Ispitivanje antiproliferativnog i in vitro antimetastatskog potencijala pikolinato rutenijum(II)-cimenskog kompleksa : poređenje sa serijom rutenijum(II)-arenskih kompleksa sa sličnom strukturom

Abstract

Cilj ove disertacije bio je ispitivanje potencijala antiproliferativnog i in vitroantimetastatskog dejstva serije novosintetisanih rutenijum(II)-arenskih kompleksa. Upitanju je serija Ru(II)-arenskih kompleksa sledeih strukturnih formula: [(6-pcimen)Ru(L1–3)Cl2], gde je L1–3: 3-acetilpiridin (1), 4-acetilpiridin (2) i 2-amino-5-hloropiridin (3), kao i [(6-p-cimen)Ru(HL4,5)Cl2], gde HL4 i HL5 odovaraizonikotinskoj kiselini (4) i nikotinskoj kiselini (5) i [(6-p-cimen)Ru(HL6–9)Cl], gdeH2L6–9 predstavlja 2,3-piridindikarboksilnu kiselinu (6), 2,4-piridindikarboksilnukiselinu (7), 2,5-piridindikarboksilnu kiselinu (8) i 2,6- piridindikarboksilnu kiselinu(9), i [(6-p-cimen)RuCl(L11)], gde je HL11 pikolinska kiselina (11). Kompleks (10) jepolazni kompleks [(6-p-cymene)2RuCl2]2 koji je korišen za sinteze navedenihkompleksa u reakciji sa odgovarajuim ligandima. Antiproliferativna aktivnost Ru(II)-arenskih kompleksa je ispitana na: šest tumorskih elijskih linija (HeLa, MDA-MB-361,MDA-MB-453, FemX, B16, LS-174), na dve transformisane endotelijalne linije (EA.hy926, MS1) i na jednoj normalnoj humanoj liniji (MRC-5). Za dalja ispitivanja poreenjapovezanosti strukture i aktivnosti odabrana su dva kompleksa sa monodentatno vezanimpiridinskim ligandom (1 i 3) i dva kompleksa sa bidentano vezanim piridinskimligandom (6 i 7), koji nisu imali znaajnu citotoksinu aktivnost i pikolinatorutenijum(II)-cimenski kompleks (11), kao kompleks sa znaajnom aktivnošu.Potencijal ispitivanih kompleksa da indukuju promene na nivou elijskog ciklusaodreen je korišenjem protonog citometra nakon bojenja tretiranih elija sapropidijum-jodidom. Takoe, korišenjem testa za detekciju rane faze apoptoze,dvokolornim bojenjem elija sa aneksinom i propidijum-jodidom i analize naprotonom citometru ispitan je potencijal kompleksa 11 da indukuje apoptozu.Distribucija rutenijuma(II) u proteinskoj i DNK frakciji HeLa elija tretiranih saispitivanim kompleksima utvrena je korišenjem indukovano kuplovane plazme sa optiko emisionom spektrometrijom (ICP-OES)...

The aim of this thesis was to investigate antiproliferative and in vitro antimetastaticpotential of series of newly synthesized ruthenium(II)-arene complexes. It is a series ofRu(II)-arene complexes of general formula: [(6-p-cymene)Ru(L1–3)Cl2], where L1–3is 3-acetylpyridine (1), 4-acetylpyridine (2) and 2-amino-5-chloropyridine (3),correspondingly, [(6-p-cymene)Ru(HL4,5)Cl2], where HL4 i HL5 are respectivelyisonicotinic acid (4) and nicotinic acid (5) and [(6-p-cymene)Ru(HL6–9)Cl], whereH2L6–9 represent 2,3-pyridinedicarboxylic acid (6), 2,4-pyridinedicarboxylic acid (7),2,5-pyridinedicarboxylic acid (8) and 2,6- pyridinedicarboxylic acid (9), and [(6-pcymene)RuCl(L11)], where HL11 is picolinic acid (11). Complex [(6-pcymene)2RuCl2]2 (10) was starting complex used for the synthesis of complexes of thisseries with the corresponding ligands. Analysis of cell growth inhibition caused byRu(II)-arene complexes was performed on: six tumor cell lines (HeLa, MDA-MB-361,MDA-MB-453, FemX, B16, LS-174), on two transformed endothelial lines (EA.hy 926,MS1) and on one normal human cell line (MRC-5). For further examination ofcomparison of structure and activity we used two complexes with monodentate bondedpyridine ligand (1 an 3) and two with bidentate bonded pyridine ligand (6 and 7), whichdidn´t have any important cytotoxic activity and picolinate ruthenium(II)-cymenecomplex (11), as complex with important activity. Potential of investigated complexesto induce cell cycle perturbations was determined after staining of treated cells withpropidium iodide (PI) on flow cytometer. As well as determination of induction of early apoptotic changes by complex 11, after two colors staining with Annexin V-FITC andPI and analysis on flow cytometer. Ru(II) distribution among the DNA and proteinfractions in HeLa cells treated with investigated complexes was determined usinginductive coupled plasma with optical emissione spectrometry (ICP-OES). In this studywe evaluated whether DNA-repair-dependent signaling, as a result of interaction withDNA, which includes components of NER or MMR is utilized in cell response to ruthenium(II)-p-cymene complexes, by following expression of ERCC1 (mRNA andprotein level) and MSH2 (protein level) using Quantitative Real-Time PCR (RQ-PCR)and Western blot...