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Experimental Therapeutics |
Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558 [F. U., T. F., Y. T., S. O., K. K., M. T., T. O., Y. N., N. T.], and Tsukuba Research Institute, Novartis Pharma K. K., Ibaraki 300-2611 [M. N.], Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heparanase is an endoglucuronidase that cleaves HS at specific interchain sites, thus facilitating the release of several protein modulators of cell function, including migration, adhesion, and angiogenesis (5) . Heparanase activity correlates with the metastatic potential of tumor cells in animal models (6) and, moreover, is increased in the sera and urine of human patients with metastatic cancer. Although the importance of heparanase function has long been recognized, the mammalian heparanase has remained an elusive target. However, several laboratories recently purified human heparanase and have isolated the cDNA encoding this activity. The human heparanase enzyme is expressed as a 543-amino acid precursor protein that is modified by proteolytic cleavage to remove the first 157 amino acids (7, 8, 9, 10) . Treatment with heparanase inhibitors considerably reduced the incidence of lung metastasis in animal models (11, 12, 13) .
These observations led us to hypothesize that abrogation of heparanase expression could reduce pleural dissemination by inhibiting the tumor cell invasion to the basement membrane. Here we demonstrate that an adenoviral vector expressing an antisense to a full-length human heparanase gene specifically inhibits heparanase expression as well as its invasive potential in vitro, leading to the reduction of pleural dissemination in an orthotopic murine model in vivo.
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MATERIALS AND METHODS |
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Recombinant Adenoviruses.
Replication-deficient, E1- and E3-deleted recombinant Ad5 was used as the viral backbone. A human heparanase cDNA of 1758 bp was inserted, as a EcoRI/XhoI fragment, in sense and antisense orientation into the cosmid vector pAxCAt (Adenovirus Expression Vector Kit; TaKaRa Biomedicals, Tokyo, Japan) to generate the pAxCAt-S/hep and pAxCAt-AS/hep constructs, respectively. These constructs were cotransfected with restriction enzyme-digested DNA-TPC into 293 cells to isolate recombinant adenovirus. The resultant adenoviruses were named Ad-S/hep and Ad-AS/hep (Fig. 1A)
. The E1A-deleted adenovirus vector d1312 was used as the control vector. Recombinant virus was purified by ultracentrifugation in cesium chloride step gradients, and its titer was determined by pfu on the 293 cells.
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Western Blot Analysis.
Cells were collected by trypsinization and washed twice in cold PBS. Cells then were dissolved in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 µg/ml aprotinin). The lysis was carried out at 4°C for 30 min and centrifuged at 15,000 rpm. The protein concentration of the supernatant was determined using the Bio-Rad protein determination method (Bio-Rad, Hercules, CA). Equal amounts (60 µg) of proteins were electrophoresed under reducing conditions on 12% (w/v) polyacrylamide gels. Proteins were electrophoretically transferred to a Hybond- polyvinylidene difluoride transfer membranes (Amersham, Arlington Heights, IL) and incubated with primary antibodies against heparanase and then peroxidase-linked secondary antibody. An Amersham enhanced chemiluminescence chemiluminescent Western system (Amersham, Tokyo, Japan) was used to detect secondary probes.
Immunohistochemistry.
Immunohistochemical staining was performed using a HISTOFINE SAB PO kit (Nichirei, Tokyo, Japan) according to the instructions provided by the manufacturer. Formalin-fixed, paraffin-embedded tissue sections were mounted on silanized slides, deparaffinized in xylene for 20 min, and rehydrated in graded ethanol solutions. Endogenous peroxidase was blocked by incubating the sections in 3.0% H2O2 in methanol for 15 min. Antigen retrieval on paraffin sections was performed by heating three times in 10 mM citrate buffer solution (pH 6.0) in a microwave. After the blocking of nonspecific reactivity with rabbit serum for 10 min at room temperature, sections were incubated overnight at 4°C with the antihuman heparanase mouse monoclonal antibody raised against recombinant human heparanase. After rinsing, the slides were incubated with biotinylated rabbit antimouse IgG and then with avidin-biotin-peroxidase complex. Peroxidase activity was determined using DAB/H2O2 solution (Histofine DAB substrate kit; Nichirei). The slides were counterstained with methyl green with Mayer’s hematoxylin. As a negative control, some sections were subjected to normal serum blocking and omission of the primary antibody.
Quantitative Real-Time RT-PCR.
Heparanase mRNA copy number in surgical specimens and cell lines were determined by real-time quantitative RT-PCR using a LightCycler instrument and a LightCycler DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Indianapolis, IN). Amplifications were done in glass capillary tubes using 20-µl reaction containing 3 mM MgCl2, 0.5 µM each primer, and 2 µl of 10x LightCycler FastStart DNA Master SYBR Green I. PCR amplification began with a 60-s denaturation step at 95°C and then 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 10 s, and extension at 72°C for 9 s. The oligonucleotides used as specific primers were 5'-GCG GTT ACC CTA TCC TTT TT-3' and 5'-GCA GCA ACT TTG GCA TTT C-3'. Copy numbers of mRNA were calculated from serially diluted standard curves (Fig. 3B)
generated from purified cDNA template, which consisted of a human heparanase cDNA of 1758 bp inserted into the expression vector. Data analysis was performed using LightCycler Software (Roche Molecular Biochemicals).
