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1 University of Manchester Gynaecological Oncology Laboratory, St. Mary’s Hospital, Manchester, United Kingdom; 2 Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Manchester, United Kingdom; and 3 University of Manchester Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor invasion is influenced by multiple factors and can be subdivided into different types such that effective blockade of one type of metastatic spread causes tumors to switch to alternative mechanisms in response to different conditions (11) . Hypoxic conditions are also known to promote metastatic spread by a variety of mechanisms (12) such as induction of the activity of vascular endothelial growth factor (13) , fibroblast growth factor (14) , and urokinase-type plasminogen activator receptor (15 , 16) .
There is accumulating evidence that the heparanase enzyme plays a key role in tumor dissemination. For example, transfection of the heparanase gene into poorly metastatic cell lines is associated with a significant increase in metastasis in vivo, and clinical studies have correlated increased heparanase activity with invasion and poor prognosis (5 , 6 , 17) . Interestingly, recent work has shown that heparanase promotes invasion via two independent mechanisms, acting as a cell adhesion receptor and through cleavage of extracellular matrix (ECM) HSGAG (18) . These data clearly indicate that blocking heparanase activity is a good potential therapeutic intervention strategy for the treatment of metastatic malignant disease. For the first time, we now report: (a) the activity of heparanase is increased by hypoxic conditions and that this is associated with increased tumor cell invasion; and (b) heparanase-mediated invasion can be blocked by treatment with a neutralizing antibody.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human ovarian cancer cell line, OC-MZ-6, used in this study was established (passaged between 100 and 300 times) from a patient with advanced serous cystadenocarcinomas of the ovary (provided by Dr. Georg Brunner, The University of Munster, Germany). Cells were cultured in DMEM containing 5% FCS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM L-glutamine, 116 mg/l arginine, and 36 mg/l asparagine (Sigma, Dorset, United Kingdom; Ref. 19 ). Hypoxia was generated in an oxygen-regulated incubator (CoyLab Products, Inc.). Cells were incubated at either 1% O2 (hypoxia) or 20% O2 (atmospheric air, normoxia).
Isolation and Sequencing of Full-Length Human Heparanase cDNA.
Full-length human heparanase cDNA was isolated by reverse transcription-PCR. Total RNA was prepared from human ovarian tumor tissue by acid guanidinium thiocyanate-phenol-chloroform extraction (20)
. RNA was treated with DNase I for 1 h at 37°C and was preheated with 50 pM random hexamers for 3 min at 70°C, quickly chilled on ice, and reverse transcribed with 20 µl of a reaction mix, containing the following: 50 units of murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals, Lewes, United Kingdom); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 5 mM MgCl2; 1 mM dNTPs; and 20 units of RNase inhibitor (Roche Molecular Biochemicals, Lewes, United Kingdom). After incubation for 10 min at room temperature, the mixture was submitted to one cycle of 42°C for 30 min and 95°C for 5 min.
PCR was performed in 50 µl of a reaction mixture containing 1 µl of reverse-transcribed product, 1x Taq Master, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM NTPs, 0.3 µM of each primer, and 1.5 units of HF TaqDNA polymerase (Roche Molecular Biochemicals, Lewes, United Kingdom) that were then denatured for 5 min at 94°C and amplified in 35 cycles, each consisting of a 20-s denaturation at 94°C, 30-s annealing at 55°C, 1.5-min extension at 72°C, followed by a single 7-min extension at 72°C. PCR primers (5'-AGATGCTGCTGCGCTCGAAG-3', 5'-TCAGATGCAAGCAGCAAC-3') were designed for human heparanase according to Gene Bank sequence (accession no. AF155510).
Generation of Heparanase Antisense cDNA Transfectants.
The heparanase antisense construct was generated by cloning the full-length 1.6-kb heparanase cDNA in antisense orientation into the pcDNA3.1 vector (InVitrogen, Leek, United Kingdom). The construct was verified by DNA sequencing (ABI PRISM; Applied Biosystems, Warrington, United Kingdom).
