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Tumor Biology |
Discovery Research Department, EntreMed, Inc., Rockville, Maryland 20850 [T. M. L., X. H. Z., M. S. J., C. J. H., G. S., W. A. H., S. J. G., V. S. P.], and Department of Immunology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855 [M. S. W.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß, does not use an ER for its antiproliferative activities, and neither ER agonists nor antagonists influence the activity of 2ME2 (3)
. The antiproliferative activities of 2ME2 have been attributed to several mechanisms, including effects on tubulin polymerization and depolymerization (4, 5, 6)
, up-regulation of p53 (7
, 8)
, and inhibition of superoxide dismutase enzymatic activity (9)
. Although some of these mechanisms are sufficient to explain the activity of 2ME2 in certain cases, there is presently no evidence for a common mechanism of action operative in all cells sensitive to 2ME2. Apoptosis is an intracellular suicide program that is executed by the activation of caspases, a family of cytoplasmic cysteine proteases. Two major pathways, the intrinsic and the extrinsic apoptotic pathways, induce the caspase signaling cascade. The intrinsic pathway involves the cell sensing stress that triggers mitochondria-dependent processes, resulting in cytochrome c release and activation of caspase-9 (10) . The extrinsic pathway involves activation of DRs such as Fas, TNF receptor 1, DR4, and DR5. Interaction with their respective ligands leads to a signal transduction cascade initiated by the recruitment of DR-associated molecules such as FADD and subsequent activation of caspase-8 (11) . This caspase then catalyzes a series of proteolytic events that result in the characteristic biochemical and morphological changes associated with apoptosis.
In this study, we show that 2ME2 induces apoptosis through engagement of the extrinsic pathway. 2ME2 treatment of a variety of tumor cells as well as endothelial cells results in up-regulation of DR5 in vitro and in vivo. It also renders the cells more sensitive to the DR5 ligand TRAIL, which acts synergistically with 2ME2 in inducing apoptosis in vitro. The findings that cell death induced by 2ME2 requires the activation of caspases, and the early activation of caspase-8 before caspase-9 and caspase-3, are also consistent with 2ME2 activation of the extrinsic pathway. Finally, blocking DR signaling by stable transfection of a DN-FADD construct results in severe impairment of the apoptotic response induced by 2ME2.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(R&D Systems); anti-Fas clone CH-11 (Upstate Biotechnology, Lake Placid, NY); zVAD-fmk (Alexis Biochemicals); and cycloheximide (Sigma, St. Louis, MO).
Cell Culture.
Human breast carcinoma cells MDA-MB-231 and MDA-MB-435, cervical carcinoma cells HeLa, prostate carcinoma cells PC-3, and glioma cells U87-MG were maintained in DMEM:Ham’s F-12 (1:1) medium containing 10% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, UT) and 1x antibiotic-antimycotic (BioWhittaker, Walkersville, MD).
HUVEC were obtained from Clonetics (San Diego, CA). HUVEC cultures were maintained for up to five passages in endothelial growth medium containing bovine brain extract (Clonetics) and 1x antibiotic-antimycotic.
Apoptosis Assays.
Apoptosis was measured by examination of the presence of cytoplasmic histone-associated DNA fragments. Briefly, cells were seeded in a 24-well plate and after 24 h were exposed to drug for the indicated times. Detection of cytoplasmic histone-associated DNA fragments was performed according to the manufacturer’s instructions (Roche, Indianapolis, IN).
Immunoblot Analysis.
