Journal of Neuro-Oncology 52: 1–9, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Suppression of matrix metalloproteinase-2 and -9 mediated invasiveness by a novel matrix metalloproteinase inhibitor, BE16627B Kunihiro Watanabe, Daizo Yoshida, Masahiro Noha and Akira Teramoto Department of Neurosurgery, Nippon Medical School, Tokyo, Japan

Key words: BE16627B, cell invasion, malignant glioma, matrix metalloproteinase, zymogram Summary Cell invasion is a nature of malignant gliomas, demeriting to many efforts of the treatment. Matrix metalloproteinase (MMP) is acknowledged as a key factor in this complicated process. The aim of this study was to investigate whether inhibition of MMP activity in malignant glioma cells could be achieved by a novel agent, BE16627B (BE). Malignant glioma cell lines, U87MG, U251MG, and U373MG, were employed to evaluate inhibitory effect on zymogram, type IV collagenolysis assay, and haptoinvasion assay for 24 h exposure of BE, following preliminary MTT assay to establish non-cytotoxic dose range. MTT assay revealed that doses of 1, 5,10, or 50 µM were non-cytotoxic. Zymog ram disclosed that expressions of MMP-2 and MMP-9 were diminished in a dose-dependent fashion. Reflecting that type IV collagenolysis assay revealing that the digestion of type IV collagen was significantly depressed along with elevated dose concentration, haptoinvasion also revealed significantly suppressive effect, meanwhile both 1 µM of BE did not give significant effect. Non-cytotoxic level of BE, ranging from 5 to 50 µM, suppressed MMP-2 and MMP-9 mediated cell invasiveness in the malignant glioma cell lines. This is the first report for cytostatic effect of this agent in glioma cells. This study might be highly implicative of BE16627B to be much meriting to support the other treatment against malignant gliomas. Introduction Malignant gliomas usually invade the adjacent normal brain tissue as one of propensities. Then, this invasive nature from the tumor periphery, in particular, interferes with many efforts of treatment involving surgical resection and the other treatment, because the satellite lesion, distant from the primary site migrating into the normal brain tissue, could be hardly exposed to local drug or gene delivery and sometimes is left even after gross removal. If this malignant tumor could be localized within the primary site, these therapeutic attempts must be more effective also by irradiation. Cell invasion is a complex phenomenon that involves disruption of the extracellular matrix [1] and penetration by the tumor cell into the normal adjacent tissues [2]. Degradation of the extracellular matrix is mediated by glioma cell-derived proteolytic enzymes, such as heparinase, serine proteinase, cathepsin and matrix metalloproteinase (MMP) [3–7]. As studied in numerous neoplastic cells, glioma cells also have been known to release MMP enzymes, mainly MMP-2

(gelatinase A) and MMP-9 (gelatinase B) [3,6]. It must be plausible to consider that cell invasiveness, a propensity of malignant glioma, could be inhibited by a MMPinhibitory agent to accomplish beffer prognosis and also enhance effects by the other anti-glioma strategies. BE16627B (BE), inhibitor for zinc matrix metalloproteinases, was established by Naito et al., who has demonstrated its efficacy to diminish the cell invasion on HT1080, human fibrosarcoma cell [8]. The purpose of the current study is to investigate whether this antiMMP agent, BE16627B, could also be efficacious for invasive behavior of human malignant glioma cell lines (U87MG, U251MG, and U373MG), mainly focusing on the activities of MMP-2 and MMP-9 and capability of digestion of their substrate, collagen type IV. Materials and methods Cell preparation Human malignant glioma cell lines, U87MG, U251MG, U373MG (American Type Culture

