title: The 1,4 benzoquinone-featured 5-lipoxygenase inhibitor RF-Id induces apoptotic death through downregulation of IAPs in human glioblastoma cells.
abstract: Embelin is a potent dual inhibitor of 5-lipoxigenase (5-LOX) and microsomal prostaglandin E2 synthase (mPGES)-1 that suppresses proliferation of human glioma cells and induces apoptosis by inhibiting XIAP and NF-κB signaling pathway. Synthetic structural modification yielded the derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id), an embelin constrained analogue, with improved efficiency against 5-LOX in human neutrophils and anti-inflammatory activity in vivo. Taking into account that lipoxygenase (LOX) metabolites, from arachidonic acid and linoleic acid, have been implicated in tumor progression, here, we determined whether RF-Id was able to hinder glioblastoma (GBM) cancer cell growth and the related mechanisms.
  
  
  
  U87MG and LN229 cells were plated in 96-wells and treated with increasing concentrations of RF-Id. Cell viability was evaluated by MTT assay. The effects of the compounds on cell cycle, apoptosis, oxidative stress and autophagy were assessed by flow cytometry (FACS). The mode of action was confirmed by Taqman apoptosis array and evaluating caspase cascade and NFκB pathway by western blotting technique.
  
  
  
  Here, we found that RF-Id induced a stronger inhibition of GBM cell growth than treatment with embelin. Flow cytometry analysis showed that RF-Id induced about 30 % apoptosis and a slight increase of autophagy after 72 h on U87-MG cells. Moreover, the compound induced an increase in the percentage of cells in G2 and S phase that was paralleled by an increase of p21 and p27 expression but no significant changes of the mitochondrial membrane potential; array analysis showed a significant upregulation of CASP8 and a downregulation of IAP family and NFκB genes in cells treated with RF-Id. RF-Id induced a significant cleavage of caspases 8, 9, 3 and 7, blocked c-IAP2/XIAP interaction by inducing XIAP degradation and inhibited NFκB pathway.
  
  
  
  RF-Id induced a caspase-dependent apoptosis in GBM cells by inhibiting IAP family proteins and NFκB pathway and represents a promising lead compound for designing a new class of anti-cancer drugs with multiple targets.
authors: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via L. De Crecchio 7, Naples, 80138, Italy.
authors: S
authors: S
authors: Zappavigna
authors: Department of Experimental Medicine, Second University of Naples, Via L. De Crecchio, 7, Naples, 80138, Italy.
authors: M
authors: M
authors: Scuotto
authors: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via L. De Crecchio 7, Naples, 80138, Italy.
authors: A M
authors: AM
authors: Cossu
authors: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via L. De Crecchio 7, Naples, 80138, Italy.
authors: D
authors: D
authors: Ingrosso
authors: Department of Experimental Medicine, Second University of Naples, Via L. De Crecchio, 7, Naples, 80138, Italy.
authors: M
authors: M
authors: De Rosa
authors: Department of Experimental Medicine, Second University of Naples, Via L. De Crecchio, 7, Naples, 80138, Italy.
authors: C
authors: C
authors: Schiraldi
authors: Department of Experimental Medicine, Second University of Naples, Via L. De Crecchio, 7, Naples, 80138, Italy. rosanna.filosa@unina2.it.
authors: R
authors: R
authors: Filosa
authors: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, via L. De Crecchio 7, Naples, 80138, Italy. michele.caraglia@unina2.it.
authors: M
authors: M
authors: Caraglia
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D001343:Autophagy; D016227:Benzoquinones / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism*; D019787:Linoleic Acid / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D016718:Arachidonic Acid; D019787:Linoleic Acid; D001094:Arachidonate 5-Lipoxygenase; C583212:ALOX5 protein, human; C010945:embelin
keywords: Apoptosis; Glioblastoma; IAPs; NFκB; RF-Id