Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

SummaryGaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme ® ) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme ® . In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-␤-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 Å resolution ( -349 and 394 -399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394 -399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.

Cancer cells utilize glucose as their main energy source and convert it to lactate at high rates, even in the presence of high oxygen concentrations. 1 Previous studies have indicated that the enhanced rate of glucose uptake in tumors might be related to an overexpression of the facilitated glucose transporter proteins. 2,3 Further studies of inducing malignant transformation of fibroblast cells by transfection with ras or src demonstrated upregulation of the glucose transporters as well. 4,5 More recently transformation of cells by c-myc transfection of rat fibroblasts showed specific induction of the glucose transporter 1 (GLUT1) expression. 6 Enhanced glycolysis and high expression of the glucose transporters were specifically identified in human breast cancer cells. [7][8][9][10][11] In addition, the expression of GLUT1 was found to be a characteristic feature in breast cancer biopsies, with a positive correlation between this expression and tumor grade, extent of proliferation and invasiveness. [12][13][14][15][16] Recently, Kang et al. 17 investigated the clinical significance of GLUT1 expression in human breast carcinoma and found a significant correlation between GLUT1 expression and estrogen and progesterone receptor status. These results are in accord with studies of human breast cancer cells in vitro, where induction of cellular differentiation of human breast cancer cells resulted in decreased expression of protein GLUT1. 9 The above findings emphasize that malignant transformation is involved in the deregulation of the expression of the glucose transporters and consequently the augmentation of glucose metabolism and glycolysis.A large number of breast tumors express receptors for estrogen, which regulates various cellular activities by modulating metabolism in the course of tumor growth and progression. 18,19 The estrogen receptors (ER ␣ and ␤), which are ligand-dependent transcription factors, 20 play a key role in the development and progression of breast cancer, as well as in the treatment of breast cancer patients. 21 Tamoxifen, the leading antiestrogenic drug, has been proven to be effective in improving overall survival in both pre-and postmenopausal women. 22 Furthermore, tamoxifen also has been shown to reduce cancer incidence and prevent new tumor formation and growth. 23 Estrogen binding to its receptor induces critical metabolic and physiologic activities via activating various ER-responsive genes. Specifically, estrogen was shown to stimulate glucose metabolism and glycolysis in various target organs, including the uterus-the classic estrogen target organ. 24,25 Furthermore, in the uterus and primate cerebral cortex, it was found that estrogen regulation of glycolysis is mediated by altering the expression levels of GLUT1. 26,27 In breast cancer, kinetic studies of perfused cells in vitro have demonstrated induction of glycolysis by estrogen and its inhibition by tamoxifen. 7,8 The prevalent role of estrogen in breast cancer progression and the need for developing objective surrogate ma...

In mammalian cells, glucosylceramide (GlcCer), the simplest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid beta-glucosidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantly decreased owing to one of approximately 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme replacement therapy (ERT), in which patients are given intravenous injections of recombinant human GlcCerase; the Genzyme product Cerezyme has been used clinically for more than 15 years and is administered to approximately 4000 patients worldwide. Here we review the crystal structure of Cerezyme and other recombinant forms of GlcCerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme, which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease.