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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [J. M., R. L. D. C., D. E. B., A. J. P. K-S.]; Departamento de Genética, Universidad de Navarra, Pamplona, Spain 31080 [J. S. C.]; and The Vollum Institute, Oregon Health Sciences Center, Portland, Oregon 97201 [G. T.]
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ABSTRACT |
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1-antitrypsin Portland (PDX) cDNA. Experimental Design: This furin inhibitor was transfected into two tumorigenic astrocytoma cell lines. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, and proliferation assays, as well as by investigating impairment of furin substrate processing.
Results: Expression of PDX prevented the s.c. growth of the transfected cells. Invasion assays demonstrated that PDX-transfected cells exhibited a reduced invasive ability in vitro and in vivo. Furthermore, s.c. growth of PDX transfectant xenotransplants showed a significant reduction in size that coincided with a significant decrease of the in vitro doubling time and of the in vivo cell proliferation ability. Additional studies showed that the furin substrates insulin-like growth factor IR, transforming growth factor ß and membrane type 1-matrix metalloprotease were not activated in PDX-expressing astrocytoma cells.
Conclusions: PDX expression in astrocytoma cells demonstrated a direct mechanistic link between furin inhibition, and decreased astrocytoma proliferation and invasive ability. Because furin inhibition inhibits both invasiveness and cell growth in astrocytoma, furin should be considered a promising target for glioblastoma therapy.
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Introduction |
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or RR, although basic residues at 4th and 6th upstream positions also contribute to substrate recognition (2)
.
The up-regulated expression of PACE4 and furin in some types of cancer supports a possible functional role in tumorigenesis (reviewed in Ref. 3
). Enhanced expression of the PC PACE4 was found in invasive mouse skin squamous cell carcinomas, whereas levels remained low in the less aggressive tumors (4)
. Among the most attractive substrates of PCs that might link proprotein processing with progression of cancer disease are MMPs, synthesized as inactive precursors, because of their role in the proteolytic digestion of the ECM that anticipate tumor invasion. Stromysin-3 is processed into its active form by furin (5
, 6)
, and the group of MT-MMPs also contains insertions between the propeptide and the catalytic domains that include the paired basic amino acid recognition site for furin processing (7)
. Recently, a variant 1-antitrypsin was constructed, which contains in its reactive site Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (8)
. This furin-specific inhibitor called PDX, unlike other related serine protease inhibitors, does not inhibit either elastase or thrombin. PDX is a potent competitive inhibitor of furin (IC50 = 0.6 nM), and when expressed in cells (either by stable or transient transfection), blocks the processing of HIV-1 gp160 and measles virus-Fo inhibiting virus spread. PDX is also 10-fold more effective than currently used antiherpetic agents in cell-culture models. The requirement of furin for the processing of envelope glycoproteins from many pathogenic viruses and for the activation of several bacterial toxins suggests that selective inhibitors of furin have potential as broad-based antipathogens (8)
. Furthermore, furin substrate processing was inhibited by PDX in four (furin-overexpressing) epithelial-derived malignant cell lines resulting in reduced tumorigenicity and invasiveness (9
, 10)
.
Although to our knowledge, furin expression levels have not been investigated in brain tumors, low levels of expression have been detected in neurons and glial cells of murine and human normal brain by in situ hybridization (11, 12, 13) .
High grade astrocytomas are characterized by enhanced proliferation and invasiveness, and their lethality is because of local brain tissue destruction rather than distant metastases. Invasion of glioma cells in the brain is preceded by a process of proteolysis and solubilization of the ECM that enables tumor cells to spread (14) . Because local invasiveness is the major cause of glioma mortality and because furin is expressed in the nervous tissue together with several MMPs that contain sequences for cleavage and activation by furin, astrocytoma cells are excellent candidates to investigate the effects of furin inhibition as a possible new approach to astrocytoma therapy.
Herein we demonstrate a very significant reduction in cell proliferation, tumorigenicity, and invasiveness after introduction of a cDNA plasmid for expression of the furin inhibitor PDX into furin-expressing anaplastic astrocytoma cell lines U87MG and U118MG.
