EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shah, S.
Right arrow Articles by Sylvester, P. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shah, S.
Right arrow Articles by Sylvester, P. W.
Experimental Biology and Medicine 229:745-755 (2004)
© 2004 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Tocotrienol-Induced Caspase-8 Activation Is Unrelated to Death Receptor Apoptotic Signaling in Neoplastic Mammary Epithelial Cells

Sumit Shah and Paul W. Sylvester1

School of Pharmacy, 700 University Avenue, University of Louisiana at Monroe, Monroe, Louisiana 71209-0470

1To whom requests for reprints should be addressed at School of Pharmacy, 700 University Avenue, University of Louisiana at Monroe, Monroe, LA 71209-0470. E-mail: sylvester{at}ulm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µM {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 µg/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 µM {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 µM {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.

Key Words: tocotrienols • breast cancer • apoptosis • caspase-8 • FLIP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Previous investigations have demonstrated that tocotrienols, a subgroup in the vitamin E family of compounds, have potent apoptotic activity in neoplastic mammary epithelial cells (1–4). These findings are particularly interesting because they are observed using treatment doses that have little or no effect on normal mammary epithelial cell growth or viability (5, 6). Apoptosis is a highly regulated process executed by the activation of specific enzymes called caspases, a family of cysteine-dependent aspartyl proteases (7, 8). Caspases are constitutively present in cells as inactive proenzymes that must be cleaved and processed for activation (7, 8). Caspases can be classified as either initiator caspases (caspase-2, caspase-8, caspase-9, and caspase-10) that subsequently activate effector caspases (caspase-3, caspase-6, and caspase-7), which are responsible for most of the proteolytic cleavages associated with cellular self-destruction (7, 8).

Activation of initiator caspases can occur through two general mechanisms. The first mechanism involves death receptor activation and apoptotic signaling. Death receptors represent a subgroup in the tumor necrosis factor (TNF) superfamily of receptors that includes TNF-R1, Fas, and TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2 (9, 10). Specific ligands to these death receptors include TNF-{alpha} that activates TNF-R1, Fas ligand (FasL) that activates Fas, and TRAIL that activates TRAIL-R1 and TRAIL-R2 (9, 10). Following activation, death receptors complex with adapter proteins such as Fas-associated via death domain (FADD) and initiator procaspases such as procaspase-8 to form the death-inducing signaling complex (DISC) at the membrane (9, 10). Within the DISC, procaspase-8 is cleaved to form the active p20 subunit of caspase-8 that subsequently cleaves and activates effector caspases, such as caspase-3, caspase-6, or caspase-7 (9, 10). The second mechanism mediating caspase activation is the "mitochondrial stress" apoptotic signaling pathway. Various stresses such as chemicals and radiation destabilize the outer mitochondrial membrane and cause the release of apoptotic proteins such as cytochrome-c and apoptosis-inducing factor (AIF) into the cytoplasm (11). Cytochrome-c then interacts with apoptotic protease activation factor-1 (APAF-1) and initiator procaspases, such as procaspase-9, to form the apoptosome. Following apoptosome formation, procaspase-9 is cleaved to form active caspase-9 subunit that is then released into the cytoplasm and activates downstream effector caspases (12).

Recent studies have shown that tocotrienol-induced apoptosis in neoplastic +SA mammary epithelial cells is mediated by specific intracellular signaling mechanisms that lead to sequential increase in activated caspase-8 and caspase-3 levels and activity (2, 4, 13). These studies also showed that tocotrienol-induced programmed cell death was not associated with an increase in activated caspase-9 levels or activity (2, 4, 13). Since caspase-8 processing and activation is associated with death receptor, whereas caspase-9 activation is associated with mitochondrial stress apoptotic signaling, these findings strongly suggest that tocotrienol-induced apoptosis is mediated by receptor-dependent caspase activation. However, the specific death receptor(s) and ligand(s) that mediate tocotrienol-induced caspase activation and apoptosis have not yet been determined (2, 4, 13).

Recently, it has been established that some cancer cells are resistant to death receptor–induced apoptosis (14–19). Death receptor resistance can result from a reduction in death receptors and/or ligand expression, death receptor gene mutation, or increased FLICE-inhibitory protein (FLIP) expression (14–19). FLICE-inhibitory protein is an endogenous inhibitor of caspase-8 activation that prevents the recruitment of procaspase-8 to the DISC and inhibits the processing of procaspase-8 to the active p20 subunit of caspase-8 (18–23). Studies have shown that activation of various growth factors and cytokine receptors stimulates the phosphatidylinositol 3-kinase (PI3K)/PI3K-dependent kinase 1 (PDK-1)/Akt mitogenic pathway, increases FLIP expression, promotes cell proliferation, and enhances cell survival (24–28). Phosphatidylinositol 3-kinase activates PDK-1 by phosphorylating Ser241 (29, 30). PDK-1 activates Akt by phosphorylating Thr308 and Ser473 (24,29, 30). Although the exact mechanism by which Akt regulates FLIP expression is unknown, suppression of PI3K/PDK-1/Akt mitogenic signaling leads to a decrease in FLIP levels and a corresponding increase in caspase-8 activity (24, 25). Therefore, studies were conducted to determine the specific death receptor(s) and ligand(s) that are involved in mediating tocotrienol-induced caspase-8 activation and apoptosis in neoplastic +SA mammary epithelial cells. Additional studies examined the effects of tocotrienols on PI3K/PDK-1/Akt mitogenic signaling and FLIP expression in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Line and Culture Conditions.
The highly malignant +SA mammary epithelial cell line was derived from an adenocarcinoma that developed spontaneously in a BALB/c female mouse (31–33). Cell culture and experimental procedures have been previously described in detail (1, 3–6, 34, 35). Briefly, +SA cells were maintained in serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 control media containing 5 mg/ml bovine serum albumin (BSA), 10 ng/ml epidermal growth factor (EGF), and 10 µg/ml insulin. For subculturing, cells were rinsed twice with sterile Ca2+-free and Mg2+-free phosphate buffered saline (PBS) and incubated in 0.05% trypsin containing 0.025% EDTA in PBS for 5 mins at 37°C. The isolated cells were washed once and then resuspended in serum-free media, and cells were counted by hemocytometer. Cells were plated at a density of 1 x 105 cells/well in 24-well culture plates (viability studies) or 1 x 106 cells/ 100-mm culture plates (Western blot and Akt kinase activity studies).

