Cyclometalated Ru(II)–NHC complexes with phenanthroline ligands induce apoptosis mediated by mitochondria and endoplasmic reticulum stress in cancer cells

Electronic Supplementary Material (ESI) for Dalton Transactions. This journal is © The Royal Society of Chemistry 2022 Electronic Supplementary Information (ESI) Cyclometalated Ru(II)-NHC Complexes with Phenanthroline Ligand Induce Apoptosis Mediated by Mitochondria and Endoplasmic Reticulum Stress in Cancer Cells Chao Chen,*a,b He Lv,b Hao Xu,a Dancheng Zhu,a and Chao Shen*a a Key Laboratory of Pollution Exposure and Health Intervention of Zhejiang Province, College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China. E-mail: chenczju@163.com. b College of Life Sciences, Huzhou University, Huzhou, 313000, PR China. A. ORTEP drawing of Ru1-Ru4 and selected bond lengths and angles............S2-S3 B. Table S1. X-ray Crystallographic data of Ru1-Ru4 ...........................................S4 C. Fig.S1. UV-vis spectra and emission spectra of Ru1-Ru4 in CH3CN.................S5 D. Fig.S2. MTT curves of Ru1 -Ru4, cis-Pt, and HL·PF6 gainst cancer cells ........S5 E. Cell experiment...............................................................................................S6-S8 F. Fig.S6. log po/w of Ru1-Ru4 and intracellular accumulation of Ruthenium in HeLa cells................................................................................................... S8 G. UV-vis spectra change of Ru1-Ru4 in 1640 medium..........................................S9 H. 1H and 13C NMR Spectrum of Ru1-Ru4...................................................S10-S15 1 A. ORTEP drawing of Ru1-Ru4 and selected bond lengths and angles. ORTEP drawing of Ru1 showing atomic numbering scheme at 50% probability ellipsoids. Selected bond lengths (Å) and angles(deg): Ru(1)-C(6) 1.993(5), Ru(1)-N(7) 2.041(4), Ru(1)-N(4) 2.049(5), Ru(1)-N(5) 2.057(5), Ru(1)-N(1) 2.075(4), Ru(1)-N(6) 2.152(5), C(6)-Ru(1)-N(7) 89.97(17), C(6)-Ru(1)-N(4) 90.63(17), N(7)-Ru(1)-N(4) 179.37(15), C(6)-Ru(1)-N(5) 101.20(17), N(7)-Ru(1)-N(5) 88.61(15), N(4)-Ru(1)-N(5) 91.49(16), C(6)-Ru(1)-N(1) 78.27(16), N(7)-Ru(1)N(1) 89.21(14), N(4)-Ru(1)-N(1) 90.70(15), N(5)-Ru(1)-N(1) 177.76(16), C(6)-Ru(1)-N(6) 174.30(17), N(7)-Ru(1)-N(6) 91.19(17), N(4)-Ru(1)-N(6) 88.20(17), N(5)-Ru(1)-N(6) 84.42(15), N(1)-Ru(1)-N(6) 96.15(15). ORTEP drawing of Ru2 showing atomic numbering scheme at 50% probability ellipsoids. Selected bond lengths (Å) and angles(deg): Ru(1)-C(6) 1.975(5), Ru(1)-N(7) 2.028(5), Ru(1)-N(6) 2.041(5), Ru(1)-N(1) 2.053(4), Ru(1)-N(4) 2.063(4), Ru(1)-N(5) 2.141(4), C(6)-Ru(1)-N(7) 99.88(19), C(6)-Ru(1)-N(6) 90.53(19), N(7)-Ru(1)-N(6) 88.16(18), C(6)-Ru(1)-N(1) 78.5(2), N(7)-Ru(1)-N(1) 175.35(16), N(6)-Ru(1)-N(1) 87.51(18), C(6)-Ru(1)-N(4) 95.44(18), N(7)Ru(1)-N(4) 90.70(17), N(6)-Ru(1)-N(4) 174.03(17), N(1)-Ru(1)-N(4) 93.78(16), C(6)-Ru(1)-N(5) 170.61(19), N(7)-Ru(1)-N(5) 87.69(17), N(6)-Ru(1)-N(5) 95.29(17), N(1)-Ru(1)-N(5) 94.40(17), N(4)-Ru(1)-N(5) 78.81(16). 