The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression

Gene expression levels of the microsomal glucose-6-phosphate transporter (G6PT), as well as of the two glucose-6-phosphatase α and β isoforms, were first assessed. Total RNA was extracted from HEP-G2 hepatoma and U-87 glioma cells, and then RT-PCR was performed as described in the Methods section. As expected for a cell line derived from a neoglucogenic tissue, we show that HEP-G2 cells express all three components of the G6Pase system with G6Pase-β being predominant amongst these three (Fig. 1a). RT-PCR was also performed on RNA extracted from U-87 glioma cells. In contrast to HEP-G2, only G6PT and G6Pase-β transcripts were significantly expressed, with very low to undetectable G6Pase-α present (Fig. 1a). This is in agreement with previous reports demonstrating the lack of G6Pase-α expression in brain-derived cells [32]. We also monitored the levels of G6PT in a brain tumor cell line derived from a pediatric medulloblastoma (DAOY), as well as in the U-118 and U-138 glioma cell lines. Interestingly, among the adult brain tumor-derived cell lines, U-87 glioma cells expressed the highest level of G6PT (Fig. 1b), while DAOY cells expressed very low to undetectable G6PT transcripts. This suggests that G6PT expression in brain tumor cells may be regulated during development.

We next assessed the potential functions that G6PT may affect in the invasive phenotype of U-87 glioma cells. Based on the assumption that CHL is able to efficiently penetrate inside the cells [33], U-87 glioma cells were treated with different concentrations of CHL and the secretion of matrix metalloproteinase (MMP)-2 was monitored by zymography. CHL significantly inhibited latent proMMP-2 secretion (Fig. 2a) and another tea-derived compound, epigallocatechin gallate (EGCg), was just as efficient (Fig. 2a and 2b). CHL was found not to induce any early/late apoptotic or necrotic processes in U-87 glioma cells as assessed by Annexin-V/Propidium iodine double staining and flow cytometry (not shown). Since EGCg has also been reported to inhibit membrane type (MT)1-MMP from activating the latent proMMP-2 in glioblastoma cells [34], we further assessed the possible effects of CHL on MT1-MMP-mediated proMMP-2 activation. Cells were incubated with an exogenous source of proMMP-2 and were treated (or not) with concanavalin-A in order to trigger proMMP-2 activation [35]. While EGCg clearly inhibited MT1-MMP-mediated activation of proMMP-2, CHL had no such inhibitory effect (Fig. 2c).

The role of G6PT in U-87 glioma cell migration was next assessed. Cells were transfected, or not (Mock), with G6PT cDNA [36] and basal cell migration assay was performed on gelatin-coated filters using modified Boyden chambers as described in the Methods section. Transfection of G6PT cDNA specifically increased G6PT transcript levels, while levels of G6Pase-β remained unaffected (Fig. 3a). Interestingly, the sole effect of G6PT overexpression was to increase the basal migration of U-87 glioma cells (Fig. 3b, white bars), while CHL inhibited both basal and G6PT-induced cell migration (Fig. 3b, black bars). This suggests that G6PT functions may, in part, regulate the invasive phenotype of U-87 glioma cells.

Sphingosine-1-phosphate (S1P) is a bioactive lipid which is present at high levels in brain tissue [36] and which acts as a potent mitogen for glioblastoma multiform cells [38]. More recently, S1P was shown also to potently enhance the in vitro motility of glioblastoma cells [39]. We thus assessed whether CHL could interfere with both basal and S1P-induced cell migration in U-87 cells, and if it could antagonize the G6PT-mediated regulation of cell migration. Control (Mock) or G6PT-transfected U-87 glioma cells were harvested and seeded on top of gelatin-coated filters as described in the Methods section. Cell migration then occurred in the presence of either S1P, CHL, or a combination of both. Representative images of stained cells that had migrated through the filters are shown (Fig. 4a). We observed that S1P induced basal cell migration by 2.6-fold, and that CHL was able to inhibit both the basal and S1P-induced cell migration (Fig. 4b, Mock). When cells were transiently transfected with the G6PT cDNA, basal cell migration increased by approximately 2-fold, in accordance with Fig. 3b. Interestingly, the effect of S1P was additive to that of the overexpression of G6PT, but CHL efficiently antagonized migration that was induced either by G6PT overexpression or by S1P in G6PT-transfected cells (Fig. 4b, G6PT). These results suggest that G6PT potentially regulates the intracellular signalling that triggers basal and S1P-mediated cell migration, and that functional inhibition of this signalling by CHL could explain some of its chemopreventive effects at the molecular level.