Subconfluent monolayers of cells were treated with the indicated concentrations of 2ME2. Cellular supernatant was removed and collected, cells were scraped in cold PBS, and the total cellular content was collected by centrifugation. Cell pellets were lysed in 50 µl of cold lysis buffer [10 mM Tris (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM sodium vanadate], and the lysate was clarified by centrifugation at 4°C. Protein concentration was determined by method of Bradford. The cytosol was mixed with an equal volume of NuPage LDS sample buffer (Invitrogen, Carlsbad, CA) containing 10% 2-mercaptoethanol (Sigma) and heated at 95°C for 5 min. Equal protein concentrations of cytosol lysate were subjected to 4–20% Tris-glycine SDS-PAGE (precast NOVEX; Invitrogen). Subsequently, protein was electrophorectically transferred to nitrocellulose membranes and blotted with either a rabbit anti-DR5 polyclonal antibody (Cell Sciences, Inc., Norwood, MA) or a rabbit antiactin polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a rabbit anti-PARP polyclonal antibody (Santa Cruz Biotechnology, Inc.). Proteins were visualized by the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ) using a horseradish peroxidase-conjugated antibody against rabbit or mouse IgG (Bio-Rad, Hercules, CA).
Immunofluorescence.
MDA-MB-231 cells were plated on chamber slides (35 x 103 cells/chamber, Falcon Culture Slides) and after 24 h were treated with media alone or containing 1 µM 2ME2 for 24 h. Attached cells were fixed with 4% paraformaldhyde for 25 min at 4°C. Slides were then washed two times in PBS for 5 min. After incubation for 5 min on ice with 0.2% Triton X-100 in PBS, the grid housing was removed and slides were washed twice in PBS. Chamber slides were then stored at -20°C until analysis. Chamber slides were warmed to room temperature and rehydrated in dH2O for 20 min were blocked with 1% fish skin gelatin (Sigma), 0.2% Triton X-100 in PBS for 50 min, rinsed twice in PBS followed by Avidin and Biotin Block (Vector Laboratories, Burlingame, CA). The preparations were then incubated with 1:100 dilution of rabbit antiactive Caspase-3 monoclonal antibody (PharMingen, San Diego, CA) at 4°C overnight, rinsed three times sequentially in dH2O, PBS with gelatin/Triton X-100 and PBS, and finally incubated with biotinylated antirabbit secondary antibody for 40 min (1:200 dilution), followed by dark room incubation with Fluorescein Avidin D (1:100 dilution) 40 min (Vector Laboratories). Chamber slides were then incubated with 1% fish gelatin, 0.2% Triton X-100 in PBS for 50 min followed by a rabbit polyclonal anti-DR5 (1:100 dilution; Cell Sciences, Inc.) for 1.5 h in the dark at room temperature followed by direct conjugation with antirabbit Texas Red Alexia 594 for 30 min (Molecular Probes, Inc., Eugene, OR). After the final wash, the slides were mounted and stained with DAPI that produces blue fluorescence. Immunofluorescence microscopy was performed using a BX60 microscope (Olympus).
Tumor Models and Immunohistochemistry.
Female 8–10 week-old BALB/c nu/nu mice (The Jackson Laboratory, Bar Harbor, ME) received injections in the mammary fat pad with 2 x 106 MDA-MB-231 cells. Daily oral administration of 2ME2 was initiated when the tumors became palpable (4–5 mm diameter). A suspension of 2ME2 was prepared by either homogenization in an aqueous solution of 0.5% methylcellulose (Sigma) using a Kinematica polytron (Brinkmann, Westbury, NY), or 2ME2 was incorporated into liposomes composed of dioleoyl phosphatidylcholine with a molar ratio of phospholipid:2ME2 of 2:1.5. Control animals were given vehicle alone or liposomes composed of dioleoyl phosphatidylcholine alone. Tumor volumes were calculated as width2 x length x 0.52. Upon termination of the dosing regimen, animals were euthanized with CO2, and tumors were excised and fixed in 10% buffered formalin. Paraffin-embedded tissue samples were sectioned (5 µm) and mounted on Superfrost slides (Fisher Scientific, Co., Houston, TX). Sections were deparaffinized in xylene, subsequently immersed in graded alcohols (100%, 95% ethanol) and rehydrated in dH2O. Sections were then treated with EDTA buffer for heat-induced epitope retrieval (NeoMarkers, Inc., Union City, CA). Samples were washed twice in dH2O and blocked for endogenous peroxidase activity by use of 3% hydrogen peroxide in methanol for 20 min. After washing sections in dH2O, samples were incubated with 5% normal rabbit serum for 40 min and then incubated with the primary antibody, polyclonal goat anti-DR5 antibody (1:100 dilution; Alexis Biochemicals), for 18 h at 4°C. Slides were rinsed in dH2O, washed twice in PBS, incubated with biotinylated antigoat secondary antibody (1:200 dilution), washed twice in PBS, and finally incubated with Elite ABC Peroxidase Kit (Vector Laboratories). After a final wash in PBS, positive reaction was visualized by incubating the slides with 3,3'-diaminobenzidine for 5 min (Vector Laboratories). The slides were then rinsed in dH2O, counterstained with Gills Hemotoxylin III (Sigma), rinsed, and immersed briefly in 1% ammonia solution for nuclei binding. Slides were hydrated and mounted with cytoseal (VWR International, West Chester, PA). For negative controls, the same procedure was performed with the omission of either the primary or secondary antibody.
