Ru(II)-Naphthoquinone complexes with high selectivity for triple-negative breast cancer.

View Article Online Dalton View Journal Transactions An international journal of inorganic chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: K. Oliveira, E. J. Peterson, M. Carroccia, M. R. Cominetti, V. M. Deflon, N. P. Farrell, A. A. Batista and R. S. Correa, Dalton Trans., 2020, DOI: 10.1039/D0DT01091J. This is an Accepted Manuscript, which has been through the Volume 46 Number 10 Dalton 1 P 4 a g M e a s r c 3 h 0 7 2 3 0 - 1 3 7 412 Royal Society of Chemistry peer review process and has been accepted for publication. Transactions Accepted Manuscripts are published online shortly after acceptance, An international journal of inorganic chemistry rsc.li/dalton 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. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event ISSN 0306-0012 shall the Royal Society of Chemistry be held responsible for any errors CDNan o- O dHuM eg tetlM ea llrs u U o r Wc N eyn .I cy CSllit A ce c Tp act hI ai O o erbn n N e s en ( teX a sH l.t +a, bXil i=z eSd, Spea,r Teen)t sulfenyl, selenenyl, or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/dalton Page 1 of 12 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Ru(II)-naphthoquinone complexes with high selectivity for triple- negative breast cancer t p Katia M. Oliveiraa,f*, Erica J. Petersonbc, Murilo C. Carrocciad, Marcia R. Cominettie, Victor M. Deflond, i Received 00th January 20xx, r Accepted 00th January 20xx Nicholas P. Farrellbc, Alzir A. Batistaa, Rodrigo S. Correaf* c s DOI: 10.1039/x0xx00000x Six new ruthenium(II) complexes with lapachol (Lap) and lawsone (Law) with the general formula [Ru(L)(P-P)(bipy)]PF, 6 u www.rsc.org/ where L = Lap or Law, P-P = 1,2’-bis(diphenylphosphino)ethane (dppe), 1,4’-bis(diphenylphosphino)butane (dppb), 1,1’- n bis(diphenylphosphino)ferrocene (dppf) and bipy = 2,2’-bipyridine, were synthesized, fully characterized by elemental analysis, molar conductivity, NMR, cyclic voltammetry, UV-vis, IR spectroscopies and three of them by X-ray crystallography. a All six complexes were active against breast (MCF-7 and MDA-MB-231) and prostate (DU-145) cancer cell lines with lower M IC values than cisplatin. Complex [Ru(Lap)(dppe)(bipy)]PF (1a) showed significant selectivity for MDA-MB-231, a model of 50 6 triple-negative breast cancer (TNBC), as compared to the “normal-like” human breast epithelial cell line, MCF-10A. Complex d (1a) inhibited TNBC colony formation and induced loss of cellular adhesion. Furthermore, the complex (1a) induced e mitochondrial dysfunction and generation of ROS, what is involved in the apoptotic cell death pathway. Preferential cellular t uptake of complex (1a) was observed in MDA-MB-231 cells compared to MCF-10A cells, consistent with the observed p selectivity for tumorigenic vs non-tumorigenic cells. Taken together, these results indicate that ruthenium complexes e containing lapachol and lawsone as ligand are promising candidates as chemotherapeutic agents. c named as lapachol (2-hydroxy-3-(3-methylbut-2-en-1- c Introduction yl)naphthalene-1,4-dione) and lawsone (2-hydroxy-1,4- A naphthoquinone) are examples of naphthoquinones that present In the search for new anticancer drugs, several promising anticancer properties of the utmost interest9. In the pursuit of s discoveries have used the class of natural products as potent anticancer agents, naphthoquinone derivatives have contributed to n anticancer agents, such as naphthoquinone derivatives1. This well- the design of new active compounds, including those containing known compound class, either synthetic or natural, exhibits a large o metal ions10,11. Recent studies have shown that the combination of variety of biological properties, which include antiallergic2, antiviral3, i metal with biologically active ligands can lead to a successful rational t antibacterial3,4, antiparasitic5–7 and anticancer8,4. Both compounds design of metallodrugs12–16. c Based on the success of cisplatin, platinum has long been the a metal most widely explored for the development of new anticancer s drugs17–19. Unfortunately, side effects have limited the use of some n platinum-based chemotherapeutics20, and to overcome this issue, in a a.Departamento de Química, Universidade Federal de São Carlos - UFSCar, Rodovia recent years a wide variety of metal complexes have been intensively Washington Luiz, KM 235 CP 676, CEP 13561-901, São Carlos - SP, Brazil. r b.Department of Chemistry, Virginia Commonwealth University, Richmond 23284, investigated, such as palladium21, osmium22, titanium23, gold24 and T Virginia – USA. ruthenium25,26, among others. c.The Massey Cancer Center, Virginia Commonwealth University, Richmond 23294, Ruthenium has become the focus of research for metal-based n Virginia – USA. d.Instituto de Química de São Carlos, Universidade de São Paulo, CEP 13566-590, chemotherapeutics because this metal exhibits some special o São Carlos - SP, Brazil characteristics that make its compounds interesting for anticancer e.Departamento de Gerontologia, Universidade Federal de São Carlos, São Carlos – t SP, Brazil. chemotherapy. One of these characteristics is its capacity to form l f.Departamento de Química, ICEB, Universidade Federal de Ouro Preto, CEP 35400- coordination number of six, being able to easily modulate different a 000, Ouro Preto-MG, Brazil. *E-mail: rodricorrea@ufop.edu.br, Tel.: +55 D ligands in its coordination sphere and the number of possible 3135591229 † Electronic Supplementary Information (ESI) available: Crystallographic oxidation states (II, III and possibly IV) that it can assume in Information Files (CIFs) were deposited on the Cambridge Structural database physiological solution27–30. Currently, there are some ruthenium [CCDC 1947785, 1947786 and 1947787 for (1a), (2a) and (2b), respectively]. These data can be obtained free of charge via complexes in clinic phase, such as the KP133929. More recently, the http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Ru(II)-polypyridyl