LipofectAMINE (Life Technologies, Inc. Ltd, Paisley, United Kingdom) was used to deliver the plasmid DNA into the human ovarian cancer cell line, OC-MZ-6 (The University of Munster, Germany) as recommended by the manufacturer (Life Technologies, Inc.) but with some modifications. Transfection with vector control was used as a control. Cells were seeded at a density of 5 x 105 cells/well in 35-mm dishes, grown until the cells achieved 70% confluence, and then rinsed three times with serum-free medium before the transfection. The transfection solution was prepared by diluting 12 µg of plasmid DNA into 1.25 ml of serum-free medium to make solution A and diluting 37.5 µl of LipofectAMINE into 1.25 ml of serum-free medium to make solution B. The two solutions were combined, mixed gently, and incubated at room temperature for 45 min to allow DNA-liposome complex formation. Finally, the solution A/B mixture was added onto the rinsed cells. After 5 h of incubation at 37°C, 5 ml of DMEM containing 20% FCS were added, and cells then were incubated overnight at 37°C. After transfection, the DNA-lipid complexes were removed and replaced with the fresh DMEM containing 10% FCS. Forty-eight h later, the cells were incubated in the selection medium (1 g/liter G418) for 30 days to allow antibiotic-resistant colonies to form, which were isolated by cloning rings. After expansion, stable transfected cell lines were analyzed for heparanase enzyme activity.
Reverse Transcription-PCR.
Reverse transcription-PCR was used to detect heparanase antisense mRNA expression. RNA isolation and reverse transcription were carried out as described previously. PCR was performed with specific primers for heparanase antisense (5'-GGTTGATTCCTTCTTGGGATCGA-3') and pcDNA3.1 vector (5'-ACAACAGATGGCTGGCAACTAGA-3'). The PCR conditions were as follows: 94°C for 3 min; 35 cycles of 94°C for 1 min, 66°C for 1 min, 72°C for 1 min; and 72°C for 7 min. The PCR products were resolved on 1.5% agarose gels and visualized by ethidium bromide staining.
Preparation of Cell Lysates and Conditioned Media.
Cells were plated in serum-containing medium in 24-well plates (5 x 105/well) and grown overnight. After five washes with serum-free medium, cells were maintained in DMEM medium for 24 h. The conditioned media were collected, and the cells were lysed by incubation with 250 µl of heparanase lysis buffer (TaKaRa Bio Inc., Otsu, Japan) for 5 min at 37°C. The supernatant was collected by centrifugation at 12,000 x g for 10 min at 4°C. Protein concentrations were determined using the Bio-Rad protein determination method (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom).
Influence of Reducing Conditions on Heparanase Activity.
Increasing amounts of ß-mercaptoethanol were added to aliquots of heparanase conditioned media, prepared as described above, and then incubated for 30 min at room temperature. Heparanase activity in all samples was measured by the Takara assay.
Influence of Hypoxic Conditions on Heparanase Activity.
Equal amounts of OV-MZ-6 cell heparanase conditioned medium (see previous) and platelet enzyme were placed under hypoxic and normoxic conditions and incubated for time intervals from 0–8 h. Heparanase activity was assayed as follows during the course of these incubations.
Heparanase Enzyme Activity Assay.
Heparanase activities were assayed in cell culture supernatants and lysates by using a heparan-degrading enzyme assay kit according to the manufacturer’s suggestions (TaKaRa Bio Inc.).
Heparanase activities in all samples were interpolated from a standard curve performed by using an unlabeled HS as a standard substitute. The absorbance was read by a microplate reader set at 450 nm (MRX Revelation; Dynex Technologies, United Kingdom).
In addition, a specific in-house method of measuring heparanase activity was used in some experiments (21) . Plates coated with biotinylated HSGAG were incubated with 50 µl/well of the samples or heparanase standard (purified human platelet haparanase) for 1 h at 37°C. After washing five times with PBS-0.1% Tween 20, the plates were incubated with 100 µl/well of streptavidin-peroxidase (0.1% in PBS) for 1 h at 37°C and washed five times with PBS-0.1% Tween 20. The reaction was terminated by mixing with 100 µl/well of the stopping solution (0.1 M HCl). The absorbance was then measured by plate reader at 450 nm, and the release of biotinylated HSGAG was quantified relative to standard background. Heparanase activity in the samples was estimated by calculating the loss of biotin signal.
Immunoprecipitation and Western Blot Analysis.
Rabbit and chicken antiheparanase antibodies were produced after s.c. immunization with recombinant heparanase (gift from Oxford Glycosciences, United Kingdom) emulsified in Titermax gold adjuvant (Sigma Chemicals). After two booster injections with immunogen in saline, rabbits were bled, and the 
globulin fraction was isolated from serum by repeated (twice) ammonium sulfate precipitation at 40% saturation. Chicken antibodies were isolated from egg yolk using solvent extraction and PEG 6000 precipitation. The final products were dissolved in PBS, extensively dialyzed against PBS, filter sterilized (0.22-µm membrane), and stored at –20°C.