Measurement of Caspase Enzymatic Activity.
Caspase activation was measured using a caspase fluorometric protease assay kit as described by the manufacturer (BioVision Research Products, Palo Alto, CA). Briefly, after treatment of cells with 2 µM 2ME2 for the indicated periods of time, cells were harvested, pelleted, and frozen on dry ice. Cell lysis buffer was added to the cell pellets, and protein concentration was determined by using Bradford analysis. A total of 200 µg of cell lysates was incubated with 5 µl of a 1 mM stock of the respective fluorescently labeled tetrapeptide at 37°C for 1 h. The release of AFC was measured with a fluorometer (Wallac Victor; Perkin-Elmer Life Sciences, Boston, MA) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Caspase activities were assayed in duplicate samples. Caspase-8-, caspase-9-, and caspase-3-like enzymatic activities were assessed in parallel.
Stable Transfection.
The expression vector pcDNA3-FADD-DN [kind gift of Dr. Mark Williams (American Red Cross)], which carries the cDNA insert of human DN-FADD under the control of the cytomegalovirus promoter or the vector pcDNA3.1 (Promega) without insert, were used to generate stable transfectants. MDA-MB-231 cells were plated at 5 x 105 cells/10-cm dish, and the next day were transfected with 1 µg of plasmid using the SuperFect reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Resistant colonies were selected in 2 mg/ml G418. Ten isolated colonies were selected, and positive clones expressing the DN-FADD construct were verified by Western blot analysis using an anti-FADD monoclonal antibody (Transduction Laboratories, Lexington, KY).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The appearance of multiple polypeptides in the DR5 immunoblot has been reported previously (14
, 15)
. This up-regulation is associated with an apoptotic response, as indicated by the appearance in 2ME2-treated cells of the Mr 85,000 PARP cleavage fragment (Fig. 1B)
. The IC50 values for the antiproliferative activity of 2ME2 are in a narrow range from high nanomolar to low micromolar, and consequently, a 2-µM concentration was selected to evaluate the effects of 2ME2 on the various cell types. However, with HeLa cells, we used a concentration of 1 µM because in these cells the overall cell death is greater, although the IC50 is not particularly different from other cell types. Treatment of HeLa cells with 1 µM 2ME2 for 48 h results in an 88.5% decrease in cell number, whereas treatment of the other cell lines with 1 µM 2ME2 results in only an approximate 65% decrease (Fig. 1C)
. It is interesting that the appearance of the PARP cleavage fragment varies among cell lines. In MDA-MB-231, MDA-MB-435, and PC3 cells, 2ME2 treatment results in the appearance of the PARP cleavage fragment at 48 h, whereas in HUVEC and U87-MG cells, appearance of detectable level of PARP cleavage fragment occurs at 24 h and does not increase with additional treatment. Consistent with HeLa cells being more responsive to 2ME2, the greatest extent of PARP cleavage in response to treatment occurs with these cells (Fig. 1B)
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2ME2 Treatment Sensitizes MDA-MB-231 Cells and HUVEC to TRAIL-induced Apoptosis.
