signalnih putova IGF-1R/FAK/Src, NF-κB/TGM2 i sfingozin kinaze 1, koji imaju važnu ulogu u kolorektalnoj karcinogenezi i metastatskim procesima. S obzirom na toksične učinke na normalnim fibroblastima čovjeka, potrebno je provesti daljnju optimizaciju i modifikaciju u svrhu dobivanja kandidata za daljnja predklinička istraživanja.
Abstract (english) In this doctoral thesis, the biological activity of novel pyrimidine derivates, N-metoxymethylated (MOM) C-6 acyclic pyrimidine derivates was evaluated in vitro and in silico. Out of all tested compounds, compound 14b (6-(3-benzyloxy-2-(benzyloxymetyl)propyl-N,N-1,3-bis(metoxymethyl)-5-methylpyrimidine-2,4,dion) showed the strongest antiproliferative effect on adherent tumour cell lines, as well as significantly stronger cytostatic effect in vitro on K562 leukaemia cell line in comparison to clinically approved drug Tasigna. In silico analysis showed that compound 14b likely works as an inhibitor of RNA and DNA polymerase, as well as an inhibitor of cell adhesion. Further in vitro research confirmed inhibitory effect of 14b compound on tumour cells adhesion on the fibronectin coated surface, especially in metastatic colorectal carcinoma SW620 cells line. Inhibitory effect of 14b compound on DNA polymerase regulated processes has also been partially confirmed by analysis of cell cycle showing 14b induced reduction of percentage of SW620 cells in the G1 phase of cell cycle, and accumulation of cells in S and G2/M cell cycle phases. Compound 14b activates intrinstic (mitochondrial) pathway of apoptosis in SW620 cells. Analysis of cell metabolism also showed concentration dependant effect of 14b on mitochondrial function and glycolitic activity. At lower concentrations of 0.1 μM increased metabolic activity was observed suggesting proliferative effect, while at the higer concentrations of 1 μM compound 14b showed the effect as mitochondrial toxicant. Antiproliferative, proapoptotic and antiadhezive effects of compound 14b may be attributed to the inhibition of signal pathways IGF-1R/FAK/Src, NF-κB/TGM2, and sphingosine kinase, which all have significant role in colorectal carcinogenesis and metastatic processes. Given the toxicity on normal human fibroblasts, further optimisation and modification is needed to obtain a candidate for subsequent preclinical research.
Keywords
Metoksimetilirani pirimidin-2
4-dion
karcinom debelog crijeva
SW620
stanični ciklus
mitohondrijska respiracija
glikoliza
IGF-1R
FAK
sfingozin kinaza
fibroblasti
citotoksičnost
Keywords (english)
Methoxymethylated pyrimidine-2
4-dione
colon cancer
SW620
cell cycle
mitochondrial respiration
glycolysis
IGF-1R
FAK
sphingosine kinase
fibroblasts
cytotoxicity
Language croatian URN:NBN urn:nbn:hr:193:727947 Promotion 2021 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2021-04-26 08:48:20
