Experiments were performed on three human glioblastoma cell lines (LN18, LN229, T98G). Cells were kindly provided by Dr K. Reiss from Temple University, (Philadelphia, PA, USA). The cells were cultured in DMEM (LN18, LN229) and in MEM (T98G) (PAA), supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Polfa Tarchomin, Poland) in the presence of 10% FBS (PAA) at 37˚C, 5% of CO₂ and 95% humidity.

Geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin), a GA analogue, both insoluble in H₂O and soluble in DMSO. 17DMAG [17-(dimethylaminoethylamino)-17-demethoxygeldanamycin], a water-soluble 17-AAG analogue; 17AEP-GA [17-(2-(pyrrolidin-1-yl)ethyl)aminno-17-demethoxygeldanamycin], a water-soluble GA analogue containing an alkylamino group instead of the methoxy moiety at C17; 17DMAP-GA [17-(dimethylaminopropylamino)-17-demethoxygeldanamycin], a water-soluble GA analogue synthesized based on binding affinity to HSP90. GA, 17AAG, 17DMAG, 17AEP-GA and 17DMAP-GA were purchased from InvivoGen, Inc. (USA).

The influence of GAs (InvivoGen, Inc.) on viability/proliferation was checked by CellTiter 96^® AQueous One Solution assay (Promega). Cells were seeded in growth medium (containing 10% FBS) on 96-well plates at 1×10³/well (LN18 and LN229) and at 2×10³/well (T98G) density in the presence of different concentration of GAs (1, 10, 100, 1,000 nM). After 24, 48, 72 and 96 h, 20 μl of CellTiter 96^® Aqueous One Solution reagent was added to each well and plates were incubated for 3 h. Subsequently, plates were read at 490 nm using the ELx800 Universal Microplate Reader (Bio-Tek) and analyzed with KC4 v3.0 with PowerReports software.

The influence of GAs on glioblastoma cells was studied by staining with Annexin V according to the manufacturer’s instructions. Briefly, after drug treatments for 24, 48 and 72 h, cells were stained with propidium iodide (PI) and Annexin V-FITC in 100 μl of staining solution at room temperature for 15 min in the dark. Samples were diluted with binding buffer and were analyzed within 1 h. In some experiments cells were tested for the activity of caspase-3 (BD Pharmingen). For this staining, cells were permeabilized for 20 min, washed and subsequently stained with anti-caspase-3-PE mAb for 30 min on ice (both Abs from BD Pharmingen). Cytofluorometric analysis was performed by FACS Canto (Becton-Dickinson) and analyzed with FASC Diva software (Becton-Dickinson).

Total RNA was extracted using RNeasy mini kit (Qiagen), followed by DNAse treatment (Promega). The reverse polymerase transcription was performed using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol.

The detection of MET mRNA level was performed by quantitative real-time PCR analysis on ABI PRISM^® 7300 Sequence Detection System (Applied Biosystems, Inc.) using a commercially available TaqMan PCR Master mix and primers for human MET gene (Hs01565589_m1; Applied Biosystems, Inc.). The mRNA expression level for all samples was normalized to the housekeeping gene GAPDH (Hs99999905_m1; Applied Biosystems, Inc.).

Western blot analyses were carried out on extracts prepared from GBM cells. The cells were lysed on ice with MPER lysing buffer (Pierce) containing protease and phosphatase inhibitors (Sigma). Extracted proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE gel) and transferred to a PVDF membrane (Bio-Rad). The activation of AKT and MAPK was detected using mouse and rabbit phospho-specific antibodies (AKT, MAPK; Cell Signaling Technology, Inc.) followed by HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG as secondary antibodies (Santa Cruz Biotechnology, Inc.). The membranes were developed with an ECL reagent and exposed to HyperFilm (Amersham Life Sciences). An equal loading in the lanes was evaluated by probing with an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc.).

The direct migration of GBM cells towards HGF gradient was studied using modified Boyden’s chamber with 8-μm pore polycarbonate membrane inserts. The cells were seeded into the upper chamber of a Transwell inserts (Costar Transwell). The lower chamber was filled with medium containing 0.5% BSA and HGF (20 ng/ml). After 24 h, inserts were removed from transwells and cells were fixed with methanol. Cells that transmigrated to the lower side of the membrane were stained with Wright solution (Merck) and counted under high power field (HPF) with an inverted microscope.