The heparanase protein was immunoprecipitated from 10 ml of prepared supernatants using 15 µg of rabbit polyclonal heparanase antibody for 1 h at 4°C. The immunocomplexes were precipitated with 50 µl of protein G-agarose (Pharmacia Biotech, Little Chalfont, United Kingdom) for 1 h at 4°C. After two sequential washes using PBS-0.1% Tween 20, the resulting pellets were boiled for 5 min in reducing or nonreducing Laemmli buffer. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes by electroblotting (Biometra semi dry blotter; Anachem Ltd., Luton, Beds, United Kingdom). The efficiency of transfer was checked by back-staining gels with Coomassie Blue. Blots were blocked and probed with chicken polyclonal antibody against human heparanase in 5% nonfat dried milk in PBS for 1 h at room temperature. After two 10-min washes with PBS-0.1% Tween 20, blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) for 1 h followed by two 10-min washes. Immunoreactive bands were detected using the ECL detection system (Amersham Biosciences, Little Chalfont, United Kingdom) according to the manufacturer’s instructions. Band intensity was quantified by the use of Labimage software (Kapelan, GmbH).
Indirect Immunofluorescence and Flow Cytometric Analysis.
Cell surface expression of heparanase was evaluated by immunofluorescence using flow cytometry. Cells (105/tube) were incubated with 100 µl (20 µg/ml in PBS) of rabbit polyclonal antibody to heparanase or IgG as negative control (20 µg/ml in PBS) on ice for 1 h and washed twice with cold PBS. After incubation with biotinylated swine antirabbit secondary antibody (1:200; DAKO) for 1 h and washing twice with cold PBS, cells were incubated with Streptavidin FITC (1:200; DAKO) on ice for 30 min. Cells were washed twice with PBS, resuspended in 0.3 ml of 2% buffered formalin, and analyzed on a Becton Dickinson FACScan flow cytometer.
Cell Proliferation Assays.
These were carried out using Celltiter 96 AQueous reagent (Promega, Southhampton, United Kingdom) according to the manufacturer’s protocol. In brief, 104 OC-MZ-6 cells/well were seeded into a microtiter plate 96 allowing 3 wells/data point. Cells were allowed to attach for 24 h, after which a time zero point was determined by adding 20 µl of Celltiter 96 reagent to each well and incubating for 4 h at 37°C in 5% CO2. The absorbance was read at 490 nm using a 96-well plate reader (Dynex MRX; Dynex Technologies). Antiheparanase antibody and IgG control were added at 50 µg/ml, and the proliferation assay was repeated at 24 h.
Cell Invasion Assay.
Invasion of cells through Matrigel was determined using 24-well BD invasion chambers (8.0-µm pore size with polycarbonate membrane; BD Biosciences, Cowley, United Kingdom) in accordance with the manufacturer’s instructions with the following modifications. BD invasion chambers were prehydrated with serum-free DMEM (500 µl/well) for 2 h of incubation at 37°C in 5% CO2. After trypsinization, OV-MZ-6 transfected A1 and A3 cells (105) were suspended in complete medium (500 µl) and immediately placed onto the upper compartment of the plates. Subsequently, the lower compartment was filled with complete medium (750 µl). After 48 h of incubation at 37°C in 5% CO2, cells remaining on the upper membrane surface were removed with a cotton swab. Cells on the lower surface of the membrane were fixed in methanol and stained with H&E. Five fields of adherent cells were randomly counted in each well under a light-inverted microscope at x10 magnification, and the results were numerically averaged.
Statistics.
Data are presented as mean ± SE. Student’s test was used for statistical analysis, and a value of P < 0.05 was considered significant.
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RESULTS |
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, heparanase antisense transfectant A1 cells had approximately 20% of the heparanase activity expressed by A3 cells taken from medium and cell lysates, which indicated a significant suppression of heparanase expression in A1. The expression of heparanase antisense mRNA in both transfectants A1 and A3 was examined by reverse transcription-PCR (as described in "Materials and Methods"). As shown in Fig. 1B
, Lanes 5 and 6, A1 and A3 cells expressed heparanase antisense mRNA because reverse transcription-PCR resulted in DNA products with the expected size of 480 bp, whereas heparanase antisense mRNA expression was not detected in vector-only transfectants (Fig. 1B
, Lane 4). Product formation was not due to the amplification of contaminating genomic heparanase DNA because omission of the reverse transcription step abolished DNA amplification from A1 and A3 cells (Fig. 1B
, Lanes 2 and 3). Thus, the difference in heparanase activity between A1 and A3 (Fig. 1A)
was most likely due to different levels of heparanase antisense mRNA expression.