2 Fig. S3. ORTEP drawing of Ru3 showing atomic numbering scheme at 50% probability ellipsoids. Selected bond lengths (Å) and angles(deg): Ru(1)-C(1) 1.977(4), Ru(1)-N(7) 2.038(4), Ru(1)-N(6) 2.039(4), Ru(1)-N(4) 2.055(4), Ru(1)-N(3) 2.062(3), Ru(1)-N(5) 2.134(4), C(1)-Ru(1)-N(7) 92.76(15), C(1)-Ru(1)-N(6) 100.25(16), N(7)-Ru(1)-N(6) 90.48(14), C(1)-Ru(1)-N(4) 94.55(15), N(7)-Ru(1)-N(4) 172.59(13), N(6)-Ru(1)-N(4) 87.01(14), C(1)-Ru(1)-N(3) 78.39(16), N(7)Ru(1)-N(3) 94.47(14), N(6)-Ru(1)-N(3) 174.91(14), N(4)-Ru(1)-N(3) 88.20(14), C(1)-Ru(1)N(5) 169.20(16), N(7)-Ru(1)-N(5) 94.16(13), N(6)-Ru(1)-N(5) 87.98(14), N(4)-Ru(1)-N(5) 78.79(14), N(3)-Ru(1)-N(5) 92.79(14). Fig. S4. ORTEP drawing of Ru4 showing atomic numbering scheme at 50% probability ellipsoids. Selected bond lengths (Å) and angles(deg): Ru(1)-C(1) 1.983(6), Ru(1)-N(6) 2.035(6), Ru(1)-N(7) 2.042(6), Ru(1)-N(4) 2.056(5), Ru(1)-N(3) 2.078(5), Ru(1)-N(5) 2.129(5), C(1)-Ru(1)-N(6) 97.6(2), C(1)-Ru(1)-N(7) 88.1(2), N(6)-Ru(1)-N(7) 88.8(2), C(1)-Ru(1)-N(4) 100.1(2), N(6)-Ru(1)N(4) 89.8(2), N(7)-Ru(1)-N(4) 171.84(19), C(1)-Ru(1)-N(3) 78.7(2), N(6)-Ru(1)-N(3) 175.5(2), N(7)-Ru(1)-N(3) 88.6(2), N(4)-Ru(1)-N(3) 93.34(19), C(1)-Ru(1)-N(5) 173.9(2), N(6)-Ru(1)-N(5) 88.4(2), N(7)-Ru(1)-N(5) 93.0(2), N(4)-Ru(1)-N(5) 78.91(18), N(3)-Ru(1)-N(5) 95.4(2). B. Table S1. X-ray crystallographic data of Ru1-Ru4. 3 Ru1 Ru2 Ru3 Ru4 formula C29H30F12N8P2R u C35H29F12N7P2R u C39H37F12N7P2R u C47H37F12N7P2R u Fw. 881.62 938.66 994.77 1090.85 crystal system Monoclinic Monoclinic Triclinic Triclinic space group P2(1)/c P2(1)/c P-1 P-1 a/Å 10.494(7) 17.9763(9) 11.7572(16) 11.1717(6) b/Å 15.943(10) 11.8371(6) 13.3532(18) 12.5812(7) c/Å 22.151(15) 18.6576(12 15.822(2 18.4997(6) β/deg 92.845(12) 107.175(6) 70.759(2) 93.676(3) V/Å3 3701(4) 3793.1(4) 2334.6(5) 2454.0(2) Z 4 4 2 2 1.582 1.644 1.415 1.476 18573 17302 29121 31388 Dcalcd, Mg/m3 Refls collected Independent reflections, Rint Goodness-of-fit on F2 R1, wR2 [I > 2σ(I)] R1, wR2 (all data) Largest diff. peak and hole (e. Å-3) 6519, 0.0569 6677, 0.0520 8163, 0.0171 8632 , 0.0861 1.069 1.056 1.042 1.051 0.0495, 0.1316 0.0596, 0.1501 0.0557, 0.1448 0.0710, 0.1859 0.0758, 0.1615 0.0864, 0.1797 0.0656, 0.1511 0.1119, 0.2117 0.847 and -0.657 0.759 and -0.708 1.274 and -1.030 0.937 and -0.533 C. UV-vis spectra and emission spectra of Ru1-Ru4 in CH3CN 4 FigS1. (a) UV-vis spectra of Ru1-Ru4 in CH3CN. (b) Room-temperature emission spectra of Ru1-Ru4 in CH3CN ( excitation wavelength at 300 nm). D. MTT curves of Ru1-Ru4, cis-Pt, and HL·PF6 against cancer cells Fig.S2 The in vitro cytotoxic activities of Ru1-Ru4, cis-Pt, and HL·PF6 against cancer cells E. Cell experiment 5 Cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin), and were maintained in a humid atmosphere at 37 °C with 5% CO2. Drug solution preparation: Ru1-Ru4 were first dissolved in DMSO to form a 32 mM solution. Then take 4 μL of the solution and dilute it in 1 ml of 1640 medium to obtain a drug solution of 128 μM. The concentrations of 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM and 1 μM were obtained by multiple dilution. MTT viability assay The in vitro cytotoxicity of the Ru1-Ru4 complexes and cisplatin were measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells were plated in 96-well plates (4000-5000 cells per well) and incubated at 37 °C overnight. Then, cells were treated with a serial dilution of Ru1-Ru4 and cisplatin in various concentrations for an additional 48 h. Following exposure, 30 μL MTT solution (5 mg/mL in PBS) was added