Unexplored features of Ru(ii) polypyridyl complexes - towards combined cytotoxic and antimetastatic activity.

View Article Online View Journal M etallomics Integrated biometal science Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: I. Gurgul, O. Mazuryk, M. omzik, P. C. Gros, D. Rutkowska-Zbik and M. Brindell, Metallomics, 2020, DOI: 10.1039/D0MT00019A. This is an Accepted Manuscript, which has been through the Volume 10 Number 4 A Pa p g ri e l s 2 0 51 1 5 8 -652 Royal Society of Chemistry peer review process and has been accepted for publication. M etallomics Accepted Manuscripts are published online shortly after acceptance, Integrated biometal science rsc.li/metallomics before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. 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In no event ISSN 1756-591X shall the Royal Society of Chemistry be held responsible for any errors PBAPs Ainn et P uiHb E d eao R,c m Ltei o grin aan al s gm a Heecu rh u eag tni n aiso l.m sa o: fp sroilvteeor mnaicnso appaprtrioclaecsh in or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/metallomics Page 1 of 19 Metallomics 1 2 3 The impact of metal-based drugs on cellular processes is crucial to understand their mode of 4 action and enables to determine the key points important for improvement of their therapeutic 5 6 index. 7 8 The presented data underline that ruthenium polypyridyl complexes except well-documented 9 cytotoxic activity can have a high potency as anti-metastatic drugs. 10 11 We show that by appropriate modification of ligands it is possible to obtain compounds, 12 which strengthen cells’ adherent properties reducing the chance of metastatic cells to escape 13 14 to surrounding tissue. 15 16 For the first time, we correlate the observed influence on cell adhesion properties with t p 17 inhibition of the released and membrane-bound metalloproteinases on in vitro level as well as 18 i r 19 using isolated enzymes. c 20 s 21 We postulate that metalloproteinases might be important cellular targets for ruthenium u 22 polypyridyl complexes, which are responsible for their potency in inhibition of metastasis. n 23 24 a 25 M 26 27 d 28 e 29 t 30 p 31 e 32 c 33 c 34 35 A 36 37 s 38 c 39 i m 40 41 o 42 l 43 l a 44 45 t e 46 M 47 48 49 50 51 52 53 54 55 56 57 58 59 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 2 of 19 1 2 3 Unexplored features of Ru(II) polypyridyl complexes - towards combined 4 cytotoxic and antimetastatic activity 5 6 7 Ilona Gurgula, Olga Mazuryka,*, Michał Łomzika,b,c, Philippe C. Grosc, Dorota Rutkowska- 8 9 Żbikd, Małgorzata Brindella,* 10 11 aDepartment of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University in 12 13 Kraków, Gronostajowa 2, 30-387 Krakow, Poland 14 bDepartment of Organic Chemistry, Faculty of Chemistry, University of Łódź, ul. Tamka 12, 15 91-403 Łódź, Poland 16 t 17 cUniversité de Lorraine, CNRS, L2CM, F-54000 Nancy, France p 18 dJerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, i r 19 Niezapominajek 8, 30-239 Krakow, Poland c 20 s 21 u 22 n 23 Abstract 24 a The well-documented cytotoxic activity of coordinatively saturated and substitutionally inert 25 M 26 polypyridyl Ru(II) complexes substantiates to their high potency as antiproliferative agents 27 against primary tumors. However, the primary cause of cancer morbidity and mortality d 28 responsible for about 90% of cancer deaths is the occurrence of metastasis. Therefore, e 29 t 30 scientists have to concentrate their efforts in designing compounds affecting not only the p 31 primary tumor, but also inhibiting efficiently the metastasis. Herein, we report two families of e 32 Ru(II) polypyridyl complexes bearing 2,2’-bipyridine substituted by a semicarbazone 2- c 33 c 34 formylopyridine moiety as one of the ligands and, 4,4′-di-tert-butyl-2,2′-dipyridyl or 4,7- 35 A diphenyl-1,10-phenanthroline as auxiliary ligands. These complexes strengthen cells’ 36 37 adherent properties as well as inhibit the activity of metalloproteinases (MMPs) in vitro, s 38 which is relevant in anti-metastatic treatment. The in vitro studies were performed on human c 39 lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) cells that have a well- i m 40 documented invasive potential. The induced alteration of tumor cells’ adhesion properties 41 o 42 correlated with the high cytotoxic effect exerted by the complexes and their excellent cellular l 43 uptake. It was also proved that both complexes directly inhibit MMP2 and MMP9 enzyme l a 44 activities, which are essential for the development of tumor metastasis. The results of this 45 t e 46 study indicate that the biological properties of polypyridyl Ru(II) complexes extend beyond M 47 the standard cytotoxic activity and represent an important step towards designing new anti- 48 metastatic agents. 49 50 51 Introduction 52 Most compounds tested for their anticancer properties act as cytotoxic agents and their 53 54 activity is focused on influencing primary tumors. However, the biggest problem in the 55 treatment of cancer is the development of metastases. It is estimated that over 90% of deaths 56 due to cancer are associated with metastases.[1, 2] So far, no effective for inhibition of 57 58 metastasis formation are available on the market. Clinical trials concentrated primarily on the 59 extracellular matrix metalloproteinase inhibitors and inhibitors of angiogenesis, however none 60 1 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Page 3 of 19 Metallomics 1 2 3 of the tested compounds have exhibited the desired therapeutic effect.[3, 4] It is postulated 4 that affecting a number of other targets related to the process of metastasis, might be 5 6 beneficial. It is believed that an essential precondition for successful clinical development 7 requires targeting the correct portions of the metastatic cascade.