2 Collection, Rockville, MD, USA), were cultured in plastic flasks (Falcon, 150 cm2 ) with DME medium (Sigma, St. Louis, MS, USA) containing 10% heatinactivated fetal calf serum (Biocell, Carson, CA, USA) supplemented with 0.05% (w/v) L-glutamine, l00 µg/ml of gentacin, and 200 lU/ml of penicillin (hereafter called culture medium). The cells were maintained at 37◦ C in humidified atmosphere containing 5% carbon dioxide. Culture medium was exchanged twice a week. Upon reaching subconfluence, the cells were detached from the flask with 0.05% trypsin/0.02% EDTA. Before use, the cells were rinsed in phosphate-buffered saline (PBS), centrifuged at 1000 r.p.m., for 10 min. And the resulting pellet was resuspended in fresh culture medium. This rinse after the detachment procedure ensured that there would be no active trypsin or antibiotics to affect the artificial basement membrane; the cell density was determined with a hemocytometer. Agent BE16627B (BE) (L-N-(N-hydroxy-2-isobutylsuccinamoyl)-seryl-L-valine (molecular weight, 375) supplied by Banyu Tsukuba Research Institute, Ibaragi, Japan) (Figure 1) was dissolved in 100% dimethyl sulfoxide (DMSO) (10−2 M). The stock solution was stored at −20◦ C, and then diluted in the culture medium immediately just prior to use. MTT assay For the preliminary study, MTT assay was examined to establish non-cytotoxic dose range of BE as follows. A stock solution of monotetrazolium (MMT) (3-(4,5-dimetyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/ml in PBS) was prepared and further dissolved in PBS at 0.5 mg/ml. After incubation

Figure 1. Chemical formula of BE16627B (molecular weight; 375.2).

overnight with a cell density of 2 × 104 cells/well in 96-well plate to allow cells to attach on the well bottoms, preincubation with 0, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, or 500 µM of BE for 24 h. A total 100 µl of MTT solution (0.5 mg/ml) in normal culture medium was replaced for 4 h with drug solution in these 96-well plates. The MTT solution was removed by aspiration. To dissolve formazan crystals within cells, 100 µl of 100% DMSO was added after aspirating MTT solution from the wells. The mitochondrial reduction of MTT salt to formazan crystals was quantified spectrophotometrically. The spectrophotometric absorbance at 550 nm was determined using a scanning multiwell spectrophotometer (Biorad, Tokyo, Japan). Each determination was performed in five replicates, and the percent surviving fraction (PSF) was calculated as the following formula [9]: PSF = mean OD value of BE/ mean OD value of the control × 100. Gelatin zymogram for detection of MMP Gelatin substrate zymograms were performed, just as previously described [5,10]. We investigated the expression of MMP-2 and MMP-9 in each concentration of BE16627B by zymogram for semiqualitative determination. Gradient sodium dodecyl sulfate (SDS) polyacrylamide slab gels (5–15%) were impregnated with gelatin (5 mg/ml) (Sigma, MO, USA). Exponentially growing cells (5×105 cells) were placed in a plastic flask (75 cm2 , Falcon, USA) and allowed to attach to the bottom for 24 h in culture medium. After twice rinses with PBS, the cells were exposed to 0, 1, 5, 10, or 50 µM of BE in 10 ml of serum free conditioned medium. After 24 h incubation at 37◦ C in humidified atmosphere containing 5% carbon dioxide, the supernatant was centrifuged at 100 g, to discard cell debris and the supernatant was used for the assay. The samples (the supernatant) were not heated before electrophoresis. An aliquot (25 µl) of each sample was mixed with 25 µl of sample buffer (0.25 M Tris, 8% SDS, 40% glycerol, 0.48 mg/ml bromophenol blue). Gels were run at 150 V at 37◦ C approximately for 1–2 h and washed with 10 mM Tris buffer containing 2.5% Triton X-100. MMP was activated by incubation for 24 h in a buffer consisting of 50 mM Tris, 0.5 mM calcium chloride, and 10 mM zinc chloride. Gels were stained with Coomasie blue (1%), and destained in 10% methanol, 5% acetic acid. Transparent bands on

