Human glioblastoma T-98G cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Antibiotics, penicillin and fetal bovine serum (FBS) were obtained from Gibco-BRL (Long Island, NY, USA). Twenty-four well tissue culture plates were obtained from Costar (Cambridge, MA, USA). Gelatinase zymography was performed on 10% Novex pre-cast SDS polyacrylamide gel (Invitrogen Inc., Carlsbad, CA, USA) with 0.1% gelatin in non-reducing conditions. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), doxycycline, epigallocatechin gallate (EGCG), actinomycin D, cyclohexamide, retinoic acid and dexamethasone were purchased from Sigma (St. Louis, MO, USA). The nutrient mixture (NM), prepared by VitaTech (Hayward, CA, USA), was composed of the following ingredients in the relative amounts indicated: vitamin C (as ascorbic acid and as Mg, Ca and palmitate ascorbate) 700 mg; L-lysine 1,000 mg; L-proline 750 mg; L-arginine 500 mg; N-acetyl cysteine 200 mg; standardized green tea extract (80% polyphenol) 1,000 mg; selenium 30 µg; copper 2 mg; manganese 1 mg. All other reagents used were of high quality and were obtained from Sigma, unless otherwise indicated.

Glioblastoma cells were grown in DME, supplemented with 15% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in 24-well tissue culture plates. The cells were plated at a density of 1×10⁵ cells/ml and grown to confluency in a humidified atmosphere at 5% CO₂ at 37̊C. Serum-supplemented media were removed and the cell monolayer was washed once with phosphate-buffered saline (PBS) and with the recommended serum-free media. The cells were then incubated in 0.5 ml of serum-free medium with various cytokines, mitogens, inducers and inhibitors in triplicates, as indicated: PMA (10, 25, 50 and 100 ng/ml); TNF-α and IL-1β (0.1, 1, 10 and 25 ng/ml); LPS (10, 25, 50 and 100 µg/ml); EGCG (10, 25, 50 and 100 µM) without and with PMA 100 ng/ml; doxycycline (10, 25, 50 and 100 µM) without and with PMA 100 ng/ml; NM (10, 50, 100, 500 and 1,000 µg/ml) without and with PMA 100 ng/ml, retinoic acid (50 µM); dexamethasone (50 µM); actinomycin D and cyclohexamide (2 and 4 µg/ml). The plates were then returned to the incubator. The conditioned medium from each treatment was separately collected, pooled and centrifuged at 4̊C for 10 min at 3,000 rpm to remove cells and cell debris. The clear supernatant was collected and used for gelatinase zymography, as described below.

Gelatinase zymography was utilized due to its high sensitivity to gelatinolytic enzymatic activity and ability to detect both pro and active forms of MMP-2 and MMP-9. Upon renaturation of the enzyme, the gelatinases digest the gelatin in the gel and reveal clear bands against an intensely stained background. Gelatinase zymography was performed using 10% Novex pre-cast SDS polyacrylamide gel in the presence of 0.1% gelatin under non-reducing conditions. Culture media (20 µl) were mixed with sample buffer and loaded for SDS-PAGE with Tris-glycine SDS buffer, as suggested by the manufacturer (Novex). Samples were not boiled before electrophoresis. Following electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS. The gels were then incubated at 37̊C overnight in substrate buffer containing 50 mM Tris-HCl and 10 mM CaCl₂ at pH 8.0 and stained with 0.5% Coomassie Blue R250 in 50% methanol and 10% glacial acetic acid for 30 min and destained. Protein standards were concurrently run and approximate molecular weights were determined by plotting the relative mobilities of known proteins. Gelatinase zymograms were scanned using CanoScan 9950F Canon scanner at 300 dpi. The intensity of the bands was evaluated using the pixel-based densitometer program Un-Scan-It, version 5.1, 32-bit, by Silk Scientific Corporation (Orem, UT, USA), at a resolution of 1 Scanner Unit (1/100 of an inch for an image that was scanned at 100 dpi).

Microsoft Excel 2010 linear trend analysis was utilized to determine the linear trend analyses of the densitometry results.