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, conditioned media from hypoxic A1 cells showed 5-fold more heparanase enzyme activity than that from normoxic cells (incubated under 20% O2), and hypoxic A3 cells released 2-fold more enzyme activity than normoxic A3 cells. To determine whether hypoxia stimulates heparanase expression, secreted protein expression was analyzed by immunoprecipitation and Western blotting from A1 and A3 cells grown under normoxic and hypoxic conditions (Fig. 2B)
. In contrast to enzyme activity, heparanase protein expression in hypoxic cells (area 50723) remained unchanged compared with normoxic cells (area 48148; Fig. 2B
). The Mr 65,000 latent form of heparanase was not detected in either culture system. Indeed, fluorescence-activated cell sorter analysis of cells immunostained for heparanase was also consistent with this result, because this showed very little difference in the amount of cell surface heparanase from cells grown under normoxic or hypoxic conditions (Fig. 2C)
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are consistent with the observed enhanced activity under low-oxygen conditions because heparanase activity was increased by reducing conditions. These findings are consistent with the observation that hypoxia also up-regulates the activity of secreted-heparanase.
Heparanase Is Activated by Hypoxic Conditions.
Comparison of heparanase activity between equal amounts of OV-MZ-6 heparanase-conditioned medium under hypoxic and normoxic conditions produced a large increase (2.5 times, P < 0.01) in activity after 6 h of hypoxic treatment (Fig. 3A)
. Platelet-derived purified heparanase also showed an increase in activity (1.6 times, P < 0.05) under hypoxic conditions after 8 h of incubation (Fig. 3B)
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shows an example of a Matrigel invasion assay using A1 and A3 cells. It can be seen that the invasion of A1 cells was approximately 43% that of A3 cells (Fig. 4B)
. The lower invasive ability of A1 cells thus correlates well with the reduced heparanase activity found in this clone, whereas A3 cells were more invasive and had higher heparanase activity (Fig. 1A)
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shows that the invasiveness of both A1 and A3 cells was significantly increased under hypoxic conditions. Quantification of this (Fig. 4B)
showed that the invasion of A1 cells doubled and A3 cells increased by one-third when grown in 1% O2 rather than standard 20% O2 conditions.
Tumor Cell Invasion Was Inhibited by Antiheparanase Antibody.
The in vitro activity of heparanase was inhibited by antiheparanase antibodies (Fig. 5)
. These were then added to invasion assays to evaluate the contribution of heparanase to this process. Antiheparanase antibody (50 µg/ml) was added into A1 cell cultures placed onto the upper compartment of BD chambers and exposed to 20% and 1% O2 for 48 h. In the presence of the antiheparanase antibody, cell invasiveness was inhibited by 80% compared with the addition of control purified IgG (Fig. 6, A and B)
. We also analyzed the A3 cell invasion under normoxic and hypoxic condition in the presence of different doses of antiheparanase antibody and IgG control. As shown in Fig. 6D
, invasiveness of A3 cells was inhibited by antiheparanase antibody in a dose-dependent fashion. The number of invading cells was not significantly altered by the addition of 10 µg/ml antiheparanase antibody, whereas invasion was blocked by 80–95% in the presence of 50–100 µg of antiheparanase antibody. This suggested that heparanase activity was required for basal levels of invasion, which was enhanced under hypoxic condition. This result is not explained by variations in cell survival or growth because the proliferation of all cells tested was not significantly changed during the incubation with antiheparanase antibody and IgG control (Fig. 6C)
. These data indicate that the hypoxia-induced cell invasion can be inhibited by antiheparanase antibodies.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As previously discussed, hypoxia has already been linked to metastatic invasion (12)
. We initially observed that exposure to hypoxia (1% oxygen) for 24 h produces a significant increase in heparanase activity from conditioned media from both A1 and A3 cells, whereas the level of protein expression remains unchanged. Western blot analysis of immunoprecipitated, secreted heparanase shows no difference in the size or level of this protein from cells grown under normoxic or hypoxic conditions. These data are supported by fluorescence-activated cell sorter analysis of cells immunostained for heparanase (Fig. 2C)
, which show very little difference in the amount of cell-surface heparanase from cells grown under normoxic or hypoxic conditions. Our results are consistent with the observed enhanced activity under low oxygen conditions because heparanase activity is also increased by reducing conditions, which could result in thiol group reduction producing an altered conformation in the heparanase enzyme (Fig. 2D)
. These data are supported by the observed hypoxia-dependent increase in heparanase activity of both cell-free conditioned medium and platelet enzyme when incubated under hypoxic and normoxic conditions (Fig. 3, A and B)
. These findings are the first evidence to suggest that hypoxia enhances the activity of secreted heparanase and are consistent with the hypothesis that the activity of this endoglucuronidase may be regulated by microenvironmental redox conditions (24)
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Comparison of the invasive properties of A1 and A3 cells, using Matrigel invasion chambers, confirmed that higher levels of heparanase activity were associated with increased invasion. In addition, the data show that hypoxia increases the in vitro invasive capacities of both A1 and A3 cells, thus implying that hypoxia-enhanced heparanase activity may be linked to the invasive behavior of tumor cells. These results suggest that hypoxia may increase metastatic spread by the activation of heparanase.