complex, TLD-1433, entered Phase IB clinical trials Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk). Figure S1 to S18 (31P{1H}, 13C{1H} and 1H NMR spectra for to treat non-muscle invasive bladder cancer with photodynamic complexes 1a – 3b). Figure S19 and S20 (31P{1H} spectra of stability study for therapy31. The ruthenium compounds, imidazolium(imidazole)- complexes 1a and 2a). Figure S21 (Effect of the complex 1a on cell cycle (dimethylsulfoxide)tetrachlororuthenate(III) (NAMI-A), indazolium distribution). Table S1-S3 (X-ray data collections and refinement details for complexes 1a, 2a and 2b). trans-tetrachlorobis(1H-indazole)ruthenate(III) (KP1019) and This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 2 of 12 ARTICLE Journal Name (KP1339) (the water-soluble sodium salt), which are well-known for simultaneous inductively coupled plasma mass spectrometer (ICP- their remarkable anticancer properties, were the first ruthenium MS) at 265 nm (Varian Inc.). complexes to entered human clinical investigations32,33. Thus, the Synthesis of the complexes NAMI-A showed success in phase I clinical studies but, unfortunately, Lapachol used in this work was obtained according to the did not advance beyond phase II clinical trials. Afterwards, the procedure described in the literature40. The synthesis of the KP1019 showed solubility limited in phase I and resulted in the failure ruthenium(II) complexes was performed from precursors of general of clinical studies29,34. formula cis-[RuCl (P-P)(bipy)], where P-P = 1,2- In addition, the introduction of organometallic ruthenium(II)- 2 bis(diphenylphosphino)ethane (dppe), 1,4- arene compounds have shown an interesting contribution for the bis(diphenylphosphino)butane (dppb), 1,1’- development of new anticancer drugs. One of these compounds, the t bis(diphenylphosphino)ferrocene (dppf) and bipy = 2,2’-bipyridine, RAPTA-C, is renowned for interesting biological properties in vivo, p which were obtained according to the procedure described in the with impressive antiangiogenic effects35,36. More recently, the study i literature41,42. All the ruthenium complexes with Lap or Law were r of RAPTA-C in combination with erlotinib have shown a strong synthesized using the generalized procedure shown in Scheme 1. The c antitumor efficacy using preclinical tumor models32,37. Lap (0.082 mmol, 20 mg) was dissolved in 50 mL of a mixture of s The coordination of naphthoquinones to ruthenium is an dichloromethane/methanol (1:2 v/v) with 40 μL of triethylamine, the u interesting alternative to obtain new compounds with good cis-[RuCl (P-P)(bipy)] (0.055 mmol) and KPF (0.11 mmol, 20 mg). The pharmacological properties, such as those highlighted in the reports 2 6 n initially red solution became dark purple. The reaction was stirred for of new compounds containing naphthoquinones as ligands, a 12 h, after which its volume was reduced to about 3 mL and exhibiting high cytotoxic activity against cancer cell lines22,38,39. M maintained under stirring until a dark purple precipitate formed. The Therefore, as part of our ongoing effort to gain more insights into the precipitate was filtered off and washed in distilled water, ethyl ether anticancer properties of this class of compounds, a new series of and dried under vacuum. The same procedure was carried out to d ruthenium complexes containing the naphthoquinones lapachol and containing the lawsone complex. It is worth mentioning that the e lawsone were synthesized, characterized and their biological properties investigated against cancer cell lines. compounds containing the dppf were precipitated with water. t p [Ru(Lap)(dppe)(bipy)]PF (1a): Yield: 46.60 mg (65 %). Elemental 6 Materials and methods analysis: found (calculated) (%) for: C H F N O P Ru.C H O: C, e 51 45 6 2 3 3 4 10 59.19 (59.22); H, 5.27 (5.51); N, 2.51 (2.43). Λ = 39.8 Ω−1 cm2 mol−1, c Materials and physical measurements M in 1.0 mM CH Cl solution. IR (Selected bands, cm-1): v(C =O) 1598, c 2 2 1 The chemicals used to prepare complexes and buffer solutions v(C 4 =O) 1606, v(C 2 O) 1103. 31P{1H} NMR (162 MHz, CH 2 Cl 2 , 298 K): A are from analytical grade or chemically pure grade. The RuCl ∙3H O 3 2 (ppm) (d, 75.3; 71.2/ 2J = 16.2 Hz). 1H NMR (400 MHz, DMSO-d , 298 6 and the ligands 1,2-bis(diphenylphosphino)ethane (dppe), 1,4- K): (ppm) 8.6 - 6.6 (m, 32 H, overlapped signals of dppe (20H), bipy s bis(diphenylphosphino)butane (dppb), 1,1’- (8H) and Lap (4H)), 4.8 (m, 1H, CH aliphatic of Lap), 3.1 and 2.9 (m, n bis(diphenylphosphino)ferrocene (dppf), 2,2’-bipyridine (bipy) and 6H, CH aliphatic of Lap (2H) and chain aliphatic of dppe (4H)), 1.4 (m, 2 o lawsone (Law) were purchased from Sigma-Aldrich and used as 6H, 2CH of Lap). 13C{1H} NMR (125.74 MHz, DMSO-d , 298 K): (ppm) received. 3 6 i 199.2 (C 1 =O), 181.1 (C 4 =O), 167.0 (C 2 O). t The FTIR spectra were measured as CsI pellets on a Bomen– c [Ru(Law)(dppe)(bipy)]PF (1b): Yield: 45.85 mg (70%). Elemental 6 Michelson FT MB-102 instrument between 4000 and 200 cm. The a analysis: found (calculated) (%) for C H F N O P Ru.C H O: C, 46 37 6 2 3 3 4 10 elemental analyses (C, H and N) were determined on Fisons EA 1108 57.31 (57.69); H, 4.52 (4.26); N, 2.67 (2.64). Λ = 44.5 Ω−1 cm2 mol−1, s M model (Thermo Scientific) equipment. The electronic absorption in 1.0 mM CH Cl solution. IR (Selected bands, cm-1): v(C =O) 1601, n 2 2 1 spectra of the samples were recorded using a Hewlett Packard diode v(C 4 =O) 1616, v(C 2 O) 1099. 31P{1H} NMR (162 MHz, CH 2 Cl 2 , 298 K): a array - 8452A spectrophotometer. The fluorescence measurements (ppm) (d, 78.9; 75.2/ 75.8; 70.8, 2J = 19.4/ 14.6 Hz). 1H NMR (400 r were measured on a SpectraMax M3 spectrofluorometer using an MHz, DMSO-d , 298 K): (ppm) 8.6 – 6.5 (overlapped signals, 32H T 6 opaque 96-well plate. Conductivity values were obtained from 1.0 aromatic hydrogen of dppe (20H), bipy (8H) and Law(4H)), 5.8 (s, 1H, mM solutions of complexes in CH 2 Cl 2 , using a Meter Lab CDM2300 CH of Law), 3 – 2.4 (m, 4H, chain aliphatic of dppe). 