[4, 5] 8 The detachment of cancer cells from their parent tumors is an initial event in 9 metastasis. It often requires losing cell-cell and cell-matrix contact, followed by migration 10 11 through the basement membrane (involving remodeling of actin cytoskeleton). Cancer cells 12 must also degrade the surrounding tissue to move outside the primary organ. This can be 13 achieved by an increased production of matrix metalloproteinases (MMPs), which are actively 14 15 involved in many other steps of the metastatic cascade like intravasation, angiogenesis or 16 t formation of an appropriate metastatic niche.[6] In particular, it was shown that p 17 overexpression of MMP-2 and MMP-9[2, 7, 8] was frequently associated with the invasive 18 i r 19 and metastatic potential of tumor cells[7] and is a bad prognostic factor in a tumor.[9] c 20 Affecting any of the mentioned processes or ideally influencing several of them can bring s 21 benefits in inhibition of metastasis at its initial phase. u 22 n 23 For many years there has been an increased interest in developing new metal based 24 a drugs using coordinatively saturated polypyridyl Ru(II) compounds bearing inert 25 M substituents.[10-13] The recent research has been directed particularly towards designing site- 26 27 specific or targeted drugs that can be obtained either through appropriate modification of d 28 ligands by conjugation of specific molecules for targeting cancer cells or by application of e 29 stimuli-responsive prodrugs.[12, 13] Most of the in vitro studies were focused on the t 30 p 31 determination of the cytotoxic activity of Ru polypyridyl complexes as well as on the e 32 elucidation of the mechanisms responsible for the observed biological effects (cell death c 33 mechanism, induction of reactive oxygen species formation, the uptake and localization in c 34 35 cell, etc.).[11] There are only a handful of studies, that considered their potential A 36 antimetastatic activity. It was shown that Ru polypyridyl complexes can influence the cell– 37 s matrix adhesion properties[14, 15], disrupt microtubule function by acting as microtubule 38 c 39 stabilizing agents[16] as well as decrease cell migration capability.[14, 17-19] Inspired by i m 40 these findings, we have decided to thoroughly investigate the in vitro behavior of a group of 41 Ru(II) polypyridyl complexes containing 2,2’-bipyridine ligand substituted by a o 42 43 semicarbazone2-formylopyridine moiety (L1, Fig.1 and Scheme 1), focusing on their impact l l a 44 on the alteration of tumor cells’ adhesion properties that is relevant in anti-metastatic 45 t treatment. e 46 47 Complexes Ru1 and Ru4 were recently prepared by our group and the mechanism of M 48 their cytotoxicity was reported.[20] Herein, we also describe the synthesis and 49 characterization of the three new Ru(II) complexes from this series Ru2, Ru3 and Ru5, 50 including their photophysical properties as well as their structure and characterization of 51 52 frontier orbitals obtained by applying Density Functional Theory (DFT) approach. In this 53 study we examine the effect of auxiliary ligands in Ru(II) complexes on the cytotoxicity, cell 54 adhesion properties, activity of matrix proteinases (MMPs) and other cellular responses. For 55 56 in vitro studies human lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) 57 cell lines have been chosen due to their well-documented invasive potential[21, 22] and their 58 expression of MMP-2 and MMP-9 largely involved in tumor invasion and metastasis. [2, 7, 8] 59 60 2 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 4 of 19 1 2 3 Our study is aimed to speculate that the ruthenium complexes impact on cancer cells 4 can be tuned towards their combined cytotoxic and antimetastatic activity by a selection of 5 6 appropriate substituents in the polypyridyl ligands. 7 R 8 9 10 R 11 R 12 R Me Me 13 N N 14 N N N N Ru2+ Ru2+ 15 N N N N 16 t N N 17 R p N O N O 18 N R N r i 19 R Ru1,R=H N H NH 2 N H NH 2 c 20 Ru2,R=Me Ru4,R=H s 21 Ru3,R=t-Bu Ru5,R=SO -,Na+ R 3 u 22 n 23 Figure 1. The studied ruthenium complexes. 24 a 25 M 26 Results and discussion 27 d 28 e 29 Synthesis and photophysical properties t 30 The target complexes Ru1 to Ru5 were prepared in acceptable yields by reacting p 31 e 32 semicarbazone (SC)-based ligand L1, prepared according to the published reference[23], with c 33 the appropriate Ru(NN) Cl complex in absolute ethanol under reflux (Scheme 1). The 2 2 c 34 Ru(NN) Cl complexes were prepared from ruthenium(III) chloride and the proper ligand in 35 2 2 A DMF under microwave irradiation. Interestingly, the coordination of ruthenium occurred 36 37 selectively on the bipyridine side of ligand L1 and no trace of ruthenium coordination by the s 38 c SC moiety was observed. 39 i R m 40 NH 41 2 o 42 O 43 NH R N l l 44 N a N Cl 45 t Ru e 46 N M 47 N Cl 48 N R 49 50 51 Me Ru1,45%9 R 52 Ru2,79% Ru(NN) Cl 53 2 2 Ru3,76% 54 N N EtOH,Ar, Ru4,47%9 55 reflux,22h Ru5,42% 56 L1 57 Scheme 1. Synthesis of complexes Ru1 to Ru5. 58 59 60 3 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Page 5 of 19 Metallomics 1 2 3 Despite all our efforts, the growing suitable crystals for Ru2, Ru3 and Ru5 was 4 unsuccessful and thus no X-Ray diffraction-based structure of the complexes could be 5 6 obtained. Thus, we focused on quantum chemical calculations within DFT approach for the 7 structural determination of the newly synthesized compounds as well as the characterization 8 of their frontier orbitals. Structural data are given in Supplementary Information while plots 9 of HOMO and LUMO of all studied compounds are given in Fig. S1.[23] In Ru1, Ru2, and 10 11 Ru3 compounds, the HOMO spanned over the L1 ligand, whereas LUMO was located on the 12 bpy ligands. Similarly, for Ru4, HOMO is located on the L1 moieties, while LUMO on the 13 bpy residue of the L1 ligand. In Ru5, HOMO is located on one of the SO - groups and LUMO 14 3 15 on the bpy of the L1 moiety. 