S1P is thought to trigger intracellular signalling, in part, through the MAPK pathway and the release of intracellular calcium pools [38, 40]. We thus monitored the extent of ERK phosphorylation that is triggered by S1P in U-87 glioma cells and whether CHL could interfere with that process. S1P triggered a rapid phosphorylation of ERK within the first 15 seconds of incubation (Fig. 5, left panel). Interestingly, pre-incubation of the cells with 100 μM CHL for 30 minutes prevented that rapid phosphorylation of ERK and even delayed it until a time of 60 seconds (Fig. 5, right panel). This inhibitory effect of CHL on S1P-induced ERK phosphorylation suggests that intracellular inhibition of microsomal G6PT activity may antagonize crucial events taking place in the ER.

Besides its classical role in recognizing and translocating G6P from the cytoplasm into the lumen of the ER, G6PT has been attributed a possible role in regulating calcium flux responses [41, 42]. Such a role in regulating intracellular calcium pools was elegantly demonstrated in neutrophils isolated from G6PT-deficient mice [43]. Interestingly, a functional link has been proposed between G6P and calcium such that cytoplasmic G6P is thought to enhance ATP-dependent sequestration of calcium in the ER [30]. In light of this, we sought to investigate the effects of the ATP-depleting agents and synthetic analogs of glucose, the glucose 5-thioglucose (5-TG) and the 2-deoxy-d-glucose (2-DG), on proMMP-2 secretion and S1P-induced ERK phosphorylation. U-87 glioma cells were serum-starved and treated with different concentrations of 2-DG and 5-TG. Conditioned media was isolated and the activity of secreted proMMP-2 was measured. ProMMP-2 secretion was significantly decreased in both 2-DG- and 5-TG-treated cells (Fig. 6a). Half-maximal inhibition constants (IC₅₀) were calculated to be 2 mM and 19 mM, respectively, for 2-DG and 5-TG (Fig. 6b). S1P-induced ERK phosphorylation was also monitored in 2-DG- and 5-TG pre-treated cells, and both ATP-depleting agents were able to inhibit S1P-induced ERK phosphorylation (Fig. 6c). Although these data are indirect, altogether they suggest that the inhibition of microsomal G6PT functions (inhibition of ER G6P uptake or of ATP-dependent calcium sequestration) leads to decreased proMMP-2 secretion and a potential decrease in calcium-mediated intracellular signalization.

Agarose, (-)-epigallocatechin 3-gallate (EGCg), sodium dodecylsulfate (SDS), gelatin, and bovine serum albumin (BSA) were purchased from Sigma (Oakville, ON). The TRIzol reagent was from Life Technologies (Gaithersburg, MD). FUGENE-6 transfection reagent was from Roche Diagnostics Canada (Laval, QC).

The U-87, U-118 and U-138 glioma cell lines, the DAOY medulloblastoma cell line and the HEP-G2 hepatoma cell line were purchased from American Type Culture Collection and cultured in their recommended media. Specifically, U-87 cells were maintained in Eagle's Minimum essential medium (MEM) containing 10% (v/v) fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin, and were cultured at 37°C under a humidified atmosphere containing 5% CO₂. The G6PT plasmid was validated and generously provided by Dr Christopher Newgard [35]. U-87 cells were transiently transfected with the cDNA construct using the non-liposomal formulation FUGENE-6 transfection reagent. Transfection efficiency was confirmed by RT-PCR. All experiments involving these cells were performed 36 hrs following transfection. Mock transfections of U-87 cultures with pcDNA (3.1+) expression vector alone were used as controls.

