NDRG2 Overexpression Suppresses Hepatoma Cells Survival During Metabolic Stress Through Disturbing The Activation Of Fatty Acid Oxidation

A cancerous cell must pass through multiple steps to form a solid tumor body,and this process is often accompanied by insufficient nutrients and oxygen supplies.Even after the tumor increases in size,the microenvironment of tumor cells often becomes heterogeneous.Some regions of large tumors display a significant gradient of critical nutrients,such as glucose,because of poor blood supply [2].In this situation,cancer cells must reprogram metabolism to adapt to stressful microenvironment,thus exhibiting different metabolic dependencies compared to their normal counterparts [3].A well-known metabolic reprogramming in tumor cells is Warburg effect,which is characterized by an increased glycolytic flux with lactate production from pyruvate.In addition,irregular glutamine and lipid metabolism are also frequently observed in tumor cells [4],for instance,tumor cells shift their metabolic pattern from glycolysis to fatty acid oxidation(FAO)to maintain ATP level and satisfy nutrient demands under a series of stressful conditions such as glucose deficiency [5,6].Furthermore,several types of cancer cells have been reported to increase FAO for cell survival due to compromised glucose uptake in the case of loss of attachment to the extracellular matrix(ECM)[12].Being competent to adapt to stressful conditions,tumor cells even exhibit an increased local invasion and distant metastasis after the anti-angiogenic therapies [7].Therefore,it is of great importance to clarify the molecular mechanism of metabolism reprogramming in cancer cells,so as to discover new therapeutic molecule targets for cancer treatment.NDRG2 is a newly identified tumor suppressor gene by our group.NDRG2 expression is down-regulated in various human tumor tissues and in vitro,NDRG2 overexpression inhibits the proliferation,migration and invasion of cancer cells [8].In addition,NDRG2 is also involved in cellular response to different stresses [9].For instance,In hepatoma Hep G2 and Huh7 cells,NDRG2 expression was induced by hyperthermia,and synergism between hyperthermia and NDRG2 effectively inhibits invasion and reduces cytotoxicity [13].Importantly,increasing evidences indicate that NDRG2 is involved in the metabolism of tumor cells.Our previous study demonstrated that NDRG2 inhibits glucose uptake in breast cancer cells by regulating GLUT1 stability,and inhibits glycolysis and glutaminolysis in colorectal cancer cells by repressing c-Myc [10,11].However,whether NDRG2 participates in cancer lipid metabolism remains unmasked.Based on this,we sought to clarify the role of NDRG2 in hepatoma cells during metabolic stress,which occurs in solid tumor due to microenvironment heterogeneity or matrix detachment.Aim:1.To clarify the effect of NDRG2 on hepatoma cell survival and death under metabolic stress.2.To explore the effect of NDRG2 on cellular energy and redox balance in hepatocellular carcinoma cells upon glucose limitation.3.To explore the molecular mechanism through which NDRG2 plays its role in hepatocellular carcinoma cells under glucose limitation.Method:1.Huh7 and Sk-hep-1 hepatoma cells that stably express NDRG2 or Cherry were established by lentiviral infection and NDRG2 expression in these cells were measured by q PCR and Western blot analysis;cell viability under normal condition and metabolic stress were measured by CCK8 assay;colony-forming ability of these cells under normal or low glucose culture conditions were measured by Colony formation assay;cell apoptosis in the presence or absence of glucose were examined using flow cytometry analysis and TUNEL staining.2.Intracellular ATP levels,NADPH content,NADP+/NADPH ratio,and ROS levels in NDRG2-overexpressing and control hepatoma cells,in the presence or absence of glucose,were measured using the ATP assay kit,NADP/NADPH quantification colorimetric kit and the ROS detection kit.3.The relative mRNA levels of the key molecules involved in FAO in NDRG2-overexpressing and control hepatoma cells,under normal condition or glucose deprivation,were measured by q PCR analysis.Intracellular ATP,NADPH level and cell viability were determined by using the ATP assay kit,NADP/NADPH quantification colorimetric kit and CCK8 assay in the presence of PPAR? inhibitor BEZA(400?M)and CPT1 A inhibitor ETO(100?M).4.The activation of AMPK/ACC pathway(p-AMPK?,AMPK?,p-ACC and ACC)in NDRG2-overexpressing and control hepatoma cells cultured in the presence or absence of glucose were measured by Western blot.Upon forced expression of the constitutively active form of AMPK? in NDRG2-overexpressing Huh7 cells,AMPK/ACC signaling was measured by Western blot analysis and cell apoptosis were determined by flow cytometry analysis.Results:1.The results of CCK-8,colony formation assay and cell apoptosis assay demonstrated that NDRG2 overexpression significantly reduced hepatoma cells viability and colony-forming ability and enhanced glucose-deprivation induced apoptosis of hepatoma cells under glucose deficiency.2.The results of ATP,NADPH and ROS detection assay demonstrated that NDRG2 overexpression remarkably aggravated ATP reduction and significantly decreased NADPH contents,leading to increased NADP+/NADPH ratio and ROS levels,in the absence of glucose.3.The inductin of FAO genes(PPAR?,CPT1 A,and ACADM)by glucose-starvation were compromised markedly in NDRG2-overexpressing hepatoma cells.BEZA,an activator of PPAR? that can enhance intracellular ATP and NADPH generation,attenuated growth inhibition by NDRG2 under glucose limitation.Conversely,an inhibitor of CPT1 A,ETO,reduced intracellular ATP and NADPH content and decreased cell survival to much more extent in control cells than in NDRG2-overexpressing cells.4.Glucose-deprivation induced phosphorylation of AMPK? and ACC was suppressed in NDRG2-overexpressing hepatoma cells.The expression of AMPK?–CA reversed the inhibition of p-ACC by NDRG2 and partially abrogated glucose-deprivation induced apoptosis in NDRG2-overexpressing Huh7 cells.Conclusion:NDRG2 overexpression impaired FAO activation and ATP and NADPH maintenance through suppressing AMPK activation,and thereby rendered hepatoma cells sensitive to cell apoptosis induced by glucose-deficiency.