| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Division of Nutrition [W. Y., K. I., Q. Y. L., K. K.] and Department of Zoology [W. Y., Q. Y. L., B. G. S.], University of Texas at Austin, Austin, Texas 78712; and The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 [C. M. A.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by 
50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopheryl succinate), a derivative of vitamin E, is currently being characterized for its chemopreventive and chemotherapeutic potential (1)
. VES has been shown to inhibit the proliferation of several transformed cell types, including human breast cancer cells (2, 3, 4, 5, 6)
. Previous studies have shown VES to inhibit tumor cell growth by a variety of mechanisms, including: induction of apoptosis, G1 cell cycle blockage, induced cellular differentiation, DNA synthesis arrest, induced secretion and activation of TGF-ßs, and enhanced expression of TGF-ß type II receptors (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
. VES is a potent inducer of apoptosis in human breast cancer cells, and it appears that a minimum of two apoptotic triggering pathways may be involved. For example, previous studies by our laboratory have demonstrated a partial role for TGF-ß signaling and involvement of JNK and c-Jun (15, 16, 17) . Turley et al. (18) have proposed a role for Mr 48,000 Fas signaling in VES-induced apoptosis in human breast cancer cells.
Fas, also called Apo-1 or CD95, is a member of the tumor necrosis factor receptor and nerve growth factor receptor superfamily, which can trigger programmed cell death upon trimerization with Fas ligand (CD95L) or upon cross-linking with Fas-specific antibodies (also called agonistic anti-Fas antibodies). Fas is expressed on the cell surface of a variety of cell types, including T cells, B cells, and macrophages as well as cells of the liver, spleen, lung, testis, heart, brain, intestine, and breast (19, 20, 21, 22) . Although Fas-induced apoptosis has been studied most in T lymphocytes, recent studies show Fas expression in a wide range of tissues, including breast (18 , 19 , 22 , 23) . Furthermore, studies show that Fas signaling is altered in some breast, prostate, colon, and central nervous system cancer cells (18 , 19 , 23, 24, 25, 26, 27, 28, 29) .
Apoptosis or programmed cell death is a well-established phenomenon in tissue development and homeostasis (30) . Apoptotic pathways can generally be classified into three distinct phases: the initiation phase, which may differ, depending on the apoptosis triggering stimulus; the common effector phase, in which the decision to die is made; and the degradation phase, in which the cell dies and exhibits the morphological and biochemical characteristics of apoptosis (31, 32, 33, 34) . It appears that the degradation phase uses the same cascading molecular events and produces similar morphological characteristics; however, apoptotic initiating or triggering events use several pathways, and the utilization of a specific pathway(s) may vary, depending on environmental context of cells and apoptotic triggering agent (30 , 32 , 35) . Defects in key components of apoptotic pathways provide a survival advantage to cells and have been implicated as important factors in tumorigenesis (26 , 32) . Because therapeutic drug-induced apoptosis is a critical component in the treatment of most cancers, alterations in key apoptotic pathways may be critical to drug resistance (36) . For example, Fas signaling has been demonstrated to be defective in certain cancer cell types, and studies show that sensitivity to Fas-mediated apoptosis can be restored (23, 24, 25 , 35) . Furthermore, studies show enhanced apoptosis when immunochemotherapy is given in combination with CD95 Fas ligand (37) .
Here, we examine the apoptotic inducing abilities of VES and Fas agonistic antibody, administered separately and in combination, on non-Fas-responsive MCF-7, MDA-MB-435, and MDA-MB-231 breast cancer cells and on Fas-sensitive T47D breast cancer cells, HMECs, and immortalized but nontumorigenic MCF-10A human mammary cells. With regard to Fas signaling, we show that VES has no major effects on Fas-sensitive cells but is capable of converting Fas-resistant human breast cancer cells to Fas-sensitive cells. Whole-cell extracts from Fas-sensitive cells constitutively express high levels of Mr 43,000 Fas, whereas whole-cell extracts from Fas-resistant cells expressed lower levels of Mr 43,000 Fas, which were confined to the cytosolic fraction. VES sensitization of the three Fas-resistant cell lines is associated with a decrease in cytosolic Mr 43,000 Fas, with a concomitant increase in Mr 43,000 cell surface membrane Fas. Functional knockout of Fas signaling via Fas-neutralizing antibodies or Fas antisense oligomers partially inhibits VES-triggered apoptosis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopheryl succinate, succinic acid, and trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO). All reagents for morphological analyses of apoptosis were purchased from Boehringer Mannheim (Indianapolis, IN).
Cell Culture and VES Treatment.