Chemicals.
All chemicals were purchased from Sigma (Sigma Chemical Co., St. Louis, MO) unless otherwise stated. Purified {gamma}-tocotrienol was generously provided by Dr. Abdul Gapor of the Malaysian Palm Oil Board (Kuala Lumpur, Malaysia). Caspase-8 inhibitor II (z-IETD-fmk), caspase-3 inhibitor V (z-DQMD-fmk), and LY294002 were purchased from Calbiochem-Novabiochem Corporation (La Jolla, CA); nonspecific caspase inhibitor (z-Asp-CH2-DCB) and geldanamycin were purchased from Biomol Research Laboratories (Plymouth Meeting, PA); recombinant mouse TNF-{alpha} and Fas activation antibody were purchased from Chemicon International (Temecula, CA); recombinant human FasL plus and recombinant human TRAIL were purchased from Oncogene Research Products (San Diego, CA); antibodies for caspase-8, Fas, and FasL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); caspase-3 antibody was purchased from Trevigen (Gaithersburg, MD); antibodies for FADD and FLIP were purchased from Upstate Biotechnology (Lake Placid, NY); antibodies for phospho-PDK-1 (Ser241), total phosphatase and tensin homologue deleted from chromosome 10 (PTEN), phospho-PTEN (Ser380/Thr382/383), total Akt, and phospho-Akt (Ser473) were purchased from Cell Signaling Technology (Beverly, MA); and anticytochrome oxidase subunit IV (COX) was purchased from MolecularProbes (Eugene, OR).

Experimental Treatments.
In order to dissolve {gamma}-tocotrienol in aqueous culture media, a stock solution of {gamma}-tocotrienol was first prepared in sterile 10% BSA solution as previously described (1, 3–6, 34, 35). This solution of {gamma}-tocotrienol/BSA was then used to prepare 0–20 µM {gamma}-tocotrienol–supplemented treatment media. Sterile stock solutions for the caspase inhibitors, LY294002, and geldanamycin were prepared in dimethyl sulfoxide (DMSO) and added to the culture media to obtain the final concentration. Stock solutions for TNF-{alpha}, Fas activation antibody, FasL, and TRAIL were prepared in sterile water and then added to the culture media to achieve the final concentration.

Measurement of Viable Cell Number.
+SA cell number was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay as described previously (1, 3–6, 34, 35). Briefly, at the end of treatment exposure, cells were incubated at 37°C for 4 hrs with fresh control media containing 0.83 mg/ml MTT. After this incubation, the media was removed, MTT crystals were dissolved in 1 ml isopropanol, and the optical density of each sample was read at 570 nm on a microplate reader (SpectraCount, Packard BioScience Company, Meriden, CT).

Terminal dUTP Nick End Labeling (TUNEL) Assay.
Apoptosis was determined using the in situ cell death detection kit, POD (Roche Diagnostics Corporation, Indianapolis, IN) as previously described (4). Briefly, +SA cells were grown in serum-free control media to 60%–70% confluency on Lab-Tek chamber slides (Nalgene Nunc International, Naperville, IL), treated for 24 hrs with 0–20 µM {gamma}-tocotrienol, washed once with PBS, and then fixed with 4% paraformaldehyde. Slides were rinsed with PBS and air dried. TUNEL assay was performed, and slides were counterstained with hematoxylin. Photomicrographs were taken at 100x magnification. Positive TUNEL staining was quantified by image analysis using the Metamorph Analysis Software (Universal Imaging Corp., Downingtown, PA). The percentage of positive TUNEL cells was determined by counting the TUNEL positive cells versus the total number of cells in five photomicrographs (80,000 µm2 area per photomicrograph) for each treatment group.

Cell Lysis and Fractionation.
+SA cells in the various treatment groups were isolated with trypsin and washed once with PBS as described above. Whole cell lysates were resuspended in lysis buffer, boiled for 5 mins, sonicated, and microcentrifuged at 4°C and supernatant was collected (1, 3–6, 34, 35). Subcellular fractionation was performed by resuspending cells in fractionation buffer and cells were disrupted by 30 strokes of Dounce homogenizer in ice as described previously (36). The lysates were then incubated on ice for 5 mins and centrifuged at 14,000 g for 1 hr at 4°C. Supernatants were collected as the cytosolic fractions. The remaining membrane fraction pellets were then dissolved in lysis buffer, boiled for 5 mins, sonicated, and microcentrifuged at 4°C, and supernatants were collected.

