Metabolic remodeling of malignant gliomas for enhanced sensitization during radiotherapy: an in vitro study

Abstract

Objective: To investigate a novel method to enhance radiosensitivity of gliomas via modification of metabolite flux immediately before radiotherapy. Malignant gliomas are highly glycolytic and produce copious amounts of lactic acid, which is effluxed to the tumor microenvironment via lactate transporters. We hypothesized that inhibition of lactic acid efflux would alter glioma metabolite profiles, including those that are radioprotective. H magnetic resonance spectroscopy (MRS) was used to quantify key metabolites, including those most effective for induction of low-dose radiation-induced cell death.

Methods: We inhibited lactate transport in U87-MG gliomas with alpha-cyano-4-hydroxycinnamic acid (ACCA). Flow cytometry was used to assess induction of cell death in treated cells. Cells were analyzed by MRS after ACCA treatment. Control and treated cells were subjected to low-dose irradiation, and the surviving fractions of cells were determined by clonogenic assays.

Results: MRS revealed changes to intracellular lactate on treatment with ACCA. Significant decreases in the metabolites taurine, glutamate, glutathione, alanine, and glycine were observed, along with inversion of the choline/phosphocholine profile. On exposure to low-dose radiation, ACCA-pretreated U-87MG cells underwent rapid morphological changes, which were followed by apoptotic cell death.

Conclusion: Inhibition of lactate efflux in malignant gliomas results in alterations of glycolytic metabolism, including decreased levels of the antioxidants taurine and glutathione and enhanced radiosensitivity of ACCA-treated cells. Thus, in situ application of lactate transport inhibitors such as ACCA as a novel adjunctive therapeutic strategy against glial tumors may greatly enhance the level of radiation-induced cell killing during a combined radio- and chemotherapeutic regimen.

FIGURE 1

Cytograms showing that ACCA imparted significant cell death in U87-MG gliomas at concentrations of 10 mmol/L and above. Flow cytometric analyses of U87-MG gliomas exposed to 0- to 25-mmol/L ACCA at 5-mmol/L increments. Percent cell number for each quadrant is indicated at the corners of each cytogram. Cell populations with high FITC and low PI (lower right quadrant) are defined as apoptotic. Those with low FITC and high PI (upper left quadrant) and high FITC and high PI (upper right quadrant) are defined as necrotic.

FIGURE 2

Cellular morphology of U87-MG gliomas exposed to ACCA (cellular morphology 72 h after exposure to ACCA). A, at 0- and 5-mmol/L ACCA, the cells did not show significant morphological changes. B, at 10-mmol/L ACCA, cells were still attached to culture vessels, but displayed a rounded morphology. C, at 20-mmol/L ACCA, all cells had detached from the culture vessel (original magnification, ×100).

FIGURE 3

Graph showing that ACCA is not internalized by U87-MG glioma cells. Spectrometric analyses of whole-cell lysates of U87-MG gliomas exposed to 10-mmol/L ACCA for 0 minutes to 18 hours in the absence of digitonin, and 20 minutes in the presence of digitonin. The spectra are overlaid with a solution spectrum for ACCA (0.05 mmol/L) for peak identification.

FIGURE 4

Graph showing that ACCA inhibits lactate efflux and glycolytic metabolism. MRS analysis of U87-MG glioma intracellular lactate profile (doublet of peaks centered at 1.35 parts per million) for control cells (green), 6 hours after exposure to 10-mmol/L ACCA (black), 18 hours after exposure to ACCA (blue), and 24 hours after exposure to ACCA (red).

FIGURE 5

Key metabolites of U-87 MG gliomas are altered after ACCA treatment. MRS analyses of taurine, glutathione, and glycine (A); glutamate (B); alanine (C); and choline and phosphocholine (D). Stacked plots are shown with the chemical shift on the x-axis, and successive spectra are outlined along an oblique y-axis. Notations for arrows are indicated above the spectra profiles. Samples were analyzed in quadruplicate for the 0-hour time point (control), and 6- and 24-hour samples were analyzed in triplicate. The x-axis indicates the chemical shift in parts per million.

FIGURE 6

Illustration demonstrating key metabolites and the metabolic pathways that are altered after exposure of U-87 MG gliomas to ACCA. The key metabolites examined by MRS are outlined (green background). The primary glycolytic metabolic pathway (A ▶ B) is inhibited on exposure of cells to ACCA (red background), which blocks lactate efflux. The metabolism can then shift to the pentose-phosphate pathway (D) and towards mitochondrial respiration (C). Concomitant biosynthetic pathways for amino acids can be up-regulated (E–G) at least during the initial period of exposure to ACCA and before cells undergo metabolic crisis because of long-term exposure to ACCA. Key reducing cofactors (NADPH and NADH) are indicated (yellow background).

FIGURE 7

Graph showing clonogenic survival of irradiated U-87 MG glioma alone and in combination with previous exposure to ACCA. Clonogenic survival was measured as described in the Methods section for cells exposed to low-dose radiation alone (●) or treated with ACCA before low-dose radiation (○). The data were normalized to calculate the survival fraction.