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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Neurosurgery [J. H. U., N. P. D., J. S. R.] and Neuro-Oncology [J. H. U., A. P. K.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Department of Pathology-Neuropathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 [C. L. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß3, ß5) were capable of mediating this protective effect, because ligation of either of the two classic integrins conferred chemoresistance to Topo; and (e) chemoresistance observed with VN was associated with an increase in expression of two antiapoptotic proteins, Bcl-2 and Bcl-XL, with a consequent increase in the ratios for Bcl-2:Bax and Bcl-XL:Bax. VN, an ECM protein preferentially expressed at the tumor-brain interface in vivo, may confer a survival advantage to glioma cells at the advancing tumor margin and may thus, in part, underlie the high level of tumor recurrence at this interface. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the central nervous system, VN expression is abundant in the developing retina, where it functions as a positive modulator of neurite outgrowth (3)
and in the developing brain (4)
. Although the normal adult cortex and white matter are devoid of VN, under certain pathological conditions, VN is reexpressed in the brain parenchyma as well as in the endothelial cell basement membrane (2)
. In this regard, we have reported previously that glioma cells express VN and its cognate receptors (the ß3 and ß5 integrins) at the tumor-brain interface (2)
. Moreover, this expression correlates with tumor grade, being most abundant in gliomas of high-grade histology (glioblastoma), less in intermediate-grade gliomas, and virtually absent in low-grade lesions (2
, 5)
. With respect to its putative role at the advancing glioma margin, numerous studies have shown that VN and its receptors enhance glioma motility and invasiveness (5
, 6)
.
In this report, we addressed the novel hypothesis that a potential alternate role for VN is to confer a survival advantage for glioma cells. This postulate is based on several recent observations that suggest that the interaction between a cell and its surrounding ECM serves not only as a substrate for adhesion and motility, but that this interaction also constitutes a signal that inhibits apoptotic cell death (7)
. With respect to VN, its role as a cell survival factor has been illustrated in endothelial cells, in which ligation of the VN receptor (Vß3 integrin) dramatically inhibits apoptotic death, whereas antagonism of this receptor is lethal (8)
. In the present study, we used an in vitro model in which glioma cells were treated with the Topo I inhibitor, topotecan, an agent known to cause apoptotic death of these cells, to determine what effect the presence of VN would have on drug-induced programmed cell death as well as on the expression of the Bcl-2 family of antiapoptotic proteins.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Coating with ECM and Anti-Integrin Antibodies.
Tissue culture plastic surfaces were coated with either human FN or VN (Life Technologies, Inc.) to a surface concentration of 0–500 ng/cm2 with modifications of methods reported previously (9)
. FN was chosen as a control ECM protein, because it is recognized as a ligand by a different integrin receptor. Both VN and FN localize to the focal adhesion sites in glioma cells in vitro and are equipotent as substrates for glioma cell adhesion and migration (6)
. Moreover, both FN and VN receptors have been shown to inhibit apoptosis of endothelial cells (7
, 8) . An important difference, however, is that VN is expressed to a greater extent by glioma cells in vivo(2
, 5)
and as such may mediate a function that is distinct from FN, such as chemoresistance. Topotecan, a Topo I inhibitor, was chosen as a chemotherapeutic stressor, because it is known to cause programmed cell death in glioma cells and the doses of Topo necessary to achieve apoptotic cell death have been well established (10)
. Thus, whether VN was capable of altering the sensitivity to topotecan-induced apoptosis constituted the primary area of focus in this study. In this report, we compared the effect of plating glioblastoma cell lines on VN or FN in the presence of a chemotherapeutic agent and determined the extent of apoptosis with an in vitro assay of DNA fragmentation.
For apoptosis experiments, 100 µl of ECM solutions diluted in PBS were aliquoted into the chambers (LabTek/NUNC, Rochester, NY), left at room temperature for 2 h, and incubated with 0.1% BSA in PBS for 30 min at 37°C to block noncoated areas of plastic with interspersed PBS washes. Chamber slides were then air-dried and used immediately. For immunoblot- and cell surface-labeling experiments, 100-mm dishes (Falcon) were coated with FN or VN (Life Technologies, Inc., Rockville, MD) at 500 ng/cm2 by incubating the plates with 8 ml of the ECM proteins diluted in PBS. BSA blocking and washing steps, identical to those used for chamber slide coatings (described above), were then used.