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Animal Experiments.
The human non-small cell lung cancer cell line A549 was infected with Ad-S/hep at a MOI of 5, harvested 24 h after infection, and resuspended in HBSS at a density of 4.0 x 106 cells/200 µl. Female BALB/c nu/nu mice were intrathoracically injected with 200 µl of cell suspension through a 27-gauge needle. After 24 h, either 3.0 x 108 pfu/100 µl of dl312 or 3.0 x 108 pfu/100 µl of Ad-AS/hep was injected into the thoracic space by the same technique. The procedure was repeated over 3 consecutive days. Thirty days after cell inoculation, mice killed, and their thoracic spaces were examined macroscopically for any growths. Tumors in the thoracic spaces were removed and weighed. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Okayama University School of Medicine, Okayama, Japan.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. First we examined, by strand-specific RT-PCR assay, the expression of the sense and antisense constructs in human esophageal and lung cancer cell lines T.Tn and A549, respectively (Fig. 1B)
. The antisense transcript was detected with specific primers (Hep-AS and Ad5 pax) in Ad-AS/hep-infected cells, whereas RT-PCR with another set of primers (Hep-S and Ad5 pax) detected heparanase mRNA expression in cells transduced with the Ad-S/hep vector. Both sense and antisense transcripts could be seen when Hep-AS and Hep-S primers were used. This analysis also confirmed the dose-dependent expression of the transcripts in T.Tn cells.
Heparanase Expression in Human Cancer Cells after Ad-S/hep Infection.
Western blot analysis was carried out to examine the effect of Ad-S/hep and Ad-AS/hep infection on heparanase protein levels in T.Tn and A549 cells. As shown in Fig. 2A
, proheparanase (Mr 65,000) as well as cleaved, active heparanase (Mr 50,000) protein expression was detected in both cell lines as early as 24 h after infection, but not in parental and dl312- or Ad-AS/hep-infected cells. Experiments using various concentrations of Ad-S/hep showed that infection at a MOI of 5 was sufficient to exhibit a detectable heparanase protein expression in T.Tn and A549 cells, although the protein level was much higher in A549 cells compared with that in T.Tn cells (Fig. 2B)
. In addition, infection with dl312, Ad-S/hep, or Ad-AS/hep vectors at a MOI of 30 had no apparent effect on the in vitro growth of T.Tn and A549 cells (Fig. 2C)
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, heparanase protein expression was present in the tumor cells in all of tumor specimens examined. Although some cells in the stromal regions adjacent to the tumor cells exhibited heparanase immunoreactivity, the staining was less intense than the tumor cells. These results suggest that heparanase expression in the tumor tissues is derived primarily from the tumor cells.
To compare expression levels of heparanase message between tumor tissues and cell lines, we next performed a quantitative real-time RT-PCR assay using the LightCycler instrument (Fig. 3B)
. The assay demonstrated that heparanase mRNA expression was easily detectable in clinical samples of human esophageal and lung cancers, which is consistent with previous reports; human esophageal and lung cancer cell lines, however, expressed relatively low levels of heparanase mRNA. The relative expression of heparanase mRNA in primary tumors obtained from four patients as well as in A549 lung cancer cells was shown in Fig. 3C
. The median expression was 
300-fold higher in tumor tissues than in the A549 cell line.
Infection with Ad-AS/hep Decreased Heparanase mRNA and Protein Levels in T.Tn and A549 Cells.
We next evaluated the efficacy of Ad-AS/hep in inhibiting heparanase expression. However, having verified the presence of low expression levels of heparanase in T.Tn and A549 cell lines, we selected cells infected with Ad-S/hep for these experiments. T.Tn cells were infected with Ad-S/hep at a MOI of 30 and then Ad-AS/hep infection at various MOIs. RT-PCR analysis with specific primers demonstrated expression of the antisense transcript as well as a diminution of heparanase mRNA levels in a dose-dependent manner (Fig. 4A)
. For Western blot analysis, T.Tn and A549 cells were infected with Ad-S/hep at MOI of 10 and 5, respectively, and then Ad-AS/hep infection at various MOIs. The assay showed significantly decreased levels of heparanase protein in a dose-dependent fashion with an increase in MOI (Fig. 4B)
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, A549 cells infected with Ad-S/hep at 5 MOI showed aggressive invasion characteristics compared with dl312-infected cells, which could be significantly inhibited upon simultaneous infection with 100 MOI of Ad-AS/hep vector. These results suggest that the antisense construct selectively inhibited the heparanase-induced invasive properties of A549 cells.
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. In addition, Ad-AS/hep-treated animals showed a reduced weight of tumor nodules compared with dl312-treated animals.
Inhibition of MMP-2 Expression in Heparanase-expressing A549 Cells by Ad-AS/hep Infection.