TRAIL, the ligand for DR5, has cytotoxic activity on a wide variety of transformed cell lines in vitro and is a potent antitumor agent in vivo (17
, 18)
. However, up-regulation of DR5 does not always predict susceptibility to TRAIL-induced apoptosis (19, 20, 21)
. Because 2ME2 up-regulates DR5, we investigated whether 2ME2 treatment sensitizes tumor and endothelial cells to TRAIL-induced apoptosis. Treatment of either MDA-MB-231 cells or HUVEC with 50 ng/ml TRAIL alone or 2ME2 alone induces apoptosis. Exposure of both cell types to TRAIL in combination with 2ME2 results in a marked increase in apoptosis as compared with either treatment alone (Fig. 2, A and B)
. The strong induction of apoptosis by the combination treatment makes it appear that 2ME2 alone does not significantly induce apoptosis. However, with more prolonged incubation during the color development of the ELISA, a dose-dependent increase in apoptosis in response to 2ME2 is observed (Fig. 3)
. These data are consistent with the DR5 expressed in these cells being able to induce apoptosis and indicate that 2ME2 treatment further sensitizes cells to TRAIL-induced cell death.
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, 2ME2-induced apoptosis in MDA-MB-231 cells and HUVEC was greatly attenuated in the presence of zVAD-fmk. These data suggest that both in tumor and endothelial cells 2ME2 requires the activation of caspases to induce apoptosis.
To further characterize the apoptotic pathway activated by 2ME2, we determined the kinetics of caspase activation. The activation of caspases in 2ME2-treated cells was assessed using the fluorogenic tetrapeptide substrates, IETD-AFC, LEHD-AFC and DEVD-AFC, which have been shown to be selective for caspase-8-, caspase-9-, and caspase-3-like enzymatic activities, respectively. Treatment of MDA-MB-231 cells with 2 µM 2ME2 results in the detection of caspase-8 activation as early as 2 h followed by caspase-9 activation at 4 h and caspase-3 activation at 8–14 h (Fig. 4)
. The level of caspase-8 and caspase-9 activation increased up to 16 h of treatment. Caspase-3 activation continued to intensify over time and resulted in a much greater fluorescence signal than either caspase-8 or caspase-9 (Fig. 4)
. Our observation that caspase-8 is the first caspase activated by 2ME2 is consistent with an apoptotic pathway initiated by DR activation.
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. A slightly lower concentration of 2ME2 was used in these experiments as compared with our immunoblot analysis (Fig. 1, A and B)
to increase the number of cells retained on the coverslip, however, similar results were obtained with 2 µM 2ME2. In the treated population, there is a clear decrease in the number of cells and an increase in the number of mitotic figures and condensed nuclei. Cells stained with anti-DR5 antibody followed by a rodamine-labeled secondary antibody (Fig. 5)
showed brighter staining of the treated population overall consistent with the increase in DR5 protein expression observed by immunoblot analysis (Fig. 1A)
. Staining with an antibody that recognizes the activated cleavage fragment of caspase-3 followed by a FITC-labeled secondary antibody shows that despite occasional staining of cells in the untreated population, there are significantly more activated caspase-3-positive cells in the treated cells. Because in this assay only the cells that are still attached were evaluated, this assessment underestimates the total number of cells that have activated caspase-3. Costaining with anti-DR5 and antiactivated caspase-3 antibodies shows that 2ME2 treatment results in active caspase-3 only in cells that have up-regulated DR5 as indicated by the yellow cells (Fig. 5)
, consistent with DR5-dependent caspase-3 activation.