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Materials and Methods |
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Cell Culture and Transfection.
Parental U87MG and U118MG astrocytoma cells (American Type Culture Collection, Manassas, VA) were maintained in MEM supplemented with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and DMEM, respectively, with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Both lines were stably transfected by lipofection (LipofectAMINE PLUS, Life Technologies, Inc., Gaithersburg, MD) with either pCIN.PDX or pCI.neo vector alone (pCIN). G418 (1 mg/ml)-resistant colonies were picked, cloned, and screened for high 1-PDX expression by Western analysis. Transfectants were propagated in medium supplemented with 400 µg/ml G418. Subconfluent cells were serum-starved 1 day before harvesting. Primary cultures of NHAs from Clonetics (San Diego, CA) were used as control cells.
Protein Sample Preparation and Western Blot Analysis.
For cell lysates, cell monolayers were harvested, washed, and disrupted with radioimmunoprecipitation assay (PBS containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1% NP40, plus freshly added protease inhibitors: 2 mM phenylmethylsulfonyl fluoride, 0.2 mg/ml aprotinin, and 1 mM Na3V04) for 30 min at 4°C. The soluble part was used as protein extract. Furin was first immunoprecipitated from lysates with MON-148 (Alexis Biochemicals, San Diego, CA), a monoclonal antibody raised against an epitope in the catalytic domain. Total protein (100 µg) was cleared with normal mouse IgG/protein A/G PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA), and the cleared rest was then incubated with MON-148 and protein A/G PLUS agarose.
Extracellular proteins were assessed on 24 h-conditioned medium concentrated 50-fold with Millipore centrifugal filter devices.
For expression analysis, proteins were subjected to SDS-PAGE using Tris-glycine gels, electrotransferred overnight onto nitrocellulose membranes, and probed with antibody against either furin (MON-152; Alexis Biochemicals), PDX (1-antitrypsin antibody; Sigma, St. Louis, MO), TGF-ß affinity-purified goat antihuman latent-associated peptide IgG (R&D, Minneapolis, MN), MT1-MMP (AB815; Chemicon International, Inc., Temecula CA), and IGF-IR (sc-711; Santa Cruz Biotechnology) were used as primary antibodies. Immunocomplexes were revealed with enhanced chemiluminescence based on the use of peroxidase-labeled IgG (enhanced chemiluminescence; Amersham Pharmacia Biotech).
Immunohistochemistry.
Furin immunohistochemistry was performed using paraffin-embedded material. All of the paraffin sections were subjected to antigen retrieval for 10 min in distilled water. MON 152 (Alexis) was used as primary antibody at 1/100 dilutions. An avidin-biotin-peroxidase kit (Vectastain Elite, Vector, Burlingame, CA) was then used followed by the chromagen 3'3'-diaminobenzidine to develop the immunostain. Negative controls, not incubated with furin antibodies, were incubated with normal mouse IgG. All of the sections were counterstained with hematoxylin and mounted. Normal brain tissue (frontal cortex) from three individuals, eight high-grade astrocytomas, and tracheal xenotransplants of U87MGpCIN cells (see below) were used.
Doubling Time.
Cells were grown in 12-well plates and counted every other day. Log of cell numbers versus days was plotted to obtain the doubling time for each transfected cell line.
Zymography.
Cells (1 x 106) were grown overnight in serum-free S-MEM medium containing 2 mM L-glutamine and Pen-Strep. The conditioned media were concentrated down to 200 µl using Amicon centripreps (Fisher, Springfield, NJ), and 20 µl of each sample was loaded on a 10% Novex precast zymogram (gelatin) gel. The gel was run, renatured, and developed according to the manufacturer’s instruction. Gelatinase Zymography Standards were purchased from Chemicon International, Inc.
In Vitro Invasion Assay.
The ability of cells to degrade ECM in vitro was assessed using Biocoat Matrigel invasion chambers (Becton Dickinson, Bedford, MA) according to the manufacturer’s instructions (15)
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In Vivo Invasion Assay.
