The Relationship Between The Effects Of Cripto-1 On Cell Glucose Metabolism And Proliferation

Metabolism is one of the most important features of the life activities.The main energy supply in normal cells is the aerobic oxidation of glucose.In order to maintain their own survival,tumor cells have chosen another energy metabolism method-aerobic glycolysis.Changes in energy metabolism in tumor cells(glucose metabolism reprogramming)play an important role in the development of tumors and are considered to be one of the ten characteristics of cancers.Studies have shown that oncogene activation or tumor suppressor gene inactivation can lead to metabolic reprogramming of cancer cells.Cripto-1,also known as teratocarcinoma-derived growth factor 1(TDGF-1),is mainly expressed in the early stage of embryonic development and is not expressed or low expressed in normal cells in adulthood,but is highly expressed in tumor cells,promoting cell proliferation,invasion,migration,angiogenesis and EMT,etc.,and plays an important role in tumorigenesis.However,it is not clear whether Cripto-1 is involved in the metabolic reprogramming of tumor cells,so it is important to study the effect of Cripto-1 on cellular glucose metabolism.In this study,we first screened Cripto-1 high and low expression cell lines.Western blot result shown that the expression of Cripto-1 in Huh7 and Bel7402 cells were higher,but in L02 cells was lower.Thus,in further study,we knocked down the expression of Cripto-1 in Cripto-1 high expressing cell lines Huh7 and Bel7402 and overexpression of Cripto-1 in Cripto-1 low expressing cell line L02 to analyze the effect of Cripto-1 on the cellular glucose metabolism and its molecular mechanism.1.Effects of Cripto-1 on cell glucose metabolismIn order to study the effect of Cripto-1 on cell glucose metabolism,we overexpressed Cripto-1 in Cripto-1 low expressing cell line L02 and investigate the effects of Cripto-1 on glucose uptake,lactate secretion,lactate dehydrogenase activity and the expression of intracellular glucose metabolism related protein.The results showed that overexpression of Cripto-1 could promote cell uptake of glucose,cell lactic acid production,enhance cell LDH activity and promote the expression of intracellular glucose metabolism related proteins HK2,GLUT1,PKM2 and LDHA,whether cultured with high glucose medium or low glucose medium.Whereas when knocked down the expression of Cripto-1 in Cripto-1 high expressing cell lines Huh7 and Bel7402,the glucose uptake and lactate production were inhibited,the LDH activity was reduced,and the expression of glucose metabolism related proteins HK2,GLUT1,PKM2 and LDHA were down regulated,whether cultured with high glucose medium or low glucose medium.The above results indicate that Cripto-1 promotes aerobic glycolysis.2.The mechanisms of Cripto-1 promoting cellular aerobic glycolysisThe above experiments suggest that Cripto-1 can promote cell aerobic glycolysis,but the underlying mechanism is unclear.In order to further explore the mechanism of Cripto-1 promoting cell aerobic glycolysis,we detect the signal changes of glucose-related-MAPKs,NF-κB,PI3K-AKT signaling pathway.The results showed that overexpression of Cripto-1 significantly activated the p38/MAPK signaling pathway,whereas knockdown of the expression of Cripto-1 inhibited p38/MAPK signaling.We then overexpressing Cripto-1 in Cripto-1 low expressing cell line L02 and inhibit p38/MAPK signaling with p38/MAPK inhibitor(SB203580)or p38/MAPK dominant negative mutant(DN-p38)and to detect its effect on glucose metabolism in Cripto-1 overexpressing L02 cells.The results showed that both p38 inhibitor(SB203580)or DN-p38 inhibited glucose uptake,lactate production,LDH activity and the expression of glucose metabolism-related proteins in overexpressing Cripto-1 cells.The above results suggest that Cripto-1 promotes cell aerobic glycolysis at least in part by activating the p38/MAPK signaling pathway.3.The relationship between the effects of Cripto-1 on cell aerobic glycolysis and cell proliferationPrevious study found that Cripto-1 can promote cell proliferation,so we first verify this conclusion.Cripto-1 was overexpressed in L02 cells,and the effect of Cripto-1 on cell proliferation was detected by MTT assay and BrdU incorporation method.ATP content was measured by ATP assay kit.The results showed that overexpression of Cripto-1 could enhance cell viability,increase DNA synthesis and promote cell energy production.Conversely,knockdown of Cripto-1 in Huh7 and Bel7402 cells inhibit cell DNA synthesis,reduces cell viability and inhibits cell energy production.The above results demonstrate that Cripto-1 promotes cell proliferation and energy production.To elucidate the correlation between the effects of Cripto-1 on cellular glucose metabolism and cell proliferation,we block the cell glycolysis by glycolysis blocker 2-deoxy-D-glucose(2-DG)in L02 cells overexpressing Cripto-1,then the 2-NBDG uptake,lactate production,lactate dehydrogenase activity,cell proliferation,cell viability and ATP content were examined.The results showed that the cell glycolysis was significantly inhibited by the glycolysis inhibitor 2-DG,and the cell viability and cell DNA synthesis were significantly attenuated.Furthermore,the inhibitory effect of glycolysis inhibitor 2-DG on glucose metabolism,cell viability and cell proliferation in overexpressing Cripto-1 L02 cells was significantly stronger than that in control cells.The above results suggest that Cripto-1 may promote cell proliferation by promoting aerobic glycolysis.In summary,this study explored the effect of Cripto-1 on cellular glucose metabolism and its relations with cell proliferation,in order to providing an experimental basis for tumor therapy targeting Cripto-1.