Western Blotting.
+SA cells in the various treatment groups were isolated by trypsin or whole cell lysates or cytosolic and membrane fractions were prepared, and protein concentration was determined using a BioRad protein assay kit (BioRad, Hercules, CA). Samples (20 µg for whole lysates and 10 µg for membrane/cytosolic fractions) were loaded on 10%–15% polyacrylamide minigels and electrophoresed; Western blot analysis was performed as previously described (3, 4, 6, 34, 35); and scanning densitometric analysis was performed with Metamorph Analysis software (Universal Imaging Corp.).

Akt Kinase Activity Assay.
Akt kinase activity in +SA cells was measured using an assay kit purchased from Cell Signaling Technology. Briefly, +SA cells were treated with 20 µM {gamma}-tocotrienol for 0–8 hrs. At the end of treatment exposure, cells were rinsed once with PBS and lysed on ice for 10 mins with the cell lysis buffer provided in the kit, and protein concentration was determined as described above. Akt was immunoprecipitated with immobilized Akt antibody from 200 µg of protein per sample, pelleted by centrifugation, and then incubated for 30 mins at 30°C in 40 µl kinase buffer supplemented with 200 µM ATP and 1 µg glycogen synthase kinase-3 (GSK-3) fusion protein. The reaction was terminated with 20 µl sodium dodecyl sulfate (SDS) sample buffer, and the supernatant was isolated by microcentrifugation and then boiled for 5 mins. Samples (20 µl) were electrophoresed on 12% polyacrylamide minigels, and phospho-GSK-3{alpha}/ß (Ser21/ 9) band was visualized by Western blot analysis.

Statistical Analysis.
Differences among the various treatment groups in cell viability studies were determined by analysis of variance (ANOVA) followed by Dunnett’s t test. Differences were considered statistically significant at a value of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Viability and Apoptosis.
The effects of 24-hr exposure to various concentrations of {gamma}-tocotrienol on +SA cell viability are shown in Figure 1Go (top). Treatment with 0–20 µM {gamma}-tocotrienol resulted in a dose-dependent decrease in +SA cell viability, with 20 µM {gamma}-tocotrienol inducing approximately 80% cell death (Figure 1Go, top). The effects of similar treatments on positive TUNEL immunocytochemical staining in +SA cells are shown in Figure 1Go (bottom). Positive TUNEL staining in +SA cells was characterized by dark brown nuclear staining, in contrast to the light blue hematoxylin counterstaining observed in nonapoptotic cells (photomicrographs not shown). The number of +SA cells displaying positive TUNEL staining versus the total number of cells present in each sampling was determined for each treatment group (five samples/group). Treatment with 4–20 µM {gamma}-tocotrienol induced a dose-responsive increase in positive TUNEL staining (Figure 1Go, bottom). Treatment with 20 µM {gamma}-tocotrienol resulted in approximately 65% of the +SA cells displaying positive TUNEL staining, indicating that the majority of cells in this treatment group are actively undergoing apoptosis (Figure 1Go, bottom).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Effects of 24-hr exposure to 0–20 µM {gamma}-tocotrienol on +SA cell viability are shown in the top panel. Cells were plated at a density of 1 x 105 cells/well (6 wells/group) and maintained on serum-free control media for 3 days. Afterward, cells were exposed to treatment media, and 24 hrs later viable cell number was determined using the MTT assay. The effects of similar treatments on the percentage of +SA cells displaying positive TUNEL staining are shown in the bottom panel. Cells were fixed with 4% paraformaldehyde prior to TUNEL assay. Cells were counted in five photomicrographs (80,000 µm2 area per photomicrograph) for each treatment group. Vertical bars indicate the mean ± SEM. *P < 0.05, as compared with controls.

 
Caspase Activation.
The effects of 20 µM {gamma}-tocotrienol on intracellular levels of caspase-8, caspase-3, and FLIP during a 24-hr treatment period in +SA cells are shown in Figure 2Go. Western blot and scanning densitometric analyses showed that {gamma}-tocotrienol treatment induced an increase in the relative levels of cleaved (active) p20 subunit of caspase-8 (Figure 2Go) and cleaved (active) p18 subunit of caspase-3 (Figure 2Go) throughout the 24-hr treatment period. In contrast, {gamma}-tocotrienol treatment decreased the relative levels of FLIP, an antiapoptotic protein that inhibits caspase-8 activation, during this same time period (Figure 2Go).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Western blot and scanning densitometric analysis of the relative levels of active, cleaved caspase-8; FLIP; and active, cleaved caspase-3 in +SA cells treated with 20 µM {gamma}-tocotrienol for 0–24 hrs. Cells were grown in culture for 3 days in serum-free control media prior to treatment exposure. At various time points, whole cell lysates were prepared for subsequent fractionation by SDS–polyacrylamide gel electrophoresis (PAGE) (20 µg/well), followed by Western blot analysis. Scanning densitometric analysis was performed for each blot and shown as bar graphs below the Western blots. Vertical bars in the densitometric graphs indicate the integrated optical density of bands visualized in each lane.