Tracheal transplants were prepared as described previously (10
, 15) . Cells (5 x 105) from each transfected cell line were inoculated into de-epithelialized rat trachea (Zivic-Miller, Pittsburgh, PA). Six to ten tracheas were used for each cell line. After cell inoculation, the tracheas were sealed and transplanted into the dorsal s.c. tissues of Scid mice. Tracheal transplants were removed surgically at 8 weeks, sectioned into 3-mm thick rings, and fixed in 10% formalin. After H&E staining, the degree of invasion of the tracheal wall was determined by measuring the length of maximum penetration of the tumor cells into the tracheal wall (10
, 15)
. All of the microscopic images of cross-sections of tracheal transplants were digitized at a magnification of x40. Using an adequate reference micrometric scale, the lengths were determined by measuring the distance between the luminal center and the most distant point of tumor invasion. If the lumen was obliterated, the distance measured was between the geometric center of the tumor mass inside the tracheal lumen and the most distant point of tumor invasion either in or outside the tracheal wall. Each tracheal transplant was represented by two to six measurements corresponding to the number of available cross-sections containing tumor cells. A mean was calculated for each tracheal transplant and for each group of transfected cells. The results were expressed in µm of penetration depth. Paraffin sections of tracheal transplants containing transfected tumor cell lines were used for the immunohistochemical detection of Ki-67 (Mib-1). A mouse monoclonal Mib-1 (Immunotech, Westbrook, ME) and an avidin-biotin-peroxidase kit (Vectastain Elite; Vector) were used. Negative controls were incubated with normal mouse IgG. Labeling indices (percentage of Mib-1-positive cells) were calculated by counting stained and unstained cells in tracheal transplants containing either vector alone- or 1-PDX transfected cells (at least 500 cells/tracheal transplant were counted).
In Vivo Tumorigenicity.
Either PDX-transfected or vector alone-transfected cells (5 x 106) were injected into the s.c. tissues of Scid mice. Tumors were measured twice a week after the appearance of the first tumor for each pair of PDX- and vector alone-transfected cells using a Vernier caliper. Volumes (V) of the tumors were obtained using the following equation: V = [(L1 + L2)/2] x L1 x L2 x 0.526, where L1 and L2 are the length and width of the s.c. tumor.
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. The expression of PDX was confirmed by Western analysis of astrocytoma cell lysates (Fig. 1B)
. PDX did not affect cell survival and was secreted into the culture medium (data not shown) as a product of Mr 
60,000. The transfection of PDX into the astrocytoma cell lines did not change their respective furin expression levels (Fig. 1C)
.
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. Conversely, six of eight high-grade astrocytomas examined showed moderate to intense immunostain (Fig. 2B)
. Control-transfected U87MG cells grown in tracheal xenotransplants showed intense furin immunostain (Fig. 2C)
. PDX transfection did not change this staining pattern (data not shown).
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105,000) corresponding to the processed form of the receptor. Conversely, the PDX-transfected astrocytoma cells showed the predominance of a higher molecular weight band (Mr 200,000) corresponding to the proform and low expression of the processed form (Fig. 3A)
. Similarly, Western analysis of TGF-ß showed that the processed form of this growth factor (Mr 40,000) was present in the vector alone-transfected astrocytoma cells, whereas the PDX transfectants did not express this lower molecular weight form and expressed the higher molecular weight band corresponding to the unprocessed form (Mr 50,000; Fig. 3B
).
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. PDX-transfected astrocytoma cells retained the latent MT1-MMP proform and failed to exhibit the mature form of Mr 63,000. Conversely the vector alone-transfected cells showed both the proform and the mature form of MT1-MMP (Fig. 3C)
. Because MT1-MMP intervenes in the activation of MMP2, the possible effects of furin inhibition on the extracellular gelatinase activity of astrocytoma transfectants were evaluated by zymography (Fig. 3)
. Processing of Mr 72,000 gelatinase-A (MMP-2) into the mature active form was inhibited by PDX transfection, as evidenced by the low expression levels of the processed forms of MMP2.
