Molecular mechanisms of the antiproliferative activity of ruthenium(II)-arene complex with isoquinoline-3-carboxylic acid as ligand under in vitro conditions

Još od otkrića cisplatine sintetisan je veliki broj kompleksa prelaznih metala, čija je antikancerska aktivnost ispitana. Naročitu pažnju zaslužuju jedinjenja zasnovana na rutenijumu kao metalu koja su pokazala antitumorsku i antimetastatsku aktivnost, sa potencijalnom primenom kod tumora rezistentnih na cisplatinu, ili kao alternative cisplatini. Cilj ove disertacije je ispitivanje molekularnih mehanizama antiproliferativne aktivnosti rutenijum(II)-arenskog kompleksa sa izohinolin-3-karboksilnom kiselinom kao ligandom. Rezultati ispitivanja citotoksičnog potencijala sedam novih jedinjenja opšte formule [Ru(η6-p-cimen)(L1-7)Cl] su pokazali da je kompleks sa L = izohinolin-3-karboksilnom kiselinom (RuT7) u odnosu na ostale komplekse iz serije, ispoljio izraženu citotoksičnu aktivnost, sa IC50 vrednostima u opsegu od 18,5 do 84,2 μM. Ovaj kompleks je pokazao dva puta veću aktivnost na HeLa ćelijama nego na normalnoj ćelijskoj liniji MRC-5, što potvrđuju i IC50 vrednosti određene nakon inkubacionog perioda od 48 h (45,4 ± 3,0 i 84,2 ± 5,7 μM). Analiza ćelijskog ciklusa HeLa ćelija koje su tretirane RuT7 kompleksom pokazala je zaustavljanje ćelija u S fazi ćelijskog ciklusa, kao i povećanje broja ćelija u sub-G1 populaciji. Aneksin V-FITC/PI esej, kao i morfološka analiza ćelija na fluorescentnom mikroskopu, pokazali su da ispitivano jedinjenje ima potencijal indukcije apoptoze. Ispitivanje akumulacije rutenijuma unutar ćelija je pokazalo prisustvo rutenijuma u ćelijskoj unutrašnjosti nakon 6 h inkubacije (8,9 ng Ru/106 ćelija). U cilju detaljnog ispitivanja mehanizma dejstva RuT7 kompleksa na HeLa ćelijama, urađena je mikroerej analiza promena u genskoj ekspresiji na celom transkriptomu HeLa ćelija. Analiza funkcionalnih kategorija i signalnih i biohemijskih puteva koji se menjaju tokom odgovora HeLa ćelija na tretman RuT7 kompleksom je pokazala da se pod uticajem tretmana aktiviraju molekularni mehanizmi koji vode ćelije u apoptozu i to preko unutrašnjeg (mitohondrijskog) signalnog puta. Do aktiviranja mitohondrijskog apoptotskog puta dolazi usled direktnog vezivanja RuT7 kompleksa za DNK molekul, kao i indirektnim oštećenjem DNK usled dejstva reaktivnih kiseoničnih vrsta. Statistička analiza promena u ekspresiji genskih setova uključenih u poznate puteve toksičnosti koje su izazvane lekovima je pokazala manju predviđenu toksičnost RuT7 kompleksa u poređenju sa cisplatinom. Sveobuhvatno, dobijeni rezultati predstavljaju osnovu za dalja istraživanja RuT7 kompleksa u okviru studija na životinjama i prekliničkim ispitivanjima kao potencijalnog kandidata za hemioterapeutika.Since the discovery of cisplatinum, many transition metal complexes have been synthesized and assayed for antineoplastic activity. In recent years, ruthenium-based compounds have emerged as promising antitumor and antimetastatic agents with potential uses in platinum-resistant tumors or as alternatives to platinum. The aim of this thesis was to investigate molecular mechanisms of the antiproliferative activity of ruthenium(II)-arene complex with isoquinoline-3-carboxylic acid as ligand. The results of investigation of the cytotoxic potential of seven new compounds of the general formula [Ru(η6-p-cymene)(L1-7)Cl] showed that the complex with L = isoquinoline-3-carboxylic acid (RuT7) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC50 values determined after 48 h of incubation (45,4 ± 3,0 vs. 84,2 ± 5,7 μM, respectively). Cell cycle analysis of HeLa cells treated with RuT7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8,9 ng Ru/106 cells after 6 h of incubation. To gain further insight in the molecular mechanism of action of RuT7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT7. Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT7-treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT7 in animal and pre-clinical studies as a potential drug candidate

