| The rapid growth and proliferation of malignant cancer cells need enough supply of nutrient substance and energy. The metabolic characters of cancer cells are different from that of the normal cells and are adapted to tumorigenesis and tumor progression.The characteristics of tumor metabolism reprogramming are: increased aerobic glycolysis, increased glutaminolysis and enhanced synthesis of fat and nuclear acid. Besides of Warburg effect, glutaminolysis is another important metabolic pathway in cancer cells. The rapid proliferating cancer cells consume glutamine to support energy and biological macromolecular materials and to maintain the redox homeostasis in the cells; Furthermore, glutamine is also involved in intracellular signal transduction pathways. Tumor suppressor gene NDRG2(N-myc down-stream regulated gene 2) suppresses the growth and proliferation of colorectal cancer cell, but the effect of NDRG2 in glutaminolysis is still not known.Human NDRG2 was discovered in our lab and we found that NDRG2 expression is quite low in a wide variety of tumor tissues. NDRG2 inhibits cell proliferation and promotes cell differentiation, which suggested NDRG2 acts as tumor suppressor gene.Previously, we found that NDRG2 could decrease glucose consumption and lactate(main metabolite in glycolysis) production in colorectal cancer cell, and therefore NDRG2 inhibits glycolysis in cancer cells.On this basis, this topic wants to explore the role of NDRG2 in glutaminolysis.Aim: 1. Clarify whether NDRG2 plays an important role in tumor cell glutaminolysis; 2. Investigate the molecular targets regulated by NDRG2 in glutamine catabolism pathway;3. Explore the molecular mechanism of glutamine catabolism targets regulated by NDRG2; 4. Explore the clinical significance of NDRG2.Method: 1. Analyze the metabolites change(quantity and variety) of NDRG2 overexpression cells by the method of metabonomics analysis. 2. Measure the concentration of glutamine and glutamate in NDRG2 overexpression and knockdown cells with special kit; make sure that NDRG2 have an effect on glutamine intake and decomposition.3. Analyze the expression level of ASCT2 and GLS1 in NDRG2 overexpression and knockdown cells by real-time PCR and western blot. Using the same method,analyze the relationship between NDRG2 and c-myc, β-catenin.4. Test the relationship between NDRG2 and β-catenin by western blot through the separation of cell cytoplasm and nucleus.5. Analyze the expression level of NDRG2 and c-myc in colorectal tumor by immunohistochemical staining of tissue microarray and analyze the expression level of NDRG2, c–myc, β-catenin, ASCT2 and GLS1 in clinical colorectal tumor tissue specimens by western blot.6. Analyze the effect of NDRG2 on cell proliferation through plate clone formation assay and nude mice tumor formation assay.Results: 1. Metabonomics analysis showed that compared with the control group, glutamine and glutamate levels in NDRG2 overexpression cell line decreased significantly.2. Metabolites detection showed that compared with the control group, glutamine consumption and the production of glutamate decreased in NDRG2 overexpression cell line, while in NDRG2 knockdown cell line, glutamine consumption and the yield of glutamate were increased. All these results suggested that NDRG2 inhibited glutaminolysis by inhibiting glutamine intake and decomposition.3. The results of transcription and translation level showed that compared with control group, the expression of ASCT2 and GLS1 decreased dramatically in the NDRG2 overexpression cell line, while the expression of ASCT2, GLS1, c-myc and β-catenin increased significantly in NDRG2 knockdown cell line. More importantly,knock down of c-myc attenuated the inhibitory effect of NDRG2 on glutaminolysis. All data suggested NDRG2 inhibits ASCT2, GLS1 and this inhibitory effect was mediated by c-Myc.4. Cytoplamic/nuclear separation test showed that the expression of β-catenin in both cytoplasm and nucleus were decreased dramatically in NDRG2 overexpression cell line, which suggested NDRG2 plays an inhibitory role in glutamine catabolism by inhibiting the β-catenin in WNT pathways.5. Immunohistochemical results showed that compared with normal tissue adjacent totumor, the NDRG2 expression in colorectal cancer was low,while the c-myc expressionwas high, showing a negative correlation. Western blot showed that compared with normaltissue adjacent to tumor, clinical colorectal tumor tissue specimens had a lower level ofNDRG2 expression and a higher level of c-myc, β-catenin, ASCT2 and GLS1, showing asignificant negative correlation. All of above suggested that NDRG2 may play animportant role in tumorigenesis and tumor progression.6. Plate clone formation assay and nude mice tumor formation assay showed that compared with control group, clone formation reduced in NDRG2 overexpression cell lines and tumor volume and weight decreased significantly, suggested that NDRG2 inhibits the proliferation of cancer cell significantly through glutaminolysis inhibition.Conclusion: This study reveals that NDRG2 plays an inhibitory role on glutaminolysis in colorectal cancer cells by inhibiting the oncogene c-myc, and this inhibitory effect was proved to be achieved by inhibiting β-catenin in WNT pathway.This research provides a clue for the deep understanding of the molecular mechanism of NDRG2 in inhibiting the occurrence and development of tumor and discovering new targets that regulated tumor metabolism. |
