No abstract

The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and g-tocotrienol, but not a-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and g-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 mM TRF or 20 mM g-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

Tocotrienols, a subgroup within the vitamin E family of compounds, have been shown to display potent anticancer activity and inhibit preneoplastic and neoplastic mammary epithelial cell proliferation at treatment doses that have little or no effect on normal cell growth and function. However, the specific intracellular mechanisms mediating the antiproliferative effects of tocotrienols are presently unknown. Because Akt and nuclear factor kappaB (NFkappaB) are intimately involved in mammary tumor cell proliferation and survival, studies were conducted to determine the effects of gamma-tocotrienol on Akt and NFkappaB activity in neoplastic +SA mammary epithelial cells in vitro. Treatment with 0-8 microM gamma-tocotrienol for 0-3 days caused a dose-responsive inhibition in +SA cell growth and mitotic activity, as determined by MTT colorimetric assay and proliferating cell nuclear antigen immunocytochemical staining, respectively. Studies also showed that treatment with 4 microM gamma-tocotrienol, a dose that inhibited +SA cell growth by more than 50% compared with that of untreated control cells, decreased intracellular levels of activated phosphotidylinositol 3-kinase-dependent kinase (PI3K)-dependent kinase 1 (phospho-PDK-1) and Akt, and reduced phospho-Akt kinase activity. Furthermore, these effects were not found to be associated with an increase in either phosphatase and tensin homologue deleted from chromosome 10 (PTEN) or protein phosphatase type 2A phosphatase activity. In addition, gamma-tocotrienol treatment was shown to decrease NFkappaB transcriptional activity, apparently by suppressing the activation of IkappaB-kinase-alpha/beta, an enzyme associated with inducing NFkappaB activation. In summary, these findings demonstrate that the antiproliferative effects of gamma-tocotrienol result, at least in part, from a reduction in Akt and NFkappaB activity in neoplastic +SA 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.

UBE1L is the E1-like ubiquitin-activating enzyme for the IFN-stimulated gene, 15-kDa protein (ISG15). The UBE1L-ISG15 pathway was proposed previously to target lung carcinogenesis by inhibiting cyclin D1 expression. This study extends prior work by reporting that UBE1L promotes a complex between ISG15 and cyclin D1 and inhibited cyclin D1 but not other G 1 cyclins. Transfection of the UBE1L-ISG15 deconjugase, ubiquitin-specific protein 18 (UBP43), antagonized UBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation. A lysineless cyclin D1 species was resistant to these effects. UBE1L transfection reduced cyclin D1 protein but not mRNA expression. Cycloheximide treatment augmented this cyclin D1 protein instability. UBE1L knockdown increased cyclin D1 protein. UBE1L was independently retrovirally transduced into human bronchial epithelial and lung cancer cells. This reduced cyclin D1 expression and clonal cell growth. Treatment with the retinoid X receptor agonist bexarotene induced UBE1L and reduced cyclin D1 immunoblot expression. A proof-of-principle bexarotene clinical trial was independently examined for UBE1L, ISG15, cyclin D1, and Ki-67 immunohistochemical expression profiles in pretreatment versus post-treatment tumor biopsies. Increased UBE1L with reduced cyclin D1 and Ki-67 expression occurred in human lung cancer when a therapeutic bexarotene intratumoral level was achieved. Thus, a mechanism for UBE1L-mediated growth suppression was found by UBE1L-ISG15 preferentially inhibiting cyclin D1. Molecular therapeutic implications are discussed.