Abstract
BackgroundCancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. PDAC is one of the most lethal cancers and often associated with a high accumulation of ROS. Recent studies identified gold(I) NHC complexes as potent TrxR inhibitors suppressing cell growth in a wide spectrum of human malignant cell lines at the low micromolar concentration. However, the mechanism of action is not completely elucidated yet.MethodsTo understand the biological function of gold(I) NHC complexes in PDAC, we used a recently published gold(I) NHC complex, MC3, and evaluated its anti-proliferative effect in four PDAC cell lines, determined by MTT and SRB assays. In further detailed analysis, we analyzed cellular ROS levels using the ROS indicator DHE and mitochondrial membrane potential indicated by the dye JC-1 in Panc1. We also analyzed cell cycle arrest and apoptosis by FACS. To elucidate the role of specific cell signaling pathways in MC3-induced cell death, co-incubation with ROS scavengers, a p38-MAPK inhibitor and siRNA mediated depletion of ASK1 were performed, and results were analyzed by immunoblotting, ELISA-microarrays, qRT-PCR and immunoprecipitation.ResultsOur data demonstrate that MC3 efficiently suppressed cell growth, and induced cell cycle arrest and apoptosis in pancreatic cancer cells, in particular in the gemcitabine-resistant cancer cells Panc1 and ASPC1. Treatment with MC3 resulted in a substantial alteration of the cellular redox homeostasis leading to increased ROS levels and a decrease in the mitochondrial membrane potential. ROS scavengers suppressed ROS formation and rescued cells from damage. On the molecular level, MC3 blocked the interaction of Trx with ASK1 and subsequently activated p38-associated signaling. Furthermore, inhibition of this pathway by using ASK1 siRNA or a p38 inhibitor clearly attenuated the effect of MC3 on cell proliferation in Panc1 and ASPC1.ConclusionsOur results confirm that MC3 is a TrxR inhibitor and show MC3 induced apoptosis in gemcitabine-resistant PDACs. MC3 mediated cell death could be blocked by using anti-oxidants, ASK1 siRNA or p38 inhibitor suggesting that the Trx-ASK1-p38 signal cascade played an important role in gold(I) NHC complexes-mediated cellular damage.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-221) contains supplementary material, which is available to authorized users.
Highlights
Cancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive Reactive oxygen species (ROS) resulting in the compensation for intrinsic oxidative stress and therapeutic resistance
We evaluated the cytotoxicity of the gold(I) metal-complexes MC2, MC3 and MC4 in four pancreatic cancer cell lines, namely Bxpc3, Miapaca2, Panc1 and ASPC1 using MTT assay
After 72 h treatment we observed that Gem was sufficient to block 50% of cell growth in Bxpc3 and Miapaca2 cells at 10 nM, whereas IC50 values were more than 20 μM in Panc1 and ASPC1 (Figure 1B)
Summary
Cancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. The rapid proliferation of cancer cells requires high metabolic activity, which includes increased glycolysis and an elevation of other metabolic reactions. Due to this increase in metabolic rate, cancer cells, in particular, those in advanced stage are prone to high oxidative stress caused by abundant reactive oxygen species, considered to mainly originate from electronic leakage of mitochondrial respiratory complexes [14,15]. There are strong evidences that adaptive mechanisms enable cancer cells to escape from oxidative damage [18,19] by means of overexpressing ROS scavengers including Thioredoxin (Trx) and/or Glutathione (Glu) and pro-survival proteins like Bcl-xl [20]. Activation of both, redox control and antiapoptotic signaling will help cancer cells to cope with lethality in response to aberrant ROS levels
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