3 the background of the Coomasie blue-stained slub gels indicated the presence of gelatinolytic enzymes. Type IV collagenolysis assay Type IV collagenolysis assay, as previously described [3,11]. Briefly, Matrigel (Collaborative Research, Lexington, MA, USA) were diluted to achieve a final concentration of 1 mg/ml in serum-free DME medium. One hundred microliters of this diluted Matrigel was placed in each well of 96-well culture plate and left in the laminar airflow hood at room temperature to dry out overnight. Exponentially growing cells (5×105 cells) were placed in the Matrigel-coated well in a solution of 0, 1, 5, 10, or 50 µM BE in 10 ml with serum-free conditioned medium. After 24 h incubation at 37◦ C in humidified atmosphere containing 5% carbon dioxide, the supernatant was used for this assay. One hundred microliters of the supernatant was placed in each 96-well plate, coated with Matrigel as above, mixed with 100 µl of MMP activation buffer (50 mM tris, 0.5 mM calcium chloride, and 10 mM zinc chloride). The 96-well plate was incubated for another 24 h at 37◦ C. Then, 100 µl of supernatant mentioned above in the 96-well was analyzed protein concentration by Protein assay kit (Biorad, California, USA) in another, 96-well plate, and was measured by spectrophotometer at the absorbance 560 nm. Preliminary study to establish the standard line indicated as following formula: type IV collagen concentration = OD volume times 40.3 + 0.041 (µg/ml). Haptoinvasion assay In vitro invasiveness was assessed by the method presented by Albini et al. [12]. Reconstituent basement membrane substance, Matrigel, diluted at a concentration of 1 mg/ml in serum-free medium, was coated onto transwell polycarbonate filters with 8 µm pores (1000 µl) (Costar Corp. Cambridge, MA, USA) to form a thin continuous layer on top of the filter, allowed to gel for overnight room temperature in a humidified atmosphere of 5% CO2 , and placed in transwell chambers (Nunc, USA) with six-well plates. The upper chambers (2.5 cm in a diameter) were filled with 2000 µl of cell suspension (105 cells/ml) in BE solution in serumfree culture medium (0, 1, 5, 10, or 50 µM). The lower chambers were coated with 100 µl of fibronectin

(20 µg/ml) as a chemoattractant. The chambers were incubated at 37◦ C in a humidified atmosphere of 5% CO2 for 24 h. After incubation, cells remaining on the upper surface of the micropore filter were removed by a cotton swab and cells that had invaded across the filter were fixed with 100% methanol and stained with Giemsa stain. The determination of cell invasion was performed by counting the cells that had migrated to the lower side of the filter, using a light microscope at 100× magnification. Six random fields were counted for each estimates. For statistical analysis, differences between mean values were tested using Student’s t-test. Statistical significance was taken as p < 0.01 (Statworks, version 1.2, Cricket Software, Philadelphia, PA, USA).

Results MMT assay The result of MMT assay was summarized in Figure 2; as the y-axis was addressing the PSF value and x-axis was the concentration of BE16627B. This assay disclosed that significant reduction of PSF values in the three cell lines was commonly seen below 100 µM (100 µM: U87MG, 84.5%; U251MG, 84.7%; U373MG, 81.7%; 50 µM: U87MG, 96.8%; U251MG, 94.7%; U373MG, 98.6%). Therefore, we employed non-cytotoxic dose concentration of BE16627B with 0, 1, 5, 10, and 50 µM for the following examinations. Gelatin zymogram Expressions of the bands addressing MMP-2 (72 kDa) and MMP-9 (92 kDa) in zymogram were diminishing along with elevated dose concentration of BE within the non-cytotoxic level common to all three cell lines (Figure 3). Haptoinvasion assay As was summarized in Figure 4, BE16627B, ranging from 5 to 50 µM, inhibited cell invasion with statistical significance (U87MG: 0 µM, 81.00 ± 2.84, 50 µM, 19.33 ± 2.86 (p < 0.001); U251MG: 0 µM, 75.83 ± 5.68, 50 µM, 20.16 ± 2.86 (p < 0.001); U373MG: 0 µM, 147.83±12.48, 50 µM, 11.50±2.40 (p < 0.001)). Meanwhile 1 µM of BE16627B did not

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Figure 2. MTT assay. A significant reduction of percent surviving fraction was seen in dose concentrations more than 100 µM. The y-axis was addressing the PSF value, and the x-axis was the concentration of BE16627B. PSF = mean OD value of BE/mean OD value of the control ×100.

show any significant reduction in the numbers of invading cells through the Boyden chamber. This inclination was common to three cell lines. Light microscopic views at the lower surface of the filter were much demonstrable to show the diminishing cell density after an exposure of BE16627B (Figure 5). Type IV collagenolysis assay Digestion of the coated collagen type IV by each supernatant containing activated MMP was statistically suppressed in the dose-concentrations ranging from 5 to 50 µM of BE16627B in a dose-dependent fashion, while all the supernatants from 1 µM BE solutions dissolved collagen type IV not significant with the control supernatants (Figure 6). Discussion Cell invasion of malignant glioma Malignant gliomas have an invasive nature with a dismal prognosis despite aggressive therapeutic

