Synthesis, Characterization, Cytotoxic Activity, and Interactions with CT-DNA and BSA of Cationic Ruthenium(II) Complexes Containing Dppm and Quinoline Carboxylates

The complexes cis-[Ru(quin)(dppm)2]PF6 and cis-[Ru(kynu)(dppm)2]PF6 (quin = quinaldate; kynu = kynurenate; dppm = bis(diphenylphosphino)methane) were prepared and characterized by elemental analysis, electronic, FTIR, 1H, and 31P{H1} NMR spectroscopies. Characterization data were consistent with a cis arrangement for the dppm ligands and a bidentate coordination through carboxylate oxygens of the quin and kynu anions. These complexes were not able to intercalate CT-DNA as shown by circular dichroism spectroscopy. On the other hand, bovine serum albumin (BSA) binding constants and thermodynamic parameters suggest spontaneous interactions with this protein by hydrogen bonds and van der Waals forces. Cytotoxicity assays were carried out on a panel of human cancer cell lines including HepG2, MCF-7, and MO59J and one normal cell line GM07492A. In general, the new ruthenium(II) complexes displayed a moderate to high cytotoxicity in all the assayed cell lines with IC50 ranging from 10.1 to 36 µM and were more cytotoxic than the precursor cis-[RuCl2(dppm)2]. The cis-[Ru(quin)(dppm)2]PF6 were two to three times more active than the reference metallodrug cisplatin in the MCF-7 and MO59J cell lines

Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells

<div><p>Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. The high rate of metastasis associated to the fact that these cells frequently display multidrug resistance, make the treatment of metastatic disease difficult. Development of antitumor metal-based drugs was started with the discovery of cisplatin, however, the severe side effects represent a limitation for its clinical use. Ruthenium (Ru) complexes with different ligands have been successfully studied as prospective antitumor drugs. In this work, we demonstrated the activity of a series of biphosphine bipyridine Ru complexes <b>(1)</b> [Ru(SO₄)(dppb)(bipy)], <b>(2)</b> [Ru(CO₃)(dppb)(bipy)], <b>(3)</b> [Ru(C₂O₄)(dppb)(bipy)] and <b>(4)</b> [Ru(CH₃CO₂)(dppb)(bipy)]PF₆ [where dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2’-bipyridine], on proliferation of TNBC (MDA-MB-231), estrogen-dependent breast tumor cells (MCF-7) and a non-tumor breast cell line (MCF-10A). Complex <b>(4)</b> was most effective among the complexes and was selected to be further investigated on effects on tumor cell adhesion, migration, invasion and in apoptosis. Moreover, DNA and HSA binding properties of this complex were also investigated. Results show that complex <b>(4)</b> was more efficient inhibiting proliferation of MDA-MB-231 cells over non-tumor cells. In addition, complex <b>(4)</b> was able to inhibit MDA-MB231 cells adhesion, migration and invasion and to induce apoptosis and inhibit MMP-9 secretion in TNBC cells. Complex <b>(4)</b> should be further investigated <i>in vivo</i> in order to stablish its potential to improve breast cancer treatment.</p></div

Effect of complex 4 on MDA-MB-231, MCF-7 and MCF-10A cells.

<p><b>(A)</b> Cell morphology was examined after 2 h and 24 h of treatment. <b>(B)</b> Clonogenic assay of untreated MDA-MB-231 cells (control) or cells treated with complex <b>(4)</b>. A photograph of a representative experiment is shown along with graph quantifications of colony number and size. Significant at the *<i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p

Absorption spectral titration.

<p><b>(A)</b> Absorption spectral titration of complex <b>(4)</b> in the presence of increasing concentrations of <i>ct</i>-DNA at 298 K and fluorescence emission spectra of HSA at 37°C. Inset graph represents the plots of (ε_a-ε_f)/(ε_b-ε_f) versus [DNA] for the titration of DNA with Ru(II) complexes. <b>(B)</b> CD spectrum of <i>ct</i>-DNA (100 μM) in the presence of complexes <b>(1–4)</b> in Tris-HCl buffer after incubating by 18 h at 37°C. <b>(C)</b> Electrophoretic mobility pattern of pBR322 plasmid DNA incubated with metal complexes <b>(1–4)</b> by 18 h at 37°C, at indicated concentrations (μM). Ri = ratio complex/DNA; MM = molecular marker. <b>(D)</b> Fluorescence quenching spectra of HSA with different concentrations of complex (4) with the excitation wavelength at 270 nm at 37°C in a Trizma buffer, pH 7.4. The arrow shows the intensity changes upon increasing the concentration of the quencher (0 to 50 μM, orange line to light blue line, respectively).</p

Interactions with DNA and HSA.