Glioblastoma T-98G cells expressed a band corresponding to MMP-2. Table I shows the quantitative densitometry results from the effects of PMA, TNF-α, IL-1β and LPS on MMP-2 and MMP-9 expression in the T-98G cells. Upon gelatinase zymography, T-98G cells demonstrated strong expression of MMP-2 and induction of MMP-9 with PMA treatment. PMA treatment showed increased MMP-2 secretion from 0–25 ng/ml and decreased secretion at 50 and 100 ng/ml with no significant overall effect on expression of MMP-2 (linear trend R²=0.153). PMA showed increased MMP-9 secretion from 10 to 25 ng/ml followed by decreased secretion at 50 and 100, again with no significant overall effect on secretion of MMP-9 (linear trend R²=0.281). See Fig. 1 for PMA results. T-98G cells demonstrated strong expression of MMP-2 and slight induction of MMP-9 with TNF-α treatment. TNF-α treatment showed increased MMP-2 secretion from 0–1 ng/ml and decreased secretion at 10 and 25 ng/ml with no significant overall effect on expression of MMP-2 (linear trend R²=0.0.066). TNF-α showed dose-dependent increased MMP-9 secretion (linear trend R²=0.721). See Fig. 2 for TNF-α results. IL-1β demonstrated a slight dose-dependent increase in T-98G secretion of MMP-2 (linear trend R²=0.529), but no induction of MMP-9. See Fig. 3 for IL-1β results. LPS treatment showed dose-dependent decreased inactive MMP-2 secretion (linear trend R²=0.473) and increased active MMP-2 secretion (linear trend R²=0.977) (Fig. 4).

Table II shows the quantitative densitometry results from the effects of chemical inhibitors doxycycline, actinomycin D, cyclohexamide and dexamethasone on MMP-2 and MMP-9 expression in the glioblastoma T-98G cell line. Doxycycline inhibited T-98G cell MMP-2 secretion in a dose-dependent manner with 73% blockage at 100 µM (linear trend R²=0.899). Following treatment with PMA 100 ng/ml, doxycycline downregulated the expression of T-98G cell MMP-2 in a dose-dependent manner, with 73% blockage at 100 µM (R²=0.898) and total blockage of MMP-9 at 10 µM (linear trend R²=0.500). See Fig. 5 for doxycycline effects on untreated and PMA-treated T-98G cells. Actinomycin D had moderate inhibitory effect on MMP-2 secretion (R²=0.725) with ~25% inhibition at 2 and 4 µM, as shown in Fig. 6. Cyclohexamide had a potent dose-dependent inhibitory effect on MMP-2 secretion by T-98G cells with 93% inhibition at 4 µM (linear trend R²=0.787), as shown in Fig. 7. Dexamethasone had a moderate inhibitory effect on MMP-2, with inhibition of 23% at 50 µM compared to the control (Fig. 8).

Table II shows the quantitative densitometry results from the effects of natural inhibitors EGCG, the NM and retinoic acid on MMP-2 and MMP-9 expression in glioblastoma T-98G cells. EGCG potently downregulated T-98G expression of MMP-2 in a dose-dependent manner, with total blockage at 50 µM (linear trend R²=0.712), as shown in Fig. 9A and C. EGCG showed dose-dependent inhibition of PMA (100 ng/ml)-induced MMP-9 secretion (linear trend R²=0.866) and of MMP-2 (linear trend R²=0.877) with total blockage of both at 50 µM, as shown in Fig. 9B and D. NM inhibited secretion of MMP-2 by uninduced T-98G cells in a dose-dependent manner (linear trend R²=0.8706) with virtual total block at 1,000 µg/ml (Fig. 10A and C). NM showed dose-dependent inhibition of MMP-2 and MMP-9 expression in PMA-treated T-98G cells with total blockage of MMP-2 at 1,000 µg/ml and MMP-9 at 100 µg/ml (linear trends R²=0.863 and 0.742, respectively), as shown in Fig. 10B and D. Retinoic acid inhibited T-98G MMP-2 secretion by 54% at 50 µM (Fig. 8).