The effect of antitumor glycosides on glioma cells and tissues as studied by proton HR-MAS NMR spectroscopy

Abstract

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning ((1)H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the (1)H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death.

Figure 1. Chemical structures of glycoside 1 and thioglycoside 2.

Figure 2. Representative ¹H HR-MAS NMR spectra of C6 cells.

(A) Control C6 cells, with an insert corresponding to the total choline (^tCho) region shown on the top (B) ^tCho region of the spectrum of C6 cells treated with thioglycoside 2 at 20 and 40 µM (C) ^tCho region of the spectrum of C6 cells treated with glycoside 1 at 20 and 40 µM.

Figure 3. Schematic representation of the possible mechanism for deregulated choline cancer metabolism with drug treatment.

Black arrows in Cho and PC indicate a rise in the levels of these metabolites detected in this study.

Figure 4. Representative ¹H HR-MAS NMR spectra of C6 tumor core tissue.

(A) Tumor of control mice (B) Tumor of 2-treated mice (1 mg/Kg) (C) Tumor of 2-treated mice (10 mg/Kg). The complete aliphatic region is shown on the left panels, and expansion of the taurine and creatine regions on the right, asterisks: ethanol contamination.

Figure 5. Histograms summarizing the percentage differences detected in several metabolites between C6 tumor core tissue treated with glycoside 1 (10 mg/Kg/day) and 2 (1 or 10 mg/Kg/day) and the non-treated control.

Each value in the histogram represents the mean ± SD of six separate experiments. Positive values indicate the increased presence of a metabolite in tumors with respect to controls. Asterisks indicate statistically significant differences in a particular metabolite evaluated at p < 0.05. *, p ≤ 0.05; **, p ≤ 0.01.