Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells

Phytother Res. 2014 Mar;28(3):387-94. doi: 10.1002/ptr.4999. Epub 2013 May 3.


Although beta-sitosterol has been well known to have anti-tumor activity in liver, lung, colon, stomach, breast and prostate cancers via cell cycle arrest and apoptosis induction, the underlying mechanism of anti-cancer effect of beta-sitosterol in multiple myeloma cells was never elucidated until now. Thus, in the present study, the role of reactive oxygen species (ROS) in association with AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK) pathways was demonstrated in beta-sitosterol-treated multiple myeloma U266 cells. Beta-sitosterol exerted cytotoxicity, increased sub-G1 apoptotic population and activated caspase-9 and -3, cleaved poly (ADP-ribose) polymerase (PARP) followed by decrease in mitochondrial potential in U266 cells. Beta-sitosterol promoted ROS production, activated AMPK, acetyl-CoA carboxylase (ACC) and JNK in U266 cells. Also, beta-sitosterol attenuated the phosphorylation of AKT, mammalian target of rapamycin and S6K, and the expression of cyclooxygenase-2 and VEGF in U266 cells. Conversely, AMPK inhibitor compound C and JNK inhibitor SP600125 suppressed apoptosis induced by beta-sitosterol in U266 cells. Furthermore, ROS scavenger N-acetyl L-cysteine attenuated beta-sitosterol-mediated sub-G1 accumulation, PARP cleavage, JNK and AMPK activation in U266 cells. Overall, these findings for the first time suggest that ROS-mediated activation of cancer metabolism-related genes such as AMPK and JNK plays an important role in beta-sitosterol-induced apoptosis in U266 multiple myeloma cells.

Keywords: AMPK; JNK; ROS; U266; apoptosis; beta-sitosterol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Acetyl-CoA Carboxylase / metabolism
  • Acetylcysteine / pharmacology
  • Anthracenes / pharmacology
  • Apoptosis / drug effects*
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism
  • Cell Cycle Checkpoints
  • Cell Line, Tumor
  • Cyclooxygenase 2 / metabolism
  • Humans
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • MAP Kinase Signaling System
  • Membrane Potential, Mitochondrial / drug effects
  • Multiple Myeloma / metabolism*
  • Phosphorylation
  • Poly(ADP-ribose) Polymerases / metabolism
  • Reactive Oxygen Species / metabolism*
  • Sitosterols / pharmacology*


  • Anthracenes
  • Reactive Oxygen Species
  • Sitosterols
  • pyrazolanthrone
  • gamma-sitosterol
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • Poly(ADP-ribose) Polymerases
  • JNK Mitogen-Activated Protein Kinases
  • AMP-Activated Protein Kinases
  • CASP3 protein, human
  • CASP9 protein, human
  • Caspase 3
  • Caspase 9
  • Acetyl-CoA Carboxylase
  • Acetylcysteine