<p>Binding constants for the interaction between Ru(II) complexes <b>(1–4)</b> and calf thymus <i>ct</i>-DNA and stern-Volmer quenching constant (K_sv, M^-1), biomolecular quenching rate constant (Kq, M^-1s^-1), binding constant (Kb, M^-1), the number of binding sites (n), ΔG(KJ.mol^-1), ΔH (KJ.mol^-1) and ΔS (J.mol^-1K) values for the complex-HSA system at different temperatures.</p

Effects of complex (4) on MDA-MB-231 cell adhesion and cytoskeleton structure.

<p><b>(A)</b> Type I collagen, fibronectin, laminin and vitronectin were coated on the wells of a 96-well plate. Complex <b>(4)</b> was incubated with MDA-MB-231 cells, which were subsequently plated on the ECM coatings. After washes, remaining cells were quantified by colorimetry. <b>(B)</b> MDA-MB-231 cells were incubated with complex <b>(4)</b> and then with Phalloidin and DAPI. Significant at the *<i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p

Effect of complex (4) on apoptosis in MDA-MB-231 breast tumor cells and MCF-10A non-tumor breast cells.

<p><b>(A)</b> After treatment with the indicated concentrations of complex <b>(4)</b>, cells were incubated with PE-Annexin-V and 7AAD for 15min, harvested and then analyzed by cytometry. <b>(B)</b> The percentage of apoptotic and necrotic cells was plotted in a graph for MDA-MB-231 and MCF-10A cells. The fluorescence of 7AAD is detected in the FL3-A channel and the fluorescence of PE-Annexin-V is detected in the FL2-A channel. Camptothecin (campto) was used as a positive control for apoptosis. <b>(C)</b> Nuclear fragmentation promoted by complex <b>(4)</b> in MDA-MB-231 cells was investigated using DAPI staining. Staurosporine was used as a positive control for nuclear fragmentation. White arrows show fragmented nuclei. The expression of apoptotic and anti-apoptotic molecules was investigated by <b>(D)</b> qRT-PCR and <b>(E)</b> Western blotting analysis. Significant at the *<i>p</i><0.005, **<i>p</i><0.001, ***<i>p</i><0.0001 levels using ANOVA and Bonferroni tests.</p

IC₅₀, SI and Log <i>P</i> values for complexes (1–4) and cisplatin in each cell line, after 24 h of incubation.

<p>IC₅₀, SI and Log <i>P</i> values for complexes (1–4) and cisplatin in each cell line, after 24 h of incubation.</p

Crystal structures of the complexes.

<p><b>(A) (1)</b> [Ru(SO₄)(dppb)(bipy)], <b>(B) (2)</b> [Ru(CO₃)(dppb)(bipy)], <b>(C) (3)</b> [Ru(C₂O₄)(dppb)(bipy)] and <b>(D) (4)</b> [Ru(CH₃CO₂)(dppb)(bipy)]PF₆, showing the atoms labelling and the 30% probability ellipsoids. For 4, the PF₆^- anion was omitted, for sake of clarity.</p

Effects of complex (4) on MDA-MB-231 cell migration and invasion.

<p><b>(A)</b> Complex was incubated with MDA-MB-231 and cells were allowed to migrate in Boyden chambers. Migrated cells were fixed and quantified by manual counting. Representative images of the inserts are represented above each condition. The wells were photographed under a light microscope under 40× magnifications. Positive control (C+) represents migrating cells without any treatment and negative control (C-) was cells migrating toward an FBS-free medium. <b>(B)</b> Wound healing assay at 0, 24 and 48 h of treatment with the complex. <b>(C)</b> Effect of complex <b>(4)</b> on MDA-MB-231 cell invasion through matrigel. <b>(D)</b> Zymography in 1% gelatin-SDS-PAGE. A photograph of a representative zymography gel is shown. Gels were analyzed by densitometry, and data were normalized in percentage compared to untreated control cell lysate. Significant at the * <i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p