The ability of GBM cells to invade was examined by BD BioCoat™ Matrigel invasion inserts (Becton-Dickinson) where cells migrated through Matrigel layer towards HGF gradient. After 24 h, cells that invaded the Matrigel were counted on the undersides of filters after fixation and staining with Wright solution. As a control of invasion the same number of control inserts (no GFR Matrigel coating) were used.

The presence of MET receptor on the surface of target cells were demonstrated by staining with rabbit anti-MET-FITC antibody (R&D Systems) and analyzed by FACS. Briefly, 1×10⁵ cells were suspended in 100 μl of staining buffer (PBS, 2% FBS) and antibody was added. Next, the cells were incubated in the dark for 30 min at 4˚C. The stained cells were washed, collected using FACSCanto cytometer (Becton-Dickinson, USA) and analyzed with FACS Diva software (Becton-Dickinson, USA).

All results are mean ± SD. Statistical analysis was performed using the two-tailed Student’s t-test. P-values <0.05 were considered as significant, unless otherwise indicated.

Several in vitro studies indicate that GAs inhibit the proliferation of a wide range of cancer cells (18,19). The effects of GAs on the growth rate of GBM cell lines (LN18, LN229, T98G) were determined using the CellTiter 96^® Aqueous One Solution assay at 96 h. GAs were used in 1 and 10 nM concentrations. As shown in Fig. 1, GAs inhibited growth of all investigated cells already at the concentration of 1 nM. However, the level of inhibition differs between cell lines and geldanamycin analogs. We observed the strongest inhibition for LN18 and LN229 cell lines where all investigated analogs were able to inhibit cell growth (Fig. 1A and B). Moreover, we noted similar level of inhibition after 96-h incubation for higher concentration of GAs, 100 and 1,000 nM for these two cell lines (data not shown). However, T98G seemed to be less sensitive to investigated analogs at the concentration of 1 and 10 nM (Fig. 1C). Growth of T98G cell line was inhibited by 50% in comparison to untreated control after incubation with 100 and 1,000 nM of GAs (data not shown). The best results for all investigated cell lines were obtained using 17-AEP-GA.

We became interested whether the inhibition of cell growth is due to the induction of apoptosis. Therefore, in order to answer this question we used Annexin V and activated caspase-3 staining to detect apoptotic cells after GAs treatment. After incubation with 10 and 100 nM of 17-AAG and 17-AEP-GA cells became Annexin V positive (Fig. 2A). These data were confirmed with caspase-3 staining. We observed a high number of cells positive for activated caspase-3 after 17-AAG and 17-AEP-GA treatment at the concentration of 100 nM (Fig. 2B).

The expression of MET receptor mRNA was estimated by real-time PCR and we have observed the expression of MET gene for all tested cell lines (Fig. 3A). LN229 cell line, which is the most malignant one, had the highest expression of mRNA for MET receptor. LN18 and T98G cell lines had a similar level of expression. There were no significant differences between tested cell lines. To determine whether MET receptor is functional on investigated cell lines we stimulated them by HGF and checked phosphorylation of MAPK 42/44 and AKT Ser 473 by western blot analysis (Fig. 3B). For all tested cell lines we observed strong phosphorylation of AKT and MAPK after 5 min of stimulation by HGF. However, LN18 cell line did not show any differences in AKT phosphorylation between control cells and cells after stimulation. It might suggest constitutive activation of AKT.

It has been shown that MET receptor is a client protein for HSP90 (14,19). Thus, we checked if incubation of GBM cells with GAs had any effect on the surface expression of MET receptor. Cells were grown in the presence of GAs for 24 h and the level of MET receptor expression was evaluated by cytofluorometric analysis. The downregulation of MET receptor expression was observed (Fig. 3C). The strongest reduction was in LN229 cell line. These results confirm that the expression of MET receptor is dependent on the presence of HSP90 protein.

Since GBM expresses high level of MET receptor and its expression can be modulated by GAs, we tested the influence of GAs on chemotactic response of GBM cells towards HGF. The GBM cells were subjected to 10 and 100 nM concentrations of GAs and their ability to migrate was evaluated. We observed decreased migration of treated GBM cells towards HGF gradient (Fig. 4A). This effect was further enhanced when cells were preincubated for 6 h with GAs (Fig. 4B).

We also evaluated invasive properties of GBM cells after GAs treatment. LN229 cell line was treated with inhibitors in the concentration of 100 nM. We observed a strong decrease in the ability of GBM to transmigrate through extracellular matrix proteins after HGF stimulation (Fig. 4C).