13C{1H} NMR n instrument. Cyclic voltammetry (CV) experiments of the complexes (125.74 MHz, DMSO-d , 298 K): (ppm) 200/196.5 (C =O), o 6 1 in solution were promoted in an electrochemical analyzer BAS model 182.5/181.7 (C 4 =O), 171.6/170.2 (C 2 O). t 100B. These experiments were carried out at room temperature in l CH Cl containing 0.10 M tetrabutylammonium perchlorate (PTBA) [Ru(Lap)(dppb)(bipy)]PF 6 (2a): Yield: 39.80 mg (60%). Elemental a 2 2 (Fluka Purum) as the support electrolyte. The working and auxiliary analysis: found (calculated) (%) for D C H F N O P Ru.0.8CH OH.0.2CH Cl : C, 56.42 (56.68); H, 4.21 electrodes were stationary Pt and the reference electrode was 53 49 6 2 3 3 3 2 2 (4.53); N, 2.52 (2.45). Λ = 44.13 Ω−1 cm2 mol−1, in 1.0 mM CH Cl Ag/AgCl, 0.10 M PTBA in CH Cl . All the NMR experiments (1H, 31P{1H} M 2 2 2 2 solution. IR (Selected bands, cm-1): v(C =O) 1548, v(C =O) 1603, and 13C{1H}) were recorded on a 9.4T Bruker Avance III spectrometer 1 4 v(C O) 1098. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) (d, 47.7; with a 5mm internal diameter indirect probe with ATMATM 2 2 2 44.5/ 2J = 34.9 Hz). 1H NMR (400 MHz, DMSO-d , 298 K): (ppm) 8.5 (Automatic Tuning Matching). The splitting of proton, carbon and 6 - 6 (overlapped signals, 32H aromatic hydrogen of dppb (20H), bipy phosphorus resonances was reported as s = singlet and m = multiplet. (8H) and Lap (4H)), 5.3 (m, 1H, CH of Lap), 3.1 (m, 2H, CH aliphatic The cellular uptake experiments were performed on a Varian 820-MS 2 of Lap), 2.35 – 1.4 (m, 8H, chain aliphatic of dppb), 1.7 (s, 6H, 2CH of 3 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J Page 3 of 12 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Lap). 13C{1H} NMR (125.74 MHz, DMSO-d , 298 K): (ppm) 195.6 displacement parameter of the atom to which each one was bonded, 6 (C =O), 180.5 (C =O), 168.5 (C O). and C–H bond lengths were fixed at 0.97 Å. H atoms, bound to the 1 4 2 methyl of the Lap ligand, were located from an electron-density [Ru(Law)(dppb)(bipy)]PF (2b): Yield: 42.53 mg (64%). Elemental 6 difference and refined as riding on their parent atoms with U (H) analysis: found (calculated) (%) for C H F N O P Ru.0.3CH Cl : C, iso 48 41 6 2 3 3 2 2 values of 1.5U (Csp3) for methyl H atoms. 53.36 (53.14); H, 3.80 (3.95); N, 2.62 (2.51). Λ = 44.93 Ω−1 cm2 mol−1, eq M in 1.0 mM CH Cl solution. IR (Selected bands, cm-1): v(C =O) 1581, Biological Assays 2 2 1 v(C =O) 1619, v(C O) 1006. 31P{1H} NMR (162 MHz, CH Cl , 298 K): 4 2 2 2 Cell Culture (ppm) (d, 47.3; 44.64/ 2J = 34.7 Hz). 1H NMR (400 MHz, DMSO-d , 6 Human breast cell lines MCF-7 (ATCC: HTB-22) and MDA-MB-231 298 K): (ppm) 8.5 - 6 (overlapped signals, 32H aromatic hydrogen of (ATCC: HTB-26), human prostate tumor cell line DU-145 (ATCC: HTB- dppb (20H), bipy (8H) and Law (4H)), 5.6 (s, 1H, CH of Law), 2.6 – 1.5 t 81) and non-tumor breast cells, MCF-10A (ATCC: CRL-10317) were p (m, 8H, chain aliphatic of dppb). 13C{1H} NMR (125.74 MHz, DMSO- d , 298 K): (ppm) 195.6 (C =O), 180.6 (C =O), 168.5 (C O). maintained at 37 ºC in a humidified incubator under atmosphere i 6 1 4 2 r containing 5% CO . MCF-7 and DU-145 cells were routinely [Ru(Lap)(dppf)(bipy)]PF (3a): Yield: 54.53 mg (82%). Elemental 2 c 6 maintained in Roswell Park Memorial Institute medium (RPMI-1640) analysis: found (calculated) (%) for C H F FeN O P Ru: C, 57.51 s 59 49 6 2 3 3 supplemented with 10% fetal bovine serum (FBS), and cells MDA- (57.41); H, 3.99 (3.66); N, 2.25 (2.48). Λ = 45.8 Ω−1 cm2 mol−1, in 1.0 u M MB-231 cells were maintained in Dulbecco’s modified Eagle’s mM CH Cl solution. IR (Selected bands, cm-1): v(C =O) 1599; v(C =O) 2 2 1 4 medium (DMEM) supplemented with 10% FBS. MCF-10A cells were n 1615; v(C O) 1091. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) 2 2 2 cultivated in DMEM/F12 medium containing horse serum (HS) 5%, a (d, 45.4; 41.4/ 42.3; 39.1/ 2J = 30.8/ 27.5 Hz); 1H NMR (400 MHz, EGF (0.02 mg/mL), hydrocortisone (0.05 mg/mL) and insulin (0.01 M DMSO-d , 298 K): (ppm): 8.5 - 6.8 (m, 32H, an overlap of aromatic 6 mg/mL). All the media contained penicillin (100 UI/mL), streptomycin protons of phenyl groups of dppf (20H), bipy (8H) and Lap (4H)), 5.3 (100 mg/mL) and L-glutamine (2 mM). (m, 1H, CH of Lap), 5.0 - 4.3 (m, 8H, aromatic C-H ferrocene group of d MTT Growth Inhibition Assay dppf), 3.01 (m, 2H, CH of Lap), 1.8 and 1.3 (s, 6H, 2CH group of Lap). e 2 3 13C{1H} NMR (125.74 MHz, DMSO-d 6 , 298 K): (ppm) 200.6/195.7 1.5 × 104 cells per well were seeded in 200 μL of complete t (C =O), 180.9/180.6 (C =O), 169.6/165.9 (C O). medium in 96-well plates (Corning Costar). The plates were p 1 4 2 incubated at 37ºC in 5% CO for 24 h to allow cell adhesion, and e [Ru(Law)(dppf)(bipy)]PF (3b): Yield: 50.36 mg (80%). Elemental 2 6 analysis: found (calculated) (%) for C H F FeN O P Ru: C, 59.35 afterwards the compounds were added. The ruthenium complexes c 54 41 6 2 3 3 were dissolved in DMSO. After dilution, the maximum final (59.16); H, 4.32 (4.12); N, 2.24 (2.34). Λ = 48 Ω−1 cm2 mol−1, in 1.0 c M concentration used in the assay was 100 µM ruthenium complex in mM CH 2 Cl 2 solution. IR (Selected bands, cm-1): v(C 1 =O) 1601; v(C 4 =O) A 0.5% DMSO. Cisplatin solubilized in DMF was used for comparison. 1615; v(C O) 1096. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) 2 2 2 The percentage of DMSO and DMF used was 0.5%. Cells were (d, 46.6; 43.4/ 43.8; 40.1/ 2J = 30.8/ 22.7 Hz); 1H NMR (400 MHz, s incubated with compounds for 48 h, at 37 ºC in 5% CO . Thereafter, DMSO-d 6 , 298 K): (ppm): 8.6 - 6.8 (m, 32H, an overlap of aromatic 2 n the medium was removed and the MTT solution (0.5 mg/mL, 50 protons of phenyl groups of dppf (20H), bipy (8H) and Law (4H)), 5.4 o µL/well) was added to the cells and incubated for 3 hours. 