MDA-MB-435 (provided by Dr. Janet E. Price, Department of Cell Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX; Ref. 38
) and MDA-MB-231 (American Type Culture Collection, Manassas, VA) are non-estrogen-responsive breast cancer cell lines. T47D (American Type Culture Collection) and MCF-7 (provided by Dr. Suzanne Fuqua, University of Texas Health Science Center, San Antonio, TX; Ref. 39
) are estrogen-responsive breast cancer cell lines. MCF-10A (American Type Culture Collection) is an immortalized nontumorigenic breast cell line. HMECs (provided by Dr. C. Marcelo Aldaz, University of Texas M. D. Anderson-Science Park Research Division, Smithville, TX) are primary cultures of mammary cells derived from normal mammoplasty specimens. MDA-MB-435 cells were cultured in MEM with Earle’s balanced salts (Life Technologies, Inc., Grand Island, NY) supplemented with 5% FBS (Hyclone Laboratories, Logan, UT) plus 2 mM glutamine, 100 µg/ml streptomycin, 100 IU/ml penicillin, 1x (v/v) nonessential amino acids, 2x (v/v) MEM vitamins, and 1 mM sodium pyruvate (Sigma). MDA-MB-231 and MCF-7 cells were cultured in MEM with Earle’s balanced salts (Life Technologies, Inc.) supplemented with 10% FBS (Hyclone Laboratories) plus 2 mM glutamine, 100 µg/ml streptomycin, 100 IU/ml penicillin, 1x (v/v) nonessential amino acids, and 10 mM HEPES (Sigma). T47D cells were cultured in DMEM containing 10% FBS plus 2 mM glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin. MCF-10A cells were cultured in DMEM/F12 medium (Life Technologies, Inc.) supplemented with 5% horse serum plus 2 mM glutamine, 100 µg/ml streptomycin, 100 IU/ml penicillin, 0.25 µg/ml ampicillin B, 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, and 10 µg/ml insulin. HMECs were cultured with basal serum free mammary epidermal cell growth medium supplemented with 0.4% bovine pituitary extract, 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 0.5 ng/ml hydrocortisone, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B (Clonetics, San Diego, CA). All experiments with primary cultures of normal mammary epithelial cells were conducted with second-passage HMECs.
For experiments, the percentage of serum was reduced to 2% for MDA-MB-435 cells and 5% for the other cell lines (HMEC experiments were conducted in serum-free medium). In these media, growing cells were plated at 5 x 106 cells per T-75 flask for Western blotting and FACS analyses and at 1.5 x 105cells/well in 12-well plates for apoptosis analyses. Treatments were conducted at 5, 10, or 20 µg/ml VES in 0.2% ethanol (F.C. v/v) or VEH, which consisted of an equivalent or highest amount of sodium succinate in 0.2% ethanol used in the experiments.
Determination of Apoptosis.
Apoptosis was assessed based on nuclear morphology using the fluorescent DNA dye DAPI, as described previously (13
, 15)
. Briefly, cells were cultured at 1.5 x 105 cells/well in 12 well plates overnight to permit attachment. Next, the cells were treated with VEH or VES. After treatment, the cells were washed twice with PBS, and floating cells plus trypsin-released adherent cells were pelleted, washed, and stained with 2 µg/ml DAPI (Boehringer Mannheim, Indianapolis, IN) in 100% methanol for 20 min at 37°C. Cells were viewed at x400 magnification with a Zeiss ICM 405 fluorescent microscope using a 487701 filter. Cells in which the nucleus contained clearly condensed chromatin or cells exhibiting fragmented nuclei were scored as apoptotic. Apoptotic data are reported as percentage apoptosis, obtained by determining the numbers of apoptotic versus nonapoptotic cells, as visualized by DAPI staining, within a cell population by counting a minimum of five different locations/slide and counting a minimum of 100 cells per location for a minimum of 500 cells counted per slide. Apoptotic data are presented as mean ± SD for three independent experiments.
Cell Fractionation.
Membrane and cytosol fractions were isolated as described by Koli and Arteaga (40)
. Cells were washed with cold PBS and resuspended in fractionation buffer [20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 25 mM sodium fluoride, 1 mM DTT, 2 mM sodium orthovanadate, 1 µg/ml of aprotinin, and 1 µg/ml leupeptin (Sigma)]. The cells were sheared by passing through a 26-gauge needle five times and then incubated on ice for 5 min. After centrifugation at 100,000 x g for 1 h at 4°C, the supernatants (cytosol fraction) were transferred to a clean tube, and Triton X-100 was added to a final concentration of 0.8%. The pellets (membrane fraction) were resuspended in fractionation buffer and 0.8% Triton X-100, incubated for 20 min on ice, and centrifuged at 15,000 x g for 15 min at 4°C. Whole-cell protein extracts were prepared as described previously (3)
. After washing twice with PBS, the cells were lysed with lysis buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) plus 1 µg/ml of aprotinin and leupeptin, 1 mM DTT, and 2 mM sodium orthovanadate, and incubated on ice for 30 min. Lysates were then centrifuged at 15,000 x g for 15 min at 4°C. Protein concentrations were determined using the Bio-Rad Dye Binding protein assay (Bio-Rad Laboratories). Membrane and cytosolic preparations were analyzed by Western/ECL analyses for the presence of the cytosolic enzyme of glycolysis, GAPDH. Cytosolic preparations exhibited readily detectable high levels of GAPDH protein, whereas GAPDH protein in the membrane fractions was detectable only after prolonged exposure times (data not shown).
Western Blot Analyses.
Fas protein was detected in membrane, cytosol, and whole-cell lysates by Western blot analyses. Fifty to 100 µg of protein were loaded per well, separated using SDS-PAGE on a 10% gel, and electroblotted onto a nitrocellulose membrane (Optitran BA-S supported nitrocellulose for 0.2 µM pore; Schleicher and Schuell, Keene, NH). Equal loading was verified using GAPDH antibody (a gift from Dr. D. Hwang, Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA; Ref. 41
) and by staining the membrane with Ponceau red stain. Immunoblotting was performed using primary rabbit antihuman Fas polyclonal antibody (Santa Cruz Biotechnology) and peroxidase-conjugated goat antirabbit as the secondary antibody (Jackson Immunoresearch Laboratory, West Grove, PA) followed by detection with ECL (Pierce, Rockford, IL).