 
Caspase Inhibition and Cell Viability.
Figure 3Go shows the effects of combined treatment of {gamma}-tocotrienol with caspase inhibitors on +SA cell viability following a 24-hr treatment exposure. Treatment with 20 µM {gamma}-tocotrienol alone decreased +SA cell viability by approximately 80% as compared with untreated controls (Figure 3Go). Combined treatment with {gamma}-tocotrienol and 5 µM of z-IETD-fmk (specific caspase-8 inhibitor, I-8), z-DQMD-fmk (specific caspase-3 inhibitor, I-3), or z-Asp-CH2-DCB (nonspecific caspase inhibitor, I-All) blocked tocotrienol-induced apoptosis and corresponding decrease in viable +SA cell number (Figure 3Go).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Effects of 24 hrs exposure to 0 µM (closed bars) or 20 µM (open bars) {gamma}-tocotrienol alone or in combination with caspase inhibitors on +SA cell viability. Cells were plated at a density of 1 x 105 cells/well and maintained in culture for 3 days on serum-free control media. Afterward, media were replaced with fresh treatment media containing either 0 or 20 µM {gamma}-tocotrienol and vehicle (control); 5 µM z-DQMD-fmk, a specific caspase-3 inhibitor (I-3); 5 µM z-IETD-fmk, a specific caspase-8 inhibitor (I-8); or 5 µM z-Asp-CH2-DCB, a nonspecific caspase inhibitor (I-All), and 24 hrs later cell viability was determined using the MTT assay. Vertical bars indicate mean ± SEM for six replicates in each treatment group. *P < 0.05, as compared with 20 µM {gamma}-tocotrienol treated controls.

 
Death Ligands, PI3K/Akt Inhibitors, and Cell Viability.
Since caspase-8 activation is associated with death receptor (TNF-R1, Fas, TRAIL-R1, and TRAIL-R2) activation, studies were conducted to determine the susceptibility of +SA cells to death receptor ligands. Furthermore, since suppression of PI3K/PDK/Akt signaling is associated with decreased cellular survival and the induction of apoptosis, additional studies were also conducted to determine the effects of the PI3K inhibitor, LY294002, and indirect Akt inhibitor, geldanamycin, on +SA cell viability. Treatment for 24 hrs with a high dose (100 ng/ml) of death receptor ligand, TNF-{alpha}, FasL, or TRAIL had no effect on +SA cell viability as compared with untreated controls (Figure 4Go). Similarly, treatment for 24 hrs with 1 µg/ml of Fas activating antibody also had no effect on +SA cell viability (Figure 4Go). In contrast, +SA cell viability was significantly decreased following a 24-hr exposure to 20 µM {gamma}-tocotrienol, 20 µM LY294002, or 1 µM geldanamycin (Figure 4Go).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Comparative effects of 24-hr treatment exposure to {gamma}-tocotrienol, various death receptor ligands, LY294002 (PI3K inhibitor), or geldanamycin (indirect Akt inhibitor) on +SA cell viability. Cells were plated at a density of 1 x 105 cells/well and maintained in culture for 3 days on serum-free control media. Afterward, media were replaced with fresh treatment media containing vehicle (control), 100 ng/ml TNF-{alpha}, 100 ng/ml FasL, 100 ng/ml TRAIL, 1 µg/ml Fas activating antibody (FasAb), 20 µM {gamma}-tocotrienol ({gamma}T3), 20 µM LY294002 (LY), or 1 µM geldanamycin (GD), and 24 hrs later cell viability was determined using the MTT assay. Vertical bars indicate mean ± SEM for six replicates in each treatment group. *P < 0.05, as compared with vehicle treated controls.

 
Death Receptor Apoptotic Signaling.
The effects of 20 µM {gamma}-tocotrienol on total, membrane, or cytosolic levels of proteins associated with Fas receptor signaling in +SA cells over an 8-hr treatment period are shown in Figure 5Go. The 0–8-hr treatment period was selected based on the previous finding that maximal caspase-8 processing and activation was observed within 8 hrs after {gamma}-tocotrienol treatment exposure (4). Treatment with 20 µM {gamma}-tocotrienol had no effect on the relative levels of Fas, FasL, or FADD in whole cell lysates (total) or cytosolic and membrane fractions (Figure 5Go). The FADD protein band was undetectable in the membrane fractions of +SA cells. Fas activation is associated with the translocation of FasL and FADD from the cytosolic to the membrane fraction of the cell. Fas activation can also be restored by enhanced Fas translocation from the cytosolic to the membrane fraction. However, {gamma}-tocotrienol treatment was not found to induce Fas, FasL, or FADD translocation at any time during the 8-hr treatment period (Figure 5Go). In contrast, treatment with 20 µM {gamma}-tocotrienol decreased the relative levels of FLIP in whole cell lysates and cytosolic fractions throughout the 8-hr treatment period while reducing FLIP in the membrane fraction between 2 and 4 hrs after treatment exposure (Figure 5Go). Total FLIP levels decrease within 1 hr of treatment with {gamma}-tocotrienol, whereas the FLIP levels in the cytosolic and membrane fractions start decreasing only 2 hrs after treatment (Figure 5Go). Although the reason for this reproducible finding is unknown, the fact remains that {gamma}-tocotrienol treatment causes a large relative reduction in total, cytosolic, and membrane levels of FLIP in malignant +SA mammary epithelial cells. Western blot analysis of cytochrome c oxidase subunit IV (COX), an enzyme located on the mitochondrial membrane, was used as a marker for subcellular fractionation purity. The relative levels of COX in the membrane fraction were high prior to and after {gamma}-tocotrienol treatment exposure, while cytosolic levels of COX were very low throughout the experiment (Figure 5Go). These results demonstrate that subcellular fractionation of +SA cells was relatively pure and that the cytosolic fraction was free of membrane fraction contamination.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 5. Western blot and scanning densitometric analysis of the relative total, membrane, or cytosolic levels of FasR, FasL, FADD, FLIP, and COX in +SA cells treated with 20 µM {gamma}-tocotrienol for 0–8 hrs. Cells were grown in culture for 3 days in serum-free control media prior to treatment exposure. At various time points, whole cell lysates or membrane and cytosolic fractions were prepared for subsequent fractionation by SDS-PAGE (20 µg/well for whole cell lysates, and 10 µg/well for membrane/cytosolic fractions), followed by Western blot analysis. Scanning densitometric analysis was performed for each blot and is presented as bar graphs adjacent to the corresponding Western blot. Vertical bars in the densitometric graphs indicate the integrated optical density of bands visualized in each lane.