intervention, irradiation, and chemotherapy. Invasion is accompanied by remodeling of the surrounding vasculature and the destruction of the adjacent normal brain tissue [2]. The arrest of tumor invasion in glioma might be one of reasonable targets, because these tumors do not usually metastasize to distinct organs and could be regarded as a localized disease [11]. If malignant glioma could be localized in the primary site, it would contribute much to surgical removal of the tumor and more exact targeting for irradiation, and local delivery to the tumor margins. Cell invasion is a complex matter involving several steps; (1) escape from the primary tumor, (2) the initially generating motion to attach the adjacent tissue, and (3) the activation of matrix degrading enzymes secreted by invading cells themselves to induce remodeling of the membrane layer [13]. We have been targeting on these mechanisms in which we have highlighted on the other MMP inhibitor, D-Penicillamine on tumor angiogenesis, and inhibition of cell motility by microtubule depolymerizing agent [9,14,15]. To our best knowledge, few studies have been described with respect to anti-MMP agent on gliomas [15]. To penetrate Matrigel by various malignant calls has been regarded

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Figure 3. Geratin zymogram: the expressions of MMP-2 (72 kDa) and MMP-9 (92 kDa) were indicated in a dose-dependent manner in the non-cytotoxic level of BE16627B.

Figure 4. Haptoinvasion assay. Bars denote the standard errors of the mean (SEM). NS, not significant; *p < 0.01; **p < 0.005; ***p < 0.001. BE16627B, ranging from 5 to 50 µM, inhibited cell invasion statistically significantly (U87MG: 0 µM, 81.00 ± 2.84, 50 µM, 19.33 ± 2.86 (p < 0.001); U251MG: 0 µM, 75.83 ± 5.68, 50 µM, 20.16 ± 2.86 (p < 0.001); U373MG: 0 µM, 147.83 ± 12.48, 50 µM, 11.50 ± 2.40 (p < 0.001)).

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Figure 5. Light microscopic findings in haptoinvasion (Giemza stain, 200× magnification): (a) control; (b) 10 µM of BE16627B for 24 h.

as a hallmark of their metastatic potentials [16–19], and glioma cells also showed this invasive behavior. It was often one reason for fatal prognosis. In general, the basement membrane consists of collagen type IV, laminin, heparin surfate, and proteogrycans, that were found in the systemic blood vessels [19] and also in normal astrocytes [5,7]. Therefore, the haptoinvasion assay, utilizing Matrigel, could be reflecting the invasive nature of glioma cells in the normal brain tissue.

Matrix metalloproteinase and malignant gliomas Rapidly mounting evidence has shown that proteinases and proteinase inhibitors derived from tumor cells play crucial roles in tumor invasion. The past studies highlighted mainly on the production of plasminogen activators [20,21] and MMP in human glioma cells [5,7]. Matrix metalloproteinase represents one of gene families of zinc metalloproteinases that are capable of digestion almost all extracellular matrix macromolecules.

Thirteen of these MMP families have recently been classified [5,22]. These enzymes have different substrate specificity: MMP-1 degrades collagen type I, II, and III; MMP-2 degrades denatured collagen (gelatin), collagen type IV and V, fibronectin, and laminin; MMP-3 digests collagen type IV, gelatin, fibronectin, laminin, and proteoglycans; MMP-9 degrades gelatins collagen type III, IV, and V, and alpha-2 chain of collagen type I. In addition to these MMPs, MMP-7 (matrilysin), MMP-8 (neutrophil collagenase), MMP-10 (stromelysin), MMP-11 (stromelysin-2), MMP-12 (stromelysin-3), MMP- 13 (collagenase-3), and membrane-type metalloproteinase (MT-MMP, activator of pro-MMP-2) have also been further reported [4,22,23]. This enzymatic activity is regulated by natural inhibitors in the extracellular milieu, of which three related, still distinct inhibitors, designed as TIMP-1, TIMP-2, and TIMP-3, are considered to be major regulators of MMP [6]. As previously analyzed, TIMPs inhibit MMPs, and additionally TIMP-2 reduces the level of MMP-2 and MMP-9 that expressed

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Figure 6. Type IV collagenolysis assay. Bars denote the standard errors of the mean (SEM). NS, not significant; *p < 0.005; **p < 0.001. The y -axis was type IV collagen concentration of each supernatants, and the x -axis was the concentration of BE16627B.The standard line: concentration of type IV collagen (µg/ml) = OD value times 40.3 + 0.041 (µg/ml). Suppression of the absorbance of type IV collagen by activated MMP in each samples was denoted.

prominently in glioma cells more than TIMP-1 [13]. Apodaca et al. described that human glioma cell in vivo mainly secreted MMP-1, MMP-2, and MMP-9, as well as TIMPs [24]. In the other literature, human normal glial cell in vitro produced MMP-1, MMP-2, MMP-3, and MMP-9, as well as TIMP-1. Tumor cell-derived metalloproteinases have also been shown to be responsible for the invasion of cultured brain tissues by rat glioma cells [25].

