This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have previously reported that cholesterol secoaldehyde (ChSeco), a putative product of ozone reaction with cholesterol in aqueous environments, induces apoptosis in H9c2 cell line. The present study further investigated the involvement of apoptotic-related proteins and gene expression using quantitative real-time (RT) PCR and western blot analyses, and appropriate biochemical assays. The RT-PCR analysis revealed that ChSeco activates the expression of genes involved in the death receptor (extrinsic) pathway. The significance of this pathway was also evident based on increased activity of caspase-8. The overexpression of Apaf-1, loss of mitochondrial transmembrane potential and subsequent release of cytochrome c, and increased activity of caspase-9, further provide strong evidence for the involvement of mitochondrial (intrinsic) pathway. Thus, it appears that a combined activation of the intrinsic and extrinsic pathways resulted in the activation of effector caspase-3/7. Prior treatment of H9c2 cells with caspase-8- and -9-specific inhibitors decreased the activity of caspase-3 and the extent of cytotoxicity was significantly lowered by Z-Val-Ala-Asp-fluoromethyl ketone, a pancaspase inhibitor (PCI). Western blot analysis showed that, in H9c2 cells exposed to ChSeco, the expression of neither Bcl-2 or Bax was altered, although there was a decrease in the phosphorylated Bcl-2. A time-course analysis of the ChSeco-treated H9c2 cells showed an upstream rapid increase in the generation of reactive oxygen species (ROS) and the associated decrease in intracellular glutathione. N-Acetyl-L-cysteine and Trolox significantly attenuated the ChSeco-induced ROS formation and cytotoxicity and also down-regulated the expression of genes of all the players of either pathway. This study clearly demonstrated that ChSeco induces apoptosis in H9c2 cells through ROS generation and activation of both the intrinsic and extrinsic pathways.
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