title: Carnosic Acid Induces Apoptosis Through Reactive Oxygen Species-mediated Endoplasmic Reticulum Stress Induction in Human Renal Carcinoma Caki Cells.
abstract: BACKGROUND
  Carnosic acid, which is one of extract components of rosemary, has anti-inflammatory, anti-oxidant, and anti-cancer effects. However, the anti-cancer effect of carnosic acid in human renal carcinoma cells is unknown.
  
  
  METHODS
  Flow cytometry analysis was used to examine the effects of carnosic acid on apoptosis, and Asp-Glu-Val-Asp-ase activity assay kit was used to investigate the involvement of caspase activation. To determine protein expression of apoptotic and endoplasmic reticulum (ER) stress-related proteins, we used Western blotting. Intracellular accumulation of reactive oxygen species (ROS) was determined using the fluorescent probes 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA).
  
  
  RESULTS
  Carnosic acid induced sub-diploid DNA content, sub-G1, population and poly (ADP-ribose) polymerase (PARP) cleavage and activated caspase-3. A pan-caspase inhibitor, a benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone, markedly reduced apoptosis in carnosic acid-treated cells. Carnosic acid promoted intracellular ROS production, and pretreatment with the ROS scavengers (N-acetyl-L-cysteine and glutathione ethyl ester) inhibited carnosic acid-induced apoptosis. Furthermore, carnosic acid also induced expression of ER stress marker proteins, including activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-homologous protein (CHOP), in a dose- and time-dependent manner. Down-regulation of ATF4 and CHOP by small interfering RNA (siRNA) markedly reduced carnosic acid-induced sub-G1 population and PARP cleavage. In addition, carnosic acid induced apoptosis in human breast carcinoma MDA-MB-361 and human hepatocellular carcinoma SK-HEP1 cells, but not in normal human skin fibroblast cells and normal mouse kidney epithelial TMCK-1 cells.
  
  
  CONCLUSIONS
  Carnosic acid induced apoptosis through production of ROS and induction of ER stress in human renal carcinoma Caki cells.
authors.affiliation: Department of Immunology, School of Medicine, Keimyung University, Daegu, Korea.
authors.forename: Kyoung-Jin
authors.initials: KJ
authors.lastname: Min
authors.affiliation: Department of Immunology, School of Medicine, Keimyung University, Daegu, Korea.
authors.forename: Kyong-Jin
authors.initials: KJ
authors.lastname: Jung
authors.affiliation: Department of Immunology, School of Medicine, Keimyung University, Daegu, Korea.
authors.forename: Taeg Kyu
authors.initials: TK
authors.lastname: Kwon
keywords: Activating transcription factor 4; CCAAT/enhancer-binding protein-homologous protein; Carnosic acid; Reactive oxygen species