16 t All the studied Ru(II) complexes exhibited broad and intense absorption bands in the p 17 visible region of the spectrum (400-500 nm), which is predominated by metal-to-ligand 18 i r 19 charge transfer (1MLCT) character. Additionally, the studied ruthenium complexes exhibited c 20 a very intense absorption band in the UV region of the spectrum (250-300 nm). These bands s 21 were assigned to the intraligand  → * transition. Electronic absorption spectra of the u 22 n 23 studied complexes are presented in Fig. S3 and the main absorption bands wavenumbers with 24 a molar absorption coefficients are collected in Table 1. The Time-Dependent DFT (TD-DFT) 25 M calculations confirmed that the most intensive bands in the visible part of the spectrum show 26 27 the MLCT character, and that they can be attributed to the excitation from a mixture of Ru d d 28 orbitals (mixed to a low extent with  orbitals of –SC-py fragment of L1 ligand) to the * of e 29 bpy fragments of all ligands (for the schematic representation of the most intense absorption t 30 p 31 bands and their assigment as computed with TD-DFT see Fig. S2) e 32 The studied Ru(II) complexes showed luminescence after excitation into the MLCT c 33 band and the emission bands are observed in the 610 - 640 nm range (Fig. S3, Tab. 1). In c 34 35 comparison to the excitation wavelength, a significant red shift of the emission energies (ca. A 36 200 nm) is observed along with a quite high quantum yield and relatively long emission 37 s lifetime (Table 1). This should make them perfectly detectable by fluorescent microscopy 38 c 39 when uptaken by cells. i m 40 41 o 42 Table 1. Photophysical data for all studied Ru(II) complexes in air-equilibrated and l 43 l deoxygenated aqueous solutions. Absorption band maxima (), molar extinction coefficients a 44 45 (ε) emission wavelengths ( ) and quantum yields ( ) were measured at 25 °C while t em em e 46 emission lifetimes ( ) at room temperature. M 47 em 48  𝜋→𝜋∗ [𝑛𝑚]  MLCT [nm]  em  em [%]  em [µs] 49 (  103[M-1cm-1]) (  103[M-1cm-1]) [nm] Air Air 50 (Anaerobic) (Anaerobic) 51 52 Ru1 286 (108.6 ± 0.4)a,b 427 (12.3 ± 1.8)a,b 617a 0.92 ± 0.09a 0.27 ± 0.03a 53 455 (14.8 ± 2.1)a,b (1.62 ± 0.02)a (0.36 ± 0.06)a 54 Ru2 286 (107.9 ± 0.3) 431 (11.6 ± 1.1) 626 0.68 ± 0.01 0.26 ± 0.02 55 56 459 (13.5 ± 1.3) (1.16 ± 0.03) (0.36 ± 0.04) 57 Ru3 286 (91.2 ± 0.5) 431 (9.6 ± 0.8) 624 0.74 ± 0.04 0.29 ± 0.05 58 458 (11.0 ± 0.9) (1.03 ± 0.02) (0.39 ± 0.02) 59 60 Ru4 279 (204.8 ± 0.8)a,b 435 (21.8 ± 1.7)a,b 623a 1.18 ± 0.03a 0.65 ± 0.03a 4 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 6 of 19 1 2 3 457 (22.2 ± 1.6)a,b (2.62 ± 0.04)a (1.72 ± 0.07)a 4 Ru5 279 (159.0 ± 0.9) 440 (18.0 ± 0.2) 618 2.21 ± 0.13 0.86 ± 0.04 5 6 464 (17.4 ± 0.2) (8.10 ± 0.09) (2.90 ± 0.12) 7 a taken from ref. [23], b the absorption coefficient were corrected considering hygroscopic 8 properties of compounds 9 10 11 Cytotoxicity and uptake 12 The viability of human lung adenocarcinoma cells (A549) was inhibited by the studied 13 14 Ru(II) complexes in a dose-dependent manner. The measured IC 50 values are listed in Table 15 2. The parent compound with two 2,2’-bipyridyl (bpy) ligands (Ru1) exhibited a low 16 t cytotoxicity, while the introduction of tert-butyl groups on the pyridine rings (Ru3) p 17 18 dramatically increased complex’s potency in inhibition of the cell growth. Ru4, which has i r 19 two 4,7-diphenyl-1,10-phenanthroline (dip) ligands, was the most cytotoxic compound. c 20 Further modification of phenyl rings in dip with sulfonate groups (Ru5) led to a change in the s 21 u 22 overall charge of Ru(II) complex from positive to negative and resulted in a substantial n 23 decrease in cytotoxicity. Our previous studies showed that the cytotoxicity of L1 was at the 24 a same level as Ru1.[20] Under the tested conditions all Ru(II) complexes were more cytotoxic 25 M than cisplatin (IC 1630 ± 60 µM). Another cancer cell line (human pancreas carcinoma cells 26 50 27 (PANC-1) was even more sensitive towards the studied Ru(II) complexes than A549 cells d 28 (see Table S1). e 29 t 30 p 31 e 32 Table. 2. Cytotoxicity (IC ) and uptake ([Ru ]/[Ru ])a of the studied Ru(II) complexes 50 cell medium c 33 evaluated for A549 cells along with determined lipophilicity (logP )b. c 34 o/w 35 IC /µM [Ru ]/[Ru ]a logP b A 50 cell medium o/w 36 Ru1 >240 ~2 -1.54 ± 0.08 37 s 38 Ru2 158 ± 15 ~18 -0.86 ± 0.02 c 39 i Ru3 14.1 ± 0.3 ~90 -0.11 ± 0.02 m 40 41 Ru4 10.7 ± 0.7 ~85 0.36 ± 0.01 o 42 Ru5 >240 ~15 -0.90± 0.10 l 43 l a 44 45 a The average value was obtained after the calculation the ratio between [Ru] in cell t e 46 (determined by ICP-MS) and [Ru] in cell medium used for cell treatment independently M 47 determined for three concentrations: 3.5, 7 and 14 µM. 48 49 blogP , where P is 1-octanol/water partition coefficient o/w o/w 50 51 The cytotoxicity of the studied Ru(II) complexes was found to be strongly dependent 52 53 on their propensity to accumulate in the cells. The cell uptake of all the studied complexes 54 was dose-dependent (Fig. 2). The direct quantification of the ruthenium concentration inside 55 live A549 cells using ICP-MS revealed an equally excellent uptake of Ru3 and Ru4, which 56 57 was manifested in an approximately 90-fold or 85-fold increase in the concentration of 58 ruthenium accumulated in cells in relation to the concentration in the cell medium for Ru3 59 and Ru4, respectively (Table 1 and Fig. 2A). Cytotoxicity and uptake of the Ru complexes 60 5 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A are positively correlated with their lipophilicity (Pearson’s r are 0.971 and 0.936, respectively; p< 0.05, Fig. S5). Such correlation between lipophilicity and IC has been already reported 50 for several Ru complexes.[24, 25] Ru(II) complexes with the highest values of logP (0.36 o/w for Ru4 and -0.11 for Ru3) showed the highest uptake and the lowest IC values. On the 50 other hand, Ru1 with the lowest logP value exhibited poor accumulation and cytotoxic o/w parameters. Even though the lipophilicity of Ru4 was much higher than that of Ru3 its uptake and cytotoxicity were at a similar level pointing out that the structure is an another critical factor affecting the accumulation. Ru1, Ru2, Ru3 and Ru4 are +2 charged, so they enter the cells most probably via a passive diffusion facilitated by the membrane potential difference.[14] Ru5 has a similar lipophilicity and uptake values as Ru2 despite the overall charge 2, indicating that its accumulation goes either via energy-dependent pathway (endocytosis) or that its charge is at least partially compensated during accumulation with a suitable counter ion. Despite similar accumulation levels, Ru2 and Ru5 exhibited different cytotoxicity values, further confirming the relevance of structure on the biological activity of Ru complexes. All studied Ru(II) complexes were luminescent, so their cellular uptake was followed using flow cytometry assuming that the emitted light by the Ru treated cells was proportional to the amount of the accumulated Ru(II) complexes (Fig. 2B). Since the luminescence quantum yield for these compounds is different (see Table 1) and strongly influenced by the environment[24] their concentration cannot be compared between each other but only separately for each of them. It is a fast and simple method for checking the internalization of luminescent Ru(II) complexes. 1000 800 600 400 200 0 3.5 7 14 [Ru ][M] medium 6 ]M[]llecuR[ Page 7 of 19 Metallomics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 24 a 25 M 26 27 d 28 e 29 t 30 p 31 e 32 c 33 c 34 35 A 36 37 A Ru1 s 38 Ru2 c 39 Ru3 i 40 Ru4 m Ru5 41 o 42 l 43 l a 44 45 t e 46 M 47 48 49 50 51 52 53 54 55 56 57 58 59 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A 1400 1200 1000 800 600 400 200 18 16 14 12 10 8 6 4 2 0 0 1.75 3.5 7 14 [Ru ][M] medium 7 ].u.a[ lortnoc .sv esaercni ecnecsenimuL Metallomics Page 8 of 19 1 2 3 B Ru1 4 Ru2 5 Ru3 6 Ru4 7 Ru5 8 9 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 Fig. 2. Ruthenium accumulation in A549 cells determined after 24 h incubation with Ru1 24 a 25 (green), Ru2 (red), Ru3 (blue), Ru4 (purple) and Ru5 (orange) presented as A) a M 26 concentration in a single cell (obtained from ICP-MS measurements) or B) luminescence 27 d increase vs. control (determined by flow cytometry, λ = 488 nm, λ = 700 nm). 28 ex em e 29 t 30 Impact of Ru complexes on cell adhesion properties p 31 e 32 33 To check the changes in adhesion properties of A459 and PANC-1 cells upon c c 34 exposure to the Ru(II) complexes their sensitivity to trypsin was evaluated. Cells, after 24 h of 35 A incubation with the tested compounds, were exposed to a diluted trypsin solution for a short 36 37 time to avoid cells damage, and the amount of remaining trypsin-resistant cells was quantified s 38 using a resazurin assay. As shown in Figs. 3 and S6 all studied Ru(II) complexes significantly c 39 i decreased the cells susceptibility to detachment by trypsin, on both plastic and collagen m 40 surfaces, and the effect was more pronounced for plastic. The observed effect was not 41 o 42 concentration dependent, however, it can be directly correlated to the uptake, cytotoxicity and l 43 lipophilicity of the studied compounds. Ru4 had the strongest impact on cells adhesion. It is a l a 44 45 result of a synergistic effect of the hydrophobic, bulky dip ligands and quite elongated L1 t e 46 ligand with its hydrophilic end. Neither L1 ligand alone nor [Ru(dip) (bpy)]2+ induced such a 2 M 47 strong resistance against trypsin (Fig. 3). The in vivo studies proved that trypsin-sensitive 48 subpopulation of cancer cells generated more metastases than the trypsin-resistant 49 50 subpopulation.[26] Therefore, by decreasing the ability of cancer cell to detach using Ru(II) 51 treatment we may block cancer cells ability to leave the primary site and in this way decrease 52 their metastatic potential. Recently, it was shown that genistein (derivative of isoflavone), can 53 54 inhibit cell detachment by increasing cell adhesion in vitro and in vivo as well as in human 55 clinical trials. Together with its inhibitory effect on protease production and cellular invasion 56 made this compound a potent anti-metastatic agent.[27] 57 58 59 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A 600 500 400 300 200 100 0 1 2 4 8 8 ]%[ sllec tnerehdA Ru1 A Ru2 Ru3 Ru4 Ru5 [Ru(dip)(bpy)]2+ 2 L1 [Ru ][M] medium 600 500 400 300 200 100 0 1 2 4 8 ]%[ sllec tnerehdA Page 9 of 19 Metallomics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 24 B Ru1 a Ru2 25 Ru3 M Ru4 26 Ru5 27 [Ru(dip) 2 (bpy)]2+ d 28 L1 e 29 t 30 p 31 e 32 c 33 c 34 35 A 36 37 s 38 c 39 i [Ru ][M] m 40 medium 41 Fig. 3. A549 cell adherence to A) plastic or B) collagen coated surface evaluated as the o 42 l 43 percentage of remained adherent cells upon controlled trypsin treatment. Cells were treated l a 44 for 24 h with Ru1 (green), Ru2 (red), Ru3 (blue), Ru4 (purple), Ru5 (orange), 45 t [Ru(dip) (bpy)]2+ (pink) and L1 (grey). Untreated cells were used as control (100%). e 46 2 M 47 48 Change in the activity of matrix proteinases 49 50 51 Inhibition of matrix metalloproteinases (MMPs) suppresses the invasive potential of 52 tumors. MMPs take part in physical barriers breakdown, which is a crucial step in tumor 53 migration. They are involved in among others the degradation and remodeling of basement 54 55 membrane and extracellular matrix (ECM) as well as cell adhesion properties.[28, 29] To 56 determine if the observed changes in cells adhesion might arise from Ru complexes effects on 57 MMPs, their cellular enzymatic activity was monitored using a fluorogenic substrate FS-6 58 59 (Mca–Lys–Pro–Leu–Gly–Leu–Dpa–Ala–Arg–NH 2 ; Mca = methoxycoumarin-4-acetyl). 60 Some of MMPs, are only partly released into a soluble form whereas, the majority remains .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A attached to the cell surface. Furthermore, the six-membrane types of MMPs (MMP-14, -15, - 16, -17, -24, and -25) are not released at all.