| Lung carcinoma,among which 85%are non-small-cell lung carcinoma(NSCLC),is one of the most common causes of cancer-related death in the world.It accounts for 27 percent of cancer-related death in the world.NSCLC has higher tolerance to chemotherapy and radiotherapy than small cell lung cancer.Therefore,drug resistence is a severe problem to be solved clinically.GCN2(general control nonderepressible 2)is a serine/threonine-protein kinase that senses amino acid deficiency through binding to uncharged transfer RNA(tRNA).In our study earlier,GCN2 phosphorylation play a key role in promoting cisplatin tolerance of NSCLC.Block GCN2 phosphorylation enhance the sensiti-vity of lung cancer to cisplatin.Ouabain,one of endogenous cardiac glycosides,is higher in patients with non-small-cell lungcarcinomathan health people.It induce cisplatin resistance via phosphorylate GCN2.However,the molecular mechanism remained unknown.Na+/K+-ATPase is the specific receptor for ouabain.Ouabain up-regulated Ca2+intracellular and active SRC/EGFR by binding to Na+/K+-ATPase specifically.We treat cells with SRC inhibitor PP2 and Ca2+chelant BAPTA-AM respectively,BAPTA-AM could inhibit GCN2 phosphorylation significantly induced by ouabain but PP2 cannot.We confirmed calcium take part in the process of GCN2 phosphorylation.Furthermore,extracellular calcium influx through L-type calcium channel and calcium release from endoplasmic reticulum/sarcoplasmic reticulum are the primary source of calcium waves.Calcium ion is an important signaling messenger that is widely present in cells and participates in the activation and regulation of many signal transduction pathway.It has been reported that ouabain can active MAPK signal pathway through Ca2+-PKC MAPK pathway regulate the proliferation,differentiation and growth of tumor cells.We pretreat cells with MEK1 inhibitor PD0325901,L-type calcium channel blocker-nifedipine and Ca2+chelant BAPTA-AM.Interestingly,the expression of activated GCN2 and ERK1/2 was suppressed simultaneously.These results indicate that Ca2+-MAPK take part in the process of GCN2 phosphorylation induced by ouabain.GCN2 phosphorylation has close relationship to amino acid intracellular.We found in our study that ouabain reduced total amino acid in the cell notably.Detected by LC-MS,low dosage ouabain increased the levels of various amino acids.However,high concentration ouabain reduced the levels of amino acids in the cell almostly and result in amino acid deprivation and GCN2 phosphorylation.For further investigation,we construct recombinant plasmid MEK1-CA mutation,which is continuous activation.Overexpression the plasmid activated MAPK pathway effectively and upregulated the amino acid catabolic enzymes BCAT1,BCAT2 and downregulated amino acid transporters SNAT2,CAT1.The research confirmed MAPK pathway is involved in the uptake and metabolism of amino acids.Collectively,our study found for the first time that ouabain phosphorylate GCN2 via active ATP1A1-Ca2+-MAPK signal transduction pathway as well as induce amino acid deprivation.What’more,MAPK signal pathway negative regulat the uptake and metabolism of some of the amino acids.GCN2 phosphorylation induced tolerance for cisplatin in NSCLC.This paper has explored the underlying mechanism of ouabain phosphorylate GCN2 from the theoretical point of view.It provides a theoretical basis for cisplatin resistance in NSCLC. |
