The Inhibition Of NDRG2 On Glucose Metabolism Of Cancer Cells

As one of the most important substance metabolism, Glucose metabolism is vital for organisms to maintain homeostasis. Meanwhile, Glucose is also regarded as the major energy resource for supporting proliferation of cells. Compared with normal cells, tumor cells are characteristic of rapid proliferation, fast growth and huge demand for calories. As a kind of major energy resource Glucose is more important for tumor. So modulation of glucose metabolism in tumor cells must be significant for anticancer therapy.Human NDRG2 gene (also named SYLD/KIAA1248) belongs to a family of N-Myc downstream-regulated genes (NDRG). It was first cloned in our laboratory from a normal human brain cDNA library by subtractive hybridization (GenBank Accession No. AF159092). The NDRG2 gene is located at chromosome 14q11.2, including 16 exons and 15 introns. The entire mRNA of NDRG2 is 2024bp in length and it encodes 357 amino acids with a calculated molecular mass of 41 kDa. There is a high expression level of human NDRG2 in many normal tissues. But it has a low expression level in various cancer tissues. This predicts that NDRG2 is a candidate tumor suppressor gene(TSG). Many studies show that NDRG2 can make an impact on cell differentiation, Alzheimer's disease and the metabolism of sodium in rat renal collecting ducts through mineralocorticoid. NDRG2 protein is phosphorylated by Akt and PKC in the insulin signal pathway. However, the function of NDRG2 is not yet fully understood. NDRG2 has also been reported to play roles in other functions, such as insulin action, aldosterone-mediated epithelial sodium channel (ENaC) function. These data indicate that NDRG2 not only has the function of controlling transcription, but also involves in materials transportation.Does NDRG2 affect the proliferation of cancer cells through regulating the glucose intake. To exploit this hypothesis, we have done the following studies that are investigating NDRG2 expression level of several tumor cell lines, detecting the alteration of cell proliferation in high level and low level glucose situation, measuring the variation of intracellular glucose concentration caused by the alteration of expression of NDRG2 in cells and checking out the mechanism about impact of NDRG2 on glucose intake in cancer cells.Firstly, we screened kinds of breast cancer cell lines and liver cancer cell lines to see the expression level of NDRG2 through western blotting. Then we chose SK-BR-3, T-47D, HHCC and HepG2 for our following study.Secondly, we cultured cell lines in high glucose medium and low glucose medium separately, and then measured the intracellular glucose. We found the level of intracellular glucose had significant difference in different groups. We detected the multiplication capacity of cells through MTT and plate colony formation assay, indicating that the cells cultured in high level glucose grew faster than the cells cultured in low level glucose.Thirdly, we transfected cells with NDRG2-specific siRNA and NDRG2 recombinant adenovirus separately. We demonstrated that the expression of NDRG2 was downregulated and upregulated successfully through western blotting, then we measured the intracellular glucose levels of cells cultured in high glucose medium and low glucose medium. We found that the alteration of expression level of NDRG2 in cells cultrured in high glucose medium was more significant than that in cells cultrured in low glucose medium. With the expression level of NDRG2 increasing, the intracellular glucose level decreased, and vice versa. While in low glucose level medium, the phenomenon was not as significant as that in high level glucose medium.Forthly, we exploited the mechanism of the phenomenon. As the main glucose transporter in mammalian, GLUT1 mainly located in cell membrane and had a high expression level in many tumors. GLUT1 plays an important role in glucose intake of cancer cells. Recently, it is reported that NDRG2 could regulate the transportation of substance through influencing the function of some transporters in cell membrane. In order to further investigate the function of NDRG2, we hypothesize that NDRG2 regulate the glucose intake of cancer cells through interacting with GLUT1.Lastly, we measured the GLUT1 expression levels of cells transfected with NDRG2-specific siRNA through Real-time PCR and western blotting. We found that there was no significant alteration in the mRNA and protein levels of GLUT1 when the expression of NDRG2 was downregulated. But we observed that NDRG2 and GLUT1 colocated in cells through immunofluorescence staining. In order to verify that whether they interacted with each other directly, we perform the co-immunoprecipitation assay. The result showed that NDRG2 and GLUT1 combined with each other directly in cells. These data indicated that the regulation of NDRG2 on the glucose intake of cancer cells may result from the direct interaction between NDRG2 and GLUT1 influencing transportation,location,function and degradation of latter.In summary, we first defined an important role of NDRG2 in the regulation of the glucose metabolism of cancer cells. NDRG2 could inhibit the glucose intake and proliferation of cancer cells via combination with GLUT1. The function of the interaction is needed for further investigation. As a new gene, NDRG2 has a lot of unknown functions. We focused our studies on the role of NDRG2 in glucose metabolism of caner cells, which would enrich the understanding of this gene`s function and tumor metabolism.