 
PI3K/PDK-1/Akt Mitogenic Signaling.
Since stimulation of the PI3K/PDK-1/Akt mitogenic signaling pathway is associated with increased FLIP expression, it was of interest to determine the effects of {gamma}-tocotrienol on PI3K/ PDK/Akt signaling in +SA cells. Additional studies were also conducted to determine the effects of {gamma}-tocotrienol on PTEN phosphatase activity, an enzyme known to suppress PI3K activity. Western blot analysis showed that treatment with 20 µM {gamma}-tocotrienol resulted in a large time-dependent decrease in the relative levels of phospho-PDK-1 (active) during the 8-hr treatment period (Figure 6Go). However, {gamma}-tocotrienol treatment had no effect on total or phospho-PTEN levels in +SA cells (Figure 6Go). This same treatment only marginally decreased the total (active and inactive) levels of Akt but did cause a large decrease in the relative levels of phospho-Akt (active) starting at 4 hrs and continuing for at least 8 hrs following {gamma}-tocotrienol exposure (Figure 6Go). Treatment effects on Akt activity, as determined by phospho-GSK-3 (Ser 9 and Ser 21) protein levels, are shown in Figure 6Go. Western blot analysis showed that treatment with 20 µM {gamma}-tocotrienol induced a large decrease in the relative levels of phosphorylated GSK-3 protein after 4–8 hrs of treatment exposure, indicating a reduction in Akt activity (Figure 6Go).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 6. Western blot and scanning densitometric analysis showing the effects of 0–8 hrs of 20 µM {gamma}-tocotrienol treatment on total relative levels of phospho-PDK-1 (active form, Ser241), total PTEN, phospho-PTEN (inactive form, Ser380/Thr382/383), total Akt, phospho-Akt (active form, Ser473), and Akt kinase activity in +SA cells. For phospho-PDK-1, PTEN, phospho-PTEN, Akt, and phospho-Akt experiments, cells were grown in culture for 3 days in serum-free control media prior to treatment exposure. At various time points, whole cell lysates were prepared for subsequent fractionation by SDS-PAGE (20 µg/well), followed by Western blot analysis. Cells were prepared in a similar manner for assay of Akt activity, except that Akt was immunoprecipitated from whole cell lysates and incubated with GSK-3 fusion protein and ATP prior to Western blot analysis for phospho-GSK-3-{alpha}/ß (Ser21/9). Scanning densitometric analysis was performed for each blot and is presented as bar graphs below the Western blots. Vertical bars in the densitometric graphs indicate the integrated optical density of bands visualized in each lane.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results in this study demonstrate that {gamma}-tocotrienol–induced apoptosis in malignant +SA mouse mammary epithelial cells is mediated by caspase-8 and caspase-3 activation. Since processing and activation of caspase-8 is generally associated with death receptor apoptotic signaling, it was initially hypothesized that {gamma}-tocotrienol stimulates death receptor (TNF-R1, Fas, TRAIL-R1, and/or TRAIL-R2) activation. However, treatment with high doses of FasL or Fas activating antibody did not induce apoptosis in +SA cells. Similarly, treatment with high doses of additional death receptor ligands, such as TRAIL and TNF-{alpha}, was also found to have no adverse effect on+SA cell viability. Experimental findings also showed that treatment with apoptosis-inducing doses of {gamma}-tocotrienol (20 µM) did not cause increased expression of Fas, FasL, or FADD and failed to induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction in +SA cells. These findings indicate that Fas, TRAIL-R1, TRAIL-R2, and TNF-R1 death receptors are nonfunctional in +SA cells, and {gamma}-tocotrienol–induced caspase activation and apoptosis does not result from a restoration in apoptotic signaling by Fas. However, additional studies showed that treatment with 20 µM {gamma}-tocotrienol decreased PI3K/PDK-1/Akt mitogenic signaling and FLIP expression in these cells. Taken together, these findings suggest that {gamma}-tocotrienol–induced caspase-8 activation and apoptosis in +SA cells does not result from enhanced death receptor activation but is instead associated with tocotrienol-induced suppression of PI3K/PDK-1/Akt mitogenic signaling and the subsequent downregulation of FLIP, an endogenous inhibitor of caspase-8 processing and activation.

