# Protection against Shiga Toxins
## 2 Compounds that Protect Cells against Stx
### The Glucose Analogues 2-Deoxy-D-glucose and 2-Fluoro-2-deoxy-D-glucose
For FDG, we have also tested whether the protective effect is observed in non-cancer originated cells, and found that immortalized human brain microvascular cells (HBMEC) also became less sensitive to Shiga toxin after 4 h and 24 h pretreatment with 1 mM FDG [48]. However, all these toxicity assays have been performed with a relatively short 3 h challenge with the toxin. Thus, we have now also tested whether FDG protects HEp-2 cells against 24 h challenge with Stx2, and observed an essentially similar protection as shown for 3 h incubation with the toxin (Figure 5). Interestingly, although both 2 DG and FDG were found to reduce cellular Gb3 levels by 50% following 24 h treatment (Figure 6), it was only FDG that also led to reduction in Shiga toxin binding [48; 99]. The mechanisms by which 2 DG and FDG inhibit Gb3 synthesis are still not clear and seem to be different, as 2 DG has been shown to inhibit the transcription of the Gb3 synthase gene [99; 135], while FDG has no effect on the expression of Gb3 synthase [48]. In addition, we have found that cell treatment with FDG also reduces cellular levels of LacCer and glucosylceramide (GlcCer) (Figure 6) [48], indicating that FDG inhibits the synthesis of GlcCer and thus depletes cells for the precursors required for Gb3 synthesis, rather than inhibiting the synthesis of Gb3 directly. Figure 5: Cell protection against Stx2 by FDG. HEp-2 cells were treated with or without 1 mM 2-fluoro-2-deoxy-d-glucose (FDG) in complete growth medium for 4 h or 24 h prior to incubation with 10-fold serial dilutions of Stx2 for 24 h in the presence or absence of FDG. The cells were then incubated in the presence of [3H]leucine for 20 min, and protein synthesis was measured as described in [98]. The left panel shows relative protein synthesis as a percentage of the samples without Stx2 added. The right panel shows relative fold protection against Stx2. The protection was calculated as an increase in IC50 for treated samples compared to control. In the samples treated with FDG for 24 h, the highest toxin concentration tested (1 ng/mL) did not reduce protein synthesis down to 50%, therefore the fold-protection could not be calculated and was estimated to be more than 100-fold (marked as #). The error bars show SEM for 4 h treatment (\(n=4\)) and the deviation from the mean of two independent experiments for 24 h treatment. One sample \(t\)-test was used for statistical analysis of the protection data for 4 h treatment, and the obtained \(p\) value is given in the figure. Although the inhibition of Gb3 synthesis might be an important factor for the protection against Stx by 2 DG and FDG after long-term treatment, the protection observed after 4 h treatment with drugs does not seem to be mediated by changes in Gb3 [48; 99]. We have shown that following 4 h treatment with either 10 mM 2 DG or 1 mM FDG the intracellular transport of Shiga toxin is changed and most likely accounts for the protection observed at this time point. Four hours pretreatment with 10 mM 2 DG almost completely blocked the release of Shiga toxin A\({}_{1}\)-moeity in the ER in HEp-2 cells, which correlated well with the depletion of calcium from the ER [99]. Although it has been proposed that 2 DG induces release of calcium from the ER via induction of ER stress [142], combined treatment with mannose, which rescues 2 DG-mediated ER stress [143], does not prevent calcium leakage from the ER upon 2 DG treatment and does not rescue cell sensitivity to Shiga toxin [99]. Three ER chaperones, HEDJ (also called ERdj3), BiP (also called GRP78) and GRP94 (glucose-regulated protein of 94 kDa), have been shown to bind the A-moiety of Stx1 [120], and thus are suggested to be involved in the release of StxA\({}_{1}\) in the ER. Substrate binding to GRP94 and BiP has been shown to be regulated by calcium [144; 145], suggesting that 2 DG might prevent the release of StxA\({}_{1}\) in the ER by inhibiting Shiga toxin interaction with chaperones.