PDX Effects on Proliferative, Tumorigenic, and Invasive Potential of Astrocytoma Cells.
PDX-transfected cells exhibited a marked increase in doubling time in regard to their vector alone-transfected counterparts. This effect of the furin inhibitor on cell division was more obvious in transfectants of U87MG, which exhibited an increase of 50% (from 27 h to 42 h doubling time), whereas an increase of 30% was noted in U118MG cells (from 55 h to 72 h doubling time). The tumorigenic ability of the transfectants was evaluated in s.c. xenografts using Scid mice (Fig. 4)
. Growth of s.c. transfected U87MG cells was almost completely prevented by PDX expression. The growth of the vector alone-transfected cells was nearly exponential during the first week after inoculation. In the remaining 4 weeks the tumors increased very rapidly in size, forcing the termination of the in vivo experiment at 5 weeks after cell inoculation. Conversely, the cells transfected with furin inhibitor grew very little and remained practically unchanged during the course of the experiment (Fig. 4A)
. This inhibitory effect of PDX on s.c. tumor growth was also observed in tumors derived from U118MG cells (Fig. 4B)
. However, tumor masses produced after inoculation of U118MGpCIN.PDX cells shrunk and subsequently disappeared 15 days after cell inoculation. The vector-alone-transfected U118MG.pCIN cells tumors reached volumes of 0.3–0.6 cm3 4 months after inoculation, whereas no tumor was noticed in animals injected with U118MG.pCIN.PDX cells even after 6 months of observation.
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. An in vivo invasion assay was performed to assess the invasive ability of PDX-transfected astrocytoma cell lines grown as xenotransplants in Scid mice. As shown in Fig. 4C
, the PDX transfectants of each astrocytoma cell line exhibited a markedly decreased invasive ability when compared with their respective vector alone-transfected counterparts. U87MG cells had remarkably dissimilar invasion patterns depending on whether or not PDX was expressed. Control vector alone-transfected cells penetrated deeply into the tracheal wall and reached the outer part of the trachea (adventitia; Fig. 5A
), whereas the PDX-transfected cells lacked an invasive front, and the cells remained in the tracheal luminal area (Fig. 5B)
. Vector alone-transfected U118MG cells grew with difficulty in the tracheal transplants and remained either in the luminal area or in a few cases invaded the pars membranacea (Fig. 5C)
. PDX-transfected U118 MG cells grew very little or not at all in tracheal transplants (Fig. 5D)
. Evaluation of Mib-1 LIs in the astrocytoma cells growing in tracheal transplants showed that U87 MG cells expressing PDX exhibited a reduction of >50% in their proliferative ability when compared with U87MG transfected with vector alone (Fig. 5, E and F)
. U118 MGpCIN cells had a lower LI than the other astrocytoma cells. Nevertheless the decrease in LI of PDX-transfected cells was also remarkable (Table 1)
.
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Discussion |
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Furin is detected at very low levels in most normal tissues, suggesting that the potential deleterious effects of furin on the maintenance of cellular homeostasis under physiological conditions are avoided by very low cellular levels of expression. In certain types of cancer, expression of furin above that level is associated with the acquisition of a more aggressive phenotype (18 , 19) .
In this study we detected furin expression in primary glial cell cultures and even higher expression in two tumorigenic astrocytoma cell lines.
The two tumorigenic astrocytoma cell lines with demonstrable furin overexpression were transfected with the potent furin inhibitor PDX resulting in a decrease in cell growth, and an inhibition of tumorigenicity and invasive ability. These effects were associated with a decreased activation of MMPs combined with lower proliferative rates of the PDX-transfected cells.
MT1-MMP participates in the activation of the Mr 72,000 type IV collagenase, MMP-2 (20) , a major ECM-degrading enzyme that is of paramount importance in human gliomas (21 , 22) . Although furin-dependent activation of MT1-MMP seems to vary among different cell types (23) , astrocytoma cells effectively express the activated form (24) . Our results demonstrate conclusively that PDX-transfected astrocytoma cells are unable to activate MT1-MMP. Consequently, activation of MMP-2 proenzyme into active ECM-degrading forms was also inhibited by PDX transfection. These effects of PDX strongly suggest that the impairment of furin-mediated metalloprotease processing results in the decreased invasive ability observed in PDX transfectants.