Chamber slides were coated with antibodies capable of ligating specific VN receptors by methods described elsewhere (11)
. In brief, the slides were pretreated with goat anti-mouse IgG (100 µl of 50 µg/ml solutions) for 1 h, followed by incubation with 1% BSA for 30 min. Onto slides coated with anti-mouse IgG, the following mouse-derived purified monoclonal antibodies were added (100 µl of 20 µg/ml solutions; 1 h at room temperature): (a) control mouse IgG; (b) anti-5ß1 integrin (ligates FN receptor); (c) anti-ß3 integrin (ligates VN receptor; this antibody is the LM609 clone); or (d) anti-ß5 integrin (ß5 is also a VN-binding receptor). All antibodies were purchased from Chemicon (Temecula, CA). All incubations were performed at room temperature; between each step, the chambers were rinsed three times with PBS. The chambers were allowed to air dry and used immediately for cell seeding.
Apoptosis Assay.
Cells were seeded onto ECM or antibody-coated chamber slides by the following method: (a) flasks of cells grown in the presence of serum were rinsed twice with PBS and harvested in suspension with buffered 2 mM EDTA (trypsin was not used to avoid proteolytic cleavage of cell surface receptors for VN or FN); (b) cells were resuspended in serum-free F-12 medium supplemented with ITS (see above), and 1.5 x 104 cells in 200 µl were seeded to each chamber; (c) after 12 h, cells were treated with topotecan, and 48 h later, they were assayed for apoptosis with an in situ method for detecting DNA fragmentation (TUNEL) by methods described previously (12)
.
In brief, the TUNEL assay was performed by first washing cells in chamber slides with PBS and then fixing them in acetone:methanol (1:1 mixture) for 20 min at -20°C. Cells were then rehydrated for 30 min at room temperature. Each coverslip was then incubated in a humidified chamber at 37°C for 30 min in the following: 0.5 µl of TdT enzyme (Promega Corp., Madison, WI), 10 µl of 5x TdT reaction buffer, 1 µl of biotin-dUTP (Boehringer Mannheim, Indianapolis, IN), and double-distilled H2O to make up 50 µl total volume. Coverslips were then washed and subsequently incubated in streptavidin conjugated to FITC (1:100 dilution) for another 30 min at 37°C. Cells were washed again in PBS and then incubated for 10 min at room temperature in 10 µg/ml PI, which counterstains all nuclei. Coverslips were mounted using glycerol/PBS and visualized by fluorescence microscopy. Apoptotic cells were defined as those cells whose DNA was labeled by TUNEL and that demonstrated other morphological characteristics of apoptosis, including nuclear blebbing and eccentrically displaced and condensed nuclei. To quantitate the percentage of cells demonstrating DNA fragmentation, the number of TUNEL-labeled cells were divided by the number cells counterstained with PI present in five random microscopic fields.
Treatment with RGD Peptides.
In these experiments, topotecan was not added to the glioma cell cultures; rather, the ability of VN receptor antagonists to independently cause tumor cell apoptosis was assessed. To determine the effect of VN receptor antagonism on cell survival, glioma cells were treated with one of three peptides (Life Technologies, Inc.) used at a concentration of 100 µM: (a) cycRGD [G-Pen-GRGDSPC (RGD tri-amino acid sequence is italicized)], a peptide that has been shown to selectively antagonize the VN receptor [the VN receptor-inhibitory concentration of 100 µM is close to an order of magnitude below the threshold for inhibition of the FN receptor (13)
]; (b) GRGDSP, which typically can inhibit all RGD-mediated integrin interactions (including FN or VN receptor) but is significantly less effective than cycRGD in inhibiting the VN receptor (6
, 13)
; or (c) GRGESP, a control peptide that does not interfere with integrins. Cells were suspended medium containing 10% fetal bovine serum, which is a rich source of VN. While in suspension, the cells were treated with peptides for 5 min, following which the mixtures were seeded onto coverslips precoated with FN (500 ng/cm2). By this approach, we were able to control for cell adhesion, because cells were seeded onto chamber slides in conditions that would permit adhesion/spreading via either VN, or alternately, FN, a ligand that is as equally adhesive as VN for glioma cells (6)
. After 48 h, cells were assayed by TUNEL to determine whether peptide treatment influenced susceptibility to apoptosis. To confirm that cell adhesion was not adversely affected in the cycRGD-treated cells, adhesion assays were performed as described previously (6)
.