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, Fas, TNF, and TRAIL-mediated apoptosis were markedly reduced by stable overexpression of DN-FADD protein as compared with the treated cells transfected with vector alone. These death-inducing cytokines have been shown to mediate apoptosis through a DR-mediated pathway that activates caspase-8 through a FADD-dependent pathway (22
, 23)
. Similarly, 2ME2 treatment of two independent clones of cells transfected with DN-FADD resulted in a marked reduction in the amount of apoptosis observed as compared with the cells expressing the vector alone (Fig. 6B)
. These data indicate that a DR-mediated pathway is used in the mechanism of 2ME2-induced apoptosis. Although these data do not exclude a role of other DRs, only DR5 is up-regulated in these cells in response to 2ME2 treatment.
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. At the termination of study the tumor burden was reduced 
65% in the animals treated with 150 mg/kg 2ME2. Consistent with data using cultured cells, immunohistochemistry analysis of MDA-MB-231 tumors demonstrated that 2ME2 treatment results in up-regulation of DR5 protein expression (Fig. 7B)
but not DR4 protein expression (data not shown). Similar results were obtained using U87-MG in a xenograft tumor model (24)
. These data show that treatment of tumors with doses of 2ME2 that produce a reduction in tumor burden in two animal models results in the up-regulation of DR5 protein.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our evaluation of which DRs were induced in response to 2ME2 treatment, we found that DR5 was consistently up-regulated. DR4 protein up-regulation was observed in treated PC3 cells in vitro; however, there was no significant increase in DR4 protein expression in response to 2ME2 treatment in vivo. Consistent with previous reports, 2ME2 treatment resulted in up-regulation of Fas in HUVEC, but this treatment did not result in up-regulation of Fas in tumor cell lines (data not shown). Similarly, the ligand for DR5 TRAIL was up-regulated in response to treatment of HUVEC but not of tumor cell lines. Our in vivo analysis showed a mixed response in that only some of the treated animals showed a slight increase of TRAIL protein expression. Thus, whereas DR4, Fas, and TRAIL up-regulation varied as a function of cell type, DR5 was consistently up-regulated in response to 2ME2 treatment. Alternatively, TRAIL produced in the tumor vasculature could exert its effects in the surrounding tumor cells. Currently, this cannot be assessed in these models because antibodies recognizing murine TRAIL are not available.
The cell type-specific Fas and TRAIL up-regulation suggest that some aspects of the mechanisms by which 2ME2 induces apoptosis in tumor and endothelial cells may be distinct. Two antiangiogenic proteins, pigment epithelium-derived factor and thrombospondin 1, have recently been reported to selectively induce apoptosis in proliferating endothelial cells by up-regulating FAS ligand. Fas was shown to be up-regulated in only proliferating endothelial cells and, thus, allows these antiangiogenics agents to selectively inhibit the growing or remodeling endothelium (25) . Whether these endogenous angiogenesis inhibitors also up-regulate DR5 is unknown. The finding that several 2ME2 analogs show preferential inhibition of endothelial cells over tumor cells (26 , 27) additionally suggests that the mechanisms of 2ME2-induced apoptosis in endothelial cells versus tumor cells may be different.
There are several possible mechanisms by which 2ME2-induced apoptosis in tumor cell lines in vitro could be mediated through DR5. TRAIL protein is present and it is possible, as has been suggested for other agents that up-regulate DR5 without up-regulating TRAIL, that the newly generated DR is able to use the constitutively expressed ligand. However, we were unable to affect 2ME2-induced apoptosis with a soluble DR5 alone or in combination with an antagonist antibody against Fas and soluble TNF receptor (data not shown). These data suggest either that the interaction with the endogenous ligand is not readily accessible to exogenously added inhibitors or that the up-regulated DR5 does not require additional activation. This has been shown in transfected cell lines in which overexpression of DR5 can induce apoptosis in a ligand-independent manner (23) . The up-regulation of DR5, the kinetics of caspase activation, the coexpression of activated caspase-3 only in cells expressing DR5, and the attenuation of apoptosis mediated by expression of DN-FADD are all consistent with DR5 involvement in 2ME2-mediated cell death, irrespective of the mechanism underlying this process.
The inhibitory effects of 2ME2 have been attributed to several mechanisms, including disruption of microtubule function (4, 5, 6) , inhibition of superoxide dismutase (9) , inhibition of angiogenesis (28) , and induction of apoptosis (16 , 29) . 2ME2 has been demonstrated to cause G2-M arrest, to bind to tubulin and microtubules inhibiting both polymerization and depolymerization (4 , 5) , and to cause abnormal spindle formation in cultured cells (30) . In contrast, it has been also reported that treatment of cells with antiproliferative concentrations of 2ME2 cause microtubule stabilization in some cases (6) and does not result in inhibition of mitotic spindles in others (31) . We have only observed destabilization of microtubules on cells in culture at concentrations of 2ME2 significantly higher than those used in this study to see effects on DR5 up-regulation. However, effects on microtubule dynamics occur at concentrations that do not affect stability (32) . We found that treatment of tumor-bearing animals with vinblastine, a drug that destabilizes microtubules, also resulted in up-regulation of DR5 protein (data not shown). Paclitaxel and docetaxel, two agents that stabilize microtubules, have been shown to up-regulate DR4 and DR5 protein expression and sensitize prostate cell lines to the cytotoxic effects of TRAIL (33) . However, these effects may not represent a general cellular observation because paclitaxel did not up-regulate DR4 and DR5 protein or enhance the activity of TRAIL in non-small cell lung cancer or breast cancer cell lines (34, 35, 36) . In the case of 2ME2 it is still uncertain how up-regulation of DR5 is related to its effects on microtubule stability and this requires additional investigation.
In the current study, we demonstrate that 2ME2 up-regulates DR5, uses the extrinsic apoptotic pathway, and makes cells more sensitive to TRAIL. Similar to 2ME2, TRAIL exerts potent antitumor activity in vivo without affecting normal tissue, suggesting that a combination of 2ME2 and TRAIL may be an effective antitumor therapy. Furthermore, in the absence of dose-limiting toxicity for 2ME2, DR5 expression may serve as a surrogate marker for biological response in the clinic. DR5 may also serve as a marker for assessing and discriminating 2ME2-like molecules from other related antiproliferative agents because structurally related molecules could induce antiproliferative responses through a variety of mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Phone: (240) 864-2600; Fax: (240) 864-2601; E-mail: theresal{at}entremed.com 
2 Present address: LabBook, Inc., 8260 Greensboro Drive, Suite 100, McLean, VA 22102.
3 The abbreviations used are: 2ME2, 2-methoxyestradiol; AFC, 7-amino-4-trifluoromethyl coumarin; DAPI, 4',6-diamidino-2-phenylindole; DN-FADD, dominant-negative Fas-associated death domain; DR, death receptor; ER, estrogen receptor; HUVEC, human umbilical vein endothelial cells; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor, TRAIL, TNF-related apoptosis-inducing ligand.
Received 6/17/02. Accepted 11/14/02.
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indicates the cleaved 85 kDa polypeptide fragment. The figure is a composite of three separate gels. C, to determine the antiproliferative activity, cells were treated with the increasing concentrations of 2ME2 for 48 h, and proliferation was determined by cell counts. The IC50s for the various cell lines are 0.67 µM (MDA-MB-231), 0.70 µM (PC3), 0.27 µM (MDA-MB-435), 0.50 µM (U87-MG), and 0.27 µM (HeLa).
) or presence (
) of 50 ng/ml TRAIL for 24 h, and apoptosis was determined by the cell death detection ELISA (Roche). The results are presented as the average of triplicates ± SD.
, respectively) or presence (
, respectively) of 50 µM zVAD-fmk for 48 h, and apoptosis was determined using the cell death detection ELISA (Roche). The results are presented as the average of triplicate samples ± SD.
) or with DN-FADD expression vector (
) were treated with 100 ng/ml anti-Fas IgM (clone CH-11) or 100 ng/ml TNF