title: Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells.
abstract: Tocotrienols and tocopherols represent the two subgroups that make up the vitamin E family of compounds. However, tocotrienols display significantly more potent apoptotic activity in neoplastic mammary epithelial cells than tocopherols. Studies were conducted to determine the intracellular mechanism(s) mediating tocotrienol-induced apoptosis in neoplastic +SA mouse mammary epithelial cells in vitro. An initial step in apoptosis is the activation of 'initiator' caspases (caspase-8 or -9) that subsequently activate 'effector' caspases (caspase-3, -6 and -7) and induce apoptosis. Treatment with cytotoxic doses of alpha-tocotrienol (20 microM) resulted in a time-dependent increase in caspase-8 and caspase-3 activity. Combined treatment with specific caspase-8 or caspase-3 inhibitors completely blocked alpha-tocotrienol-induced apoptosis and caspase-8 or caspase-3 activity, respectively. In contrast, alpha-tocotrienol treatment had no effect on caspase-9 activation, and combined treatment with a specific caspase-9 inhibitor did not block alpha-tocotrienol-induced apoptosis in (+)SA cells. Since caspase-8 activation is associated with the activation of death receptors, such as Fas, tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL) receptors, studies were conducted to determine the exact death receptor(s) and ligand(s) involved in mediating tocotrienol-induced caspase-8 activation and apoptosis. Treatment with Fas-ligand (FasL), Fas-activating antibody, or TRAIL failed to induce cell death in (+)SA neoplastic mammary epithelial cells, suggesting that these cells are resistant to death receptor-induced apoptosis. Moreover, treatment with cytotoxic doses of alpha-tocotrienol did not alter the intracellular levels of Fas, FasL, or Fas-associated death domain (FADD) in these cells. Western blot analysis also showed that alpha-tocotrienol did not induce FasL or FADD translocation from the cytosolic to membrane fraction in these cells. Finally, treatment with Fas-blocking antibody did not reverse the tocotrienol-induced apoptosis in (+)SA cells. These data demonstrate that tocotrienol-induced caspase-8 activation and apoptosis is not mediated through death receptor activation in malignant (+)SA mammary epithelial cells. Resistance to death receptor-induced apoptosis has been shown to be associated with increased expression of apoptosis-inhibitory proteins, such as FLICE-inhibitory protein (FLIP), and enhanced signalling of the phosphatidylinositol 3-kinase (PI3K)/PI3K-dependent kinase (PDK)/Akt mitogenic pathway. Additional studies showed that treatment with cytotoxic doses of alpha-tocotrienol decreased total, membrane, and cytosolic levels of FLIP, and reduced phosphorylated PDK-1 (active) and phosphorylated-Akt (active) levels in these cells. In summary, these findings demonstrate that tocotrienol-induced caspase-8 activation and apoptosis in malignant (+)SA mammary epithelial cells is not mediated through the activation of death receptors, but appears to result from the suppression of the PI3K/PDK/Akt mitogenic signalling pathway, and subsequent reduction in intracellular FLIP expression.
authors.affiliation: College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209, USA. sylvester@ulm.edu
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sylvester
authors.forename: Sumit
authors.initials: S
authors.lastname: Shah
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D015674:Mammary Neoplasms, Animal / Q000201:enzymology* / Q000473:pathology*; D008562:Membrane Glycoproteins; D051379:Mice; D010766:Phosphorylation; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand; D024508:Tocotrienols / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha
chemical_list: D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506387:Cflar protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505654:Tnfsf10 protein, mouse; D024508:Tocotrienols; D014409:Tumor Necrosis Factor-alpha; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases