Exploiting Co(III)-Cyclopentadienyl Complexes To Develop Anticancer Agents

Abstract

In recent years, organometallic complexes have attracted much attention as anticancer therapeutics aiming at overcoming the limitations of platinum drugs that are currently marketed. Still, the development of half-sandwich organometallic cobalt complexes remains scarcely explored. Four new cobalt(III)-cyclopentadienyl complexes containing N,N-heteroaromatic bidentate, and phosphane ligands were synthesized and fully characterized by elemental analysis, spectroscopic techniques, and DFT methods. The cytotoxicity of all complexes was determined in vitro by the MTS assay in colorectal (HCT116), ovarian (A2780), and breast (MDA-MB-231 and MCF-7) human cancer cell lines and in a healthy human cell line (fibroblasts). The complexes showed high cytotoxicity in cancer cell lines, mostly due to ROS production, apoptosis, autophagy induction, and disruption of the mitochondrial membrane. Also, these complexes were shown to be nontoxic in vivo in an ex ovo chick embryo yolk sac membrane (YSM) assay.

Scheme 1. Synthesis of the New Complexes of General Formula [CoCp(PPh₃)(NN)][(CF₃SO₃)₂] (Cp = η⁵-C₅H₅; NN = N,N-Heteroaromatic Ligands): 1 (NN = bipy = 2,2′-Bipyridine); 2 (NN = Me₂bipy = 4,4′-Dimethyl-2,2′-bipyridine); 3 (NN = phen = 1,10′-Phenanthroline); 4 (NN = NH₂phen = 5-Amino-1,10′-phenanthroline)

Figure 1

Electronic spectra of [CoCp(PPh₃)(bipy)][(CF₃SO₃)₂] (1, ––), its precursor [CoCp(CO)I₂] (- - -), and free bipy ligand (^···) in dichloromethane.

Figure 2

Evaluation of the stability of complex 1 in 90% D₂O/10% DMSO-d₆ solution (2.5 mM) over 24 h, by ¹H NMR (left) and ³¹P{H} NMR (right).

Figure 3

DFT optimized structures of complexes 1–4. For 4, the possible pair of diastereoisomers was considered (denoted as 4′ and 4″). Relevant distances are given in Angstrom.

Figure 4

TD-DFT excitations plotted against the UV–visible absorption spectra for complexes 1–4. The excitations reported in Table S2 are highlighted (*). Both calculated and experimental spectra were obtained in dichloromethane.

Figure 5

Representative natural transition orbital pairs along with the respective coefficients for the TD-DFT calculated low-energy excitations of complexes 1-4. For 4, only 4″ is represented for simplicity.

Figure 6

Cellular viability (%) in A2780, HCT116, MDA-MB-231 and MCF-7 after 48 h of exposure to complexes 1 (A), 2 (B), 3 (C), and 4 (D). Data normalized against the control (0.1% (v/v) DMSO) and expressed as the mean ± SEM of at least two independent assays. The symbols ** and * represent p < 0.0005 and p < 0.05, respectively. Statistical analysis was performed by two-way ANOVA method.

Figure 7

Cellular viability (%) in A2780 after 12 h of exposure to 20× IC₅₀ concentrations of complexes 2 – 4 at 37 and 4 °C. Data normalized against the control (0.1% (v/v) DMSO) and expressed as the mean ± SEM of at least two independent assays. The symbols ** and * represent p < 0.05 and p < 0.5, respectively. Statistical analysis was performed by the two-way ANOVA method.

Figure 8

Apoptosis induction in the A2780 cell line exposed to IC₅₀ of the complexes 2–4 for 48 h by flow cytometry. Annexin V/PI double staining was used along with DMSO 0.1% (v/v) as negative control (solvent control) and 0.4 μM DOX and 5 μM cisplatin (Cis) as positive controls. Data expressed as the mean ± SD of three independent assays. The symbols ***, **, and * represent p < 0.0005, p < 0.005 and p < 0.05, respectively. Statistical analysis was performed by the two-way ANOVA method.

