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

Glioblastoma multiforme (GBM) is the most common of all primary brain tumors. It is also the most aggressive, with patient survival being dismal with conventional therapies. Despite numerous clinical approaches, little progress in prolonging patient survival has been achieved in the past 20 years. Similar to most highly malignant tumors, these aggressive gliomas are highly glycolytic, producing large amounts of lactic acid as a metabolic by-product. This aberrant metabolic phenotype is known as aerobic glycolysis because it occurs even in the presence of oxygen (2, 27, 30, 39). In contrast, normal cells produce lactate mainly under anaerobic conditions (anaerobic glycolysis). Thus, such malignant tumors require an effective mechanism for the rapid disposal of accumulating lactic acid to the tumor microen-vironment. Tumors, as well as normal tissue, use a family of transmembrane transporters (monocarboxylate transporter isoforms 1 to 9) (14, 15) for this purpose, in which isoforms with different substrate affinities are expressed in a tissue-specific manner throughout the organism. Previous studies from our laboratory (26) indicated differential expression of these isoforms in normal brain versus malignant glioma, in which the Type 3 isoform was predominantly expressed in normal tissue, whereas Type 1 and 2 isoforms were highly expressed in GBMs. Our in vitro studies using small interfering ribonucleic acids indicated that selective blocking of lactate efflux (by targeting glioma-expressed mono-carboxylate transporter isoforms) induced critical metabolite changes in the tumors, resulting in cell death (26). Intracellular pH changes were observed (a fourfold increase in H+ concentrations, resulting in a pH change from 7.4 to 6.8) as well as indications of altered cofactor profiles (oxidized nicotinamide adenine dinucleotide [NAD+]/reduced nicotinamide adenine dinucleotide [NADH] and oxidized nicotinamide adenine din-ucleotide phosphate [NADP+]/reduced nicotinamide adenine dinucleotide phosphate [NADPH]). Thus, tumor-specific blocking of these transporters presented an attractive anticancer therapeutic target that may debilitate such malignant tumors, either via disruption of energy flux through the tumor cells or via metabolite remodeling within the tumors. α-cyano-4-hydroxycinnamic acid (ACCA; CHC; mw 189.2) is a known competitive inhibitor of mammalian lactate transporters (13, 36) that also inhibits pyruvate entry into mitochon-dria (13). We hypothesized that application of ACCA to malignant gliomas might disrupt energy-generating metabolic cascades (both aerobic glycolysis and oxidative phosphorylation), promoting both induction of apoptosis and disruption of pyruvate-derived antioxidant biosynthesis via mitochondria-derived NADH. Disruption of glycolysis would also affect the pentose phosphate pathway-derived NADPH/glutathione-generating mechanisms and NADH synthesis at the glycer-aldehydes-3-P-dehydrogenase step of the glycolytic pathway, thus depleting the overall capacity of the tumor to defend against free radical-induced cell damage. We reasoned that a combined experimental strategy of inhibiting lactate efflux with concomitant treatment with radiation would result in the targeted gliomas being highly radiosensitized towards free radical-induced damage and also energetically debilitated because of the disruption of key metabolic pathways. Thus, a combined protocol of chemotherapy and radiotherapy should provide a more effective GBM-targeting strategy. Radiotherapy has been one of the more effective modalities for therapy of malignant glioma, and dosages of 60 Gy are routinely used (10, 21). However, these can result in toxicity to the surrounding normal brain tissue, including leukoencephalopathy and necrosis (40). Therefore, strategies to enhance radiosensitivity of targeted glioma would permit lower radiation doses to be used, thereby vastly minimizing the frequently observed normal tissue damage while maintaining the efficacy of radiotherapy treatment. Low-dose radiotherapy using doses between 0.5 and 1.0 Gy has been suggested as a potential strategy to develop improved radiotherapeutic regimens while minimizing excessive radiation-induced injury (3, 17, 22, 25). In the present investigation, we first established that ACCA acts only as a cell-surface inhibitor of lactate transporters in the model U-87MG glioma cells and that it does not internalize. Thus, we reasoned that a combined strategy of first using ACCA as an inhibitor to disrupt glioma metabolism would reduce cellular radioprotectant generation and that subjecting the targeted cells to low-dose irradiation would then provide an avenue for an improved chemoradiotherapy regimen against malignant glioma while significantly minimizing damage to normal tissues. Using in vitro studies, we first used magic angle spinning (MAS) 1H magnetic resonance spectroscopy (MRS) to demonstrate that radioprotective metabolites are significantly reduced when lactate efflux is blocked in glioma cells and that the treated cells are highly susceptible to low-dose radiation-induced cellular damage, resulting in cell death.