These results confirm and extend previous investigations demonstrating tocotrienol-induced apoptosis is mediated by caspase-8 and caspase-3 activation and is independent of caspase-9 activation (4, 13). Although previous investigations did not determine the specific death receptor(s) and ligand(s) involved in mediating tocotrienol-dependent caspase activation, it is well established that FasL activation of the death receptor, Fas, stimulates procaspase-8 activation to caspase-8 (9, 10). Previous studies have also shown that Fas activation plays a role in normal mammary gland remodeling and involution, as well as in the removal of neoplastic mammary epithelial cells during the early stages of tumorigenesis (37). In addition, treatment with the succinate derivative of RRR-{alpha}-tocopherol, RRR-{alpha}-tocopheryl succinate, has been shown to induce DNA synthesis arrest and apoptosis in breast cancer cells, and these effects are associated with Fas apoptotic signaling (36, 38). The present study demonstrates for the first time that the death receptor, Fas, is nonfunctional in malignant +SA mammary epithelial cells and that tocotrienol-induced caspase-8 and caspase-3 activation occurs independently of Fas activating and apoptotic signaling. Experimental data showed that stimulation of Fas with very high doses of Fas activation antibody or FasL had no effect on +SA cell viability. Likewise, treatment with 20 µM {gamma}-tocotrienol (cytotoxic dose) had no effect on intracellular Fas, FasL, and FADD levels. Previous studies have shown that RRR-{alpha}-tocopheryl succinate restores Fas sensitivity and apoptotic signaling in Fas-insensitive human breast cancer cells (36). However, the present study demonstrates that tocotrienol treatment does not restore Fas receptor signaling in Fas-insensitive malignant +SA mammary epithelial cells, as indicated by the absence of Fas, FasL, or FADD translocation from the cytosolic to membrane fractions of +SA cells.

It has also been shown that treatment with TRAIL induces caspase-8 activation by stimulating TRAIL-R1 and TRAIL-R2 apoptotic signaling in several cell types (9, 10). Likewise, TNF-{alpha} has been shown to stimulate TNF-R apoptotic signaling and caspase activation (9, 10). However, treatment with high doses of TNF-{alpha} or TRAIL was found to have no effect on +SA cell viability, suggesting that tocotrienol-induced caspase activation in these cells occurs independently of TNF-R and TRAIL-R apoptotic signaling. Although these data provide strong evidence, additional knockdown studies, such as those involving the use of death receptor neutralizing antibodies, antisense oligomers, or small interfering RNA (siRNA), are required to conclusively determine whether tocotrienol-induced caspase activation occurs independently of death receptor apoptotic signaling in malignant +SA mammary epithelial cells. Nevertheless, the results from this present study suggest that tocotrienol treatment might also be effective in inducing apoptosis and reducing viability in death receptor resistant human breast cancer.

Resistance to death receptor–induced apoptosis is associated with enhanced tumorigenesis, multidrug resistance, and enhanced survival in a number of tumor cell type cancers (14, 15, 36). A reduction in the expression of death receptors and/or their ligands, as well as mutations within genes encoding for death receptors, is associated with insensitive or nonfunctional death receptors in various types of tumor cells (14–17). In addition, resistance to apoptotic stimuli can be acquired through upregulation and enhanced activity of various mitogen-dependent signaling pathways, particularly the PI3K/PDK/Akt mitogenic signaling pathway (24–30, 39, 40). Stimulation of the PI3K/PDK/Akt pathway has been shown to increase expression of antiapoptotic protein, FLIP, which inhibits procaspase-8 cleavage and activation (24–30, 39, 40). Previous investigations have also shown that overexpression of FLIP is associated with tumor cell resistance to death receptor activation and apoptotic signaling and that the ratio between caspase-8 and FLIP levels is critical in determining tumor cell sensitivity to apoptotic stimuli (18–23). The present study shows that treatment with apoptosis-inducing doses of {gamma}-tocotrienol increases the ratio of caspase-8 versus FLIP in malignant +SA mammary epithelial cells. Treatment with 20 µM {gamma}-tocotrienol resulted in a large and prolonged decrease in intracellular FLIP levels and a corresponding increase in intracellular cleaved (active) caspase-8 levels within 24 hrs of treatment.

It is well established that various intracellular signaling pathways and second messengers associated with cellular proliferation and survival, such as Ras/Raf/MEK/ERK, PI3K/PDK-1/Akt, and NF{kappa}B, modulate FLIP expression (24, 25, 41, 42). A common characteristic of many breast cancer cells is elevated PI3K/PDK-1/Akt signaling, and pharmacologic treatments that inhibit PI3K/PDK-1/Akt signaling have been shown to reduce intracellular FLIP expression, increase caspase-8 activation, and reduce cell survival (24–30, 39, 40). The present findings show that treatment with apoptosis-inducing doses of {gamma}-tocotrienol caused a relatively large decrease in phospho-PDK-1 (active form), and phospho-Akt (active form), a reduction in Akt kinase activity, and a corresponding decrease in FLIP expression in +SA cells. However, {gamma}-tocotrienol did not lead to an increase in the activity of PTEN, a lipid phosphatase known to inhibit PI3K activity, suggesting that tocotrienol-induced suppression in PI3K/PDK-1/Akt signaling does not result from an increase in PTEN activity. These data indicate that {gamma}-tocotrienol–induced caspase-8 activation and apoptosis are associated with suppression in PI3K/PDK-1/Akt signaling and a decrease in FLIP expression in +SA cells. At present, it is not known whether {gamma}-tocotrienol directly inhibits PDK-1 activity or acts at some upstream site to inhibit PDK-1 activation. However, it is known that PI3K activation is stimulated by various growth factors, particularly EGF (24, 26–28). Previous studies have shown that {gamma}-tocotrienol treatment had no effect on EGF-receptor levels or EGF-receptor tyrosine kinase activity but did attenuate EGF-dependent mitogenic signaling in normal and preneoplastic mammary epithelial cells, (2, 3, 6, 13). Further studies are needed to determine the exact site of action where {gamma}-tocotrienol acts to suppress EGF-dependent PI3K/PDK-1/Akt signaling in malignant +SA mammary epithelial cells.