Immunolabeling of Cell Surface Receptors for VN.
All immunolabeling steps were performed with live, nonfixed cells to selectively label cell surface antigens. Glioma cells, plated on 100-mm dishes (
1 x 106 cells/dish) coated previously with 500 ng/cm2 of FN or VN (serum-free condition; cells were plated for duration of 48 h), were rinsed with PBS and then harvested into suspension with buffered 2 mM EDTA without trypsin. After centrifugation at 1000 rpm in a Sorvall desktop centrifuge at 4°C, the cell pellet was gently resuspended in 1 ml of PBS, followed by addition of either control (mouse IgG) antibody or primary monoclonal antibodies (final concentration, 1 µg/ml) that specifically recognize the native human receptors for VN (ß3 and ß5 integrins) or FN (5ß1 integrin) in their heterodimeric form (Chemicon). The cells were washed three times with PBS, resuspended in 1 ml of PBS, and then incubated with biotinylated anti-mouse IgG. After three washes, streptavidin-FITC (1:150) was added to the cells. After another three washes, the cell pellet was resuspended in 2% paraformaldehyde and analyzed on an EPICS FACS. All steps were performed using Ca2+/Mg2+-free PBS.
Immunoblotting.
Chemiluminescent immunoblot analysis for the expression of Bcl-2, Bcl-XL, and Bax was performed as described previously (14)
. Glioma cells were plated onto 100-mm dishes coated with either FN or VN (500 ng/cm2) and grown in serum-free conditions as described above (with or without topotecan; see Fig. 4
). After 48 h, the cells were washed with PBS and then lysed with ice-cold triple detergent buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP40, and 0.5% sodium deoxycholate]. After incubation on ice for 1 h, the lysate was centrifuged at 10,000 x g, and the resultant supernatant was analyzed for protein by the BCA assay (Pierce, Rockford, IL) and then mixed with 4x SDS sample buffer. Forty µg of protein were electrophoresed in 15% SDS-polyacrylamide gels, transferred overnight to Immobilon membranes, blocked with 5% milk/PBS, and then incubated with primary monoclonal antibodies diluted in 5% skim milk/PBS for 2 h at room temperature. Primary antibodies consisted of anti-Bcl-2 [mouse anti-human Bcl-2, 1 µg/ml (Oncogene Research, Cambridge, MA)], anti-Bcl-XL [rabbit anti-Bcl-XL/S (Santa Cruz; Santa Cruz, CA), which recognizes both Bcl-XL (Mr 29,000) or Bcl-XS (Mr 19,000)] and anti-Bax [rabbit anti-human Bax, 0.3 58 g/ml (Santa Cruz; Santa Cruz, CA)]. Secondary antibodies (goat-derived) conjugated to horseradish peroxidase (Amersham, Piscataway, NJ) were used at 1:1000 dilution (1 h at room temperature), after which the membranes were exposed to horseradish peroxidase substrate (Blaze substrate; Pierce) for 5 min. Chemiluminescent signals were developed with Hyperfilm and quantitated using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
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As illustrated in the micrographs of Fig. 1
, the number of U251 cells that were positively labeled with the TUNEL technique in the presence of topotecan was significantly reduced in the population seeded on VN as compared with those seeded on FN at coatings of 500 ng/cm2. For both U251 and D54 cells, the antiapoptotic effect of VN was dose dependent, with an inhibition of apoptosis of up to 50% on a 500 ng/cm2 coating of VN, whereas in contrast, cells plated on FN, even at 500 ng/cm2, fared no better than those seeded onto plates with no ECM coating (Fig. 1
, middle and bottom panels). Similarly, when glioma cells were plated on a fixed ECM coating concentration of 500 ng/cm2 but treated with escalating doses of topotecan, the proportion of cells manifesting apoptotic features at each dose of drug was significantly less in the VN-plated cells (Fig. 2
, row A). For U251 cells, the median lethal dose (LD50) on VN was 130 ng/ml of topotecan, almost 4-fold higher than the LD50 on FN (35 ng/ml).
