Inhibition of matrix metalloproteinase-9 reduces in vitro invasion and ...

3 downloads 0 Views 2MB Size Report
Abstract. The expression of matrix metalloproteinases. (MMPs), particularly MMP-9, is significantly increased during tumor progression and is thought to play a ...
INTERNATIONAL JOURNAL OF ONCOLOGY 25: 1407-1414, 2004

Inhibition of matrix metalloproteinase-9 reduces in vitro invasion and angiogenesis in human microvascular endothelial cells UNMESH JADHAV, SRINIVASULU CHIGURUPATI1, SAJANI S. LAKKA and SANJEEVA MOHANAM Program of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA Received May 3, 2004; Accepted June 8, 2004

Abstract. The expression of matrix metalloproteinases (MMPs), particularly MMP-9, is significantly increased during tumor progression and is thought to play a major role in mediating angiogenic process. Since microvasculature plays an important role in controlling tumor growth, we investigated the effects of MMP-9 inhibition on endothelial cell migration and tube formation, two determinants of angiogenesis. Adenoviral-mediated MMP-9 downregulation inhibited endothelial cell migration in cell wounding and spheroid migration assays. To determine the effects of MMP-9 reduction in glioblastoma/ endothelial co-cultures, we used a three-dimensional co-culture assay of glioblastoma spheroids and endothelial spheroids. Untreated controls showed invasion of both cell populations into each other whereas treatment of the co-cultures with adenoviral antisense MMP-9 particles resulted in reduced invasion. Next, inhibition of MMP-9 by adenoviral vectors in endothelial cells was assessed for in vitro capillary-like structure formation either by co-culture with glioblastoma cells or exposure to glioblastoma-conditioned medium. Addition of conditioned medium from human glioblastoma cells to endothelial cells treated with antisense MMP-9 adenoviral vectors or co-cultures of glioblastoma cell lines with MMP-9reduced endothelial cells resulted in reduced capillary-like tube formation demonstrating the key role of MMP-9 in endo­ thelial cell network organization. Examination of in vitro capillary-like tube structure formation using Matrigel showed

Correspondence to: Dr Sanjeeva Mohanam, Program of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA E-mail: [email protected] Present address: 'University of Louisiana at Monroe, 700 University Avenue, Monroe, LA 71201, USA Key words: MMP-9, invasion, angiogenesis, glioblastoma, endo­ thelial cells

a significant decrease in MMP-9 downregulated endothelial cells as compared to controls. In conclusion, the inhibition of MMP-9 is required for inhibition of endothelial cell migration and tube formation and is likely to be of importance in cerebral angiogenesis for therapeutic targets. Introduction Angiogenesis is critical for tumorigenesis. During the angio­ genic process, endothelial cells go through several steps including the loosening of matrix and intercellular adhesion, degradation of the extracellular matrix (ECM) and basement membrane by controlled proteolysis, migration, proliferation and formation of new tubes (1,2). The advancing front of the migrating endothelial cells presumably focuses proteolytic activity to create a defect in the vascular basement membrane and during this process the subendothelial basement membrane must be proteolytically disrupted to allow formation of new capillaries (3,4). Migrating endothelial cells elaborate a battery of proteolytic enzymes that mainly belong to the matrix metalloproteinase family to degrade ECM (5). MMPs are a family of secretory Zn-dependent endopeptidases with a diverse hydrolytic spectrum of extracellular proteins that have been involved in tumor invasion (6). MMP expression has been correlated with the invasive capacity of several tumor types. Expression of MMP-2 and MMP-9 is upregulated during tumor progression and contributes to tumor angiogenesis (7,8). Upregulation of secreted MMP-9 is correlated with an increase in tumor growth kinetics and angiogenesis as compared to cells that express low MMP-9 levels (9). Endothelial cells have been described to produce MMP-1, MMP-2, MMP-9, MT1-MMP, which are all implicated in the regulation of angiogenesis (10,11). The importance of these enzymes in angiogenesis is proved by evidence that endogenous (12,13) and synthetic inhibitors of MMPs block the process (14-16) and impair angiogenic response in mice deficient for MMPs (17,18). The ability of a tumor to grow is dependent on the formation of new blood vessels. Inhibition of angiogenesis would hence be expected to have a direct impact on tumor growth and recurrence. Consequently, the application of strategies (e.g. inhibition of MMP-9 expression) that disrupt the pro-angiogenic balance between neoplastic, stromal and endothelial cells may result in inhibition of tumor growth by preventing further development of a functional vascular network.