MMP is a neutral proteinase that mediates ECM degradation and potentiates the invasiveness and metastatic potential of malignant tumors (14)
. To determine whether heparanase expression affects other invasion-related enzyme expression, we carried out semiquantitative RT-PCR analysis with specific primer pairs for type IV collagenases: MMP-2 and MMP-9. Densitometric quantification revealed that Ad-S/hep infection at a MOI of 5 caused a 2-fold increase of MMP-2 mRNA expression (Fig. 5C)
, whereas there were no changes in MMP-9 mRNA levels (data not shown). When A549 cells were simultaneously infected with Ad-AS/hep at a MOI of 50, MMP-2 mRNA expression decreased by 1.3-fold compared with that in cells transduced with 5 MOI of Ad-S/hep alone.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many compounds, such as laminarin sulfate, phosphomannopentose sulfate (17)
, and maltohexaose sulfate (18)
, are potent inhibitors of heparanase activity; these molecules are, however, difficult to target to specific tissue sites, and their pleiotropic interactions with HSPG might produce undesired effects. Therefore, to selectively block heparanase expression, we used an antisense strategy. Expression of antisense RNA can directly influence the expression of a given gene of interest, although the precise mechanism of inhibition remains unclear (19)
. In addition, to ensure efficient transfer and high expression of antisense heparanase, we generated an adenovirus construct carrying human heparanase cDNA in an antisense orientation (Ad-AS/hep; Fig. 1
). First, we examined whether Ad-AS/hep infection could suppress endogenous heparanase expression in human esophageal and lung cancer cell lines. However, unexpectedly, our quantitative real-time RT-PCR assay demonstrated that a specific heparanase transcript amplification was undetectable or extremely low in the cell lines tested in our study, although it was readily detected in surgical specimens (Fig. 3)
. We examined at least 10 clinical tissues of esophageal and lung cancer and detected heparanase expression in all of them by either RT-PCR assay or immunohistochemistry. Possible explanation for such a discrepancy is attributable to the differences of the microenvironment. Neoplastic cells may interact with surrounding cells in vivo to sustain their malignant potentials (e.g., metastatic melanoma cells and astrocytes; Ref. 20
), which supports the concept that lack of heparanase expression in established cell lines results form the absence of microenvironmental stimuli.
Several groups have reported that tumor cells transfected with heparanase cDNA acquire a highly metastatic phenotype in vivo (21)
. Consistent with these findings, tumor cells adenovirally transduced with the human heparanase gene showed heparanase protein expression (Fig. 2, A and B
) as well as heparanase activity (data not shown), which contributed to the highly invasive nature of the infected cells (Fig. 5A)
. The finding that overexpression of heparanase had no apparent effect on the proliferation of tumor cells in vitro indicates its important roles for invasive properties in the microenvironment (Fig. 2C)
. With the use of tumor cells expressing the ectopic heparanase gene, we showed successful transduction of an antisense construct with Ad-AS/hep, resulting in decreases of mRNA transcripts (Fig. 4A)
and protein contents (Fig. 4B)
. In addition, we have noted complete inhibition of enhanced invasiveness in cells expressing heparanase by Ad-AS/hep infection (Fig. 5A)
, suggesting that Ad-AS/hep has significant biological consequences.
Our data also indicate the therapeutic potential of molecular manipulation of heparanase expression in an orthotopic model of pleural dissemination. Intrathoracic administration of Ad-AS/hep vector reduced the incidence of tumor formation in the thoracic cavity (Fig. 5B)
. Our preliminary experiments revealed that injection of adenovirus expressing the wild-type p53 gene, which has been known to induce apoptosis in certain tumor cells, had no significant antitumor effect in this animal model (data not shown). Thus, approaches that selectively block expression of molecules implicated in cellular invasion may be clinically more relevant for the prevention of tumor cell dissemination than directly inducing apoptosis of tumor cells. In fact, selective inhibition of other degradative enzymes such as MMP-9 (22)
and urokinase-type plasminogen activator receptor (23)
by antisense gene transfer has been reported to impair tumor invasion as well as metastasis. We also found that Ad-AS/hep infection suppressed heparanase-induced up-regulation of MMP-2 expression (Fig. 5C)
, suggesting that Ad-AS/hep may have direct and indirect effects on the expression of heparanase and MMP-2, respectively, thereby being more clinically beneficial.
Although additional experiments using freshly isolated primary human cancer cells that consistently express endogenous heparanase are required, to the best of our knowledge, this is the first demonstration of antisense-mediated inhibition of heparanase expression as well as invasive properties by using an adenovirus construct. Our results suggest that heparanase is a promising target for the prevention of pleural dissemination of human cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Ministry of Health and Welfare, Japan [Health Sciences Research Grants (Research on Human Genome and Gene Therapy)]. 
2 To whom requests for reprints should be addressed, at Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7257; Fax: 81-86-221-8775; E-mail: toshi_f{at}md.okayama-u.ac.jp
3 The abbreviations used are: HSPG, heparan sulfate proteoglycans; ECM, extracellular matrix; HS, heparan sulfate; pfu, plaque-forming unit; RT-PCR, reverse transcription-PCR; MOI, multiplicity of infection; MMP, matrix metalloproteinase.
Received 5/30/01. Accepted 8/30/01.
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REFERENCES |
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