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Cell Surface Integrin Receptors for VN Are Capable of Conferring Protection for Apoptosis.
Although the data illustrated in Fig. 1
suggest a role for VN in enhancing the survival of glioma cells, they do not confirm that this effect is mediated by ligation receptors capable of binding VN, which include the Vß3 and Vß5 integrins as well as the receptor for urokinase (urokinase plasminogen activator receptor; Ref. 16
). To address this issue, we targeted the integrins Vß3 and Vß5, because they have been shown previously to play an important role in endothelial cell survival (8)
, using integrin-specific antibodies immobilized on culture dishes to determine whether the ligation of these integrins may confer enhanced survival to glioma cells. Cells were plated onto chamber slides coated with monoclonal antibodies specifically directed toward the FN receptor (5ß1 integrin) or the two VN receptors (ß3 or ß5 integrins) and then treated with a fixed dose of topotecan (20 ng/ml; Fig. 2
, row B). Although both cell lines were confirmed to express the 5ß1 integrin (FN receptor) by immunofluorescence (data not shown), the antibody to the FN receptor produced only a marginal reduction in apoptosis in U251 cells, while demonstrating no significant effect in D54 glioma cells (Fig. 2
, row B). This observation was in sharp contrast to U251 cells plated on either of the VN receptor antibodies (anti-ß3 or anti-ß5), where the proportion of apoptotic cells was reduced by >75% compared to cells seeded onto control IgG. Similarly, D54 cells seeded onto the anti-Vß5 antibody coating, but not the anti-ß3 antibody coating, were protected from apoptosis. This enhanced cell survival on the immobilized antibody coatings corresponded to the expression of the respective cell surface integrins as determined by FACS; although U251 immunolabeled for both cell surface VN receptors, D54 was positive for only the ß5 integrin (Fig. 2
, row C). Taken together, these results suggest that either of the two classic VN receptors is capable of mediating protection against apoptosis in glioma cells.
Inhibition of the Receptors for VN Leads to Increased Cell Death.
To further corroborate a role for VN and VN receptors in enhancing glioma survival, a specific RGD peptide (cycRGD) was used to selectively antagonize the VN receptor. It is important to note that in contrast to previous experiments illustrated in Figs. 1
and 2
, these experiments with VN receptor antagonistic RGD peptides (Fig. 3)
were performed in the absence of topotecan. As illustrated in Fig. 3
, both U251 and D54 glioma cells underwent increased apoptosis in response to treatment with cycRGD during adhesion to coverslips coated with FN and VN; the standard linear RGD peptide had no significant effect compared with the control RGE peptide treatment. At 100 µM, the cycRGD peptide is a relatively selective antagonist of VN receptors (Ref. 13
; the threshold concentration required for inhibiting the FN receptor is close to an order of magnitude greater), whereas the linear RGD peptide at this concentration is significantly less effective as an inhibitor of the VN receptor integrin compared with cycRGD (6
, 13)
. Thus, the specific antagonism of the VN receptor was associated with an increase in cell death.