Cell Surface Fas Detection by Flow Cytometry.
Cell surface Fas expression was measured by flow cytometry using a modification of methods of Keane et al. (23)
. Following VEH and VES treatment, MDA-MB-435 cells (1 x 106) were washed twice with PBS and incubated at 4°C for 60 min with 20 µg/ml antihuman Fas mouse monoclonal IgM (clone CH-11; Upstate Biotechnology, Inc., Lake Placid, NY) or 20 µg/ml irrelevant mouse IgM (Sigma) in PBS containing 1% FCS and 0.5 mM EDTA. The specificity of the primary antibody was verified by Western immunoblotting and was determined to detect a Mr 43,000 Fas molecule in whole-cell extracts of the human breast cancer cell lines. After two washings with PBS, the cells were incubated for 30 min at 4°C with constant rotation with 10 µg/ml of affinity-purified FITC-conjugated goat antimouse IgG plus IgM (Kirkegaard & Perry, Gaithersburg, MD) in PBS containing 1% FCS and 0.5 mM EDTA. After two more washings, the cells were resuspended in PBS. Immediately before analyzing, cells were filtered through a 60-µm mesh to prevent clumping and then analyzed by flow cytometry, using a 488-nm argon laser. FACScan and CELLQuest software were used to analyze data.
Blockage of Fas Signaling with Anti-Fas Neutralizing Antibody and Fas Antisense Oligonucleotides.
Cells were treated for 48 h with VEH and VES (10 or 20 µg/ml) plus 0.5 µg/ml of an apoptosis-neutralizing monoclonal Fas antibody (clone ZB4; Immunotech, Westbrook, ME) or 0.5 µg/ml control antibody (mouse IgG; Sigma). Apoptotic cells were counted after staining with DAPI.
For antisense experiments, 1.5 x 105 cells/well were transiently transfected with sense or antisense Fas oligonucleotides. Briefly, cells were allowed to adhere overnight. The next day they were washed twice with MEM culture media without serum or antibiotics, followed by addition of 0.8 ml of this media plus a mixture of 2 µg of sense or antisense oligonucleotide and 8 µg of lipofection reagent Lipofectamine (Life Technologies, Inc.) in 200 µl of serum free Opti-MEM (Life Technologies, Inc.). After 4 h, the cells were treated with VES or VEH in 2% serum-containing medium and cultured for an additional 48 h for apoptosis analyses and 18 h for Western immunoblot analyses. Apoptosis was monitored using DAPI staining; whole-cell extract Fas expression was analyzed by Western immunoblotting, and cell surface Fas was analyzed by FACS as described above. Oligomer sequences for Fas sense (ATG CTG GGC ATC TGG ACC CTC) and antisense (GAG GGT CCA GAT GCC CAG CAT) oligonucleotides were directed against the ATG translation start site in Fas mRNA and were modified with phosphorothioate at every third nucleotide (Operon Technologies, Alameda, CA).
Fas and VES Sensitivity and Synergism Assay.
For the Fas sensitivity assay, cells were treated with 0.5 µg/ml antihuman Fas mouse monoclonal IgM antibody (clone CH-11; Upstate Biotechnology, Inc., Lake Placid, NY) or 0.5 µg/ml irrelevant mouse IgM antibody (Sigma) for 2 days, and then analyzed for percentage of cells undergoing apoptosis. For VES modulation of Fas sensitivity assays, cells were pretreated with VEH or VES at 5, 10, and 20 µg/ml for 6 h; washed in medium to remove VEH and VES; treated with 0.5 µg/ml anti-Fas or 0.5 µg/ml irrelevant antibody for another 42 h; and then analyzed for percentage of apoptotic cells. For the synergism assays, cells were cotreated with VEH or VES at 5, 10, and 20 µg/ml plus 0.5 µg/ml of anti-Fas or 0.5 µg/ml of irrelevant antibody for 30 h prior to analyzing for apoptosis. Apoptosis was monitored using DAPI staining.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. In contrast, Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells were sensitive to the apoptotic inducing properties of VES, exhibiting 32, 48, and 35% apoptosis after 2 days of treatment with 10 µg/ml VES, respectively, whereas the Fas-sensitive HMECs, MCF-10A cells, and T47D cells were resistant to VES-induced apoptosis (Fig. 1B)
.
|
. MDA-MB-435 cells that were cotreated with VES plus anti-Fas for 30 h exhibited 3.6-, 2.1-, and 2.6-fold increases in apoptosis when compared to cells treated with VES plus irrelevant antibody (Fig. 2B)
. In the absence of pretreatment or cotreatment with VES, MDA-MB-435 cells do not exhibit apoptosis to anti-Fas agonistic antibody (Fig. 1A)
.
|
. MCF-7, MDA-MB-231, and MDA-MB-435 cells pretreated with VES and triggered to undergo apoptosis with anti-Fas exhibited 2.8-, 3.0-, and 6.3-fold increases in apoptosis compared to the lower levels of apoptosis induced by pretreatment of the cells with VES for 6 h followed by treatment with irrelevant antibody (Table 1)
. Levels of apoptosis induced by anti-Fas alone (no pretreatment) were less than those obtained in cultures in which cells were pretreated with VES for 6 h and then incubated with irrelevant antibody (Table 1)
. Pretreatment of the Fas-sensitive MCF-10A cells for 6 h with VES had no enhancing effect on anti-Fas triggered apoptosis compared to the levels of apoptosis induced by anti-Fas treatment alone. Pretreatment of the Fas-sensitive T47D cells with VES resulted in a modest 1.2-fold increase in anti-Fas-triggered apoptosis. As expected, the Fas-sensitive cells treated with anti-Fas resulted in Fas-triggered apoptosis (Table 1)
.
|
. MCF-7, MDA-MB-231, and MDA-MB-435 cells exhibited 1.9-, 2.0-, and 2.6-fold increases in apoptosis compared to apoptosis induced by cotreatments of VES plus irrelevant antibody. Fas-insensitive cells treated with anti-Fas only did not exhibit apoptotic characteristics. VES and anti-Fas cotreatments had little effect on levels of apoptosis induced in Fas sensitive MCF-10A and T47D cells. Although the level of apoptosis expressed by T47D cells cotreated with VES plus anti-Fas was higher than cells treated with anti-Fas alone, the increased levels were not markedly different between the two treatment groups. In an effort to see if VES might synergize with anti-Fas at lower concentrations of anti-Fas, Fas-sensitive MCF-10A cells were cotreated with a 5-fold lower concentration of agonistic anti-Fas (namely, 0.1 µg/ml) plus VES (20 µg/ml); however, no synergistic effects were observed (data not shown).
|
and because Turley et al. (18)
reported that VES-induced apoptosis involves Fas signaling, it was of interest to see whether VES-triggered apoptosis of these cell lines also involved Fas signaling. VES- (20 µg/ml) and VEH-treated MCF-7, MDA-MB-231, and MDA-MB-435 cells were cultured in the presence of 0.5 µg/ml neutralizing antibody to Fas for 48 h. Identically treated cells cocultured with 0.5 µg/ml of irrelevant antibody served as controls (Fig. 3)
. Fas neutralizing antibody reduced the ability of VES to induce apoptosis by 57, 54, and 53% for the MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively (Fig. 3)
.
|
. VEH-treated cells exhibited background levels of apoptosis (Fig. 4A)
. Western/immunoblot analyses of whole-cell extracts from MDA-MB-435 cells (after 18 h of 20 µg/ml VES treatment) transiently transfected with antisense or sense oligomers to Fas showed reduced levels of Mr 43,000 Fas in the antisense-treated cells when compared to the levels of Mr 43,000 Fas in the sense oligomer-treated control cells (Fig. 4B)
. GAPDH protein levels served to establish protein lane loads (Fig. 4B)
. A comparison of the fluorescent profile of sense-transfected (thick line) and antisense-transfected (thin line) MDA-MB-435 cells cultured with 20 µg/ml VES and analyzed for cell surface Fas expression by flow cytometry show a reduction in fluorescence by cells transfected with Fas antisense, suggesting that the antisense treatment reduced the cell surface expression of Fas (Fig. 4C)
.
|
. In contrast, whole-cell extracts from Fas-resistant cell lines (MCF-7, MDA-MB-231, and MDA-MB-435) did not express detectable levels of Mr 43,000 Fas (Fig. 5A)
. However, prolonged exposure of immunoblots of whole-cell extracts from VEH-treated and VES-treated (20 µg/ml) MCF-7, MDA-MB-231, and MDA-MB-435 cells revealed detectable levels of Mr 43,000 Fas (Fig. 5B)
. [Please note that differences between levels of Fas in Fig. 5, A and B
are due to differences in amounts of whole-cell extract proteins analyzed (50 versus 100 µg/lane, respectively) and differences in autoradiogram exposure times (10 versus 60 s, respectively)]. Fas levels in whole cell extracts were not affected by VES treatment (Fig. 5B)
. Levels of Mr 43,000 Fas expressed by T47D cells treated with VEH and VES (20 µg/ml), after prolonged immunoblot exposure, are presented for comparative purposes (Fig. 5B)
. VES treatment had little to no effect on levels of Mr 43,000 Fas expressed in T47D whole-cell extracts (Fig. 5B)
.
|
, the overall cellular content (whole-cell extracts) of Mr 43,000 Fas did not markedly change following VES treatments of Fas-insensitive MCF-7, MDA-MB-435, and MDA-MB-435 cells. In contrast, analyses of membrane (Fig. 6 B)
versus cytosolic (Fig. 6C)
preparations of Fas-insensitive cells showed a reduction in cytosolic Mr 43,000 Fas following VES treatment (Fig. 6C)
and an increase in levels of Mr 43,000 Fas in membrane preparations (Fig. 6B)
following VES treatments. Furthermore, membrane preparations from MDA-MB-435 cells treated with 20 µg/ml VES for 3, 6, 12, and 24 h showed a time-dependent increase in membrane levels of Fas expressed when compared to the 24 h VEH-treated cells (Fig. 6D)
. Taken together, these data suggest that VES treatment causes translocation of Mr 43,000 Fas from the cytosol to the membrane in Fas-resistant cell lines.
|
, left). For these studies, VES-treated cells incubated with irrelevant primary antibody served as an important control for VES-treated cells incubated with anti-Fas. As can be seen in Fig. 7
, a subpopulation of cells treated with VES plus irrelevant antibody exhibited a shift in fluorescence compared to the VEH- plus irrelevant antibody-treated control. Furthermore, VES-treated cells (no primary or secondary antibodies) showed a similar fluorescence shift (data not shown). These shifts most likely represent apoptotic cells induced by VES because dead or dying cells exhibit more natural fluorescence than nonapoptotic cells. However, the magnitude of the fluorescence shift in the VES-treated plus irrelevant antibody-assayed cells is less than the shift obtained for the VES-treated cells assayed with anti-Fas antibody (Fig. 7)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Human Fas is a transmembrane glycoprotein found on multiple cell types (20) . Also known as APO-1 (for apoptosis-1) and CD95, Fas can mediate apoptosis when it is triggered by its natural ligand, Fas ligand, or when cross-linked with specific anti-Fas agonistic antibodies (reviewed in Ref. 42 ). Fas signaling is complex, and although Fas may be expressed, it may be inactive or may even signal accelerated cell growth (43) .
