A novel ruthenium(ii) gallic acid complex disrupts the actin cytoskeleton and inhibits migration, invasion and adhesion of triple negative breast tumor cells.

Electronic Supplementary Material (ESI) for Dalton Transactions. This journal is © The Royal Society of Chemistry 2020 SUPPLEMENTARY MATERIAL A novel ruthenium(II) gallic acid complex disrupts the actin cytoskeleton and inhibits migration, invasion and adhesion of Triple Negative Breast Tumor Cells Angelica E. Graminhaa,c, João Honoratoa, Rodrigo S. Correab, Marcia R. Cominettic, Antônio C. S. Menezesd, Alzir A. Batistaa a Departamento de Química, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luís Km 235, CP 676, 13561-901, São Carlos, SP, Brazil b Departamento de Química, Universidade Federal de Ouro Preto (UFOP), CP: 35.400-000, Ouro Preto MG, Brazil c Departamento de Gerontologia, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luís Km 235, CP 676, 13561-901, São Carlos, SP, Brazil d Centro de Ciências Exatas e de Tecnologia, Universidade Estadual de Goiás – UEG, BR 153, CP 459, 75132-903, Anápolis, GO, Brazil Corresponding authors: Angelica E. Graminha (aegraminha@gmail.com) and Alzir A. Batista (daab@ufscar.br). Tel.: +55 1633518285; Fax: +55 1633518350. Table 1S. Infrared assignments (cm-1) of symmetric (νs) and asymmetric (νas) stretching of carboxyl group in free and coordinated ligand. Compound νasCOOνsCOO- Δ(cm-1) HBAC 1688 1453 235 HGAC 1666 1468 198 HEGA 1697 1432 265 [Ru(BAC) 1619 1499 120 [Ru(GAC) 1622 1497 125 [Ru(EGA) 1624 1512 112 Table 2S. Crystallographic data and structures refinement for Ru(BAC) and Ru(EGA). Ru(BAC) Ru(EGA) Empirical formula C90 H83 F12 N4 O4.50 P6 Ru2 C51 H47.50 F6 N2 O8.25 P3 Ru Formula weight 1908.56 1128.39 Temperature 293(2) K 293(2) K Wavelength 0.71073 Å 0.71073 Å Crystal system Triclinic Monoclinic Space group P -1 P21/a Volume a = 11.7551(2) Å a= 110.1960(10)°. b = 17.4930(4) Å b= 97.4780(10)°. c = 22.9633(4) Å g = 99.2730(10)°. 4284.88(15) Å3 a = 18.6440(6) Å a= 90°. b = 31.0250(8) Å b= 110.1340(10)° c = 19.4890(7) Å g = 90°. 10584.1(6) Å3 Z 2 8 Density (calculated) 1.479 Mg/m3 1.416 Mg/m3 Absorption coefficient 0.545 mm-1 0.461 mm-1 F(000) 1946 4612 Crystal size 0.20 x 0.40 x 0.40 mm3 0.12 x 0.13 x 0.34 mm3 Theta range for data collection 3.017 to 26.093°. 1.292 to 25.598°. Index ranges -14≤h≤14, -21≤k≤21, -28≤l≤28 -17≤h≤22, -37≤k≤35, -23≤l≤15 Reflections collected 72618 45924 Independent reflections 16813 [R(int) = 0.0777] 18766 [R(int) = 0.0638] Completeness to theta (25.242) 98.9 % (25.242°) 95.1 % Refinement method Full-matrix least-squares on F2 Full-matrix least-squares on F2 Data / restraints / parameters 16813 / 0 / 1072 18766 / 0 / 1265 Goodness-of-fit on F2 1.023 0.967 Final R indices [I>2sigma(I)] R1 = 0.0597, wR2 = 0.1599 R1 = 0.0780, wR2 = 0.1946 R indices (all data) R1 = 0.0931, wR2 = 0.1815 R1 = 0.1657, wR2 = 0.2515 Largest diff. peak and hole 1.009 and -0.800 e.Å-3 0.869 and -0.995 e.Å-3 Unit cell dimensions Figure 1S: 1H (A), 13C{1H} (B), 1H-1H COSY (C) and 1H-13C HSQC (D) NMR spectra of complex Ru(BAC) in chloroform-d at 298 K. Figure 2S: 1H (A), 13C{1H} (B), 1H-1H COSY (C) and 1H-13C HSQC (D) NMR spectra of complex Ru(GAC) in chloroform-d at 298 K. Figure 3S: 1H (A), 13C{1H} (B), 1H-1H COSY (C) and 1H-13C HSQC (D) NMR spectra of complex Ru(EGA) in chloroform-d at 298 K. (A) 0.3 II Ru /Ru [Ru(ABz)(dppb)(bipy)]PF6 III (B) 0,25 0.25 [Ru(AGM)(dppb)(bipy)]PF6 0,20 0.2 RuII/RuIII Current (!A) Corrente, µA Current (!A) Corrente, µA 0,15 0.15 0.1 0.0 0,10 0,05 0.05 0,00 -0,05 -0.05 -0.1 III Ru /Ru 0.0 0.5 II 1.0 RuIII/RuII -0,10 1.5 2.0 Potencial,(V) V Potential 0,0 0.0 0,2 0,4 0.4 0,6 0,8 0.8 1,0 1,2 1.2 1,4 1,6 1.6 1,8 Potencial, mV Potential (V) Figure 4S: Cyclic voltammograms for complexes (A) Ru(BAC), (B) Ru(EGA) in Pt disc electrode (0.1 M PTBA, 100 mV s1, reference electrode: Ag/AgCl). 125 ns ** * % FRS 100 HGAC Ru(GAC) **** **** 75 **** 50 **** 25 ns 0 200.00 100.00 50.00 25.00 12.50 6.25 3.13 1.56 Concentration (µmol. L-1) Figure 5S: % FRS of the ligand HGAC and the complex Ru(GAC) after 30 minutes of reaction. Data represents mean ± SD from three independent assays in triplicate. Significance at the *p < 0.05; **p < 0.01and ****p < 0.0001 levels using 2way ANOVA and Sidak's test. DMSO DMSO + RPMI (24 h) DMSO + RPMI (48 h) Figure 6S: 31P{1H} NMR spectra of stability studies of complex Ru(GAC) under physiological conditions DMSO/RPMI medium (2:1). Ru(EGA) - Albumin (A) Ru(EGA) – Albumin % Cell viability 125 100 75 50 25 0 + BSA (µg/mL) Ru(EGA) (3.0 µM) + 20 40 80 160 200 240 280 300 + + + + + + + + Ru(BA) - Transferrin Ru(BAC) – Apo-Tf Ru(EG) - – Transferrin Ru(EGA) Apo-Tf 125 125 100 100 % Cell viability % Cell viability (B) 75 50 25 0 Apo-Tf (µg/mL) Ru(BAC) (0.39 µM) 75 50 25 0 + + 1.56 3.12 6.25 12.5 25 50 75 100 + + + + + + + + Apo-Tf (µg/mL) Ru(EGA (3.0 µM) + + 1.56 3.12 6.25 12.5 25 50 75 100 + + + + + + + + Figure 7S: Transport of ruthenium complex by albumin and transferrin in MDA-MB-231. The complexes, with fixed concentrations, were incubated with increasing concentrations of bovine serum albumin (A) or apo-Tf (B) for 48 h. Viable cells were estimated by MTT assay, and results were compared to cells treated with the complexes but in the absence of the proteins. Data represents mean ± SD from two independent assays in triplicate. Significance at the *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 levels using ANOVA and Dunnet’s test.