Lipophilic Fe(III)-Complex with Potent Broad-Spectrum Anticancer Activity and Ability to Overcome Pt Resistance in A2780cis Cancer Cells

Abstract

Although iron is essential for all forms of life, it is also potentially toxic to cells as the increased and unregulated iron uptake can catalyze the Fenton reaction to produce reactive oxygen species (ROS), leading to lipid peroxidation of membranes, oxidation of proteins, cleavage of DNA and even activation of apoptotic cell death pathways. We demonstrate that Fe(hinok)3 (hinok = 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), a neutral Fe(III) complex with high lipophilicity is capable of bypassing the regulation of iron trafficking to disrupt cellular iron homeostasis; thus, harnessing remarkable anticancer activity against a panel of five different cell lines, including Pt-sensitive ovarian cancer cells (A2780; IC50 = 2.05 ± 0.90 μM or 1.20 μg/mL), Pt-resistant ovarian cancer cells (A2780cis; IC50 = 0.92 ± 0.73 μM or 0.50 μg/mL), ovarian cancer cells (SKOV-3; IC50 = 1.23 ± 0.01 μM or 0.67 μg/mL), breast cancer cells (MDA-MB-231; IC50 = 3.83 ± 0.12 μM or 2.0 μg/mL) and lung cancer cells (A549; IC50 = 1.50 ± 0.32 μM or 0.82 μg/mL). Of great significance is that Fe(hinok)3 exhibits unusual selectivity toward the normal HEK293 cells and the ability to overcome the Pt resistance in the Pt-resistant mutant ovarian cancer cells of A2780cis.

Transferrin-inspired iron delivery across the cell membrane using [(L2Fe)2(μ-O)] (L = chlorquinaldol) to harness anticancer activity of ferroptosis

“…The experimental procedure was adopted from our recent publication. 23 Briefly, cells of A2780cis were cultured in RPMI medium for 24 hours at 37 °C and in 5% CO 2 atmosphere, followed by treating them with [(L 2 Fe) 2 (μ-O)] at the concentration of 1.0 μM and incubating the treated cells for 24 hours under the same conditions. After washing with 1× PBS and fresh RPMI each 2 times, a 5 μL solution of calcein AM in anhydrous DMSO (4 mM) and a 10 μL solution of ethidium homodimer-1 DMSO/water of 1 : 4 v/v (2 mM) were consecutively added to a 10 mL RPMI cell culture medium to prepare a LIVE/DEAD working solution.…”

“…The protocol used for nuclear accumulation experiments was adopted from one of our recent publications. 23 The main experimental steps include: (i) cells of A2780cis were seeded at the density of 4 × 10 5 cells per mL in a 6-well plate and incubated for 24 hours; (ii) the above cells were treated with 8 μM of [(L 2 Fe) 2 (μ-O)], followed by incubation for 4 hours; (iii) cells were washed with 1× PBS 3 times, harvested and counted, followed by isolation of mitochondria using the mitochondria isolation kit for mammalian cells (Thermo Scientific); (iv) the isolated mitochondria were digested with HNO 3 at room temperature overnight; and (v) Fe contents were analyzed by AAS after the iron oxides formed at 620 °C for 5 hours were dissolved in aqua regia and the solution volume was adjusted to the desired value.…”

“…The protocol used for cell membrane permeabilization assay was adopted from the same publication. 23 The main experimental steps include: (i) cells of A2780cis were seeded at the density of 2 × 10 4 cells per mL in a 35 mm sterile glass bottom culture dish and incubated for 12 hours; (ii) the above cells were treated with 8 μM of [(L 2 Fe) 2 (μ-O)] as the treatment group and or FeCl 3 as the control group, followed by incubation for 24 hours; (iii) cells were washed with 1× PBS 3 times and incubated with a medium containing PI dye (2 μM) for 20 minutes at 37 °C; and (iv) after the medium was replaced with 500 μL dye-free RPMI, the cells were examined under a fluorescence microscope.…”

“…The protocol used for cell viability assay was adopted from our recent publication. 23 The keep experimental parameters are: (i) cells were first seeded at the density of 8 × 10 4 cells per mL in a 96-well plate each with a 100 μL cell suspension and then incubated for 24 hours at 37 °C in 5% CO 2 atmosphere; (ii) the above cells were then treated with different amounts of [(L 2 Fe) 2 (μ-O)] each dissolved in 100 μL of fresh medium, followed by incubation under the same conditions for 24 hours; (iii) 10 μL of MTT reagent was added to each well containing the drug-treated cells and incubated for 2 hours at 37 °C, followed by addition of 100 μL of detergent to each well; and (iv) after the plate was kept in the dark for 2 hours at 37 °C, the absorbance was measured at 570 nm with a SpectraMax M4 microplate reader.…”

“…The protocol used for intracellular ROS measurements was adopted from the same recent publication. 23 The main experimental steps include: (i) cells of A2780cis were plated at the density of 2 × 10 4 cells per mL in a in black, clear-bottomed, 96-well plate and incubated for 12 hours; (ii) the above cells were treated with varying amounts of [(L 2 Fe) 2 (μ-O)], followed by the addition of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to the cell culture to reach the final concentration of 10 µM and incubated in dark for 30 minutes. ; (iii) cells were washed with 1× PBS 2 times and resuspended in 100 µL of PBS; and (iv) the fluorescence intensity of solution was measured using a SpectraMax M4 microplate reader at the wavelength of 497/529 nm (excitation/emission).…”