title: Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB.
abstract: BACKGROUND
  Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells.
  
  
  METHODS
  In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays.
  
  
  RESULTS
  We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21(WAF1/Cip1)) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway.
  
  
  CONCLUSIONS
  According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Miroslav
authors.initials: M
authors.lastname: Genov
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Birgit
authors.initials: B
authors.lastname: Kreiseder
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Michael
authors.initials: M
authors.lastname: Nagl
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Drucker
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Martina
authors.initials: M
authors.lastname: Wiederstein
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Barbara
authors.initials: B
authors.lastname: Muellauer
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Julia
authors.initials: J
authors.lastname: Krebs
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Teresa
authors.initials: T
authors.lastname: Grohmann
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Dagmar
authors.initials: D
authors.lastname: Pretsch
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Karl
authors.initials: K
authors.lastname: Baumann
authors.affiliation: 2. Division of Chemistry of Renewables, Department of Chemistry, University of Natural Resources and Life Sciences, Konrad Lorenz Straße 24, A-3430 Tulln, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Bacher
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Alexander
authors.initials: A
authors.lastname: Pretsch
authors.affiliation: 1. SeaLife Pharma GmbH, Technopark 1, A-3430 Tulln, Austria;
authors.forename: Christoph
authors.initials: C
authors.lastname: Wiesner
keywords: 4-DACL; NF-κB; ROS; anthraquinone; apoptosis.; cell cycle arrest; melanoma; spheroids