Recent evidence suggests that tumor cells can exploit the Fas system by down-regulating the expression of functional Fas to resist Fas ligand-mediated killing by cytotoxic T cells, whereas some tumor cells, which exhibit a Fas-resistant phenotype themselves, may actually use the Fas system in mounting a "counterattack" for deletion of tumor-infiltrating T-cells via up-regulation of tumor cell-derived Fas ligand (42, 43, 44, 45) . Recent studies indicated that anticancer drugs such as doxorubicin lead to the induction of the Fas system (46) , and as tumors develop mechanisms of drug resistance, they also develop mechanisms of resistance to Fas-mediated apoptosis (36) . Due to the importance of Fas signaling of apoptotic cell death of tumor cells, characterization of the molecular events involved in Fas resistance and identification of agents that can stimulate the expression of responsive Fas are needed.
In the in vitro studies reported here, VES is demonstrated to be an effective agent for switching Fas-resistant human breast cancer cells to a Fas-sensitive phenotype. Studies by Keane et al. (23) showed both primary normal HMECs and the immortalized nontumorigenic HMEC line (MCF10A) as well as T47D tumor cells to be Fas sensitive, whereas six breast cancer cell lines (MCF-7, ZR 75-1, MDA-MB-231, MDA-MB-435, MDA-MB-468, and SKBr3) were shown to be resistant to anti-Fas antibody-triggered apoptosis. Furthermore, Keane et al. (23) demonstrated the expression of a Mr 43,000 Fas molecule by Fas-sensitive cells (HMECs, MCF10A, and T47D cells), which was not observed in Fas-resistant breast cancer cell lines. Our data are in complete agreement with the data of Keane et al. (23) , regarding the responses to anti-Fas antibody-triggered apoptosis by HMECs, MCF10A, and T47D cells; the lack of anti-Fas antibody triggered apoptosis by MCF-7, MDA-MB-231, and MDA-MB-435 breast cancer cell lines that we studied; and the high constitutive expression of a Mr 43,000 Fas molecule by the Fas-sensitive cells and the comparatively low level of expression of the Mr 43,000 Fas molecule (namely, detectable only after long autorad exposure) by the Fas-resistant cells.
Although Keane et al. (23)
showed that treatment of the Fas-resistant breast cancer cells with IFN-
for 4 days sensitized them to Fas-mediated apoptosis, they reported little or no increase in the cell surface expression of Fas following IFN- treatment. In contrast to IFN- sensitization, VES sensitization of Fas-resistant cells is correlated with an increase in cell surface Fas expression. In our studies, pretreatment of Fas-resistant cells with VES for 6 h rendered the cells sensitive to the apoptotic inducing actions of the anti-Fas agonistic antibody, and although analyses of Fas levels in whole cell extracts showed that the overall levels of Fas did not change, analyses of cytosolic and membrane enriched extracts showed cytosolic levels of Fas to decrease while membrane levels of Fas increased, suggesting that VES was inducing Fas-resistant cells to become Fas-sensitive via translocation of the Mr 43,000 Fas molecule from the cytosol to the membrane. Thus, the studies by Keane et al. (23)
and data reported here show that Fas-resistant human breast cancer cells can be rendered Fas-sensitive by two agents in vitro; namely, IFN- treatment for 4 days which appears to sensitize the cells to anti-Fas antibody-triggered apoptosis via increased interleukin-1ß-converting enzyme (caspase-1) expression, and VES treatment for 6 h which appears to sensitize the human breast cancer cells via increasing Mr 43,000 Fas cell surface expression.
Additional support for VES-induced increased levels of cell surface Fas expression comes from FACS analyses, showing that VES-treated MDA-MB-435 cells, analyzed using a monoclonal reagent to Fas, express higher levels of Fas than VES-treated cells analyzed using an irrelevant control antibody. It is important to note that cells induced to undergo apoptosis exhibit an increase in "natural" cellular fluorescence that is detectable in the 488-nm range used to detect fluorescein-tagged antibodies, thus making it critical to distinguish between an increase in fluorescence due to increased apoptotic cell fluorescence from an increase in fluorescence due to an increase in cell surface Fas detected using an anti-Fas reagent in combination with a fluorescein-tagged secondary antibody.
In addition to sensitizing Fas-resistant breast cancer cells to anti-Fas triggered apoptosis, the combined treatment of Fas-resistant cells with VES and anti-Fas agonistic antibody produced synergisms. The molecular basis of this synergism is unknown, but it is interesting to note that VES activates latent TGF-ß and up-regulates TGF-ß receptor type II expression in human breast cancer cells (2
, 3
, 15)
. Furthermore, antisense functional knockouts of either TGF-ß1 or TGF-ß receptor type II or antibody neutralization of TGF-ßs inhibits VES-induced apoptosis by 50% (15)
. Pretreatment of glioma cells in vitro with TGF-ß has been shown to enhance Fas/APO-1 antibody-induced apoptosis (24)
. More recent studies show that endogenous expression of TGF-ß1 by murine glioma cells inhibits growth and tumorigenicity and enhances Fas-mediated apoptosis and that combinations of anti-Fas plus TGFß1 result in a synergistic apoptotic effect (47)
.