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As illustrated in Fig. 4
, both U251 and D54 cells plated on VN demonstrate a significant increase in the ratios for Bcl-2:Bax and Bcl-XL:Bax, largely as a result of increases in levels of Bcl-2 and Bcl-XL, because Bax expression remained relatively unchanged. It is interesting to note that both in the presence and absence of drug, the Bcl-2/Bax- as well as the Bcl-XL:Bax ratios were greater in cells grown on VN compared with FN. As such, the up-regulation of antiapoptotic proteins such as Bcl-2 and Bcl-XL may represent a possible mechanism by which VN confers enhanced survival to glioma cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß3 and ß5) also inhibited apoptosis ( Figs. 1
and 2
).
With regards to the VN receptor, the ligation of the Vß3 integrin on endothelial cells has been associated with an increase in the expression of the antiapoptotic protein, Bcl-2 (17)
. Given that the overexpression of Bcl-2 has been correlated with a chemoresistant phenotype in numerous peripheral tumors (18)
, we pursued the hypothesis that the survival advantage conferred by VN to glioma cells may also involve an increase in the levels of Bcl-2 expression. In concordance with this postulate, Bcl-2 levels were indeed found to be higher in lysates of both U251 and D54 glioma cells plated on VN, compared with those of FN-plated cells (Fig. 4)
. Moreover, VN-plated glioma cells demonstrated increased expression of another antiapoptotic protein, Bcl-XL. The observed increases in the expression of these two antiapoptotic proteins may thus underlie the survival advantage observed with the VN substrate.
Bcl-2, Bcl-XL, and other antiapoptotic members of the Bcl family of proteins exert their protective effect, in part, by inhibiting the mitochondrial release of cytochrome c(19
, 20)
, a critical cofactor in the proteolytic activation of the caspases, the effector molecules of apoptosis. Evidence for a role for the Bcl family of proteins in the treatment-resistant phenotype in malignant gliomas has been demonstrated previously. Nagane et al.(21)
have reported recently that the resistance of glioma cells that harbor the constitutively activated mutant epidermal growth factor receptor to cisplatinum therapy was a consequence of the up-regulation of Bcl-XL. Moreover, the endogenous or ectopic overexpression of either Bcl-2 or Bcl-XL in glioma cells has been shown to be able to abrogate programmed cell death induced by radiation (22)
or chemotherapeutic agents, such as nitrosoureas (22)
, cisplatinum (21
, 23)
, and agents that target various components of the growth factor signal transduction cascade (24, 25, 26)
. However, although the antiapoptotic role of Bcl-2 and Bcl-XL in gliomas has been characterized, the regulation of these proteins by the ECM in the context of high-grade gliomas has not been assessed previously. In this report, we have demonstrated that VN, an ECM protein expressed specifically by glioma cells in vivo, increases the expression of these antiapoptotic proteins in glioma cells with a concomitant reduction in chemotherapy-induced programmed cell death (Fig. 4)
.
The mechanism by which the VN-VN receptor interaction modulates the expression of Bcl family proteins has been addressed in the context of endothelial cells, in which the ligation of the endothelial ß3 integrin is associated with a decrease in p53 levels (17)
. p53, when present, typically decreases Bcl-2 and increases Bax (27)
, thereby tipping the ratio in favor of Bax and thus cell death. Therefore, upon Vß3 ligation, the reduced p53 expression observed in endothelial cells leads to the opposite situation, in which there is an increase in the Bcl-2:Bax ratio, hence favoring cell survival (17)
. However, it is important to note that for the glioma cells evaluated in our present study, the p53 status for the D54 is wild type, whereas the p53 status in U251 is mutant. As both glioma cell lines demonstrated an increase in Bcl-2 expression when grown on VN (Fig. 4)
, our findings suggest that the increase in Bcl-2 levels and resistance to apoptosis conferred by VN in glioma cells may be independent of the p53 status.
The importance of the interaction between VN and its receptors in the survival of glioma cells is further supported by our observation that antagonism of the VN receptor sensitizes glioma cells to programmed cell death (Fig. 3)
. It is important to note that the increase in apoptosis after treatment with the VN receptor antagonist, cycRGD, occurred in the absence of a chemotherapeutic agent (i.e., topotecan), further suggesting that the VN receptor itself may serve as a potential target for novel therapeutics. The potential therapeutic utility of VN receptor antagonism has been assessed in the setting of endothelial cells, which require the VN receptor (ß3) for cell survival. Treatment of animals with a neutralizing antibody against this integrin is able to induce massive apoptosis of proliferating endothelial cells that express this integrin and thereby induce regression of tumors in vivo by an antiangiogenic mechanism (8)
. More recently, peptidomimetic inhibitors of the ß3 integrin have been developed, and encouraging preclinical results of studies conducted with animals inoculated with germ cell tumors have been reported (28)
.