Figure 9

Evaluation of the mitochondrial membrane potential (ΔΨM) changes in the A2780 cell line exposed to IC₅₀ of the 2–4 complexes for 48 h by flow cytometry. The JC-1 staining was used along with DMSO 0.1% (v/v) as a negative control (solvent control) and cisplatin (Cis) 5 μM as positive control. Data normalized against the DMSO control and expressed as the mean ± SEM of two independent assays. Statistical analysis was performed by two-way ANOVA method.

Figure 10

Relative expression of BAX (A) and BCL-2 (B) proteins in the A2780 cell line incubated for 48 h with IC₅₀ of the complexes 2–4. (C) BAX/BCL-2 ratio in A2780 exposed to the different complexes. (D) Western Blot bands used for the quantification of proteins BAX and BCL-2 in A2780 cells after their exposure to complexes 2–4 or DMSO. DMSO 0.1% (v/v) was used as negative control (solvent control). Data normalized against the DMSO control (values represented as a dotted line at the value of Y = 1) and expressed as the mean ± SEM. The symbols ****, ***, **, and * represent p < 0.0001, p < 0.001, p < 0.01, and p < 0.1, respectively. Statistical analysis was performed by the two-way ANOVA method.

Figure 11

Caspase 8 activity in A2780 cell line incubated with the complexes 2–4 for 48 h. DMSO 0.1% (v/v) was used as negative control (solvent control) and DOX 0.4 μM and cisplatin (Cis) 5 μM as positive controls. Caspase 8 activity was quantified using the caspase 8 assay kit (Abcam). Data normalized against the DMSO control (value represented as a dot line at y = 1) and expressed as the mean ± SEM of three independent assays. The symbols ** and * represent p < 0.0005 and p < 0.005, respectively. Statistical analysis was performed by two-way ANOVA method.

Figure 12

Evaluation of the autophagy induction in A2780 cell line after 48 h of exposure to the IC₅₀ of the complexes 2–4 by flow cytometry using the Autophagy assay. DMSO 0.1% (v/v) was used as negative control (solvent control) and DOX 0.4 μM, cisplatin (Cis) 5 μM, and Rapamycin 0.5 μM as positive controls. Data expressed as the mean ± SEM of two independent assays. The symbol * represents p < 0.0005. Statistical analysis was performed by two-way ANOVA method.

Figure 13

Evaluation of the production of reactive oxygen species (ROS) in A2780 cell line after 48 h of exposure to the IC₅₀ of the complexes 2–4 by flow cytometry. DMSO 0.1% (v/v) was used as negative control (solvent control) and DOX 0.4 μM, cisplatin (Cis) 5 μM, and TBHP 22.2 μM as positive controls. Data normalized against the DMSO control (value represented as a dot line at y = 1) and expressed as the mean ± SEM of two independent assays. The symbols ** and * represent p < 0.0005 and p < 0.05, respectively. Statistical analysis was performed by two-way ANOVA method.

Figure 14

Cell cycle progression in A2780 cells after 9 h, 18 h, and 24 h of exposure to the IC₅₀ concentrations of the complexes 2–4, 0.4 μM DOX, 5 μM Cisplatin or DMSO 0.1% (v/v). DMSO 0.1% (v/v) was used as a vehicle control. Data expressed as the mean ± SEM of two independent assays. The statistical analysis was done by the two-way ANOVA test against the DMSO control. The symbols *, **, *** and **** represent p < 0.1, p < 0.01, p < 0.001 and p < 0.0001, respectively.

Figure 15

Evaluation of the fibroblasts’ remission after 24 h of exposure to the IC₅₀ of the complexes 2–4 by inverted microscopy. DMSO 0.1% (v/v) was used as negative control (vehicle control) and DOX 0.4 μM was used as positive control. Data normalized against the DMSO control (value represented as a dot line at y = 100%) and expressed as the mean ± SEM of three independent assays. The symbols * represent p < 0.05.

Figure 16

Evaluation of the complexes potentiality to modulate angiogenesis. (A) RGB image of the O-ring interior; (B) Green channel of the same image used for counting the number of veins; (C) Binary of the segmented image used to calculate the number of branches; (D) Percentage of newly formed vessels in ex ovo YSMs after 24 and 48 h exposure to IC₅₀ concentrations of complexes 2–4. It was used at least 7 independent chicken embryos experiments for each condition. Data normalized against the DMSO control (100% dotted line) and expressed as the mean ± SEM of two independent assays.