New Insights into the Behavior of NHC-Gold Complexes in Cancer Cells

Abstract

Among the non-platinum antitumor agents, gold complexes have received increased attention owing to their strong antiproliferative effects, which generally occur through non-cisplatin-like mechanisms of action. Several studies have revealed that many cytotoxic gold compounds, such as N-heterocyclic carbene (NHC)-gold(I) complexes, are potent thioredoxin reductase (TrxR) inhibitors. Many other pathways have been supposed to be altered by gold coordination to protein targets. Within this frame, we have selected two gold(I) complexes based on aromatic ligands to be tested on cancer cells. Differently from bis [1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I) bromide (Au4BC), bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) inhibited TrxR1 activity in vitro. Treatment of Huh7 hepatocellular carcinoma (HCC) cells, and MDA-MB-231 triple-negative breast cancer (TNBC) cells, with Au4BC inhibited cell viability, increased reactive oxygen species (ROS) levels, caused DNA damage, and induced autophagy and apoptosis. Notably, we found that, although Au3BC inhibited TrxR1 activity, no effect on the cell viabilities of HCC and BC cells was observed. At the molecular level, Au3BC induced a protective response mechanism in Huh7 and MDA-MB-231 cells, by inducing up-regulation of RAD51 and p62 protein expression, two proteins involved in DNA damage repair and autophagy, respectively. RAD51 gene knock-down in HCC cells increased cell sensitivity to Au3BC by significant reduction of cell viability, induction of DNA damage, and induction of apoptosis and autophagy. All together, these results suggest that the tested NHC-Gold complexes, Au3BC and Au4BC, showed different mechanisms of action, either dependent or independent of TrxR1 inhibition. As a result, Au3BC and Au4BC were found to be promising candidates as anticancer drugs for the treatment of HCC and BC.

Keywords: NHC-gold(I) complexes; TrxR1 activity; antiproliferative effect; cancer cells.

Figure 1

Chemical structures of the selected complexes Au3BC and Au4BC.

Figure 2

Scheme of synthetic route of Au4BC.

Figure 3

Inhibition of TrxR1 activity by Au3BC and Au4BC in vitro. (A) recombinant rat liver TrxR1, and (B) 10 µg of protein samples extracted from Huh7 cells, were incubated with different concentrations of Au3BC and Au4BC, and then TrxR1 activity was assayed by a DTNB reduction assay. Auranofin was used as the positive control inhibitor for TrxR1. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Figure 4

Effects of Au3BC and Au4BC treatment on cell viability in HCC and BC cell lines. Huh7 and MDA-MB-231 cells were treated with different concentrations of auranofin, Au3BC, and Au4BC for 72 h. Cell viability was determined by using an MTS assay. Auranofin was used as the positive control inhibitor for TrxR1. Data are expressed as the percentage of control cells and are the means ± SD of three separate experiments, each performed in triplicate. ** p < 0.01, *** p < 0.005, **** p < 0.001.

Figure 5

Immunoblotting evaluation of apoptosis, autophagy, and DNA damage-related protein expression in HCC and BC cells after Au3BC and Au4BC treatments. Cells were exposed to the indicated Au3BC and Au4BC concentrations for 72 h. The PARP1 fragment (85 kDa) is indicated by an arrowhead. For LC3 protein, the relative protein expression levels of LC3II were calculated. The numbers in the bar plots represent the ratio of the relevant protein normalized with β-actin, with vehicle-treated control samples arbitrarily set at 1.0.

Figure 6

ROS generation in Huh7 and MDA-MB-231 cells treated with Au4BC. (A) Cells were untreated or treated with Au4BC at the indicated concentrations for 48 h, and intracellular ROS levels were evaluated using H₂DCFDA as a probe. Hydrogen peroxide (H₂O₂) was used as a positive control. Data are expressed as the percentage of control cells and are the means ± SD of two separate experiments, each performed in triplicate. * p < 0.05, ** p < 0.01. (B) Huh7 and MDA-MB-231 cells were pre-treated for 2 h with 5 mM NAC and then treated with Au4BC for 24 h in the presence or absence of NAC. Cell viability was assessed by an MTS assay. Data are expressed as in Figure 4. * p < 0.05; ** p < 0.01.

Figure 7

RAD51 knockdown sensitizes Huh7 cells to Au3BC-mediated cell death. (A) RAD51 expression levels in cells transfected for 72 h with RAD51 siRNA (siRAD51) or with control siRNA (siNC). (B) Cell viability after treatment with the indicated Au3BC concentrations for 72 h of cells transfected with siRAD51 or siNC. * p < 0.05. (C) Representative Western blotting of PARP1, γ-H2AX, RAD51, and p62 levels expressed in Huh7 cells transfected with siRAD51 or siNC. The PARP1 fragment (85 kDa) is indicated by an arrowhead. The numbers in the bar plots represent the ratio of the relevant protein normalized with β-actin, with vehicle-treated control samples arbitrarily set at 1.0.