A biscarbene gold(I)-NHC-complex overcomes cisplatin-resistance in A2780 and W1 ovarian cancer cells highlighting pERK as regulator of apoptosis

Abstract

Purpose: Cisplatin resistance is the major obstacle in the clinical treatment of ovarian cancer patients. Molecular mechanisms of cisplatin resistance are multifaceted. Gold(I)-compounds, i.e. N-heterocyclic carbene-gold(I)-complexes (NHC-Au(I)) has been regarded as promising cytotoxic drug candidates. However, their potential to overcome cisplatin resistance has hardly been addressed yet. Here we investigated the activity of the gold(I) drug auranofin and the NHC-Au(I)-compound MC3 in W1CR and A2780cis cisplatin-resistant ovarian cancer cells.

Methods: Cytotoxicity of auranofin and MC3 was detected by MTT assay, correlated with intracellular gold(I) content, analyzed by AAS, and with flow cytometric detection of the cell cycle. Insight into cellular redox balance was provided by fluorimetric ROS-formation assay and western blotting thioredoxin (Trx) and Nrf2. The role of ERK was elucidated by using the inhibitor SCH772984 and its impact on cytotoxicity upon co-treatment with cisplatin and Au(I)-compounds, respectively.

Results: MC3 overcomes cisplatin resistance in A2780cis and W1CR, and auranofin in W1CR cells completely, which is neither reflected by intracellular gold levels nor cell cycle changes. Upregulated redox balance appears as a basis for resistance. W1CR cells possess higher Trx levels, whereas A2780cis cells display strong Nrf2 expression as anti-oxidative protection. Nevertheless, overcoming redox balance appears not primary mode of activity comparing cisplatin and gold(I)-compounds. pERK emerges as a critical component and thus a promising target for overcoming resistance, regulating apoptosis differently in response to either gold(I) or cisplatin in A2780 cells.

Conclusion: These data reflect the complexity of cisplatin resistance in cell models and emphasize NHC-Au(I)-complexes as prospective cytotoxic agents for further investigations in that respect.

Keywords: Chemoresistance; Cisplatin; ERK-signaling; Gold(I)-NHC-complex; Ovarian cancer; Thioredoxin reductase.

Scheme 1

Gold(I) complexes applied and their proposed mode of action. Adapted from [10]

Fig. 1

Exemplary cytotoxicity-curves of auranofin in A2780/cis cells (A) and in W1/W1CR cells (B), and of MC3 in A2780/cis cells (C) and in W1/W1CR cells (D) compared to cisplatin display an overcoming of cisplatin resistance; tables below curves show means ± SD of biological and technical triplicates. A cellular pretreatment with gold compounds at sub-toxic concentrations prior to cisplatin treatment indicates no impact on cisplatin activity and refers to a different mode of action of cisplatin and gold compounds in at least n = 3 biological and technical samples ± SD. Tables below histograms show the resulting CI at the IC₅₀ of the combinational treatments (E and F). Intracellular gold levels of W1/CR and A2780/cis cells after treatment with 0.1 µM auranofin (G) and 0.01 µM MC3 (H) for 72 h analyzed by AAS indicate no correlation of intracellular gold concentrations and cytotoxicity for overcoming resistance. Shown data represent n = 3 samples ± SD, asterisks indicate statistical significance *p < 0.05; **p < 0.01

Fig. 2

Intracellular increase of ROS as a quotient of 24 h and 4 h measurement as n = 4 ± SD (A). Comparison of the relative Nrf2 expression in untreated cell pairs normalized to W1 or A2780 cells, respectively (B). Relative Nrf2 expression in W1/W1CR cells (C) or A2780/A2780cis cells (D) upon treatment normalized on untreated cells, respectively. Relative Trx expression in W1 and W1CR cells normalized on untreated cells (E), and of A2780 and A2780cis cells normalized on untreated cells (F) using 1 µM cisplatin, 0.1 µM auranofin and 0.01 µM MC3. All Western Blot data represent means ± SD of at least n = 3 biological samples. Asterisks indicate statistical significance *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

A Overview of investigated signaling pathways. B Flow cytometric analysis of cell cycle in A2780 and A2780cis cells upon the indicated treatments with cisplatin (cDDP), auranofin, or MC3. C The expression of ERK and pERK in untreated A2780 and A2780cis cells and the effects of cytotoxic treatment using 1 µM cisplatin, 0.1 µM auranofin and 0.01 µM MC3, indicated by a representative blot (above) and as calculated ratio pERK/ERK normalized to untreated A2780 or A2780cis cells, respectively (below). Statistical analysis was performed by one-way ANOVA following Dunnettt’s test. D The impact of blocking ERK by sub-toxic concentrations of SCH772984 on the cytotoxicity of Au(I)-compounds in A2780 cells (grey) and A2780cis cells (black); and dose-dependently on the cytotoxicity of cisplatin (E). All shown data represent means ± SD of at least n = 3 biological samples, asterisks indicate statistical significance *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001