Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells.
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.
Shah
Sumit
S
School of Pharmacy, 700 University Avenue, University of Louisiana at Monroe, Monroe, Louisiana 71209-0470, USA.
Sylvester
Paul W
PW
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000201:enzymology / Q000473:pathology; D005260:Female; D015674:Mammary Neoplasms, Animal / Q000201:enzymology* / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand; D024508:Tocotrienols / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D018124:Receptors, Tumor Necrosis Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; C485251:Tnfrsf21 protein, mouse; C505654:Tnfsf10 protein, mouse; D024508:Tocotrienols; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases