Switching Off HSP70 and i-NOS to Study Their Role in Normal and H2O2-stressed Human Fibroblasts

Life Sci. 2003 Dec 26;74(6):757-69. doi: 10.1016/j.lfs.2003.07.016.

Abstract

i-NOS and HSP70 antisense oligonucleotides were used to study the role of the two well known stress-regulated molecules on cell survival of both untreated control, and H2O2-stressed human fibroblasts. Cell survival was assessed either by LDH release or by MTT assay. The levels of cytosolic i-NOS and HSP70 were tested by using immunoblotting analysis, and reactive oxygen species (ROS) production was quantified. Compared to the values observed in untreated control cells, anti HSP70-transfected human fibroblasts showed an increase in ROS production, i-NOS level and LDH release. The addition of 0.12 mM H2O2 for 20 min. to the HSP70-deprived fibroblasts did not modify the percentage of LDH release observed in H2O2 stressed cells, but reduced cell viability increasing both ROS production and i-NOS level. Anti i-NOS-transfected fibroblasts, compared to the control untreated cells, showed no modification in ROS production, while cell survival was improved. When treated with H2O2 the i-NOS depleted cells counteracted ROS formation as well as LDH release but negatively affected cell viability and HSP70 levels, compared to the results obtained with H2O2 alone-treated fibroblasts. The data indicates that the induced decrease in HSP70 level in oxidative stress conditions makes fibroblasts more prone to oxidative injury and also increases i-NOS level. Whereas in one way the forced decrease in i-NOS expression seems to counteract ROS production stimulated by the oxidative insult in the cells, in another way, since it causes a decrease in HSP70 expression as well as in cell viability, it seems to activate some unidentified pathways affecting cell demise.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Transformed
  • Cell Survival / drug effects
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Formazans / metabolism
  • HSP70 Heat-Shock Proteins / genetics*
  • HSP70 Heat-Shock Proteins / metabolism
  • Humans
  • Hydrogen Peroxide / metabolism
  • Hydrogen Peroxide / pharmacology*
  • L-Lactate Dehydrogenase / metabolism
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II
  • Oligonucleotides, Antisense / pharmacology*
  • Oxidative Stress*
  • Reactive Oxygen Species / metabolism
  • Tetrazolium Salts / metabolism
  • Transfection

Substances

  • Formazans
  • HSP70 Heat-Shock Proteins
  • Oligonucleotides, Antisense
  • Reactive Oxygen Species
  • Tetrazolium Salts
  • MTT formazan
  • Hydrogen Peroxide
  • L-Lactate Dehydrogenase
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II