title: The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells.
abstract: METHODS
  Western blotting and flow cytometric analyses were performed using rat notochordal cells.
  
  
  OBJECTIVE
  To demonstrate the apoptotic effect of oxidative stress and the antiapoptotic effects of caspase inhibitors on rat notochordal cells.
  
  
  BACKGROUND
  Although oxidative stress causes apoptosis in many cell types, its effect on the apoptosis of notochordal cell and antiapoptotic effects of caspase inhibitors on the oxidative stress-induced apoptosis are unknown.
  
  
  METHODS
  Cultured rat notochordal cells were exposed to oxidative stress [500 micromol/L of hydrogen peroxide (H2O2)]. To determine the oxidative stress-induced apoptotic pathways, activations of caspases (-3, -8, and -9) as well as cleavages of Bid and poly (ADP-ribose) polymerase (PARP) were evaluated with Western blotting 6 hours after oxidative stress. To elucidate the antiapoptotic effects of caspase inhibitors on the oxidative stress induced-apoptosis, apoptotic rates of notochordal cells with or without treatment of specific caspase inhibitors (z-IETD-fmk for caspase-8, z-LEHD-fmk for caspase-9, and z-DEVD-fmk for caspase-3) were quantified by flow cytometry.
  
  
  RESULTS
  Oxidative stress significantly increased apoptosis of rat notochordal cells (2.1% vs. 4.75%, P = 0.008) and led to activations of initiators of intrinsic (caspases-9) and extrinsic (caspase-8) pathways as well as their common executioner (caspase-3). It also caused cleavages of Bid and PARP. Flow cytometric analysis showed that inhibition of only one of the intrinsic and extrinsic pathways by caspase-9 inhibitor (4.75% vs. 3.56%, P = 0.31) and caspase-8 inhibitor (4.75% vs. 5.24%, P = 0.84) did not significantly suppress the oxidative stress-induced apoptosis. However, inhibition of both pathways by caspase-3 inhibitor significantly reduced the oxidative stress-induced apoptosis (4.75% vs. 2.64%, P = 0.008) to the control level (2.1% vs. 2.64%, P = 0.15).
  
  
  CONCLUSIONS
  Oxidative stress caused apoptosis of rat notochordal cells via both intrinsic and extrinsic (Type I and Type II) pathways. Because caspase inhibitors are being used in clinical trials, inhibition of both pathways using caspase inhibitors might be of future therapeutic importance in oxidative stress-induced apoptosis of notochordal cells. Our results suggest that inhibition of inappropriate or premature oxidative stress-induced apoptosis of notochordal cells may delay the starting point of disc degeneration.
authors: Department of Orthopedic Surgery, St. Mary's Hospital, College of Medicine, Catholic University of Korea, Seoul, Korea.
authors: Ki-Won
authors: KW
authors: Kim
authors: Kee-Yong
authors: KY
authors: Ha
authors: Jun-Seok
authors: JS
authors: Lee
authors: Kee-Won
authors: KW
authors: Rhyu
authors: Howard S
authors: HS
authors: An
authors: Young-Kyun
authors: YK
authors: Woo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D009672:Notochord / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016877:Oxidants; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases