Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Exp Biol Med (Maywood). 2004 Sep;229(8):745-55. doi: 10.1177/153537020422900806.


Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma / enzymology
  • Adenocarcinoma / pathology
  • Animals
  • Antioxidants / pharmacology
  • Apoptosis / physiology*
  • Apoptosis Regulatory Proteins
  • Caspase 8
  • Caspases / metabolism*
  • Cell Death / drug effects*
  • Cell Line, Tumor
  • Cell Survival
  • Enzyme Activation
  • Epithelial Cells / enzymology
  • Epithelial Cells / pathology
  • Female
  • Mammary Neoplasms, Animal / enzymology*
  • Mammary Neoplasms, Animal / pathology*
  • Membrane Glycoproteins / pharmacology
  • Mice
  • Mice, Inbred BALB C
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Receptors, Tumor Necrosis Factor / drug effects
  • Receptors, Tumor Necrosis Factor / physiology
  • Signal Transduction
  • TNF-Related Apoptosis-Inducing Ligand
  • Tocotrienols / pharmacology*
  • Tumor Necrosis Factor-alpha / pharmacology


  • Antioxidants
  • Apoptosis Regulatory Proteins
  • Membrane Glycoproteins
  • Proto-Oncogene Proteins
  • Receptors, Tumor Necrosis Factor
  • TNF-Related Apoptosis-Inducing Ligand
  • Tnfrsf21 protein, mouse
  • Tnfsf10 protein, mouse
  • Tocotrienols
  • Tumor Necrosis Factor-alpha
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Casp8 protein, mouse
  • Caspase 8
  • Caspases