Inhibition of Glycolysis in Cancer Cells: A Novel Strategy to Overcome Drug Resistance Associated with Mitochondrial Respiratory Defect and Hypoxia Rui-hua Xu, 1,3 Helene Pelicano, 1 Yan Zhou, 1 Jennifer S. Carew, 1 Li Feng, 1 Kapil N. Bhalla, 4 Michael J. Keating, 2 Peng Huang, 1 Departments of 1 Molecular Pathology and 2 Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas; 3 Department of Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, China; and 4 Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida Abstract Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associ- ated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser 112 , relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environ- ment in which the cancer cells exhibit high glycolytic activ- ity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently in- duced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications. (Cancer Res 2005; 65(2): 613-21) Introduction Over 70 years ago, Warburg (1) observed that cancer cells frequently exhibit increased glycolysis and depend largely on this metabolic pathway for generation of ATP to meet their energy needs. He attributed this metabolic alteration to mitochondrial ‘‘respiration injury’’ and considered this as the most fundamental metabolic alteration in malignant transformation or ‘‘the origin of cancer cells’’ (2). During the past several decades, the Warburg effect has been consistently observed in a wide spectrum of human cancers, although the underlying biochemical and molecular mechanisms are extremely complex and remain to be defined. Among the possible mechanisms, mitochondrial malfunction and hypoxia in the tumor microenvironment are considered two major factors contributing to the Warburg effect. However, whether the increase of glycolytic activity in cancer cells is mainly due to inherent metabolic alterations or due to anaerobic environment in the tumor tissues remains controversial (3). Under physiologic conditions, generation of ATP through oxidative phosphorylation in the mitochondria is an efficient and preferred metabolic process, which produces far more ATP molecules from a given amount of glucose compared with glycolysis. However, when the ability of cells to generate ATP through mitochondrial oxidative phosphorylation is compro- mised, cells are able to adapt alternative metabolic pathways, such as increasing glycolytic activity, to maintain their energy supply. Mitochondrial respiratory function can be negatively affected by multiple factors, including mutations in mitochon- drial DNA (mtDNA), malfunction of the electron transport chain, aberrant expression of enzymes involved in energy metabolism, and insufficient oxygen available in the cellular microenviron- ment. It is known that mtDNA contains a displacement loop, and the coding gene sequence for 13 important protein components of the mitochondrial respiratory complexes without introns. Mutations in mtDNA are likely to cause alterations of the encoded protein and compromise the respiratory chain function. Thus, the frequent mtDNA mutations observed in a variety of human cancers are thought to contribute to respiratory malfunction in cancer cells (4–6). The constant generation of reactive oxygen species within the mitochondria and the increased free radical stress in cancer cells may cause further damage to both mtDNA and the electron transport chain, thus amplifying respiratory malfunctions and dependency on glycolysis (7). Hypoxia is another important factor that contributes to the Warburg effect. The fast growth of cancer cells and rapid expansion of the tumor mass usually outpace new vascular generation, resulting in an insufficient blood supply to certain area of the tumor tissues. Such a hypoxic environment within the tumor mass limits the availability of oxygen for use in mitochondrial respiration and synthesis of ATP and forces the cancer cells to up-regulate the glycolytic pathway as the main route of energy production (8). The ability of oxygen to regulate glucose metabolism is know as the Pasteur effect and is mediated through several pathways involving various kinases (9, 10). In this case, the increased glycolytic activity in cancer cells is not necessarily due to intrinsic mitochondrial defects but is induced by the tumor microenvironment through a series of metabolic adaptation processes, including preferentially increased expression of enzymes required for glycolysis (11). Although the underlying mechanisms responsible for the Warburg effect are rather complex and can be attributed to a variety of factors, such as mitochondrial defects and hypoxia, the metabolic consequences seem similar. The compromised ability of cancer cells to generate ATP through oxidative phosphorylation Requests for reprints: Peng Huang, Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Box 89, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7742; Fax: 713-794-4672; E-mail: phuang@mdanderson.org. I2005 American Association for Cancer Research. www.aacrjournals.org 613 Cancer Res 2005; 65: (2). 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