Antiproliferative effects, mechanism of action and tumor reduction studies in a lung cancer xenograft mouse model of an organometallic gold(i) alkynyl complex

Abstract

Organometallic complexes offer a wide range of properties like structural variety, reaction kinetics, tunable lipophilicity and alternate mechanisms of activation under physiological conditions compared to platinum chemotherapeutics and are thus being explored for their potential anticancer applications. In this regard, gold(i) organometallics hold a pivotal position for their ability to act on biological targets different from DNA (which is the primary target of platinum therapeutics), such as thioredoxin reductase. Here, we report on the stability, in vitro antiproliferative effects, protein binding, cellular uptake, mechanism of action, effects on mitochondrial respiration of cancer cells as well as in vivo tolerance, toxicity and tumor reduction in an A549 lung cancer xenograft mouse model of an organometallic gold(i) complex (1) bearing 4-ethynylanisole and triethylphosphane as ligands. The complex, which was stable in DMSO and reactive towards N-acetylcysteine, triggered strong antiproliferative effects in various cancer cell lines and had a protein binding of approximately 65% that reduced its generally efficient uptake into tumor cells. Antimetastatic properties were indicated for 1 in a scratch assay and strong inhibition of thioredoxin reductase (TrxR) was confirmed for the purified enzyme as well as in A549 lung cancer cells, which strongly overexpress TrxR. Real time monitoring of the oxygen consumption rate in multiple cancer cell lines, using the Seahorse Mito stress assay, demonstrated that mitochondrial respiration was severely disrupted, showing a significantly low oxygen consumption rate. Other respiratory parameters, such as proton efflux, spare respiratory capacity and maximal respiration, were also attenuated upon treatment with 1. The complex was well tolerated in vivo in mice at a dose of 10 mg kg^-1 and showed tumor reduction compared to the control group of animals in a lung cancer xenograft model of nude mice. In summary, complex 1 represents a novel organometallic anticancer drug candidate with a mechanism related to TrxR inhibition and mitochondrial respiration inhibition, showing efficient in vivo antitumor efficacy.

Fig. 1. Structures of the FDA approved antiarthritic gold(i) drug auranofin and of reported organometallic gold(i) complexes bearing an alkynyl ligand, such as 1.

Fig. 2. HPLC-MS analysis of 400 μM 1 in DMSO (left) and a 400 μM equimolar mixture of 1 and NAC at 37 °C over 96 h; retention times: 4.5 min for 1 and 2.2 min for 4-ethynylanisole.

Fig. 3. (a) Percentage of gold bound to proteins in the presence of fetal bovine serum (FBS) after incubation at different time points measured using ICP-OES; (b) cellular uptake of complex 1 in MDA-MB-231 and A549 cells after different incubation times in the presence or absence of FBS.

Fig. 4. Live cell imaging in A549 cells with and without 1 (10 μM), showing changes in the cell morphology at 0, 24 and 48 h.

Fig. 5. Effect of 1 on MDA-MB-231 cells after inducing a scratch and allowing the cells to grow for 24 h. Untreated cells showed nearly complete wound closure in 24 h while the cells treated with 1 (2 μM) showed a reduced rate of wound closure.

Fig. 6. Seahorse Mito stress assay in HT-29 cells (a) and MDA-MB-231 cells (b) showing the effect of 1 and auranofin on mitochondrial respiration. Oligomycin (ATP synthase inhibitor), FCCP (proton decoupler), rotenone (electron transport chain complex 3 inhibitor) and rotenone (electron transport chain complex 1 inhibitor) were added at the time points shown by the arrows. Comparison of the respiratory parameters between untreated cells (control) and cells treated with either 1 or auranofin: basal respiration (c), maximal respiration (d) and ATP production (e) in HT-29 and MDA-MB-231 cells as indicated in the graphs.

Fig. 7. Docking of 1 (black), (phosphine)Au⁺ (green) and alkynyl-Au⁺ (brown) into TrxR1 (PDB: 2J3N, Sec → Cys); receptor surface: H-bonding (red), mild polar (blue), and hydrophobic (grey).

Fig. 8. In vivo study in an A549 lung cancer xenograft model in animal groups treated with the vehicle or complex 1 (10 mg kg⁻¹): mean body weight (a) and tumor volume (b). Biodistribution of gold in vital organs of mice treated with 1 (n = 5) (c).