In conclusion, these findings demonstrate that {gamma}-tocotrienol–induced caspase-8 activation and apoptosis is not associated with death receptor signaling in malignant +SA mouse mammary epithelial cells. Experimental findings indicate that tocotrienol-induced caspase-8 activation results from the suppression of PI3K/PDK-1/Akt mitogenic signaling and a corresponding decrease in intracellular FLIP expression, an endogenous inhibitor of caspase-8 activation. Recent studies have shown that resistance to death receptor-induced apoptosis is an important contributor for multidrug resistance in a variety of cancer cell types, and nonfunctional death receptors are also associated with cancer cell escape and survival from normal immunosurveillance mechanisms (14, 15, 36). Therefore, it is possible that tocotrienols may provide additional therapeutic benefits when combined with traditional chemotherapeutic agents in the treatment of breast cancer in women. Understanding the molecular mechanisms involved in tocotrienol-induced apoptosis may also provide critical leads for the development of novel therapies to treat breast cancer cases that have acquired resistance to death receptor signaling.


   Acknowledgments
 
This work was performed at the School of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209-0470 and supported by a grant from the National Institutes of Health (Grant CA 86833). We thank the Malaysian Palm Oil Board for their support.


   Footnotes
 
This work was supported in part by National Institutes of Health Grant CA 86833.