[6] To determine the activity of dissolved MMPs the hydrolysis of FS-6 was measured in a cell-free conditioned medium from the A549 cells (supernatant), while cell-associated MMPs activity was investigated by addition of FS-6 into the A549 cell culture immediately after removing the supernatant. Proteolysis of FS-6 was also measured in supernatants and on the cell surface in the presence of the broad-spectrum MMPs inhibitor GM6001 (I1) and the serine protease inhibitor AEBSF (I2) as positive controls. As shown in Fig. 4 Ru3 and Ru4 exhibited the strongest inhibition of both, cell associated and dissolved MMPs in A549 cells, regardless of the applied concentration. Both of them are as potent as GM6001, which inhibition constant for several MMPs (MMP-1, 2, 3, 8 and 9) was in the low nanomolar range.[4] Ru1 also inhibited MMPs activity but only at higher concentrations (from 4 to 8 µM). No significant influence was observed for Ru2 and Ru5. Inhibition of MMPs in vitro by the studied Ru complexes correlates well with their impact on cells adhesion. 2.5 2.0 1.5 1.0 0.5 0.0 1248 1248 1248 1248 1248 9 ].u.a[ lortnoc .sv ytisnetni ecnecseroulF A Ru1 Ru2 Ru3 Ru4 Ru5 I1 I2 2.5 2.0 1.5 1.0 0.5 0.0 1 2 4 8 1 2 4 8 1 2 4 8 1 2 4 8 1 2 4 8 I1 I2 ].u.a[ lortnoc .sv ytisnetni ecnecseroulF Metallomics Page 10 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 24 a 25 M 26 27 d 28 e 29 t 30 p 31 e 32 c 33 c 34 35 A 36 37 s 38 c 39 i m 40 41 o 42 l 43 B l a 44 45 t e 46 M 47 48 49 50 51 52 53 54 55 56 57 58 Ru1 Ru2 Ru3 Ru4 Ru5 59 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Fig. 4. Hydrolysis of FS-6 (substrate for MMPs) by A549 cells A) in situ and B) by cell-free supernatant after 24 h incubation with various concentrations of Ru complexes (1, 2, 4 and 8 µM) versus control. MMPs inhibitor GM6001 (I1, 10 µM) and serine protease inhibitor AEBSF (I2, 1mM) were used as positive control. Bars represent mean and SD from triplicate experiments. A549 cells, are known to produce MMP-2 and MMP-9 [2, 7] that are key ECM-degrading enzymes in lung cancer cells. It was shown that inhibition of MMP-2 led to a decrease in tumor growth and invasiveness, so MMP-2 is an interesting target for antimetastatic therapy.[30] In turn, MMP-9 was recognized as an important enzyme involved in the formation of the metastatic niche[31], related to among others its ability to liberate vascular endothelial growth factor and thereby supporting angiogenesis.[32] To closer examine the observed inhibitory effect of Ru3 and Ru4 on MMPs in vitro, their impact on enzyme activity was evaluated on commercially available MMP2 and MMP9 enzymes (see Table S2). A potent broad-spectrum hydroxamate metalloproteinase inhibitor, GM6001, was used for comparison.[33] Ru4 was highly potent with IC as low as 0.4 and 1.8 µM for MMP2 and 50 MMP9, respectively (Fig. 5). Ru3 was ca. 25 times more potent in inhibition of MMP2 over MMP9 while Ru4 had a similar effect on both enzymes. The obtained data suggest that the inhibition of MMPs might be engaged in alteration of adhesion properties by preventing the degradation of surface adhesion receptors. The detailed mechanism by which the studied Ru complexes interact with MMPs is currently under investigation, there is also a lack of data in the literature concerning applying Ru complexes as MMPs inhibitors. 1.0 Ru4 Ru3 0.8 GM6001 1.7 0.6 34 0.4 0.2 0.0 0.1 1 10 100 10 0v/iv A MMP2 MMP-9 1.0 Ru4 Ru3 0.8 GM6001 17 0.6 1.8 0.4 0.4 0.6 0.2 0.0 0.1 1 10 100 Inhibitor [M] 0v/iv Page 11 of 19 Metallomics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 24 a 25 M 26 27 d 28 e 29 t 30 p 31 e 32 c 33 c 34 B 35 A 36 37 s 38 c 39 i m 40 41 o 42 l 43 l a 44 45 t e 46 M 47 Inhibitor [M] 48 Fig. 5. IC values for inhibition of MMP-2 (A) and MMP-9 (B) by Ru3 and Ru4 as well as 49 50 50 the reference metalloproteinase inhibitor GM6001. Experimental conditions: [FS-6] = 2.5 51 µM, [enzyme] = 0.5 nM, 0.1M Tris, pH 7.4 at 37 ºC (MMP-9 was activated using 1 mM 52 APMA, 4ºC, overnight and buffer was supplemented with 0.1M NaCl, 10mM CaCl , 0.1mM 53 2 54 ZnCl and 0.05% Brij35). Bars represent mean and SD from triplicate experiments. 2 55 56 Cellular responses upon treatment with Ru(II) complexes 57 58 To get better insight into changes induced by the treatment with Ru(II) complexes, 59 several parameters were evaluated for A549 cells. The level of oxidative stress induced by the 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 12 of 19 1 2 3 studied Ru(II) complexes was evaluated by using various fluorescent probes (Fig. S7). 4 Treatment of A549 cells with Ru4 caused a pronounced increase in general ROS production, 5 6 in particular pointing to the formation of H 2 O 2 (see the explanation in supplementary 7 material) as well as singlet oxygen and superoxide anion radical formation in a dose- 8 dependent manner (Fig. S7). The high level of oxidative stress induced by Ru4 correlates 9 with its high cytotoxicity. The cell death in this case might be related to the huge ROS 10 11 formation efficiency.[23] For Ru3, which has its toxicity and uptake similar to Ru4, only a 12 slight elevation in 1O formation was determined which pointed out a different mechanism in 13 2 cell death induction. Treatment of cells with Ru2 also caused a small increase in H O and 14 2 2 15 singlet oxygen production. Ru1 did not cause any significant ROS production most probably 16 t due to its poor accumulation. p 17 Changes in mitochondrial membrane potential caused by the treatment of A549 cells 18 i r 19 with Ru(II) complexes were evaluated using JC-1 probe (Fig. S8). A marked decrease in the c 20 mitochondrial membrane potential was determined only for cells incubated with Ru3 while s 21 other compounds (Ru2, Ru4, Ru5) had a tendency to slightly increase it. Similar disruption u 22 n 23 in the mitochondrial transmembrane potential observed for Ru3 (regardless of the applied 24 a concentration in the range of 2.2-9.0 µM) as for both highly toxic positive controls 25 M gramicidin[20] (10 µM) and valinomycin (20 µM) suggests that this feature of Ru3 might be 26 27 