100 μL of (s, 1H, Law), 5.2 - 4.4 (m, 8H, aromatic C-H ferrocene group of dppf). isopropanol was added to dissolve the precipitated formazan i 13C{1H} NMR (125.74 MHz, DMSO-d 6 , 298 K): (ppm) 201.8/197.1 t (C =O), 182.1/181.7 (C =O), 171.9/169.1 (C O). crystals. The conversion of MTT to formazan by metabolically viable c 1 4 2 cells was measured in an automated microplate reader at 595 nm. In a X-ray crystallographic studies order to analyze the effects of complexes on cell viability, the viability s Purple single crystals of complexes (1a), (2a) and (2b) were rate in the control wells (cells receiving only DMSO) was taken as a n grown by slow evaporation of a dichloromethane/ethyl ether reference (100%). The viability rates of treated cultures were then a solution. X-ray crystallography analyses were carried out using Mo- expressed as a percent of the control value, and % cell viability was Kα radiation (λ = 0.71073 Å) on a BRUKER APEX II Duo diffractometer. plotted against drug concentration (logarithmic scale) to determine r T Standard procedures were applied for data reduction and absorption the IC (drug concentration at which 50% of the cells are viable 50 correction. The structures were solved by the direct method using relative to the control), with the error estimated from the average of n SHELXS-97 and refined using the software SHELXL-9743. Crystal 3 trials. o structure representations were generated using the MERCURY 3.9 Colony Formation Assay program44. The main crystal data collections and structure t MDA-MB-231 cells were seeded (300 cells/plate) in Petri dishes l refinement parameters are summarized in Table 1S (supplementary a to form colonies. Cells were grown at 37 ºC in 5% CO overnight, and information). Non-hydrogen atoms of the complexes were located, 2 D then treated with complex (1a) (0.05, 0.15, 0.3 and 1 µM) for 48 h. and a full-matrix, least-squares refinement of these atoms with After this time, the medium was changed to a fresh one without any anisotropic thermal parameters was carried out. In all the ligands of complex. Colonies (> 50 cells) formed after 10 days were washed complexes, the aromatic C–H hydrogen atoms were positioned with PBS (pH= 7.4), fixed with methanol-acetic acid (3:1) solution for stereochemically and were refined with fixed individual 15 min, stained with crystal violet solution (1% crystal violet, 20% displacement parameters [U (H) = 1.2 U (Csp2)] using a riding iso eq ethanol) for 30 min and rinsed with water to remove extra dye. model with aromatic C–H bond lengths fixed at 0.93 Å. Methylene Finally, colonies with a minimum of 50 cells were quantified using groups of Lap, dppe and dppb were also set as isotropic with a ImageJ Software. thermal parameter 20% greater than the equivalent isotropic This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 4 of 12 ARTICLE Journal Name Morphological following 5 s exposure time. The spot intensity was quantified by densitometry using ImageJ software and normalized with the MDA-MB-231 cells were seeded (1 × 105 cells/well) into 12-well intensity of internal positive controls. Densitometry data is reported plates and treated with complex (1a) (0.01, 0.15, 5 and 15 µM) for 48 h. Changes in the morphology were monitored using an inverted as the average of two independent experiments  SD. microscope coupled with a camera (Moticam 1000 - 1.3MP Live Resolution). Cellular Uptake Mitochondrial membrane potential MDA-MB-231 cells (1 × 106 cells/plate) were seeded into 100 mm dishes and after 24 h, the complex (1a) (5 M) was added. The cells The investigation of the changes of mitochondrial potential were incubated for 3 and 6 h. After that, the cells were washed with (m) in MDA-MB-231 cells after exposure to complex (1a) was PBS, harvested with trypsin, collected and washed twice with PBS. t carried out using DBTM MitoScreen Kit with the dye JC-1 (5’,5’,6,6’- p One mL of nitric acid was added to the pellets and after 3 days 2 mL tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide). i of water was added. The ruthenium analysis was performed on a MDA-MB-231 cells (1 × 105) were seeded in 12-well plate and r Vista-MPX simultaneous inductively coupled plasma optical mass c incubated for 24 h. After that, the cells were treated with complex spectrometer (ICP-MS) ate 265 nm (Varian Inc.). s (1a) in different concentrations (IC of 24h and 2× IC of 24 h) for 4 50 50 u h. The cell samples were washed with ice-cold PBS stained with JC-1 in the dark, according to the manufacturer’s directions. The cells Results and Discussion n were analyzed using a flow cytometer BD Accurri C6 Plus. a Synthesis and characterization M Reactive oxygen species Six novel ruthenium complexes were prepared in high yield, as air stable dark purple solids, from the reaction of the precursors with MDA-MB-231 cells (6 × 105 cells/plate) were plated into 6-well d the ligands lapachol (Lap) or lawsone (Law), as summarized in plate and after 24 h, the cells were treated with complex (1a) in Scheme 1. Different phosphines were used as ligands in order to e concentration (2× IC of 24 h) and with free lapachol (10 M) for 4 50 investigate the effect of their size on the biological properties of the t h. The H 2 O 2 (10 M) was employed as positive control and for corresponding complexes. All the complexes are highly soluble in p negative control was used cells without treatment. After the drug most organic solvents and insoluble in water. The structures for the e exposure, cells were washed with PBS and then incubated with complexes were confirmed by analytical and spectroscopic c 10 M of H DCFDA [2’,7’-Dichlorodihydrofluorescein diacetate] 2 techniques described in the experimental section. Moreover, the c (Sigma-Aldrich, D6883) for 30 min at 37C. The cells were washed crystal structures for complexes (1a), (2a) and (2b) were also A with ice-cold PBS (3×), were harvested and the fluorescence intensity confirmed by single-crystal X-ray diffraction analysis. was analyzed using a Synergy H1 Hybrid Multi-Mode Microplate- Fluorimeter at a wavelength of  = 400 nm and  = 525 nm and s ex em images were taken by using an Olympus BX50 fluorescence n microscope. o Flow Cytometry Analysis for Cell Apoptosis i t MDA-MB-231 cells (0.7 × 105 cells/well) were cultured in 24-well