Most anticancer agents, which are currently used clinically, target the aerobic rapidly dividing cells of a tumor. Therefore, slow-growing anaerobic (hypoxic) cells found in necrotic centers and at the inner core of most solid tumors, will be resistant to these chemotherapeutic drugs. Thus, slow-growth may be considered another form of MDR. The fact that the most common toxicities of currently used chemotherapeutic agents in normal cells are found in the fastest dividing tissues, i.e., bone marrow, gut, and hair, provides further evidence that the selectivity of anticancer drugs in general lies not as much between tumor and normal cells as it does between rapidly dividing and slow, or nondividing cells.
The hypothesis on how to overcome these slow-growing drug-resistant tumor cells found in the hypoxic areas of tumors derives from work in which Rho123 was shown to inhibit mitochondrial oxidative phosphorylation (OxPhos) [61
]. Consequently, tumor cells treated with this drug have to rely solely on glycolysis for ATP production and thus become hypersensitized to inhibitors of glycolysis, such as 2-deoxy-d
-glucose (2-DG). In fact it was shown that cotreating human breast carcinoma cells, MCF-7, with Rho 123 and 2-DG, 100% of the colony forming units was inhibited whereas similar treatment in normal epithelial cells showed little or no toxicity [17
]. This concept was carried over to in vivo
studies in which it was found that tumor bearing animals treated in combination with 2-DG and Rho123 were cured whereas when treated with either drug alone, only partial or no responses were obtained [18
]. This latter result provides evidence that manipulation of OxPhos and glycolysis simultaneously can cure tumors in animals. Furthermore, these in vivo
data also demonstrate that 2-DG can be administered safely to animals, at doses which are effective for antitumor activity in combination with an OxPhos inhibitor. In this regard, several reports have shown that low levels of 2-DG can be safely administered to animals for various reasons including hypersensitization of tumors to irradiation [63
Since hypoxia, similar to Rho123 treatment, forces cells to rely mainly on anaerobic metabolism of glucose for survival, hypoxic tumor cells could be selectively targeted with inhibitors of glycolysis such as 2-DG [64
]. The switch from aerobic to anaerobic metabolism in cells that lie in hypoxic regions of tumors creates two windows of selectivity that we believe will ultimately prove beneficial to cancer patients when 2-DG is used in combination with cytotoxic agents. The first is that tumor cells under hypoxia upregulate both glucose transporters and glycolytic enzymes [64
], which favors increased uptake of 2-DG in these cells as compared to normal aerobic cells. The second is based on the principle that even if enough 2-DG is accumulated in normal cells to block glycolysis, they can survive by using oxygen to burn fats and proteins through their mitochondria to produce ATP. In contrast, when glycolysis is blocked in hypoxic tumor cells they die, since their mitochondria at these oxygen levels (8–57 μM) [64
] are less efficient in converting these alternative energy sources to ATP. These two windows of selectivity are based on fundamental principles of biochemistry and as such provide the basis for using glycolytic inhibitors to raise the efficacy of current chemotherapy by targeting the slow-growing hypoxic cell population found in most, if not all, solid tumors.
In order to investigate the mechanisms involved with hypersensitivity to glycolytic inhibitors, we developed three distinct models of simulated hypoxia which are referred to as chemical model A, genetic model B, and environmental model C [66
]. Model A approximates hypoxia by using chemicals such as Rho123, rotenone, antimycin A, and oligomycin to interfere with mitochondrial function thereby rendering the cell unable to produce ATP via
]. Model B uses tumor cells that have been permanently genetically altered by depletion of their mitochondrial DNA and cannot undergo OxPhos [66
]. Since models A and B are growing under oxygen, but are unable to produce ATP via
their mitochondria, they simulate hypoxic cells by relying exclusively on glycolysis for this function, whereas, model C are actually tumor cells grown under decreased atmospheric oxygen [68
]. In all three models, glycolysis is increased (as measured by lactic acid) and most importantly, they are all found to be hypersensitive to glycolytic inhibitors as compared to their aerobic counterparts [66
]. However, model C is found to be less sensitive to glycolytic inhibitors than models A and B, which suggests that certain variables influencing the cellular response to glycolytic inhibitors differ between these models. One such variable is hypoxia-inducible factor-1 (HIF-1), which is expressed only in model C. HIF-1 is known to be a key regulator of a wide range of cellular responses to lowered oxygen tension [69
]. Among the numerous genes activated by HIF-1, are glucose transporters and glycolytic enzymes [70
]. Since HIF-1 is activated in model C [69
], but not in other models, it seems likely that HIF-1 induction is contributing to the increased cellular resistance to glycolytic inhibition by 2-DG found in this model. In this regard, a recent publication demonstrated that HIF-1 induced hexokinase 2 expression confers resistance to glycolytic inhibition by 2-DG, suggesting that combining inhibitors of HIF with 2-DG may be a more effective strategy than either agent alone, particularly for targeting the slow-growing hypoxic cell populations found in most solid tumors.