1408

JADHAV et al: MMP-9 MODULATES in vitro INVASION AND ANGIOGENESIS

The targeting of proliferating endothelial cells may allow a new therapeutic strategy against malignant tumors (19). Endothelial cells are easily accessible through the bloodstream as well as being genetically stable, thereby reducing the possibility of acquiring drug resistance. Adenoviral vectors have been widely used to transfer genes based on their unique ability to efficiently deliver genes to various tissues (20). In the present study, we used a replication-deficient recombinant adenovirus containing antisense cDNA for human MMP-9 to infect human microvascular endothelial cells (HMECs) in culture and then determined the role that MMP-9 plays during migration of human endothelial cells and its putative relevance for the angiogenic process. Materials and methods Generation of recombinant adenoviral vector. Ad AS-MMP-9 was constructed as described previously (21). Briefly, an antisense 528 bp fragment of 5' MMP-9 cDNA was placed in the deleted El region of an adenovirus. The control virus Ad CMV was generated in the same manner and had a similar structure to that of Ad AS-MMP-9 with the exclusion of the MMP-9 cDNA. High titer infectious Ad AS-MMP-9 viral stocks were prepared after the replication of recombinant adenoviral vector in the human kidney cell line 293. Cell culture and transduction. HMECs were maintained in medium and infection of the cells was carried out as described earlier (22). The viral stock was diluted, different multiplicity of infection (MOI) were added to cell monolayers (1.2 ml per 60 mm dish or 3 ml per 100 mm dish) and incubated at 37°C for 30 min with a brief agitation for every 5 min. The necessary amount of culture medium was then added and cells were returned to the incubator. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from cultured cells using RNeasy Protect Mini kit (Qiagen, Valencia, CA). RNA thus obtained was further purified by digesting with DNase for 20 min at 37°C and then reverse-transcribed using the cDNA cycle kit (Invitrogen, Carlsbad, CA) with random primers (23). To amplify the cDNA, the reverse-transcribed cDNA was subjected to 30 cycles of PCR in 25 |il of PCR Master Mix (Promega, Madison, WI) containing 100 pmol of sense and antisense primers. The efficiency of cDNA synthesis was estimated by PCR with GAPDH-specific primers. The following sense (S) and antisense (AS) primers were used in the RT-PCR reactions: MMP-9 (S, 5'-TGGACGATGCCTG CAACGTG-3'; AS, 5'-GTCGTGCGTGTCCAAAGGCA-3'; amplicon size 455 bp); GAPDH (S, 5'-CGGAGTCAACGGA TTTGGTCGTAT-3'; AS, 5'-AGCCTTCTCCATGGTGGTG AAGAC-3'; amplicon size 307 bp). Samples were subjected to electrophoresis on a 1.5% agarose gel and photographed as ethidium bromide fluorescent bands. Tomographic assays. Cells were changed to serum-free medium 24 h prior to the assay. Supernatants were then resolved under nonreducing conditions on 10% SDS-PAGE gels embedded with 1 mg/ml gelatin (22). Gels were rinsed three times in 2.5% Triton X-100 for 30 min at room temperature and then

incubated in 50 mM Tris-HCl pH 7.5, 10 mM CaCl 2 overnight at 37°C. Gels were stained with Amido Black and areas of gelatinolysis were visualized as transparent bands. Wound-induced migration assay. Migration through a wound introduced in a cell monolayer was assayed as described elsewhere (24). Briefly, subconfluent monolayers of cells were wounded by scraping with a plastic pipette tip and the distance that the advancing cells had moved into the cell-free (wound) area was measured after 24 h by staining with Hema-3. Cell migration from spheroids. Migration from spheroids was assayed as described previously (24). Single multicellular spheroids were placed in the center of each well of a 96-well microplate and were cultured for 24 h, after which the spheroids were fixed and stained with Hema-3 and cellular migration from the spheroids was assessed under light microscopy. Matrigel invasion assay. Cells were plated on Matrigel-coated cell-culture inserts in Transwell chambers (Corning Inc., Corning, NY) containing 6.5-mm filters (pore size 8 \xm) as described earlier (23). Cells were added to the Matrigelcoated chamber, and after a 48-h incubation period, cells on the Matrigel-coated side of the filter were removed with a cotton swab and the migrating cells remaining on the bottom part of the filters were fixed and stained with Hema-3. Cells were counted and the percentage of cells that had migrated through the Matrigel was determined (23). Co-culture invasion assay. Multicellular tumor-cell spheroids of HMECs and human glioblastoma cells (U87 or SNB19) infected with and without Ad AS-MMP-9 were grown separately on 0.75% agar-coated plates and stained with 3,3'dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1 dioctadecyl-3.3.3'.3'-tetramethylindocarbocyanine perchlorate (Dil) respectively as described earlier (23). For the co-culture, the Dil-stained tumor spheroids and DiO-stained HMEC spheroids were placed in close contact with each other in individual wells of a 96-well plate that had been base-coated with agar. At different times thereafter, serial optical sections were obtained from the surface to the center of the co-cultures by confocal laser-scanning microscopy, and the extent of invasion was monitored. Capillary-like structure formation. Human microvascular endothelial cells were seeded onto 48-well plates and grown in the presence of serum-free conditioned medium collected from human glioblastoma SNB19 cells for 48 h. Cells were stained with Hema-3 and photographed (22). The capillary length was determined by computer-assisted image analysis with the Image-pro Discovery program (Media Cybernetics, Silver Spring, MD). In vitro angiogenesis assay. Matrigel basement membrane matrix (Becton Dickinson) was diluted 1:2 in cold DMEM medium. Diluted Matrigel was plated into flat-bottomed 96-well tissue culture plates and allowed to gel for 20 min at 37°C before cells were added. After a 24-h incubation period, images were taken with a phase contrast microscope and

INTERNATIONAL JOURNAL OF ONCOLOGY 25: 1407-1414, 2004

1409

Figure 2. Inhibition of MMP-9 activity by Ad AS-MMP-9. Endothelial cells were transfected with 100 MOI of Ad CMV and Ad AS MMP-9 vectors, maintained for 72 h and changed to serum-free medium. Conditioned medium was collected after 24 h and MMP activity was determined using gelatin zymography (top panel) and graphical representation of MMP-2 and MMP-9 activities measured by densitometric scanning (bottom panel). The conditioned medium from HT1080 cells was used as a reference for MMP-2 and MMP-9. The results are representative of three experiments done in duplicate and values are mean + SD. *p