A role for the Fas system in VES-triggered apoptosis was originally reported by Turley et al. (18) . Although Turley et al. (18) did not address VES sensitization of Fas-resistant breast cancer cells and VES plus anti-Fas synergisms, they did show that VES up-regulated both Fas and Fas ligand protein levels in two human breast cancer cell lines (MDA-MB-231 and SKBR-3 cells) and that functional knockouts of the Fas system blocked VES-mediated apoptosis. In contrast to the studies reported here and by Keane et al. (23) , Turley et al. (18) reported on a Mr 48,000 Fas molecule. Turley et al. (18) analyzed 300 µg of cell lysates per lane using rabbit polyclonal antihuman Fas (Santa Cruz Biotechnology). In our hands, this antibody (albeit a different lot number) detected a prominent Mr 48,000 band the levels of which did not change following VES treatments and a faint Mr 43,000 band. The data reported here used a different antibody to human Fas (rabbit polyclonal antibody reagent; Santa Cruz Biotechnology). This antibody reagent that was used for all of the Western immunoblotting analyses reported here detected a very prominent Mr 48,000 molecule as well as a less prominent Mr 43,000 molecule. The Mr 48,000 molecule was judged to not represent functional Fas for the following reasons. The Mr 48,000 molecule was constitutively expressed by all cells tested (with the notable exception of HMECs) at very high levels in both whole cell and membrane preparations (levels much higher than those observed for the Mr 43,000 molecule); however, expression of the Mr 48,000 protein did not correlate with Fas functionality. HMECs, which are Fas sensitive, expressed only the Mr 43,000 molecule, and functional knockout experiments using antisense oligonucleotides to Fas reduced levels of the Mr 43,000 molecule but not levels of the Mr 48,000 molecule (Note: Mr 48,000 data were not presented in the immunoblots to focus attention to the relevant Mr 43,000 Fas molecule.) Additionally, VES treatments that sensitized Fas-resistant cells to anti-Fas agonistic antibody triggered apoptosis was correlated with marked changes in the expression of cytosolic and membrane Mr 43,000 Fas, whereas no noticeable changes were observed in the expression of the Mr 48,000 molecule following VES treatments in our experiments. Furthermore, although Fas was originally reported to be a Mr 48,000 molecule in SKW6.4 human B cells using SDS-PAGE under nonreducing conditions (48) , it has since been reported to be a Mr 43,000 molecule in human fibroblasts (20) and human breast cancer cells (23) . Thus, we judged the Mr 48,000 molecule to not be relevant and chose to present data on the Mr 43,000 molecule only (i.e., Mr 48,000 molecules were cutoff).
Studies reported here indicating a role for Fas in VES-triggered apoptosis agree, in principle, with those previously reported by Turley et al. (18)
. Functional knockout (antisense oligonucleotides or neutralizing antibody) of Fas reduced VES-induced apoptosis in MDA-MB-435 cells by 50%. Experiments reported by Turley et al. (18)
showed that neutralizing antibody to Fas reversed VES-induced apoptosis by 91% in MDA-MB-231 cells and 97% in SKBR-3. Although the same monoclonal antibody reagent was used in both studies, differences in cell lines analyzed and methods of determining apoptosis may account for the differences. Although it is unclear at this time whether Fas-signaling is both necessary and totally sufficient for VES-triggered apoptosis in human breast cancer cells, our previous studies suggest that this is unlikely. We have shown that VES-triggered apoptosis involves the TGF-ß signaling pathway, because blockage of this signaling pathway inhibits VES-induced apoptosis to a similar degree as that we observed in our studies of blockage of the Fas signaling pathway; namely, 50% (6
, 15)
. Furthermore, our previous studies have indicated an important role for JNK in VES-mediated apoptosis (17)
. Although oligomerization of Fas results in the activation of JNK, the role of JNK in Fas-induced apoptosis is controversial (43
, 49, 50, 51)
. Some studies support a role for JNK in the induction of cell death by Fas because blockage of JNK signaling prevents Fas-induced apoptosis (49
, 50)
, whereas other studies appear to rule out a role for JNK in Fas-mediated apoptosis because Fas still kills cells under conditions in which JNK is blocked (43
, 51)
. A recent report may reconcile the seemingly contradictory data in that Fas-triggered apoptosis can involve two pathways, a JNK-dependent pathway that is triggered by a novel Fas binding protein called Daxx, and a JNK-independent pathway that is triggered by the FADD-mediated pathway (52)
. The potential interactions among TGF-ß, Fas, and JNK, all of which appear to be involved in VES-triggered apoptosis of human breast cancer cells, remain to be sorted out.