Sinteza i kristalna struktura 1,2,3,4-tetrahidro-9-aminoakridin-tetrahlorocinkata(II) monohidrata

In the reaction of ZnCl(2) with tacrine hydrochloride in water novel tetracoordinated (C(13)H(15)N(2))(2)[ZnCl(4)]-H(2)O complex was obtained and characterized by elemental analysis, molar conductivity and X-ray analysis. The complex crystallizes in the space group P-1 of the triclinic crystal system. The structure contains two crystallographically different molecules of protonated tacrine present as counter cations, the [ZnCl(4)](2-) complex anion and one water solvent molecule. The counter cations slightly differ in the puckering of the cyclohexene ring. The molecules of protonated tacrine are involved in different intermolecular hydrogen bonds. In the crystal, the hydrogen bonding generates a 3D assembly. In the crystal, pi center dot center dot center dot pi stacking interactions between the rings of protonated tacrine were evidenced. The [ZnCl(4)](2-) complex anion has a distorted tetrahedral geometry. Three out of the four Cl atoms are involved in intermolecular hydrogen bonding. The intermolecular H-bond interactions involving the Cl atoms affect the Zn-CI bond lengths.U reakciji ZnCl2 sa takrin-hidrohloridom u vodi, dobijen je novi tetrakoordinovani (C13H15N2)2[ZnCl4]?H2O kompleks koji je okarakterisan pomoću elementalne analize, molarne provodljivosti i rendgenske strukturne analize. Kompleks kristališe u prostornoj grupi P?1 trikliničnog kristalnog sistema. Struktura sadrži dva kristalografski različita molekula protonovanog takrina koji su prisutni kao kontra-katjoni, [ZnCl4]2 kompleksni anjon i molekul kristalne vode. Molekuli katjona se neznatno razlikuju u stepenu nabiranja cikloheksenovog prstena. Molekuli protonovanog takrina su uključeni u različite intermolekulske vodonične veze. Intermolekulsko vodonično vezivanje u kristalu generiše 3D molekulski skup pi...pi interakcije između prstenova protonovanog takrina su primećene u kristalu. [ZnCl4]2- ima distorgovanu tetraedarsku geometriju. Tri od četiri Cl atoma su uključena u intermolekulske vodonične veze. Intermolekulske vodonične interakcije koje uključuju Cl atome utiču na dužinu Zn-Cl veza

Supplementary material for the article: Čobeljić, B.; Milenković, M.; Pevec, A.; Turel, I.; Vujčić, M.; Janović, B.; Gligorijević, N.; Sladić, D.; Radulović, S.; Jovanović, K.; et al. Investigation of Antitumor Potential of Ni(II) Complexes with Tridentate PNO Acylhydrazones of 2-(Diphenylphosphino)Benzaldehyde and Monodentate Pseudohalides. Journal of Biological Inorganic Chemistry 2016, 21 (2), 145–162. https://doi.org/10.1007/s00775-015-1315-x

Supplementary material for: [https://doi.org/10.1007/s00775-015-1315-x]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1906

Supplementary data for article : Todorović, T. R.; Vukašinović, J.; Portalone, G.; Suleiman, S.; Gligorijević, N.; Bjelogrlić, S.; Jovanović, K.; Radulović, S.; Anđelković, K.; Cassar, A.; et al. (Chalcogen)Semicarbazones and Their Cobalt Complexes Differentiate HL-60 Myeloid Leukaemia Cells and Are Cytotoxic towards Tumor Cell Lines. MedChemComm 2017, 8 (1), 103–111. https://doi.org/10.1039/c6md00501b

Supplementary material for: [https://doi.org/10.1039/c6md00501b]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2418]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3092

Supplementary data for the article: Filipović, N. R.; Marković, I.; Mitić, D.; Polović, N.; Milčić, M.; Dulović, M.; Jovanović, M.; Savić, M.; Nikšić, M.; Andelković, K.; et al. A Comparative Study of In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N-Heteroaromatic Schiff Base (E)-2-[N’-(1-Pyridin-2-Yl-Ethylidene)Hydrazino]Acetate. Journal of Biochemical and Molecular Toxicology 2014, 28 (3), 99–110. https://doi.org/10.1002/jbt.21541

Supplementary material for: [https://doi.org/10.1002/jbt.21541]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1509