TGF-ß promotes the proliferation of highly malignant, aneuploid gliomas and inhibits the growth of near diploid gliomas, normal astrocytes, and microglia (25) . In addition, TGF-ß1 and 2 are expressed by glioma cell lines, and PDX inhibited the in vitro TGF-ß processing and release of two other glioma cell lines (LN-18 and T98G; Ref. 26 ). The IGF-IR has three major properties in terms of growth: it is mitogenic, it is required for the establishment and maintenance of the transformed phenotype in many cell types, and it protects cells from apoptosis (27) .
Activation of TGF-ß and IGF-IR are know to up-regulate cell proliferation in astrocytoma cells and other cell types. Inhibition of their furin-mediated processing could have a negative effect on mitogenesis. This was seen in a colon carcinoma cell line transfected with PDX (9) but could not be demonstrated in three transfectant head and neck cell lines (10) . In the two PDX-transfected astrocytoma cell lines described herein, inhibition of cell proliferation plays a significant role as seen by the impressive reduction of the proliferation rate of PDX-transfected cells (50% reduction in vitro and 2–10 fold decrease in vivo). Furthermore, in vivo invasiveness was reduced 4-fold. Hence, it would be logical to conclude that substrates involved in loss of ECM integrity (MT1-MMP) together with cell cycle regulators, such as growth factors and receptors, play a central role in PDX-induced reduction of tumorigenicity and invasiveness of astrocytoma cells.
Astrocytoma is a type of brain tumor characterized by the strong tendency of neoplastic cells to emerge from the tumor margins and invade the normal brain (14) . Because these tumors almost never metastasize, local growth and invasiveness should be the targets of innovative therapies. The present study shows that inhibition of a member of the family of PCs results in decreased tumor growth and invasiveness of astrocytoma cells through reduction of furin substrate (MMPs and growth factors) activation. Compared with carcinoma cells (10) , astrocytomas showed a marked inhibition of cell proliferation after PDX transfection (little change in carcinoma cell lines versus a several-fold decrease in astrocytoma cells). The effect on in vivo invasiveness was similar in both cell systems. The difference in proliferation inhibition is probably because astrocytoma cells may express putative furin-substrates, such as nerve growth factor or glial cell line-derived neurotrophic factor (28 , 29) , which regulate cell proliferation and are not expressed at all in carcinoma cells.
In summary, furin inhibition results in a marked reduction of cell proliferation and invasiveness in xenotransplanted astrocytoma cells. Because astrocytoma cell lines were very sensitive to the inhibitory effects of PDX, these findings provide a rationale for a new therapeutic strategy to limit astrocytoma cell growth based on the development of a small molecule targeting furin. In this cell system, in contrast to squamous cell carcinoma cells, both invasiveness and proliferation were affected by furin inhibition. Hence, this approach could have a remarkable impact on the survival of patients suffering from the usually chemoresistant glioblastomas.
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FOOTNOTES |
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1 Supported by NIH National Cancer Institute Grants CA 75028, CA 06927, and by an appropriation of the Commonwealth of Pennsylvania. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA 19111. Phone: (215) 728-3154; Fax: (215) 728-2899; E-mail: AJ_Klein-Szanto{at}fccc.edu
3 The abbreviations used are: PC, proprotein convertase; PDX, 1-antitrypsin Portland; ECM, extracellular matrix; IGF-IR, insulin-like growth factor receptor 1; LI, labeling indices; MMP, matrix metalloproteinase; MT-MMP, membrane type-MMP; TGF-ß, tumor growth factor ß; NHA, normal human astrocyte.
Received 10/ 8/01; revised 2/26/02; accepted 3/11/02.
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1) are strongly expressed in human gliomas. Acta Neuropathol., 99: 131-137, 2000.[CrossRef][Medline]This article has been cited by other articles:
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