We do not know why the Fas-sensitive cell lines used in these studies are resistant to VES-induced apoptosis. Because our previous studies have indicated that VES-induced apoptosis requires, at least in part, TGF-ß signaling (15) , it is possible that VES-insensitive cells may have defective or inhibited TGF-ß-mediated apoptotic signaling pathways. For example, T47D cells are TGF-ß resistant and do not express TGF-ß receptor type II mRNA (40) , and HMECs undergo DNA synthesis arrest but not apoptosis when treated with 10 ng of exogenous TGF-ß.4 Furthermore, VES-mediated apoptosis requires, at least in part, prolonged activation of JNK, and prolonged activation of JNK does not occur in VES insensitive HMECs and T47D cells.4
After the submission of this manuscript, a paper was published on cell surface trafficking of Fas in Science (53) . Fas trafficking in human breast cancer epithelial cells following VES treatment appears to differ from Fas trafficking reported by Bennett et al. (53) in at least two aspects. (a) Wild-type p53 is not required in VES-mediated increases of membrane Fas, because two of the cell lines (MDA-MB-231 and MDA-MB-435) studied have been reported to have p53 mutant proteins (54) . (b) In contrast to p53-mediated Fas trafficking, which was observed to be rapid and transient (cell surface Fas levels increased within 1 h of treatment and returned to baseline levels 2 h after treatment), VES induced Fas membrane expression occurs within 3 h following treatment and remains for at least 24 h.
In summary, RRR--tocopheryl succinate (VES), a derivative of vitamin E, is demonstrated for the first time to be capable of reversing the Fas-resistant phenotype exhibited by Fas-resistant human breast cancer cells. Human breast cancer cells treated with VES for as briefly as 6 h become sensitized to anti-Fas agonistic antibody triggered apoptosis, and this VES-mediated sensitization is correlated with a translocation of cytosolic Mr 43,000 Fas to the cell surface. Our hope is that further characterization of death receptor systems in human breast cancer cells and further characterization of VES-mediated proapoptotic effects will someday aid in the rationale selection of single, combined, or sequential chemotherapeutic regimes for improved treatment of human breast cancer.
|
FOOTNOTES |
|---|
1 This work was supported by Public Health Service Grant CA59739 awarded by the National Cancer Institute, the Foundation for Research, and National Institute of Environmental Health Sciences Center Grant ES 07784. 
2 To whom requests for reprints should be addressed, at the Division of Nutrition/A2703, University of Texas at Austin, Austin, TX 78712-1097. Phone: (512) 471-8911; Fax: (512) 471-5844; E-mail: k.kline{at}mail.utexas.edu
3 The abbreviations used are: VES, vitamin E succinate; TGF-ß, transforming growth factor-ß; JNK, c-Jun NH2-terminal kinase; HMEC, human mammary epithelial cell; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorting; VEH, vehicle; DAPI, 4',6-diamidino-2-phenylindole; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
4 W. Yu, B. G. Sanders, and K. Kline, unpublished data.
Received 8/ 3/98. Accepted 12/14/98.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tocopheryl succinate inhibits proliferation and enhances secretion of transforming growth factor-ß (TGF-ß) by human breast cancer cells. Nutr. Cancer, 19: 225-239, 1993.[Medline]
-tocopheryl succinate enhances TGF-ß1, - ß2, - ß3 and TGF-ßR-II expression by human MDA-MB-435 breast cancer cells. Nutr. Cancer, 26: 237-250, 1996.[Medline]
-tocopherol on human cancer cell lines in vitro. J. Oral Maxillofac. Surg., 50: 367-373, 1992.[Medline]
-tocopheryl succinate modulation of human promyelocytic leukemia (HL-60) cell proliferation and differentiation. Nutr. Cancer, 18: 201-213, 1992.[Medline]
-tocopheryl succinate inhibits DNA synthesis and enhances the production and secretion of biologically active transforming growth factor-ß (TGF-ß) by avian retrovirus-transformed lymphoid cells. Nutr. Cancer, 24: 171-185, 1995.[Medline]
-tocopheryl succinate induces apoptosis in avian retrovirus-transformed lymphoid cells. Nutr. Cancer, 25: 9-26, 1996.[Medline]
-tocopheryl succinate inhibits the proliferation of human prostatic tumor cells with defective cell cycle/differentiation pathways. Nutr. Cancer, 24: 161-169, 1995.[Medline]
-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells. Nutr. Cancer, 27: 267-278, 1997.[Medline]
-tocopheryl succinate induction of prolonged activation of c-Jun amino-terminal kinase and c-Jun during induction of apoptosis in human MDA-MB-435 breast cancer cells. Mol. Carcinog., 22: 247-257, 1998.[Medline]