responsible for its high cytotoxicity. d 28 Cytosolic calcium concentration evaluated using Fluo-8 AM probe was altered only in e 29 the presence of Ru3 and Ru4 complexes in an opposite way (Fig. S9). Ru3 greatly increased t 30 p 31 it while Ru4 decreased it. This finding further supports different mechanisms of the induced e 32 cytotoxicity by both complexes proposed based on previously discussed induced oxidative c 33 stress and changes in the mitochondrial membrane potential. c 34 35 A 36 Cellular re-adhesion properties – preliminary studies 37 s Tumor cells, which have survived the treatment with cytotoxic drugs, managed to 38 c 39 detach from the primary tumor mass, invade ECM to migrate towards blood vessels and i m 40 finally succeeded in entring into a blood vessel can circulate until they are able to attach to a 41 vessel wall and start extravasation. The attachment of cancer cells to new places is crucial in o 42 43 starting proliferation, which is the beginning of a secondary growth. Thus, the effect of the l l a 44 treatment of A549 cells with non-toxic dose of the studied Ru(II) complexes on anti-adhesion 45 t properties of cancer cells was examined by evaluating the ability of the treated cells to re- e 46 47 adhere to plastic surface. The decrease in re-adhesion of A549 cells to a plastic surface, as M 48 shown in Fig.6, occurred for cells treated with Ru2, Ru3, Ru4 and Ru5, usually at higher 49 concentrations. The most significant inhibition of cell adhesion was observed for Ru4 and this 50 effect became gradually larger as the concentration increased. A similar effect was observed 51 52 for PANC-1 cell line (Fig. S10). Similar results were obtained for parent compound 53 [Ru(dip) (bpy)]2+ (Fig. 6) as well as for compound, in which bpy was modified with a 54 2 nitroimidazole unit (4-[3-(2-nitro-1H-imidazol-1-yl)propyl]).[14] The results are very 55 56 promising, however to evaluate the relevance of this property, further studies using the in 57 vitro flow adhesion assay on endothelial cells are planned to be performed. 58 59 60 11 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A 160 140 120 100 80 60 40 20 0 1 2 4 8 [Ru ][M] medium 12 ]%[ sllec tnerehdA Page 13 of 19 Metallomics 1 2 3 4 5 Ru1 6 Ru2 7 Ru3 Ru4 8 Ru5 9 [Ru(dip) 2 (bpy)]2+ 10 11 12 13 14 15 16 t p 17 18 i r 19 c 20 s 21 u 22 n 23 24 Fig 6. Influence of Ru1 (green), Ru2 (red), Ru3 (blue), Ru4 (purple), Ru5 (orange) and a 25 [Ru(dip) (bpy)]2+ (pink) on A549 cells’ ability to re-adhere to plastic surface, measured after M 2 26 24 h incubation of cells with Ru(II) complexes. 27 d 28 e 29 Conclusions t 30 p In conclusion, among the studied series of Ru(II) polypyridyl complexes two of them Ru3 31 e 32 and Ru4 demonstrated very interesting biological properties. They exhibited high cytotoxicity c 33 against studied cancer cells that correlated very well with their excellent uptake by cells. In c 34 addition, both of them strongly influenced the adhesion properties of cells. The strengthening 35 A 36 of cells’ adherent properties upon treatment with Ru3 and Ru4 along with inhibition of 37 released and membrane-bound MMPs was observed. Prevention of the detachment of tumor s 38 c cells from cancer tissue, reduces the chance of metastatic cells to escape to the surrounding 39 i 40 tissue. Therefore, these types of compounds might be beneficial not only in the treatment of m 41 solid tumors in standard chemotherapy as cytotoxic agents but also in inhibition of metastasis o 42 development. However, due to their high toxicity the use of drug delivery systems to increase l 43 l 44 selectivity towards cancer cells for in vivo application is necessary. In addition, preliminary a 45 studies showed that cells pre-treated with non-toxic doses of the studied compounds had t e 46 lower ability to re-adhere to new place after detachment. This effect may contribute to a M 47 48 decrease in their ability for extravasation (needed to complete metastatic colonization), which 49 requires arresting of circulated cells that is supported by specific adhesive interactions. 50 We anticipate that our findings will draw the attention of scientists to unrecognized, 51 underrated properties of polypyridyl Ru(II) complexes towards their application as 52 53 antimetastatic agents. Still, there is a huge knowledge gap not only in the experimental work 54 demonstrating such properties but particularly in the understanding of the molecular 55 foundations of the observed effects, that will provide a mechanistic rationale for designing 56 57 new chemotherapeutics. 58 59 Experimental 60 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 14 of 19 1 2 3 Synthesis and photophysical characterization 4 All solvents were of analytical grade and were used without further purification. Reagents 5 6 were purchased from Sigma-Aldrich. Ru1, Ru4, and ligand L1 were obtained according to 7 the published procedures.[20] 8 Synthesis of studied complexes – general procedure 9 A solution of L1 (0.06 mmol) in absolute ethanol (3 mL) was added to a solution of cis- 10 11 Ru(NN) Cl (0.05 mmol) in absolute ethanol (5 mL). The resulting mixture was refluxed 2 2 12 under argon atmosphere for 16h. After that time, solvent was removed under reduced 13 pressure, and red-orange residue was dissolved in water (10 mL) and filtrated. Filtrate was 14 15 evaporated until dry, dissolved in a small amount of dichloromethane with a few drops of 16 t methanol and precipitated out with diethyl ether. Orange powder was washed with diethyl p 17 ether and dried to give the pure product. 18 i r 19 Synthesis of Ru2 c 20 NN = 4,4’-dimethyl-2,2’-bipyridine, yield 79% (36 mg); 1H NMR (600 MHz, DMSO-d ) 2.46 s 6 21 (d, J=6Hz, ~15H, superimposed with solvent signal), 2.94 (t, J=7.2 Hz, 2H); 3.07 (t, J=7.2 Hz, u 22 n 23 2H); 6.64 (brs, 2H); 7.19-7.24 (m, 1H); 7.27-7.30 (m, 1H); 7.32-7.34 (m, 4H); 7.43 (d, J=11.4 24 a Hz,1H); 7.46 (d, J=6.6 Hz, 1H); 7.50-7.52 (m, 3H); 7.65 (dd, J=8.4 and1.8 Hz, 1H); 8.09 (d, 25 M J=8.4 Hz, 1H); 8.31 (s, 1H); 8.68-8.73 (m, 6H); 10.59 (s, 1H). HRMS calculated for 26 27 C H N ORuCl m/z = 427.1366 (M2+ -2Cl), found m/z = 427.1370 (M+ -2Cl). 