c plates for 24 h and then treated with complex (1a) at different a concentrations (0.5; 1.25; 2.5; 5 and 10 µM) for 24 h. Then, the plate s was centrifuged, the medium collected, and the cells washed with n ice-cold PBS and stained with 7-AAD annexin V and PE (BD a Biosciences) according to the manufacturer’s instructions. The cells r were analyzed using a BD Accurri C6 Plus flow cytometer. T Apoptosis signaling antibody array Scheme 1. Synthesis of the ruthenium complexes containing n MDA-MB-231 cells (2 × 106) in 20 mL of DMEM media were lapachol or lawsone. o seeded in 100 mm dishes (two dishes for each condition) and The elemental analyses data are in agreement with the suggested maintained at 37C for 24h. After that, the cells were treated with t structures of the complexes and the values of molar conductivity l ruthenium complexes (5 M) and incubated for 6, 12, 18 and 24 h. values acquired in dichloromethane, indicating that the complexes a The cell samples were harvested, lysed in the presence of protease are electrolyte 1:1. The IR spectra of all complexes did not show a D and phosphatase inhibitors, and the protein quantified using the broad band around 3354 cm assigned to v(OH) vibrations, Bradford protein assay. 75 g of protein from each sample was indicating the deprotonation of this group and suggesting its coordination to the ruthenium atom. In addition, upon coordination, incubated overnight at 4C on array from the PathScan Stress and all the complexes showed shift of the carbonyl stretch around 100 Apoptosis Signaling Antibody Array Kit (Cell Signaling). The antibody cm (for smaller and higher frequencies for C1=O and C2‒O, array allows for simultaneous detection of 19 signaling molecules respectively) when compared with the frequencies of the free ligands that are involved in the regulation of the stress response and Lap and Law, indicating a bidentate coordination of these groups to apoptosis. The chemiluminescent array images were captured the ruthenium. In the free Lap, the C1=O and C2‒O bands were observed at 1643 and 1050 cm, respectively. For complex (1a) 4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J Page 5 of 12 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE these bands were shifted to 1598 and 1103 cm respectively, Table 1. 31P{1H} NMR and oxidation potential (RuII/RuIII) versus confirming the coordination of the ligand to the ruthenium by the Ag/AgCl (0.10 M PTBA in CH Cl ) for complexes 1a – 3b 2 2 oxygen atoms. All other complexes showed this same behavior. δ (ppm) 2J (Hz) E (mV) Furthermore, the bands around 842 cm and 557 cm are P-P ox. attributed to ν(P-F) and δ(P-F) stretching vibrations, indicating the (1a) 75.3 (d); 71.2 (d) 16.2 1274 presence of a counterion PF  in the complexes. 6 The UV-vis spectra of complexes displayed bands in the ultraviolet 78.9 (d); 75.8 (d); 75.2 (d); 19.4 and (1b) 1115 region characteristic of π  π* transitions of heterocycles and 70.8 (d) 14.6 benzene rings of ligands. Moreover, a metal-to-ligand charge- (2a) 47.7(d); 44.5(d) 34.9 1166 transfer (MLCT) absorption was observed around 450 nm. As perceived in compounds containing naphthoquinones as ligands, one t band around 564 nm, assigned to quinone carbonyls, was observed (2b) 47.3(d); 44.6(d) 34.7 1279 p for all ruthenium complexes reported here. This band corresponds to 45.4 (d); 42.2 (d); 41.5 (d); 30.8 and 1337 i nπ* transitions45–47. The same behavior was observed in previous (3a) 39.1 (d) 27.5 (996)* r c work conducted by our research group in complexes containing 45.9 (d); 42.9(d); 42.5(d); 30.8 and 1378 lawsone and triphenylphosphine as ligands48. (3b) s 38.9 (d) 22.7 (1013)* The cyclic voltammograms of the ruthenium complexes showed an u *FeII/FeIII oxidation process from phosphine dppf. irreversible process related to the oxidation of RuII/RuIII, around 1200 n mV (Table 1). These values are more positive than those for the During the synthetic procedure, monitored by 31P{1H} NMR, the a precursors (600 – 700 mV), resulted from greater stabilization of the formation of two isomers was observed and only complexes (1a), metallic center after coordination of the ligands Lap and Law to the M (2a) and (2b) could be isolated as pure isomers. Thus, for this reason, metal center. This can be explained by the substitution of two the following was observed in these three complexes: two pairs of chloride ligands (good donors) by negative monocharged chelating doublets in their 31P{1H} NMR spectra; and for the other complexes, d ligands (Lap or Law), resulting in a new complex with positive charge. four pairs of doublets in their 31P{1H} NMR spectra (Table 1). The e Effects of similar behavior have been observed for other ruthenium isomers present two different stereochemistries, where one isomer complexes previously reported in the literature49,48,50. In complexes t presents the oxygen of the carbonyl group (O1) of Lap or Law p containing dppf, oxidation and reduction process of iron was positioned trans to phosphorus P1, while the other isomer presents observed. e the oxygen of enol (labelled as O2) is located trans to phosphorus P1, The 1H NMR spectra of all complexes showed signals of Lap, Law, c as shown in Figure 1 for complex (1a). bipyridine and phosphines, but there are some overlapping signals c between the 8.6 – 6.6 ppm due to the high number of aromatic A hydrogens present in the complexes. Moreover, there were observed multiplets that corresponded to the CH groups of the 2 s aliphatic chains of dppe and Lap (3.1 - 2.9 ppm), CH group of Lap (4.8 ppm) and CH groups of Lap (1.4 ppm), for complex (1a). The 13C{1H} n 3 NMR spectrum shows signals characteristics due to the C1=O and o C2O unit, which were shifted to high field, supporting the i suggestion of O,O-coordination to the metal. In the Lap and Law free t ligands, these signals for C1=O and C2O were observed around 181 c and 155 ppm, respectively. For the complex (1a) these signals shifted a for 199.2 and 167 ppm. The same behavior was observed for the s other complexes. It