Received for publication January 14, 2004. Accepted for publication March 30, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McIntyre BS, Briski KP, Gapor A, Sylvester PW. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc Soc Exp Biol Med 224:292–301, 2000.[Abstract/Free Full Text]
  2. Sylvester PW, Theriault A. Role of tocotrienols in the prevention of cardiovascular disease and breast cancer. Curr Top Nutraceutical Res 1:121–136, 2003.
  3. Sylvester PW, Nachnani A, Shah S, Briski KP. Role of GTP-binding proteins in reversing the antiproliferative effects of tocotrienols in preneoplastic mammary epithelial cells. Asia Pacific J Clin Nutr 11:S452–S459, 2002.
  4. Shah S, Gapor A, Sylvester PW. Role of caspase-8 activation in mediating vitamin E-induced apoptosis in murine mammary cancer cells. Nutr Cancer 45:236–246, 2003.[Medline]
  5. McIntyre BS, Briski KP, Tirmenstein MA, Fariss MW, Gapor A, Sylvester PW. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells. Lipids 35:171–180, 2000.[Medline]
  6. Sylvester PW, McIntyre BS, Gapor A, Briski KP. Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C(alpha) activation. Cell Prolif 34:347–357, 2001.[Medline]
  7. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383–424, 1999.[Medline]
  8. Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 274:5053–5060, 1999.[Abstract/Free Full Text]
  9. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. Eur J Biochem 254:439–459, 1998.[Medline]
  10. Walczak H, Krammer PH. The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res 256:58–66, 2000.[Medline]
  11. Green DR, Reed JC. Mitochondria and apoptosis. Science 281:1309–1312, 1998.[Abstract/Free Full Text]
  12. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489, 1997.[Medline]
  13. Sylvester PW, Shah S. Antioxidants in dietary oils: their potential role in breast cancer prevention. Malay J Nutr 8:1–11, 2002.
  14. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 17:2941–2953, 1999.[Abstract/Free Full Text]
  15. Hughes SJ, Nambu Y, Soldes OS, Hamstra D, Rehemtulla A, Iannettoni MD, Orringer MB, Beer DG. Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma. Cancer Res 57:5571–5578, 1997.[Abstract/Free Full Text]
  16. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314–317, 1992.[Medline]
  17. Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MI, Puck JM. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935–946, 1995.[Medline]
  18. Hu JS, Hastings GA, Cherry S, Gentz R, Ruben S, Coleman TA. A novel regulatory function of proteolytically cleaved VEGF-2 for vascular endothelial and smooth muscle cells. FASEB J 11:498–504, 1997.[Abstract]
  19. Tschopp J, Irmler M, Thome M. Inhibition of fas death signals by FLIPs. Curr Opin Immunol 10:552–558, 1998.[Medline]
  20. Rasper DM, Vaillancourt JP, Hadano S, Houtzager VM, Seiden I, Keen SL, Tawa P, Xanthoudakis S, Nasir J, Martindale D, Koop BF, Peterson EP, Thornberry NA, Huang J, MacPherson DP, Black SC, Hornung F, Lenardo MJ, Hayden MR, Roy S, Nicholson DW. Cell death attenuation by ‘Usurpin’, a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. Cell Death Differ 5:271–288, 1998.[Medline]
  21. Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 274:1541–1548, 1999.[Abstract/Free Full Text]
  22. Shain KH, Landowski TH, Dalton WS. Adhesion-mediated intra-cellular redistribution of c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long confers resistance to CD95-induced apoptosis in hematopoietic cancer cell lines. J Immunol 168:2544–2553, 2002.[Abstract/Free Full Text]
  23. Tepper CG, Seldin MF. Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt’s lymphoma. Blood 94:1727–1737, 1999.[Abstract/Free Full Text]
  24. Panka DJ, Mano T, Suhara T, Walsh K, Mier JW. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells. J Biol Chem 276:6893–6896, 2001.[Abstract/Free Full Text]
  25. Suhara T, Mano T, Oliverira BE, Walsh K. Phosphatidylinositol 3-kinase/Akt signaling controls endothelial cell sensitivity to Fas-mediated apoptosis via regulation of FLICE-inhibitory protein (FLIP). Circ Res 89:13–19, 2001.[Abstract/Free Full Text]
  26. Neve RM, Holbro T, Hynes NE. Distinct roles for phosphoinositide 3-kinase, mitogen-activated protein kinase and p38 MAPK in mediating cell cycle progression of breast cancer cells. Oncogene 21:4567–4576, 2002.[Medline]
  27. Hermanto U, Zong CS, Wang LH. ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphatidylinositol 3-kinase signaling pathway in anchorage-independent growth. Oncogene 20:7551–7562, 2001.[Medline]
  28. Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253:239–254, 1999.[Medline]
  29. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol 10:262–267, 1998.[Medline]
  30. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res 253:210–229, 1999.[Medline]
  31. Anderson LW, Danielson KG, Hosick HL. Epithelial cell line and subline established from premalignant mouse mammary tissue. In Vitro 15:841–843, 1979.[Medline]
  32. Anderson LW, Danielson KG, Hosick HL. Metastatic potential of hyperplastic alveolar nodule derived mouse mammary tumor cells following intravenous inoculation. Eur J Cancer Clin Oncol 17:1001–1008, 1981.[Medline]
  33. Danielson KG, Anderson LW, Hosick HL. Selection and characterization in culture of mammary tumor cells with distinctive growth properties in vivo. Cancer Res 40:1812–1819, 1980.[Abstract/Free Full Text]
  34. McIntyre BS, Birkenfeld HP, Sylvester PW. Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic-responsiveness in normal mouse mammary epithelial cells in vitro. Cell Prolif 28:45–56, 1995.[Medline]
  35. Sylvester PW, Birkenfeld HP, Hosick HL, Briski KP. Fatty acid modulation of epidermal growth factor-induced mouse mammary epithelial cell proliferation in vitro. Exp Cell Res 214:145–153, 1994.[Medline]
  36. Yu W, Israel K, Liao QY, Aldaz CM, Sanders BG, Kline K. Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis. Cancer Res 59:953–961, 1999.[Abstract/Free Full Text]
  37. Kumar R, Vadlamudi RK, Adam L. Apoptosis in mammary gland and cancer. Endocr Relat Cancer 7:257–269, 2000.[Abstract]
  38. Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, Kline K. Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutr Cancer 33:26–32, 1999.[Medline]
  39. Varadhachary AS, Edidin M, Hanlon AM, Peter ME, Krammer PH, Salgame P. Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95. J Immunol 166:6564–6569, 2001.[Abstract/Free Full Text]
  40. Varadhachary AS, Peter ME, Perdow SN, Krammer PH, Salgame P. Selective up-regulation of phosphatidylinositol 3'-kinase activity in Th2 cells inhibits caspase-8 cleavage at the death-inducing complex: a mechanism for Th2 resistance from Fas-mediated apoptosis. J Immunol 163:4772–4779, 1999.[Abstract/Free Full Text]
  41. Yeh JH, Hsu SC, Han SH, Lai MZ. Mitogen-activated protein kinase kinase antagonized fas-associated death domain protein-mediated apoptosis by induced FLICE-inhibitory protein expression. J Exp Med 188:1795–1802, 1998.[Abstract/Free Full Text]
  42. Xiao CW, Asselin E, Tsang BK. Nuclear factor kappaB-mediated induction of Flice-like inhibitory protein prevents tumor necrosis factor alpha-induced apoptosis in rat granulosa cells. Biol Reprod 67:436–441, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
K. Nakagawa, A. Shibata, S. Yamashita, T. Tsuzuki, J. Kariya, S. Oikawa, and T. Miyazawa
In Vivo Angiogenesis Is Suppressed by Unsaturated Vitamin E, Tocotrienol
J. Nutr., August 1, 2007; 137(8): 1938 - 1943.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. S. Ahn, G. Sethi, K. Krishnan, and B. B. Aggarwal
{gamma}-Tocotrienol Inhibits Nuclear Factor-{kappa}B Signaling Pathway through Inhibition of Receptor-interacting Protein and TAK1 Leading to Suppression of Antiapoptotic Gene Products and Potentiation of Apoptosis
J. Biol. Chem., January 5, 2007; 282(1): 809 - 820.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S.-M. Jangi, J. L. Diaz-Perez, B. Ochoa-Lizarralde, I. Martin-Ruiz, A. Asumendi, G. Perez-Yarza, J. Gardeazabal, J. L. Diaz-Ramon, and M. D. Boyano
H1 histamine receptor antagonists induce genotoxic and caspase-2-dependent apoptosis in human melanoma cells
Carcinogenesis, September 1, 2006; 27(9): 1787 - 1796.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Munteanu, M. Taddei, I. Tamburini, E. Bergamini, A. Azzi, and J.-M. Zingg
Antagonistic Effects of Oxidized Low Density Lipoprotein and {alpha}-Tocopherol on CD36 Scavenger Receptor Expression in Monocytes: INVOLVEMENT OF PROTEIN KINASE B AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-{gamma}
J. Biol. Chem., March 10, 2006; 281(10): 6489 - 6497.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
S. J. Shah and P. W. Sylvester
{gamma}-Tocotrienol Inhibits Neoplastic Mammary Epithelial Cell Proliferation by Decreasing Akt and Nuclear Factor {kappa}B Activity
Experimental Biology and Medicine, April 1, 2005; 230(4): 235 - 241.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shah, S.
Right arrow Articles by Sylvester, P. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shah, S.
Right arrow Articles by Sylvester, P. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS