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Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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and ß, and the product of the C-kit oncogene, Kit (1, 2, 3)
. To date, STI571 has demonstrated efficacy in the treatment of two distinct types of human tumors, chronic myeloid leukemia and gastrointestinal stromal tumors. In chronic myeloid leukemia, STI571 exerts its activity by interrupting the intracellular signaling pathways initiated by the Bcr-Abl fusion protein, whereas in gastrointestinal stromal tumors, STI571 has been shown to block activation of the Kit tyrosine kinase (4
, 5)
. The promising results obtained with STI571 in chronic myeloid leukemia and gastrointestinal stromal tumor patient populations has prompted efforts to determine whether other malignancies critically dependent on tyrosine kinase signaling pathways may be targeted with STI571 therapy.
Recent studies suggest that PDGF-R might be an appropriate target. Some tumors may preferentially exploit this microvascular endothelial cell tyrosine kinase to regulate their blood supply and thus enhance their growth. Specifically, PDGF BB released from U87MG gliomas cells has been shown to promote the neovascularization of cerebral lesions, in part by stimulating the release of vascular endothelial growth factor from tumor-associated endothelia (6)
. In tumors growing in the s.c. space, the release of PDGF BB is reported to play a central role in the regulation of intratumoral hemodynamics by modulating the level of interstitial fluid pressure within the tumor tissue (7)
. Recent studies from our laboratory reported that human prostate cancer cells growing adjacent to murine bone tissue express and secrete high levels of PDGF AA and BB and that the tumor cells and, more so, tumor-associated endothelial cells express phosphorylated PDGF-R and PDGF-Rß (8)
. The inhibition of PDGF-R phosphorylation by STI571 coupled with administration of Taxol significantly reduced experimental prostate cancer bone metastases (8)
and spontaneous disease in man (9)
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The aforementioned studies emphasize the significant contribution of PDGF-R signaling to the blood vessels that support the progressive growth of neoplasms and suggest that selective targeting of this pathway may be a practical approach to halt the growth of some tumors. In this report, we examined the direct effects of activating and inhibiting phosphorylation of the PDGF-R on purified populations of murine bone endothelial cells that express PDGF-R. We demonstrate that STI571 effectively blocks PDGF-induced cell survival pathways and, moreover, significantly sensitizes bone-derived endothelial cells to Taxol.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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(PharMingen, San Diego, CA). The addition of IFN- was used to enhance the expression of the MHC H-2Kb class I promoter, which regulates the level of large T antigen protein in ImmortoMouse-derived cells (11)
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Tissue digests were plated into T75 flasks and supported at 33°C in a mixture of 5% carbon dioxide and 95% oxygen. Cells were expanded and then prepared for flow cytometry by stimulating primary cultures with 10 ng/ml recombinant murine tumor necrosis factor (R&D Systems, Minneapolis, MN) for 5 h and then labeling the endothelial cell fraction with 4 µg/ml phycoerythrin-conjugated rat antimouse E-selectin mAb and 2 µg/ml FITC-conjugated rat antimouse VCAM-1 mAb (both from PharMingen, San Diego, CA). EOMA cells, a murine endothelioma that expresses several endothelial markers (12)
, were stimulated with 10 ng/ml tumor necrosis factor and served as the positive control. Cell staining was evaluated with a Beckman Epics Elite flow cytometer (Beckman Coulter, Miami, FL) equipped with an air-cooled argon ion laser. Dual-positive cells were expanded and then subjected to an enrichment sort, during which cells were incubated in 10% DMEM containing 10 ng/ml tumor necrosis factor and 10 µg/ml fluorescent probe of acetylated-low density lipoprotein, DiI-Ac-LDL (Biochemical Technologies, Cambridge, MA) for 4 h. During this labeling period, the rat anti-E-selectin mAb (10E9.6) conjugated to FITC was used (replacing phycoerythrin-conjugated 10E9.6). Dual-positive cells were maintained in 10% DMEM without IFN-. Endothelial cell identity was confirmed by subjecting cells to a rigorous characterization analysis as described previously (10)
. Because the presence of the SV40 large T antigen has been shown to influence tyrosine kinase expression in some cell types (13)
, all experiments were performed when bone endothelial cells were cultured at 37°C for at least 72 h. Western blot analysis confirmed that the large T antigen was not expressed at this time point (data not shown).
Western Blot Analysis.
To evaluate expression levels of PDGF-R and its activation status on murine bone endothelial cells, cells were seeded in six-well plates at a density of 4 x 105 cells/well in 10% DMEM. The cells were allowed to stabilize for a 24-h period, at which time the medium was aspirated and replaced with serum-free DMEM for 48 h. After this incubation period, some of the wells were treated with STI571 at doses ranging from 0.1 to 2.0 µg/ml for 1 h. Medium from these plates was then aspirated and replaced with serum-free DMEM containing 5 ng/ml PDGF BB (Invitrogen, Carlsbad, CA). Cells were washed twice with PBS and then lysed with 0.1 ml of buffer [50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, and protease inhibitors].
To determine the effects of various treatments on activating kinases positioned downstream from PDGF-R, additional six-well plates containing 4 x 105 bone-derived endothelial cells/well were analyzed. After the 24-h incubation period, cells were treated with vehicle only, PDGF BB (5 ng/ml), Taxol (80 ng/ml), STI571 (2 µg/ml), or various combinations of drug and growth factor. A pilot study indicated that PDGF BB-induced activation of Akt and ERK1/2 was most pronounced 1–2 h after the addition of growth factor. Therefore, for Akt and ERK1/2 assessment, protein lysates were collected 2 h after treatment of cells.
Protein concentrations were determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA), and 50 µg of total protein resolved in 10% SDS-PAGE under reducing conditions were transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% (w/v) nonfat dried milk in 0.1% Tween 20 (Sigma) in PBS for 1 h and then incubated overnight at 4°C with one of the following antibodies: PDGF-Rß (sc-958) or phosphorylated PDGF-Rß (sc-12909) from Santa Cruz Biotechnology (Santa Cruz, CA); phospho Akt (Ser-473), Akt, phospho-ERK1/2 (Thr-202/Tyr-204), or P44/42 MAPK, all from Cell Signaling Technology (Beverley, MA); monoclonal antibodies recognizing murine-active caspase-3, Bcl-2, Bcl-XL, and Bad from PharMingen; and anti-ß-actin from Sigma. Immunodetection was performed using the corresponding secondary horseradish peroxidase-conjugated antibodies. Horseradish peroxidase activity was detected using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Immunohistochemistry.
Bone endothelial cells were seeded onto two-chamber slides at a density of 2 x 105 cells/chamber in 10% fetal bovine serum/DMEM. After a 24-h incubation, the media were replaced with 2.5% fetal bovine serum/DMEM containing vehicle only, PDGF BB (5 ng/ml), Taxol (80 ng/ml), STI571 (2 µg/ml), or various combinations of drug and growth factors for 1, 6, 12, 24, and 48 h. At each time point, slides were fixed by immersion into ice-cold acetone solution for 15 min, rinsed in PBS three times, and blocked in PBS containing 5% normal horse serum and 1% normal goat serum. Slides were incubated with a rabbit anti-active caspase-3 antibody (1:100; PharMingen) overnight at 4°C, rinsed three times with PBS, and then incubated with antirabbit Alexa 488 secondary antibody (1:500; Molecular Probes, Eugene, OR). Immunofluorescence microscopy was performed using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY), and images were captured using an air-cooled CCD Hamamatsu C5810 camera (Hamamatsu Photonics K.K., Bridgewater, NJ) and Optimas software (Media Cybernetics, Silver Spring, MD). The number of cells staining positive for active caspase-3 at different time points were counted in 10 random fields under high magnification (x400).
