title: Imaging induction of cytoprotective enzymes in intact human cells: coumberone, a metabolic reporter for human AKR1C enzymes reveals activation by panaxytriol, an active component of red ginseng.
abstract: We here present an optical method for monitoring the activity of the inducible aldo-keto reductases AKR1C2 and AKR1C3 in living human cells. The induction of these enzymes is regulated by the antioxidant response element (ARE), as demonstrated in recent literature, which in turn is dependent on the transcription factor Nrf2. The activation of ARE leads to the transcription of a coalition of cytoprotective enzymes and thus represents an important target for the development of new therapies in the area of neurodegenerative diseases and cancer. Through the use of Coumberone, a metabolic fluorogenic probe, and isoform-selective inhibitors, the upregulation of cellular stress markers AKR1C2 and AKR1C3 can be quantitatively measured in the presence of ARE activator compounds, via either a fluorimetric assay or fluorescence microscopy imaging of intact cells. The method has both high sensitivity and broad dynamic range, as demonstrated by induction studies in three cell lines with dramatically different metabolic capabilities (transfected monkey kidney COS-1 cells, human neuroblastoma IMR-32 cells, and human liver HepG2 cells). We applied the new method to examine a number of neurotrophic natural products (spirotenuipesine A, spirotenuipesine B, scabronine G-methylester, and panaxytriol), and discovered that panaxytriol, an active component of red ginseng extracts, is a potent ARE inducer. The upregulation of AKR1C enzymes, induced by chemically homogeneous panaxytriol, was partially dependent on PKC and PI3K kinases as demonstrated by the application of selective inhibitors. This cellular mechanism may account for panaxytriol's neurotrophic, neuroprotective, and anticancer properties. The protective effects of ARE inducers against tumorgenesis and neurodegeneration fuel the growing interest in this area of research and the method described here will greatly enable these endeavors.
authors.affiliation: Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, USA.
authors.forename: Marlin
authors.initials: M
authors.lastname: Halim
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Yee
authors.forename: Dalibor
authors.initials: D
authors.lastname: Sames
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D000074425:Aldo-Keto Reductase Family 1 Member C3; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D053281:Enediynes / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D005233:Fatty Alcohols / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D007213:Indomethacin / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D015394:Molecular Structure; D009410:Nerve Degeneration / Q000517:prevention & control; D018696:Neuroprotective Agents / Q000494:pharmacology; D005894:Panax / Q000737:chemistry*; D012680:Sensitivity and Specificity; D015854:Up-Regulation / Q000187:drug effects; D014580:Ursodeoxycholic Acid / Q000494:pharmacology; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
chemical_list: D015096:3-Hydroxysteroid Dehydrogenases; D000074425:Aldo-Keto Reductase Family 1 Member C3; D000970:Antineoplastic Agents; D053281:Enediynes; D005233:Fatty Alcohols; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D006911:Hydroxyprostaglandin Dehydrogenases; D006913:Hydroxysteroid Dehydrogenases; D007213:Indomethacin; D018696:Neuroprotective Agents; D014580:Ursodeoxycholic Acid; C533769:coumberone; C057682:panaxytriol; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C487341:AKR1C3 protein, human