Although these anti-ß3 agents have been shown to be effective as monotherapy, they have yet to be evaluated in combination with other drugs. With respect to gliomas, the antagonism of the VN receptors, such as the ß3 integrin, may sensitize tumor cells to standard chemotherapeutic agents to which the cells were resistant previously. For example, as illustrated in Fig. 2A
, the IC50 for topotecan-induced apoptosis for glioma cells plated on VN was 130 ng/ml, a drug concentration that is well beyond the cerebrospinal fluid levels that can be attained with the maximum tolerated dose of this drug (15)
, thereby effectively rendering these cells clinically resistant to this drug. In contrast, the IC50 of 35 ng/ml for glioma cells seeded on a non-VN substrate (FN) is achievable in the cerebrospinal fluid of test subjects. In this regard, the expression of VN by glioma cells selectively at the advancing tumor margin may underlie, in part, the observation that the great majority of tumor recurrences occur here, because the local deposition of VN may contribute to the chemoresistance of glioma cells in this location. Conversely, agents that disrupt the VN-VN receptor interaction may serve a potential role in sensitizing glioma cells to cytotoxic drugs.
In conclusion, we have presented data in this report that for the first time ascribes an antiapoptotic role to the VN-VN receptor interaction in malignant gliomas. In this regard, the VN-integrin interaction was able to protect glioma cells from drug-induced cell death, with a concomitant increase in expression of Bcl-2 and Bcl-XL, two key antiapoptotic proteins that have been associated with treatment resistance in a variety of tumor types, including gliomas. As an ECM molecule that may confer protection from apoptosis, VN expression by glioma cells at the tumor margin may underlie, in part, the relative chemoresistance of these cells. Moreover, as VN and its receptor have been shown to mediate glioma motility as well as the process of neovascularization, the recently reported agents that target this ECM-integrin interaction (28) may thus have a pleiotropic effect, potentially impacting upon multiple aspects of glioma biology that comprise motility/invasion and angiogenesis, as well as treatment resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Support for these studies was provided, in part, by NIH-National Cancer Institute Grants CA56792, CA75557, CA76350 (to J. S. R.), and CA59958 and NS34856 (project 3) (to C. L. G.) and the Clinician-Scientist Award from the Medical Research Council of Canada (to J. H. U.). 
2 To whom requests for reprints should be addressed, at Department of Neurosurgery, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 64, Houston, TX 77030. Phone: (713) 792-3266; Fax: (713) 794-4950; E-mail: JRao{at}notes.mdacc.tmc.edu
3 The abbreviations used are: ECM, extracellular matrix; VN, vitronectin; Topo, topoisomerase; TUNEL, terminal deoxynucleotidyl dUTP nick end labeling; FN, fibronectin; PI, propidium iodide; FACS, fluorescence-activated cell sorter; cycRGD, cyclic RGD.
Received 12/24/98; revised 3/11/99; accepted 3/16/99.
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G. E. Plautz, D. W. Miller, G. H. Barnett, G. H. J. Stevens, S. Maffett, J. Kim, P. A. Cohen, and S. Shu T Cell Adoptive Immunotherapy of Newly Diagnosed Gliomas Clin. Cancer Res., June 1, 2000; 6(6): 2209 - 2218. [Abstract] [Full Text]
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; B) and Bcl-XL:Bax (
; C). For each set of histograms, the Bcl-2:Bax or Bcl-XL:Bax ratios derived from cells seeded on FN coatings in the absence of topotecan were designated arbitrarily as 100%. Histograms reflect at least three separate experiments with values representing means; bars, SE.