46 44 10 2 d 28 Synthesis of Ru3 e 29 NN = 4,4’-di(tert-butyl) -2,2’-bipyridine, yield 76% (33 mg); 1H NMR (600 MHz, DMSO-d ) t 30 6 p 31 1.32 - 1.37 (m, 36H); 2.51 (s, ˜3H superimposed with solvent signal); 2.69 (t, J=6.0 Hz, 2H); e 32 3.09 (t, J=6.0 Hz, 2H); 7.38 (dd, J=6.0 and2.4 Hz,2H); 7.46-7.58 (m, 10H); 7.63 (d, J=24 Hz, c 33 1H); 7.90 (dd, J=8.4 and1.8 Hz, 1H); 8.48 (s,1H); 8.78-8.89 (m, 6H); 10.61 (s, 1H); 12.73 (s, c 34 35 1H). HRMS calculated for C H N ORuCl m/z = 1021.4537 (M2+-2Cl -H+), found m/z = A 58 68 10 2 36 1021.4468 (M2+-2Cl -H+) 37 s Synthesis of Ru5 38 c 39 NN = : 4,7-Diphenyl-1,10-phenanthroline-disulfonic acid disodium salt trihydrate, Product i m 40 was purified by flash chromatography (neutral Al O , dichloromethane/methanol 5-10% 2 3 41 (gradient). Yield 42% (22 mg); 1H NMR (600 MHz, DMSO-d ): (ppm) 2.52 (s, superimposed o 42 6 43 with solvent signal); 2.93-2.99 (m, 2H); 3.06-3.11 (m, 2H); 7.58-7.69 (m, 16H); 7.78-7.83 (m, l l a 44 10H); 7.89 (s, 4H); 8.18 (s, 2H); 8.23-8.31 (m, 3H); 8.35 (s, 1H); 8.37 (s, 2H). HRMS: 45 t calculated for C70H48N10O13S4RuNa4 m/z = 779.0454(M2+), found m/z = 779.0450 (M2+) e 46 47 Spectroscopic measurements were performed according to previously published procedures M 48 and its overview is given in the supplementary information along with the details for 49 computational characterization and other photophysical measurements.[23] 50 51 52 Cell culturing and cytotoxicity assay 53 Human lung adenocarcinoma A549 and human pancreas carcinoma PANC-1 cell were 54 routinely cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (v/v) 55 56 and 1% penicillin-streptomycin solution (100 units/ml-100µg/ml) (v/v), at 37°C in humidified 57 atmosphere with 5% CO (v/v). Cell viability upon treatment with Ru(II) complexes was 2 58 determined using the MTT assay. Cells were seeded into 96-well plate with the density of 3  59 60 104 cells per cm2 in complete medium and cultured for 24 h. Then the medium was removed 13 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Page 15 of 19 Metallomics 1 2 3 and various concentrations of Ru(II) complexes in basic medium (without FBS) were added to 4 the wells. Stock solutions of the Ru(II) complexes were prepared in DMSO. The final DMSO 5 6 concentration in a cell culture was fixed (0.1% (v/v) for Ru3 and Ru4 and 0.5% (v/v) for Ru1, 7 Ru2 and Ru5). After 24 h of incubation cells were washed with PBS and MTT solution (0.5 8 mg/ml, 100 µL) was added to each well. Cells were incubated for 3 h at 37°C. Next, MTT 9 was removed and the formed violet formazan crystals were dissolved in a 100 µL of 10 11 DMSO:methanol (1:1) mixture. The absorbance was measured using Tecan Infinite 200 12 microplate reader at 565 nm with 700 nm as a reference wavelength. Experiments were 13 performed in triplicates and repeated three times. Results are presented as mean values and 14 15 standard error of the mean. IC parameters were determined using the Hill equation 50 16 t (OriginPro 2018). p 17 18 i r 19 Cellular uptake of Ru compounds c 20 Cellular uptake of Ru complexes was determined using A549 cell line. A549 cells were s 21 seeded in a 6-well plate with the density of 4  104 cells per cm2 in complete medium and u 22 n 23 cultured for 24 h. Then the medium was removed and cells were treated with various 24 a concentrations of Ru(II) complexes in basic medium for 24 h. In order to correctly measure 25 M accumulation of Ru complexes only non-toxic concentrations were used. Afterwards the 26 27 incubated cells were washed, detached by trypsin treatment and counted. Additional attention d 28 was paid to eliminate dead cells by washing cells’ monolayer with PBS. The cells were e 29 isolated by centrifugation and digested in concentrated nitric acid overnight at room t 30 p 31 temperature. The solutions were diluted with Millipore water to a final nitric acid e 32 concentration of 10%. The Ru content in the samples was measured by inductively coupled c 33 plasma mass spectrometry (ICP-MS). Experiments were repeated three times. Results were c 34 35 calculated as ruthenium concentration per cell and presented as a mean values and standard A 36 deviation of mean. Additionally, after detachment by trypsin cells were suspended in PBS 37 s 38 buffer and analysed by BD FACSVerse cytometer (λ ex = 488 nm and λ em = 700 ± 27 nm). c 39 Experiment was performed twice and the mean values and standard error of the mean are i m 40 presented. 41 o 42 l 43 Trypsin resistance assay l a 44 The ability of A549 and PANC-1 cells to detach from the surface after their exposure to 45 t Ru(II) complexes was evaluated by checking their resistance to trypsin treatment. Cells were e 46 47 seeded into a 96-well plate on the uncoated bottom of plastic wells or on collagen coated M 48 surfaces with the density of 3  104 cells per cm2 in a complete medium and cultured for 24h. 49 Then the complete medium was removed and various concentrations of the studied complexes 50 51 were added to the wells for a 24h incubation. Afterwards the cells were washed and 30 µL of 52 a trypsin solution (0.05%) was added to each well and incubated for 10 min at 37°C. Then the 53 cells were washed with PBS and a resazurin assay was performed to quantify the adherent 54 55 cells according to the procedure published by us.[23] The obtained results were normalized 56 with respect to corresponding wells without the trypsin treatment to exclude the possible 57 toxicity of the studied compounds and are presented as a percentage of untreated cells. 