is worth mentioning that we observed the n duplication of all signals for the complexes (2b), (3a) and (3b) due to the presence of the isomers. a The 31P{1H} NMR spectra of the complexes exhibit a septet 143 r ppm, related to an ionic PF , and a pair of doublets in the range of Figure 1. Assignment of the 31P{1H} NMR spectrum of isomers of T 6 38 to 70 ppm. These signals are typical AX spin system that indicated complex (1a) in CH 2 Cl 2 /D 2 O. n the magnetic non-equivalence of the phosphorus atoms, where P1 is trans O (Lap or Law) and P2 trans N (Table 1). All complexes show a The molecular structures determined by X-ray diffraction for o significant upfield shift when compared to the precursor complexes, complexes (1a), (2a) and (2b) allow us to identify these two isomers t with displacements around 30 and 42 ppm. (Figure 2), where in the complex (1a), the O1 is trans N1 and O2 trans l P1, and for complex (2a) and (2b), the O1 is trans P1 and O2 trans a N1. D This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 6 of 12 ARTICLE Journal Name Comparing the distances of Ru(1)-P(2) [2.2974(12)-2.3218(10) Å] with Ru(1)-P(1) [2.2678(12)-2.2946(10) Å] bonds, it is observed that in all complexes the Ru(1)-P(2) bond lengths are slightly longer than the distances of Ru–P(1). This trend may be due to stronger trans influence of nitrogen atom of bipy species compared with the oxygen atoms of the Lau or Lap, for the of the ruthenium. The Ru-O bond located trans to phosphorus atoms are longer than Ru-O bond lengths with the oxygen trans to N1 nitrogen atoms (See Table 1), which is a consequence of the stronger trans influence of the phosphorus atoms. The crystal structures do not present strong intermolecular t hydrogen bonds to stabilize the crystal self-assembly, only p hydrophobic forces are present. An interesting aspect occurring in all i complexes is the presence of intramolecular π-π stacking between r the phenyl (dppe or dppb) and quinone ring (See Supplementary c information). This interaction displays an import role to stabilize the s molecular structure and conformation of the complexes (1a, 2a and u 2b). n In order to gain insights into the stability of the complexes, 31P{1H} NMR experiments of the complexes were recorded, in DMSO. a All the complexes were stable in DMSO for a period of 72 h. However, M complex (1a) demonstrated beyond the signals at 75.3 and 71.2 ppm, Figure 2. Crystal structures of complexes (1a) a very small amount (~ 8% at 72 h) of the isomer with the O1 trans [Ru(lap)(dppe)(bipy)]PF , (2a) [Ru(lap)(dppb)(bipy)]PF and (2b) d 6 6 to the phosphorus atom, (80 and 76 ppm). This is an indication that [Ru(lau)(dppb)(bipy)]PF 6 , with ellipsoids at 30% of probability. the system must be reaching dynamic equilibrium of the two e Hydrogen atoms and the counter-ion PF  were omitted for sake of 6 isomers, over time (see Figure S19-S20 in the Supporting t clarity. Information). p e Table 2. Selected bond distances (Å) and angles (°) for complexes c (1a), (2a) and (2b) Fragment (1a) (2a) (2b) Inhibition of cancer cell growth c Ru(1)-O(1) 2.115(3) 2.1684(17) 2.161(2) A The proliferation of human cancer cell lines (DU-145, MCF-7 and Ru(1)-O(2) 2.124(3) 2.0886(16) 2.101(3) MDA-MB-231) and the non-tumorigenic cell line, MCF-10A, was Ru(1)-P(1) 2.2678(12) 2.2791(7) 2.2946(10) measured using the MTT assay after treatment with the series of s Ru(1)-P(2) 2.2974(12) 2.3087(6) 2.3218(10) ruthenium complexes. As shown in Table 3, complexes containing n Ru(1)-N(1) 2.048(4) 2.077(2) 2.070(3) the Lap or Law ligands show lower IC values than their precursor 50 o Ru(1)-N(2) 2.098(4) 2.097(2) 2.097(3) complexes (1) and (2) and are 15 times more effective than cisplatin. O(1)-C(1) 1.264(5) 1.249(3) 1.245(5) i Interestingly, all six ruthenium complexes exhibited more sensitivity t O(2)-C(2) 1.290(6) 1.300(3) 1.293(5) towards MDA-MB-231 compared to MCF-7 cells. The MDA-MB-231 c O(4)-C(4) 1.232(5) 1.234(3) 1.246(5) cell line represents triple-negative breast cancer (TNBC) that lacks a C(1)-C(2) 1.481(7) 1.480(3) 1.477(5) the hormone membrane receptors required for targeted therapy O(1)-Ru(1)-O(2) 77.00(13) 76.49(6) 76.27(10) s options, and thus, is a particularly difficult cancer to treat. Most O(1)-Ru(1)-P(1) 105.05(9) 173.53(4) 176.63(8) n importantly, complex (1a) exhibits selective activity against MDA- O(2)-Ru(1)-P(2) 177.60(10) 91.85(5) 88.73(8) MB-231 compared to the non-tumorigenic cell line, MFC-10A. It is a O(1)-Ru(1)-P(2) 88.59(9) 87.08(5) 88.42(8) worth mentioning that the IC 50 value of the isolated isomer of r P(2)-Ru(1)-P(1) 83.94(5) 95.26(2) 94.04(4) complex (1a) against MDA-MB-231 was nearly identical to the T N(2)-Ru(1)-N(1) 78.45(15) 78.35(8) 78.54(12) mixture of isomers; isolated isomer IC = 0.15 ± 0.01 µM, mixture of 50 N(1)-Ru(1)-P(2) 102.29(11) 100.81(6) 102.79(9) the two isomers = 0.13 ± 0.03 µM. n The antiproliferative properties of complex (1a) against MDA- o Complexes (1a), (2a) and (2b) crystalize in the tetragonal I-4, MB-231 cells were further tested using the colony formation assay, t triclinic P-1 and monoclinic P2 1 /c (space groups, respectively. which measures the capacity of a single cell to proliferate l According to the molecular structure (Fig. 2) and the bond angles indefinitely, forming a visible colony. Complex (1a) inhibited the a collected in Table 2, the complexes (1a), (2a) and (2b) present a number and size of colony formation of MDA-MB-231 cells in a D distorted octahedral geometry. The main crystallographic data are concentration-dependent manner, consistent with results of the given in Supplementary Information (Tables S1-S3). MTT assay (Fig. 3). The MTT and clonogenic formation assays cannot As can be seen by the crystallography study, the phosphorus atom make the distinction between cytostatic (proliferative arrest) and P2 (dppb or dppe) is disposed trans to nitrogen atom of the bipy in cytotoxic (necrotic/apoptotic) effects, therefore microscopy, cell all structures. On the other hand, the other phosphorus P1 is trans cycle and apoptosis assays were performed. to O1 in the complexes (2a and 2b) and P1 is trans to O2 in the complex (1a). These structural differences justify those behavior observed in the 31P{1H} NMR study. 