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 vitro antimetastatskog dejstva serije novosintetisanih rutenijum(II)-arenskih kompleksa. U pitanju 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 odovara izonikotinskoj kiselini (4) i nikotinskoj kiselini (5) i [(6-p-cimen)Ru(HL6–9)Cl], gde H2L6–9 predstavlja 2,3-piridindikarboksilnu kiselinu (6), 2,4-piridindikarboksilnu kiselinu (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) je polazni kompleks [(6-p-cymene)2RuCl2]2 koji je korišen za sinteze navedenih kompleksa 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.hy 926, MS1) i na jednoj normalnoj humanoj liniji (MRC-5). Za dalja ispitivanja poreenja povezanosti strukture i aktivnosti odabrana su dva kompleksa sa monodentatno vezanim piridinskim ligandom (1 i 3) i dva kompleksa sa bidentano vezanim piridinskim ligandom (6 i 7), koji nisu imali znaajnu citotoksinu aktivnost i pikolinato rutenijum(II)-cimenski kompleks (11), kao kompleks sa znaajnom aktivnošu. Potencijal ispitivanih kompleksa da indukuju promene na nivou elijskog ciklusa odreen je korišenjem protonog citometra nakon bojenja tretiranih elija sa propidijum-jodidom. Takoe, korišenjem testa za detekciju rane faze apoptoze,dvokolornim bojenjem elija sa aneksinom i propidijum-jodidom i analize na protonom citometru ispitan je potencijal kompleksa 11 da indukuje apoptozu. Distribucija rutenijuma(II) u proteinskoj i DNK frakciji HeLa elija tretiranih sa ispitivanim 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 antimetastatic potential of series of newly synthesized ruthenium(II)-arene complexes. It is a series of Ru(II)-arene complexes of general formula: [(6-p-cymene)Ru(L1–3)Cl2], where L1–3 is 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 respectively isonicotinic acid (4) and nicotinic acid (5) and [(6-p-cymene)Ru(HL6–9)Cl], where H2L6–9 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 this series with the corresponding ligands. Analysis of cell growth inhibition caused by Ru(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 of comparison of structure and activity we used two complexes with monodentate bonded pyridine ligand (1 an 3) and two with bidentate bonded pyridine ligand (6 and 7), which didn´t have any important cytotoxic activity and picolinate ruthenium(II)-cymene complex (11), as complex with important activity. Potential of investigated complexes to induce cell cycle perturbations was determined after staining of treated cells with propidium 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 and PI and analysis on flow cytometer. Ru(II) distribution among the DNA and protein fractions in HeLa cells treated with investigated complexes was determined using inductive coupled plasma with optical emissione spectrometry (ICP-OES). In this study we evaluated whether DNA-repair-dependent signaling, as a result of interaction with DNA, which includes components of NER or MMR is utilized in cell response to ruthenium(II)-p-cymene complexes, by following expression of ERCC1 (mRNA and protein level) and MSH2 (protein level) using Quantitative Real-Time PCR (RQ-PCR) and Western blot...