58 59 Experiments were performed in triplicates and each experiment was repeated five times to 60 obtain mean values and the standard error of the mean. 14 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Metallomics Page 16 of 19 1 2 3 4 Activity of matrix proteinases assays 5 6 In vitro MMPs’ inhibition assays 7 The MMP enzymatic activity was quantitated using FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa- 8 Ala-Arg-NH2) fluorogenic substrate (Sigma-Aldrich). Cells were seeded into a 96-well plate 9 10 with the density of 3104 cells per cm2 in a complete medium and cultured for 24 h. Then the 11 medium was removed and various concentrations of the studied Ru complexes were added for 12 24 h incubation. To determine the soluble MMP activity, supernatant (80 µL) was transferred 13 14 to a black 96-well plate, FS-6 (final concentration 5 µM) was added to each well and 15 incubated for 30 min. The activity of cell membrane-associated MMP was measured using 16 t Ru(II) treated cells by adding FS-6 solution (final concentration 5 µM) directly to the cells p 17 18 and incubating for 30 min. After that, 80 µL of the obtained solution were transferred to a i r 19 black 96-well plate. Next the enzymatic reactions were terminated by the addition of 90 µL of c 20 3% (w/v) sodium acetate. Fluorescence of the samples was detected at 400 nm using a 345 nm s 21 excitation wavelength (Tecan Infinite 200 microplate reader). MMP inhibitor GM6001 (10 u 22 n 23 µM) and serine protease inhibitor AEBSF (1 mM) incubated with the cells for 4 h were used 24 a as a positive control. Experiments were performed in triplicates and results are presented as a 25 M mean values and standard error of the mean. 26 27 d 28 MMP-2 and MMP-9 inhibition e 29 Inhibitor potency of Ru3, Ru4 and GM6001 (as a reference) against human MMP-2 and t 30 p 31 MMP-9 was studied using the fluorogenic substrate FS-6. MMP-2 and MMP-9 enzymes were e 32 obtained from Sigma-Aldrich. MMP-9 was activated using APMA (1 mM, 4 ºC, overnight) c 33 prior to the experiments. MMP-2 assays were performed in 0.1 M Tris/HCl (pH 7.4, 37 ºC), c 34 35 while for the MMP-9 evaluation Tris/HCl buffer was additionally enriched with 0.1 M NaCl, A 36 10 mM CaCl , 0.1 mM ZnCl and 0.05% Brij35. The final concentrations of enzyme and 37 2 2 s substrate were kept constant at 0.5 nM and 2.5 µM, respectively. Kinetic fluorescence 38 c 39 measurements were performed using a Perkin Elmer LS55 spectrofluorimeter (λ = i ex/em m 40 325/400 nm). The enzyme activity in the presence of inhibitors was expressed as a fraction of 41 initial reaction rate (v/v ratio, v - initial reaction rate, v – initial reaction rate in the presence o 42 i 0 0 i l 43 of inhibitor). Experiments were repeated three times and results are presented as mean values l a 44 and standard deviation of the mean. IC parameters were calculated from a dose-response 45 50 t plot of enzyme fractional activity as a function of inhibitor concentration, using the Hill e 46 47 equation (OriginPro2018). M 48 49 Re-Adhesion assay 50 51 The effect of the studied complexes on adhesion properties of cancer cells was also examined 52 by evaluating the ability of the treated cells to re-adhere. Cells were seeded into a 6-well plate 53 with the density of 3104 cells per cm2 in complete medium and cultured for 24 h. Then, the 54 55 medium was removed and various concentrations of the studied Ru(II) complexes were added 56 and incubated with the cells for 24 h. Afterwards, the cells were washed, and incubated with a 57 fresh portion of PBS without Mg and Ca ions for 20 min. Then, the cells were detached with a 58 59 cell dissociation solution, counted and seeded into 96-well plates with the density of 6 104 60 cells per cm2. Plates were incubated for 1 h in humidified atmosphere at 37 °C and then were 15 .MP 63:65:2 0202/22/3 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 hcraM 91 no dehsilbuP View Article Online DOI: 10.1039/D0MT00019A Page 17 of 19 Metallomics 1 2 3 washed with PBS to remove non-adherent cells. Resazurin assay was performed to quantify 4 the adherent cells according to the published procedure.[23] Detachment of cells treated with 5 6 Ru4 was not possible by using the cell dissociation solution so instead a trypsin solution 7 (0.05%) was used. Experiments were performed in triplicates and each experiment was 8 repeated five times to calculate mean values and the standard error of the mean. 9 10 11 Influence of Ru(II) complexes on other cellular processes 12 Evaluation of oxidative stress, changes in mitochondrial membrane potential and cytosolic 13 calcium concentration were performed using various fluorescent probes according to the 14 15 manufacturers protocols and full experimental details are given in the supplementary 16 t information. p 17 18 i r 19 Statistical analysis c 20 For in vitro experiments, all data were expressed as the mean ± standard error of the mean s 21 (SEM). Pearson correlation coefficients were calculated using Statistica 13 software. u 22 n 23 Probabilities of p < 0.05 were considered as statistically significant. 24 a 25 M 26 27 Acknowledgments d 28 e 29 M.B. thanks for financial support from National Science Center (DEC- t 30 2016/21/B/NZ7/01081). I.G. acknowledges the support of InterDokMed project no. p 31 POWR.03.02.00-00-I013/16. The research was carried out with equipment purchased with e 32 c 33 financial support from the European Regional Development Fund within the framework of the c 34 Polish Innovation Economy Operational Program (contract no. POIG.0 2.01.00-12-0 23/08). 35 A 36 References 37 s [1] P.S. Steeg, Targeting metastasis, Nat. Rev. Cancer, 16 (2016) 201-218 38 c [2] J. Araya, M. Maruyama, K. Sassa, T. Fujita, R. Hayashi, i.S. Matsu, T. Kashii, N. 39 i 40 Yamashita, E. Sugiyama, i.M. Kobayash, Ionizing radiation enhances matrix m 41 metalloproteinase-2 production in human lung epithelial cells, Am. J. Physiol. Lung Cell. o 42 Mol. Physiol. , 30-38 (2001). l 43 [3] D.-Y. Lu, T.-R. Lu, H.-Y. Wu, New insights into individualized antimetastatic therapy., l a 44 Adv. Tech. Biol. Med. , 1 (2013) 1000106 (1000101-1000103). 45 t [4] R.E. Galardy, M.E. Cassabonne, C. Giese, J.H. Gilbert, F. Lapierre, H. 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