Are these effects specific for heparanase, or could additional factors be involved in the apparent hypoxic induction of heparanase-dependent invasion? To address this, we investigated the effects of agents with specific inhibitory activity against heparanase. It has been demonstrated that heparanase activity and experimental metastasis can be inhibited by non-anticoagulant and low-Mr species of heparin (25) . Other studies have shown that sulfated polysaccharides and antiheparanase antibodies also have inhibitory activity (24 , 26 , 27) . Our data clearly show that the in vitro activity of heparanase is inhibited by an antiheparanase antibody. Moreover, numbers of invading cells exposed to hypoxic conditions are decreased by addition of the antibody in a dose-dependent fashion, whereas control immunoglobulins had no effect. Interestingly, the antibody was more effective at blocking Matrigel invasion than was predicted from its modest effects on the in vitro activity of heparanase. What is the explanation for this? As discussed, recent work has indicated that heparanase can promote invasion by two separate mechanisms acting as adhesion receptor and an endoglucuronidase (28) . Our data are consistent with these observations because the antiheparanase antibody may block adhesion-mediated invasion in addition to endoglucuronidase activity. Nevertheless, our results indicate that heparanase may be activated by hypoxia, and this could be a key factor in the invasion process. This is the first report describing antibody reagents that specifically affect heparanase-mediated tumor cell invasion.
How can these results be explained? HS and its degrading endoglucuronidase are involved in diverse physiological and pathological processes, such as development, wound healing, and inflammation, in addition to tumor metastasis (4 , 18 , 29) . Experimentally, the mature heparanase enzyme exhibits little or no activity at normal physiological pH (7 , 30) , thus implying that its in vivo activity may be influenced by the cell microenvironment and/or by unknown stimulators/activators. The microenvironment of solid tumors contains regions of poor oxygenation and high acidity (reducing conditions; Ref. 12 ), which according to our data, may increase heparanase activity. The results presented demonstrate that the redox state of the microenvironment of both tumors and ischemic tissues may enhance heparanase activity to promote metastasis in the former and tissue repair in the latter.
Although the molecular mechanism of hypoxic regulation of heparanase activity remains to be defined, we suggest that this could be accomplished by either increasing the formation or stabilization of the active heterodimeric form of heparanase or stimulation by some unidentified hypoxia responsive activator. As previously stated, we did not see any difference in the quantity or size of immunoprecipitated heparanase from cells grown under hypoxic or normoxic conditions.
We propose a model for how heparanase degrades cell surface or ECM HS. Under normoxic conditions and neutral pH, cell surface or ECM heparanase has little activity (7 , 30) . Under hypoxic reducing conditions and/or pH, mature heparanase becomes activated to degrade HS.
Cancer therapeutics that are designed to target adhesion receptors or proteases involved in tumor invasion have not proven effective in slowing tumor progression (11) . As discussed, the explanation of this is thought to be that malignant cells can adapt their migratory mechanisms in response to different conditions (11) . The hypoxic activation of heparanase is thus likely to represent a distinct mechanism by which cancer cells promote metastatic spread. Clearly combinations of therapeutic strategies designed to target more than one feature of the metastatic process are more likely to be effective.
In conclusion, our work demonstrates that: (a) the activity of heparanase is up-regulated by hypoxic conditions, which may depend on the environmental redox state; (b) this is linked to increased tumor cell invasion; and (c) heparanase-mediated invasion can be blocked by specific neutralizing antibodies. These findings lead us to postulate that the activity of heparanase is tightly regulated by microenvironmental condition in vivo. Indeed, heparanase is likely to be an important target for anticancer therapy, supporting our further investigations into heparanase inhibitors and antibodies as therapeutic agents for metastatic diseases.
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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: Ian N. Hampson, University of Manchester Gynaecological Oncology Laboratory, St Mary’s Hospital, Whitworth Park, Manchester M13 OJH, United Kingdom. E-mail: ian.hampson{at}man.ac.uk
Received 8/29/03. Revised 3/16/04. Accepted 3/26/04.
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