Supplementary data for article: Čobeljić, B.; Pevec, A.; Turel, I.; Swart, M.; Mitić, D.; Milenković, M.; Marković, I.; Jovanović, M.; Sladić, D.; Jeremić, M.; et al. Synthesis, Characterization, DFT Calculations and Biological Activity of Derivatives of 3-Acetylpyridine and the Zinc(II) Complex with the Condensation Product of 3-Acetylpyridine and Semicarbazide. Inorganica Chimica Acta 2013, 404, 5–12. https://doi.org/10.1016/j.ica.2013.04.017

Supplementary material for: [https://doi.org/10.1016/j.ica.2013.04.017]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1367]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3539

Supplementary material for the article: Čobeljić, B.; Milenković, M.; Pevec, A.; Turel, I.; Vujčić, M.; Janović, B.; Gligorijević, N.; Sladić, D.; Radulović, S.; Jovanović, K.; et al. Investigation of Antitumor Potential of Ni(II) Complexes with Tridentate PNO Acylhydrazones of 2-(Diphenylphosphino)Benzaldehyde and Monodentate Pseudohalides. Journal of Biological Inorganic Chemistry 2016, 21 (2), 145–162. https://doi.org/10.1007/s00775-015-1315-x

Supplementary material for: [https://doi.org/10.1007/s00775-015-1315-x]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1906

Supplementary data for article : Todorović, T. R.; Vukašinović, J.; Portalone, G.; Suleiman, S.; Gligorijević, N.; Bjelogrlić, S.; Jovanović, K.; Radulović, S.; Anđelković, K.; Cassar, A.; et al. (Chalcogen)Semicarbazones and Their Cobalt Complexes Differentiate HL-60 Myeloid Leukaemia Cells and Are Cytotoxic towards Tumor Cell Lines. MedChemComm 2017, 8 (1), 103–111. https://doi.org/10.1039/c6md00501b

Supplementary material for: [https://doi.org/10.1039/c6md00501b]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2418]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3092

(Chalcogen)semicarbazones and their cobalt complexes differentiate HL-60 myeloid leukaemia cells and are cytotoxic towards tumor cell lines

Cobalt complexes with semi-and thiosemicarbazones of 8-quinolinecarboxaldehyde have been synthesized and characterized by X-ray diffraction analysis. These novel complexes and a previously synthesized cobalt complex with a selenium-based selenosemicarbazone ligand showed myeloid differentiation activity on all trans retinoic acid resistant HL-60 acute myeloid leukaemia cells. They also showed varying levels of cytotoxicity on five human tumor cell lines: cervix carcinoma cells (HeLa), lung adenocarcinoma cells (A549), colorectal adenocarcinoma cells (LS-174), breast carcinoma cells (MDA-MB-361), and chronic myeloid leukaemia (K562) as well as one normal human cell line: fetal lung fibroblast cells (MRC-5). Leukaemia differentiation was most strongly induced by a metal-free oxygen ligand and the selenium ligand, whilst the latter and the cobalt(II) complex with an oxygen ligand showed the strongest dose-dependent cytotoxic activity. In four out of five investigated tumor cell lines, it was of the same order of magnitude as cisplatin. These best compounds, however, had lower toxicity on non-transformed MRC-5 cells than cisplatin.This is the peer-reviewed version of the following article: Todorović, T. R.; Vukašinović, J.; Portalone, G.; Suleiman, S.; Gligorijević, N.; Bjelogrlić, S.; Jovanović, K.; Radulović, S.; Anđelković, K.; Cassar, A.; et al. (Chalcogen)Semicarbazones and Their Cobalt Complexes Differentiate HL-60 Myeloid Leukaemia Cells and Are Cytotoxic towards Tumor Cell Lines. MedChemComm 2017, 8 (1), 103–111. [https://doi.org/10.1039/c6md00501b]Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3093

Supplementary data for the article: Filipović, N. R.; Marković, I.; Mitić, D.; Polović, N.; Milčić, M.; Dulović, M.; Jovanović, M.; Savić, M.; Nikšić, M.; Andelković, K.; et al. A Comparative Study of In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N-Heteroaromatic Schiff Base (E)-2-[N’-(1-Pyridin-2-Yl-Ethylidene)Hydrazino]Acetate. Journal of Biochemical and Molecular Toxicology 2014, 28 (3), 99–110. https://doi.org/10.1002/jbt.21541

Supplementary material for: [https://doi.org/10.1002/jbt.21541]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1509