which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice. J. Clin. Invest., 97: 2651-2659, 1996.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Shim and T. E. Eling Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism Mol. Cancer Ther., April 1, 2008; 7(4): 961 - 971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Neuzil, M. Tomasetti, Y. Zhao, L.-F. Dong, M. Birringer, X.-F. Wang, P. Low, K. Wu, B. A. Salvatore, and S. J. Ralph Vitamin E Analogs, a Novel Group of "Mitocans," as Anticancer Agents: The Importance of Being Redox-Silent Mol. Pharmacol., May 1, 2007; 71(5): 1185 - 1199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yin, J. Ni, M. Chen, M. A. DiMaggio, Y. Guo, and S. Yeh The Therapeutic and Preventive Effect of RRR-{alpha}-Vitamin E Succinate on Prostate Cancer via Induction of Insulin-Like Growth Factor Binding Protein-3 Clin. Cancer Res., April 1, 2007; 13(7): 2271 - 2280. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Donapaty, S. Louis, E. Horvath, J. Kun, S. M. Sebti, and M. P. Malafa RRR-{alpha}-Tocopherol succinate down-regulates oncogenic Ras signaling. Mol. Cancer Ther., February 1, 2006; 5(2): 309 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kline, W. Yu, and B. G. Sanders Vitamin E and Breast Cancer J. Nutr., December 1, 2004; 134(12): 3458S - 3462S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Adams and D. Teegarden 1,25-Dihydroxycholecalciferol Inhibits Apoptosis in C3H10T1/2 Murine Fibroblast Cells Through Activation of Nuclear Factor {kappa}B J. Nutr., November 1, 2004; 134(11): 2948 - 2952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Lawson, K. Anderson, M. Simmons-Menchaca, J. Atkinson, L. Sun, B. G. Sanders, and K. Kline Comparison of Vitamin E Derivatives {alpha}-TEA and VES in Reduction of Mouse Mammary Tumor Burden and Metastasis Experimental Biology and Medicine, October 1, 2004; 229(9): 954 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shah and P. W. Sylvester Tocotrienol-Induced Caspase-8 Activation Is Unrelated to Death Receptor Apoptotic Signaling in Neoplastic Mammary Epithelial Cells Experimental Biology and Medicine, September 1, 2004; 229(8): 745 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Anderson, M. Simmons-Menchaca, K. A. Lawson, J. Atkinson, B. G. Sanders, and K. Kline Differential Response of Human Ovarian Cancer Cells to Induction of Apoptosis by Vitamin E Succinate and Vitamin E Analogue, {alpha}-TEA Cancer Res., June 15, 2004; 64(12): 4263 - 4269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Zu and C. Ip Synergy between Selenium and Vitamin E in Apoptosis Induction Is Associated with Activation of Distinctive Initiator Caspases in Human Prostate Cancer Cells Cancer Res., October 15, 2003; 63(20): 6988 - 6995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bradford, J. Atkinson, N. Fuller, and R. P. Rand The effect of vitamin E on the structure of membrane lipid assemblies J. Lipid Res., October 1, 2003; 44(10): 1940 - 1945. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yu, B. G. Sanders, and K. Kline RRR-{alpha}-Tocopheryl Succinate-induced Apoptosis of Human Breast Cancer Cells Involves Bax Translocation to Mitochondria Cancer Res., May 15, 2003; 63(10): 2483 - 2491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Lawson, K. Anderson, M. Menchaca, J. Atkinson, L. Sun, V. Knight, B. E. Gilbert, C. Conti, B. G. Sanders, and K. Kline Novel Vitamin E Analogue Decreases Syngeneic Mouse Mammary Tumor Burden and Reduces Lung Metastasis Mol. Cancer Ther., May 1, 2003; 2(5): 437 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Prasad, B. Kumar, X.-D. Yan, A. J. Hanson, and W. C. Cole {alpha}-Tocopheryl Succinate, the Most Effective Form of Vitamin E for Adjuvant Cancer Treatment: A Review J. Am. Coll. Nutr., April 1, 2003; 22(2): 108 - 117. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. Brigelius-Flohe, F. J Kelly, J. T Salonen, J. Neuzil, J.-M. Zingg, and A. Azzi The European perspective on vitamin E: current knowledge and future research Am. J. Clinical Nutrition, October 1, 2002; 76(4): 703 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Kumar, M. N. Jha, W. C. Cole, J. S. Bedford, and K. N. Prasad D-Alpha-Tocopheryl Succinate (Vitamin E) Enhances Radiation-Induced Chromosomal Damage Levels in Human Cancer Cells, but Reduces it in Normal Cells J. Am. Coll. Nutr., August 1, 2002; 21(4): 339 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Weber, M. Lu, L. Andera, H. Lahm, N. Gellert, M. W. Fariss, V. Korinek, W. Sattler, D. S. Ucker, A. Terman, et al. Vitamin E Succinate Is a Potent Novel Antineoplastic Agent with High Selectivity and Cooperativity with Tumor Necrosis Factor-related Apoptosis-inducing Ligand (Apo2 Ligand) in Vivo Clin. Cancer Res., March 1, 2002; 8(3): 863 - 869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. N. Prasad, W. C. Cole, B. Kumar, and K. C. Prasad Scientific Rationale for Using High-Dose Multiple Micronutrients as an Adjunct to Standard and Experimental Cancer Therapies J. Am. Coll. Nutr., October 1, 2001; 20(90005): 450S - 463. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. You, W. Yu, B. G. Sanders, and K. Kline RRR-{alpha}-Tocopheryl Succinate Induces MDA-MB-435 and MCF-7 Human Breast Cancer Cells to Undergo Differentiation Cell Growth Differ., September 1, 2001; 12(9): 471 - 480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yu, Q. Y. Liao, F. M. Hantash, B. G. Sanders, and K. Kline Activation of Extracellular Signal-regulated Kinase and c-Jun-NH2-terminal Kinase but not p38 Mitogen-activated Protein Kinases Is Required for RRR-{alpha}-Tocopheryl Succinate-induced Apoptosis of Human Breast Cancer Cells Cancer Res., September 1, 2001; 61(17): 6569 - 6576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kline, W. Yu, and B. G. Sanders Vitamin E: Mechanisms of Action as Tumor Cell Growth Inhibitors J. Nutr., January 1, 2001; 131(1): 161S - 163. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or VEH (
) for 48 h. Apoptosis was monitored by DAPI staining. Columns, mean percentage apoptosis of three independent experiments; bars, SD.
