The indicated cells were harvested and homogenized, and samples made up of equal amounts of proteins were subjected to an LDH-A activity assay as described in Materials and Methods

The indicated cells were harvested and homogenized, and samples made up of equal amounts of proteins were subjected to an LDH-A activity assay as described in Materials and Methods. conferred resistance to cetuximab-induced G1-phase cell cycle arrest, which could be overcome by knockdown of LDH-A expression. Inhibition of LDH-A activity with oxamate enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no apparent inhibitory effect on glycolysis in nontransformed cells. These findings provide novel mechanistic insights into cetuximab-induced cell cycle arrest from the perspective of cancer metabolism and suggest novel strategies for enhancing cetuximab response. Introduction Glucose is an important source of energy and carbon for both normal and cancer cells. Unlike most normal cellswhich metabolize glucose by a low rate of glycolysis followed by oxidative phosphorylation in the mitochondria through the tricarboxylic acid cycle (also known as the citric acid cycle or Krebs cycle)malignancy cells metabolize glucose by a high rate of glycolysis followed by lactate production in the cytosol even in the presence of abundant oxygen, a phenomenon known as aerobic glycolysis or the Warburg effect (1, 2). The Warburg effect is important for malignancy cell proliferation because this process generates building blocks and reducing power, both of which are needed for the biosynthesis that fuels cell growth and proliferation (3, 4). This altered metabolism in cancer cells is a direct result of the aberrant cell signaling caused by overexpression of growth factor receptors, activation of oncogenes, and/or inactivation of tumor suppressor genes that permits unlimited cancer cell proliferation (5C7). The transcription factor hypoxia-inducible factor-1 (HIF-1) plays a key role in reprogramming cell metabolism from oxidative phosphorylation to aerobic glycolysis. HIF-1 regulates the expression of the genes coding Rebeprazole sodium for proteins involved in various steps of cancer metabolism, from glucose uptake and subsequent glycolytic reactions to the generation of lactate and its secretion by lactate transporters (8). HIF-1 is usually a heterodimer consisting of a highly regulated HIF-1 subunit and a constitutively expressed HIF-1 subunit (9C12). A high Rebeprazole sodium level of HIF-1 protein is common in many types of solid tumors, including tumors of the colon, lung, breast, stomach, ovary, pancreas, prostate, kidney, and head and neck (13C15). The high level of HIF-1 in cancer cells is caused not only by the decreased ubiquitination and degradation of HIF-1 protein via a Rebeprazole sodium posttranslational mechanism associated with tumor hypoxia (16, 17), but also by aberrant cell signaling, which increases HIF-1 protein expression via a translational mechanism (18C22). Cetuximab is an epidermal growth factor (EGF) receptor (EGFR)-blocking monoclonal antibody approved for treating patients with head and neck cancers and colorectal cancers in combination with radiotherapy and/or chemotherapy (23, 24). We as well as others have previously shown that cetuximab binds to EGFR and blocks the ligand-induced activation of EGFR downstream cell signaling, which leads to G1-phase arrest of cell cycle traversal and even apoptosis in certain circumstances (25C40). Our previous work showed that cetuximab can downregulate HIF-1 protein by inhibiting the PI3K/Akt and MEK/Erk pathways, and this downregulation of HIF-1 Rabbit Polyclonal to COX7S is required, although may not be sufficient, for cetuximab to induce antiproliferative effects (41C45). Knockdown of HIF-1 by small interfering RNA Rebeprazole sodium (siRNA) partially overcame the resistance caused by overexpression of constitutively active Ras mutant to cetuximab-induced antiproliferative effects (43C45). These previous studies established the importance of HIF-1 downregulation in mediating cetuximab-induced antitumor effects; however, to our knowledge, no studies have carefully examined the mechanism that leads to growth inhibition after downregulation.