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J Page 7 of 12 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE t p Figure 5. (A) Effects of complex (1a) in mitochondrial membrane i r potential (m) of MDA-MB-231 cells. Gated region R1 (red) c Figure 3. (A) Representative images of colonies developed from contains cells with intact mitochondrial membranes and R2(green), MDA-MB-231 cells 10 days after treatment with complex (1a). (B) s cells with loss of m. (B) Representation of mean values for cells Quantitative data representing the colony number and size with u with loss of m (Gated region R2). Data are expressed as mean ± relation to the concentration of complex (1a). Data are expressed as SD of three independent measurements. The statistical analysis used n mean ± SD of three independent measurements. The statistical was one-way ANOVA using Tukey test; **** p < 0.0001. a analysis used was one-way ANOVA using the Tukey test; * p < 0.02 and ** p < 0.009. M Reactive oxygen species (ROS) d The morphology analysis of MDA-MB-231 cells treated for 48h Reactive oxygen species (ROS) can be generated by normal with 0.15 µM complex (1a) showed a reduced cell number, loss of metabolism or due xenobiotic exposure55. ROS are involved in many e cellular adhesion and distinct morphological changes. Higher physiological processes, for example, as mediators in signal t concentrations caused additional loss of adhesion, cell shrinkage and transduction, activation of tyrosine kinase, among others. However, p fragmentation, indicative of apoptotic or necrotic processes (Fig. 4). the excess of ROS can cause an oxidative stress, and trigger the e activation of apoptotic signaling, causing cell death56. In this context, c we investigated if the complex (1a) is capable to induce the ROS in c MDA-MB-231 cell line. The intracellular levels of ROS were measured by a fluorescence, A and as show in Fig. 6. The cells treated only with 0.5% of DMSO (Control) not showed a significantly green fluorescence. On the other s hand, the cells exposed to H 2 O 2 , lapachol and complex (1a) showed n a considerable increase in the levels of ROS, mainly for cells treated o with complex (1a). Therefore, these results revealed that the complex (1a) strongly generated ROS in MDA-MB-231 cells. i t c Figure 4. Effect of complex (1a) on MDA-MB-231 cells morphology a using of inverted microscope (10×). s n Mitochondrial membrane potential a Mitochondria is an important organelle known as the cellular r energy factory, what is responsible for producing ATP and T metabolites necessary for the cellular demands. This organelle can be a targeting in a therapy against cancer. Some compounds are n capable to disrupting the function of the mitochondria and leading o the activation of mitochondrial dependent cell death signaling t pathways51,52. Figure 6. (A) Fluorescence detected in MDA-MB-231 cells treated l Therefore, mitochondrial membrane potential (m) of MDA- with H O , lapachol and complex (1a) for 4h before staining with 10 a 2 2 MB-231 cells was investigated by flow cytometry using a dye JC-1, M of H DCFDA and (B) quantification of the green fluorescence D 2 which is cationic dye that is capable to accumulate into the generated by ROS formation. mitochondria and that can be measured by change in a fluorescence from green to red. Typical changes from red to green in the fluorescence of JC-1 in MDA-MB-231 cells treated with complex (1a) were detected as shown in Fig. 5. The complex (1a) evidently changed the m, how we observe by the increase of the green fluorescence, with the increase of the concentration of complex. The loss of m is indicative of an early event in apoptotic process53,54. This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 8 of 12 Journal Name ARTICLE Table 3. Cellular growth inhibitory effects of ruthenium complexes, Lap, Law and cisplatin t IC (µM); 48h p 50 Compounds i DU-145 MCF-7 MDA-MB-231 MCF-10A SI1 SI2 r c (1a) 0.39 ± 0.07 2.40 ± 0.07 0.15 ± 0.01 2.76 ± 0.30 18.4 1.15 s u (1b) 0.42 ± 0.17 0.87 ± 0.01 0.11 ± 0.04 0.72 ± 0.01 6.54 0.83 n (2a) 0.56 ± 0.02 0.21 ± 0.05 0.14 ± 0.01 0.33 ± 0.02 2.36 1.57 a (2b) 0.23 ± 0.01 0.30 ± 0.18 0.08 ± 0.05 0.51 ± 0.03 6.71 1.7 M (3a) 0.12 ± 0.04 1.20 ± 0.05 0.07 ± 0.03 0.77 ± 0.03 11.0 0.64 d (3b) 0.18 ± 0.01 0.70 ± 0.01 0.06 ± 0.01 0.58 ± 0.02 9.67 0.83 e t (1) 27.30 ± 1.18 8.99 ± 3.14 26.06 ± 4.84 25.51 ± 0.18 0.98 2.84 p e (2) 26.71 ± 0.66 33.84 ± 0.44 27.55 ± 1.84 11.67 ± 1.44 0.42 0.34 c Lap >100 >100 >100 >100 -- -- c Law >100 >100 >100 >100 -- -- A Cisplatin 2.00 ± 0.47 13.98 ± 2.02 2.33 ± 0.40 29.45 ± 0.85 12.63 2.11 s n *(1) cis-[RuCl 2 (dppe)(bipy)] and (2) cis-[RuCl 2 (dppb)(bipy)]. The precursor complex cis-[RuCl 2 (dppf)(bipy)] was not evaluated due the low o solubility. SI = Selectivity Index = SI1 = IC MCF-10A/IC MDA-MB-231 and SI2 = IC MCF-10A/IC MCF-7. 50 50 50 50 i t c using a chemiluminescent readout. We noticed the changes of the a levels of Hsp27, Bad, caspase 3, SAPK/JNK and Smad2. The intensity Induction of apoptosis of the signals was analyzed by ImageJ software and expression levels s The potential of complex (1a) to induce cell death by apoptosis of -tubulin were employed to normalize the signals of each n was investigated using flow cytometry and cells stained after samples. a treatment with a combination of 7-AAD, a cell impermeable The Hsp27 is a Heat Shock Protein-27 that is involved in r fluorescent DNA-binding compound, and the fluorescently labeled protection of proteins of cells, from damage as loss of protein T apoptotic marker, Annexin V. As shown in Figure 7, incubation with functionality, growth arrest or receptor mediated apoptosis57,58. The complex (1a) induced a concentration-dependent decrease in the expression of this protein is related with lethal conditions, which is n unlabeled (viable) fraction