to each well. The MTT solution was removed from the wells after 4 h and the purple MTT-formazan crystals were then dissolved by the addition of DMSO (100 μL). The absorbance in each well was measured at 490 nm using a microplate reader (Multiskan FC, Thermo Scientific). Cell proliferation as determined by EdU incorporation HeLa cells were plated in 48-well plates (2×104 cells per well) and incubated at 37 °C overnight. Then, Ru1-Ru4 and cisplatin (4 μM) were added to the cells and then incubated for 24 h at 37 °C. DNA synthesis was quantified at the end of the drug treatment using a Click-iT EdU Alexa Fluor 488 Assay Kit (Invitrogen) according to the manufacturer’s protocol. Finally, the cells were imaged by fluorescence microscopy (Olympus, IX72, Japan), n ≥ 5 regions with 1500-2000 total cells were counted to assess the presence of cell proliferation Wound healing assay HUVEC cells were seeded into 6-well plates and reached 90% confluence after 24 h of incubation at 37 °C. A pipette tip (200 μL) was used to make a line across the cell monolayer. After washing with PBS, cells were cultured in serum-free culture medium containing Ru3, Ru4 and cisplatin ( 4 μM). Images of wounds were acquired by optical microscopy (Olympus, IX72, Japan) at 0 and 24 h of the incubation. The migration ratio (%) = change in width value of each group at each time / avenge width 6 of initial wound × 100%. Measurement of mitochondrial membrane potential HeLa cells were seeded in 6-well plates (2×105 cells/well) overnight. After 12 h of treatment with Ru3, Ru4, or cisplatin (4 μM), the cells were incubated with 5 μM of 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) for another 30 min. After incubation, the cells were washed twice with prewarmed PBS, harvested, and analyzed with a flow cytometer (Cytoflex, Beckman Coulter, American). The JC-1 fluorescence data were recorded and analyzed with the CytExpert software. A parallel batch of treated cells was stained with JC-1 and washed with PBS before visualization under a fluorescence microscope (Olympus, IX72, Japan). Intracellular Reactive Oxygen Species (ROS) detection For this assay, HeLa cells were seeded in a 6-well plate (2×105 cells/well). After 12 h of growth, the cells were treated with Ru3, Ru4, or cisplatin (4 μM) for 12 h, harvested, and washed twice with PBS. Then, the cells were resuspended in 1 mL of 1640 solution with 5 μM DCFH-DA and incubated for 30 min at 37 °C. After incubation, the samples were washed with PBS and analyzed for DCF fluorescence in a flow cytometer (Cytoflex, Beckman Coulter, American) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence data were recorded and analyzed with the CytExpert software with 1×104 cells in each sample. ROS generation was expressed in terms of the percentage of cells with DCF (green) fluorescence. Apoptosis analysis by flow cytometry HeLa cells were seeded at a density of 1 × 106 cells in each well and allowed to grow overnight at 37 °C.Then, the cells were incubated with Ru3, Ru4 and cisplatin (4 μM) for another 24 h. The untreated cells were used as the control. After drug treatment, cells were centrifuged and washed with cold PBS repeatedly. The cell apoptosis analysis was determined by an Alexa Fluor 488 annexin V/PI apoptosis detect