title: A TrxR inhibiting gold(I) NHC complex induces apoptosis through ASK1-p38-MAPK signaling in pancreatic cancer cells.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  To 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.
  
  
  RESULTS
  Our 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.
  
  
  CONCLUSIONS
  Our 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.
authors.forename: Xinlai
authors.initials: X
authors.lastname: Cheng
authors.forename: Palvo
authors.initials: P
authors.lastname: Holenya
authors.forename: Suzan
authors.initials: S
authors.lastname: Can
authors.forename: Hamed
authors.initials: H
authors.lastname: Alborzinia
authors.forename: Riccardo
authors.initials: R
authors.lastname: Rubbiani
authors.forename: Ingo
authors.initials: I
authors.lastname: Ott
authors.affiliation: Institut für Pharmazie und Molekulare Biotechnologie, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. wolfl@uni-hd.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wölfl
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003720:Densitometry; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D050607:Organogold Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000473:pathology*; D011485:Protein Binding / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000093542:Gemcitabine
chemical_list: D016166:Free Radical Scavengers; D050607:Organogold Compounds; D017382:Reactive Oxygen Species; D003841:Deoxycytidine; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D000093542:Gemcitabine