Total RNA was extracted from cultured monolayers of U-87 cells using the TRIzol reagent. One microgram of total RNA was used for first strand cDNA synthesis followed by specific gene product amplification with the One-Step RT-PCR Kit (Invitrogen, Burlington, ON). Primers for G6Pase-α (forward : 5'-TTCAGCCACATCCACAGCATC-3', reverse : 5'-GGGGTTTCAAGGAGTCAAAGACG-3'), for G6Pase-β (forward : 5'-ACTCTTCCTGACTTCTTGTGTGCC-3', reverse : 5'-TTGCCTTTGCTCTTTGGGGG-3') and for G6PT (forward : 5'-CAGGGCTATGGCTATTATCGCAC-3', reverse : 5'-ATGGCTCAAACCACTTCCGCAG-3') were all derived from human sequences. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA amplification was used as an internal house-keeping gene control. PCR conditions were optimized so that the gene products were examined at the exponential phase of their amplification and the products were resolved on 1.5% agarose gels containing 1 μg/ml ethidium bromide.

Gelatin zymography was used to assess the extent of MMP-2 activity. Briefly, an aliquot (20 μl) of the culture medium was subjected to SDS-PAGE in a gel containing 0.1 mg/ml gelatin. The gels were then incubated in 2.5% Triton X-100 and rinsed in nanopure distilled H₂O. Gels were further incubated at 37°C for 20 hrs in 20 mM NaCl, 5 mM CaCl₂, 0.02% Brij-35, 50 mM Tris-HCl buffer, pH 7.6, then stained with 0.1% Coomassie Brilliant blue R-250 and destained in 10% acetic acid, 30% methanol in H₂O. Gelatinolytic activity was detected as unstained bands on a blue background. The exogenous source of proMMP-2, used to assess MT1-MMP-mediated activation of proMMP-2 by concanavalin-A, was isolated from 48 hrs serum starved-U87 glioma cells. This conditioned media contains high levels of latent proMMP-2 as well as TIMP-2 necessary to for the ternary complex with MT1-MMP and to monitor any potential MT1-MMP-mediated activation of proMMP-2 activation.

Proteins from control and treated cells were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes which were then blocked overnight at 4°C with 5% non-fat dry milk in Tris-buffered saline (150 mM Tris, 20 mM Tris-HCl, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with the primary antibodies (1/1,000 dilution) in TBST containing 3% bovine serum albumin, followed by a 1 hr incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1/10,000 dilution) in TBST containing 5% non-fat dry milk. Immunoreactive material was visualized by enhanced chemiluminescence (Amersham Biosciences, Baie d'Urfée, QC).

Cells were dislodged after brief trypsinization, washed extensively and resuspended in DMEM at a concentration of 10⁶ cells/ml [35]. Cells (7 × 10⁵) were then dispersed onto 1 mg/ml gelatin/PBS-coated chemotaxis filters (Costar; 8-μm pore size) within Boyden chamber inserts. Migration proceeded for 3 h at 37°C in 5% CO₂. Cells that had migrated to the lower surface of the filters were fixed with 10% formalin phosphate, colored with 0.1% crystal violet/20% MeOH and counted by microscopic examination. The average number of migrating cells per field was assessed by counting at least four random fields per filter using Northern Eclipse software. Data points indicate the mean obtained from three separate chambers within one representative experiment.

Data are representative of three or more independent experiments. Statistical significance was assessed using nonparametric one-way ANOVA with GraphPad Prism Version 4.0. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance in each figure.