Inhibition of MMP activity by BE16627B on the other cancers It must be plausible to propose that agents inhibiting enzyme activity of MMP might be the therapeutic strategy to prevent an invasion of gliomas, which could enhance the efficacy of irradiation and chemotherapy. Currently, the other low molecular MMP inhibitors, Marimastat (British Biotech) [26], and other agent have been aimed for clinical trials. They inhibited glioma invasion, yet little complicated with side effects. The present novel MMP inhibitor, BE16627B [8] (Banyu Tsukuba Research Institute in Collaboration, Tsukuba, Ibaragi, Japan), was screened and purified as a low molecular MMP inhibitor with

molecular weight of 375.2. Naito et al. [8] uncovered the characteristics of BE as inhibitor of MMPs. It suppressed the several zinc metalloproteinases activities (MMP-2, MMP-3, MMP-9, themolysin, bacterial collagenase and leucine aminopeptidase), while activities of non-zinc-dependent proteinases, such as trypsin, chymotrypsin, papain, pepsin and elastase, were not influenced, because BE16627B depended on Zn2+ . And since this inhibition could be reversed by dialysis, BE16627B is likely to be a reversible inhibitor. With regards to cytotoxity, this agent showed little on HT1080, human fibrosarcoma cell and HCT116, human colon carcinoma cells, where both of IC50 were over 100 µg/ml, compared with Adriamycin, 0.14–0.28 µg/ml. Both the gelatin and collagen degradation activities were reduced by the metalloproteinase inhibitors, EDTA, phenanthroline, and BE16627B. When HT1080 cells, secreting enriched MMP, were cultured in the presence of BE16637B, the gelatinand collagen-degradative activities after treatment with APMA, an activator of pro-MMP were both inhibited dose dependently [8]. Successfully in our results from the type IV collagenolysis assay, anti-MMP activity of this agent could be demonstrated also in malignant glioma cell lines. By the way, administrating this agent through osmotic pump in nude mice inoculated

8 with HT1080 cells, this significantly reduced the number and size of nodules to 24.3%, while 46.4% of those in untreated mice. These results suggested that BE16627B inhibits the growth of HT1080 in the lung of mice in dose dependent with safety. When using HCT116, barely secreting metalloproteinases in vitro, in the same methodology, BE16627B could suppress the tumor growth of HT1080 (at a dose of 2 mg/day for oral administration) [8]. These results were much demonstrable for that this agent was able to inhibit metalloproteinase-mediated expansion of human cancer cells. This is the first description to report a drug effect of BE16627B on malignant gliomas, aimed to investigate the inhibition of cell invasion within the cytostatic dose concentrations. In the type IV collagenolysis assay, digestion of type IV collagen was suppressed in a dose-dependent manner, ranging from 5 to 50 µM, meanwhile 1 µM of BE could not achieve significant inhibitory effect on cell invasion of malignant glioma cells. This result was closely related to the result in the haptoinvasion assay, in which 1 µM of BE16627B did not diminish the cell density of invaded cells significantly. Furthermore, expression of bands addressing to MMP-2, and MMP-9 in the zymogram were decreasing in the dose-dependent manner, in which MMP-2 and MMP-9 were degradating type IV collagen impregnated in the slub gels. In conclusion, BE16627B could inhibit the cell invasion of glioma cell line, mediated by deactivated MMP-2 and MMP-9, successfully in cytostatic dose range. The cytostatic effect against malignant glioma cell lines would be much beneficial to support the other anti-glioma therapies. Finally, molecular chemotherapeutic mechanism in the drug mechanism should be further investigated.

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Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture in Japan (No. 11671398).

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Address for correspondence: Kunihiro Watanabe, Department of Neurological Surgery, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-Ku, Tokyo 113-8603, Japan; Tel.: +0081-3-3822-2131; Fax: +0081-3-5685-0916