Endothelial Cell Proliferation.
Small (T25) flasks containing near confluent (80–90%) bone endothelial cells were maintained in a 37°C incubator for a 48-h period and then seeded into 96-well plates at a density of 3 x 103 cells/well in 100 µl of 10% DMEM. After an overnight incubation, the medium was aspirated and replaced with 200 µl of 2.5% DMEM containing 0 (control), 1, 10, 50, or 100 ng/ml PDGF BB. Cells were cultured for 78 h before being pulsed with 0.2 µCi of [3H]thymidine/well (ICN, Wilmington, MA) 18 h before the end of the assay. Endothelial cells were harvested, and cpm were determined using a betaplate 96-well harvester (Brandel, Gaithersburg, MD).
Cytotoxicity Assay.
Bone endothelial cells were seeded into 38-mm2 wells of flat-bottomed 96-well plates at a density of 2500 cells/well and allowed to adhere overnight. The cultures were then washed and replenished with medium containing increasing concentrations of Taxol or Taxol containing 5 ng/ml PDGF BB, 2 µg/ml STI571, or PDGF BB plus STI571. After 4 days (cultures did not reach confluence), the number of metabolically active cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol-tetrazolium bromide (MTT) assay (14)
. After a 4-h incubation in medium containing 0.42 mg/ml MTT, the cells were lysed in DMSO. The conversion of MTT to formazan by metabolically active viable cells was monitored by an MR-5000 96-well microtiter plate reader at 570 nm (Dynatech, Inc., Chantilly, VA). Growth inhibition was calculated from the formula: cytostasis (%) = [1 – (A/B)] x 100, where A is the absorbance of treated cells and B is the absorbance of the control cells.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Stimulation of bone endothelial cells with 5 ng/ml PDGF BB markedly increased the activation of both Akt and ERK1/2 (Fig. 2)
. Pretreatment of endothelial cells (1 h before addition of growth factor) with STI571 completely abrogated PDGF BB-induced activation of these downstream targets.
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. Small clusters of cells (2.2 ± 0.2 cells/field) expressing activated caspase-3 could be found in cultures that received combination treatment 6 h earlier. At 24 h, the number of endothelial cells positive for activated caspase-3 in response to combination treatment (6.7 ± 0.7 cells/field) was significantly greater than cells receiving only single agent treatment (Student’s t test, P < 0.0001).
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Consistent with the results from the immunohistochemical analysis of activated caspase-3, single or combined treatment with PDGF BB and STI571 did not induce apoptosis of bone endothelial cells at low levels of Taxol (<10 ng/ml). The addition of 2 µg/ml STI571 to bone endothelial cells that were cultured in Taxol-containing medium resulted in a 3-fold increase in cytotoxicity (Fig. 4)
. PDGF BB (5 ng/ml) did not rescue the endothelial cells from the combined effects of STI571 and Taxol. Similar to the effects observed during the Western analyses, several cells that were exposed to both STI571 and Taxol detached from the surface of the plate and floated in the medium.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Recent data suggest that paracrine PDGF signaling pathways within the microenvironment of some tumors may be critical for the neovascularization process that accompanies tumor progression. For glioblastomas and pancreatic tumors, PDGF signaling cascades appear to stabilize developing vascular networks by recruiting mural cells to the immature vessel wall (6 , 15) . However, given the sinusoidal arrangement of the bone microcirculation, it is likely that PDGF-R and its cognate ligands contribute to a different facet of angiogenesis in this tissue (i.e., migration, proliferation, etc.). Indeed, it has been known for some time that endothelial cells from some regional circulations express high-affinity PDGF-R (16, 17, 18, 19) and that PDGF is a potent endothelial cell mitogen (20) . Regardless, for prostate tumor progression in bone, paracrine PDGF signals from androgen-independent tumor cells to receptors on supporting blood vessels appear compulsory for tumor progression (8) .
The results of the present study indicate that when bone endothelial cells are exposed to PDGF BB, PDGF-Rß is rapidly phosphorylated, leading to activation of the downstream targets Akt and ERK1/2. Other studies have demonstrated that Akt signaling in response to PDGF-Rß stimulation functions as a survival mechanism for a number of cell types (21 , 22) . For example, neuronal cells exposed to a hypoxic environment survive through the autocrine activation of the PDGF-R and ensuing phosphorylation of Akt (23) . We have also observed that PDGF BB may support bone endothelial cell survival by enhancing protein levels of Bcl-2.1 In addition, PDGF BB also produced a modest increase (62%) in bone endothelial cell proliferation. Thus, a number of critical cellular processes appear to be affected by inhibiting PDGF-Rß signaling on bone endothelial cells. These cells are further compromised when STI571 is combined with the microtubule-stabilizing agent, Taxol. Together, STI571 and Taxol promote activation of caspase-3 and induce down-regulation of Bcl-2 and Bcl-xL.
Following lymph node, liver, and lung, the bone is the fourth most common site of tumor metastasis. In fact, a recent report estimates that 350,000 cancer patients will die each year with evidence of tumor in the skeleton (24) . Consequently, considerable effort has been extended toward identifying those components in the bone microenvironment that support tumor growth. Recently, we reported that human prostate cancer cells growing adjacent to mouse bone tissue (tibia) express high levels of PDGF AA and BB, whereas human prostate cancer cells growing at a distance of greater than 3 mm from the bone tissue do not (8) . Mouse endothelial cells within tumors that express PDGF AA or BB have activated PDGF-R, whereas mouse endothelial cells within tumors that do not express PDGF protein do not express PDGF-R (8) . In that model, inhibition of PDGF-R activation with STI571, when administered in combination with taxotere, produced a profound reduction in both tumor mass and lymphatic metastases in mice harboring human prostate (PC-MM2) tumors in the bone. The investigators attributed the effectiveness of the combination therapy to apoptosis of tumor-associated endothelial cells. The results of the present study both support and extend this conclusion. Indeed, by exploiting a recent advance that permits the generation of tissue-specific microvascular endothelial cells (10) , we were able to establish pure populations of murine bone endothelial cells and to define the mechanism of endothelial death in response to STI571 and Taxol. As was the case for the xenograft tumor model, agent treatment with either STI571 or Taxol alone was ineffective. Taken together, these findings add to the growing evidence that molecular targeted therapeutic approaches are most successful when combined with standard chemotherapy.
The results of the present study may have important implications for other cancers that preferentially metastasize to bone, such as those from breast, renal, and lung tumors. Before we attempt to extend our conclusions, it will be necessary to determine whether or not these tumors also operate through a PDGF-dependent pathway. For example, an orthotopic model of renal tumor bone metastasis has shown that some malignant renal cells use epidermal growth factor pathways to promote their vascularization and ensure their growth (25) . Continued investigations into the signaling networks used by other types of tumors in the bone microenvironment will likely lead to the generation of rational therapeutic interventions that alleviate the morbidity and mortality associated with skeletal metastasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Isaiah J. Fidler, Department of Cancer Biology (Unit 173), The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8577; Fax: (713) 792-8747; E-mail: ifidler{at}mdanderson.org
1 R. Langley, R. Tsan, unpublished data. 
Received 12/16/03. Revised 3/30/04. Accepted 4/12/04.
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B and Bad. J Am Soc Nephrol, 14: 1427-34, 2003.This article has been cited by other articles:
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), or Taxol containing either 5 ng/ml PDGF B (
), 2 µg/ml STI571 (
), or PDGF BB plus STI571 (
). This assay was conducted over a period of 96 h, and cytotoxicity was determined at that time using MTT assay. Data are representative of three independent experiments. Results are mean ± SD.