of cells and an increase in the 7-AAD and common when the cells show some stress. We observed the o Annexin V labeled (apoptotic/dead) fraction of cells. Furthermore, a significantly increase of the levels of phosphorylated Hsp27 (Ser82) concentration-dependent increase in fractionated DNA (sub-G1 for both complexes. t l events) was observed, as determined by flow cytometry and The proteins of the Bcl-2 family is characterized by the ability to a propidium iodide cell staining (Figure S21). Complex (1a) did not control the mitochondrial pathway. This family includes pro- D induce distinct alterations in the G /S/G -phases of the cell cycle. apoptotic (e.g., Bax, Bad) and anti-apoptotic (e.g., Bcl-2, Bcl-xL) 1 2 proteins. The pro-apoptotic proteins are capable to induce the formation of pores in the outer membrane of mitochondria that Apoptosis signaling antibody array allows the release of the cytochrome c into the cytoplasm, and To gain further insights into the mechanism of cell death we activate the apoptotic cascade59. analyzed the effects of the complex (1a) on cell signaling. For that, As shown in Fig 8a we observed a time-dependent of the the MDA-MB-231 cells were incubated for 6, 12, 18 and 24 h with e x p ression levels of Bad (Ser136), which indicates that the complexes 5 M of complex (1a), and the cell lysates collected and analyzed are capable to inhibit of its function. The imbalance of the Bad can with a PathScan Stress and Apoptosis Signaling Antibody Array kit, This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 8 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J Page 9 of 12 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE led to cleavage of caspase 3, inducing the apoptotic signal from cell Preferential cellular uptake in cancer cells membrane to mitochondria. The cellular accumulation of ruthenium complex (1a) in the Finally, we observed the increase of the phosphorylated MDA-MB-231 and MCF-10A was investigated to understand whether SAPK/JNK (Thr183/Tyr185) (Fig 8b). SAPK/JNK belongs to the family the selectivity index was related to the rate of cellular entry (Fig. 9). of mitogen-activated protein kinase (MAPK), that exert important Results indicate that the intracellular concentration of complex (1a) role in cell proliferation and apoptosis, among others60. increases over time, and that the level of accumulation of complex (1a) into MDA-MB-231 was significantly higher than that observed for MCF-10A. Therefore, the cytotoxic activity and the rate of intracellular accumulation of complex (1a) correlates well with the observed selectivity between tumorigenic and normal cell lines. t p i r c s u n a M d Figure 9. Cellular accumulation of ruthenium in MCF-10A and MDA- e MB-231 cells, after 3 and 6 h of incubation. Statistical significance t was determined using a one-way ANOVA, Bonferroni post-test. p e c Conclusions c Figure 7. (A) Apoptosis of MDA-MB-231 cells induced by complex There is reported the synthesis, characterization and some in vitro A (1a) after 24 h of incubation, examined by flow cytometry. (B) biological activities of new ruthenium complexes containing the Percentage of cell death in various concentrations of complex (1a). naphthoquinones lapachol or lawsone as ligands were reported. All s Data are expressed as mean ± SD of three independent complexes are cytotoxic against the breast and prostate cancer cell measurements. The statistical analysis used was one-way ANOVA lines with higher selectivity indexes than the cisplatin drug, used as a n using the Tukey test; ** p < 0.002, *** p < 0.0002 and **** p < reference. The [Ru(Lap)(dppe)(bipy)]PF 6 (1a) is the most selective o 0.0001. among all the six complexes synthesized here, to MDA-MB-231 cells i when compared to the “normal-like” human breast epithelial cell t line, MCF-10A. Complex (1a) is capable of changing the cell c morphology, inhibiting the size and number of colonies of the MDA- a MB-231 TNBC cells. Furthermore, complex (1a) changed the s mitochondrial membrane potential, generated ROS in MDA-MB-231 n cells leading cell death by apoptosis. The cellular uptake assay showed the significant accumulation of ruthenium in MDA-MB-231 a cells compared to the MCF-10A cells, which is in agreement with the r selectivity observed in the cytotoxic assay. The work reported in this T paper confirms the hypothesis that the coordination of the naphthoquinone lapachol and lawsone are an interesting alternative n to develop complexes with relevant anticancer properties, as we o have previously suggested based on studies developed in our t laboratory. l a D Conflicts of interest There are no conflicts to declare. Figure 8. (A) Chemiluminescent array images obtained for effects of Acknowledgements complex (1a) of signaling proteins in MDA-MB-231 cells and (B) We would like to thank the following Brazilian Agencies of Research quantification of the signaling proteins by use of ImageJ. The Agencies: CAPES, CNPq, FAPEMIG and FAPESP. K. M. Oliveira would expression levels of -tubulin were used to normalize the signals. like to thank FAPESP for a research fellowship (grant numbers This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 9 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 10 of 12 ARTICLE Journal Name 2014/04147-9 and 2016/18530-4) and CAPES for postdoctoral 19 N. P. Farrell, Chem. Soc. Rev., 2015, 44, 8773–8785. fellowship. A.A. Batista for a research grant (2016/16312-0). R.S. 20 E. Jamieson and S. 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Name., 2013, 00, 1-3 | 11 Please do not adjust margins .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J Dalton Transactions Page 12 of 12 t p i r c s u n a M d e t Ru(II)/lapachol complex shows significant selectivity for triple negative breast cancer p e (TNBC) compared to the non-tumor human breast epithelial cell line. c c A s n o i t c a s n a r T n o t l a D .MP 25:30:21 0202/41/4 no eltsacweN fo ytisrevinU yb dedaolnwoD .0202 lirpA 31 no dehsilbuP View Article Online DOI: 10.1039/D0DT01091J