Lactoferrinplaysan Important Role In AKT Signaling Pathway Induced Anoikis-resistant And Metastasis In Nasopharyngeal Carcinoma Cells

Objective : To investigate whether LF could affect the anoikis-resistant nasopharyngeal carcinoma(NPC)cells,and then to explore the relationship between LF and the anoikis-resistant of NPC cells which had been induced by AKT signaling pathway.Methods:Two strains of anoikis-resistant HNE-1 and HONE-1 NPC cells were constructed.The proliferation and survival of cells was detected by Cell Counting Kit-8(CCK8),and the cell cycle and apoptosis were detected by flow cytometry.Transwell assay was used to detect the ability of invasion and metastasis ability.The expression of matrix metallopeptidase-9(MMP-9),vascular endothelial growth factor-A(VEGF-A),E-cadherin and Vimentin were detected by Western blot.LTF plasmids and LTF shRNA plasmids were transfected into HNE-1 and HONE-1 respectively,and then to detect the expression of E-cadherin,Vimentin,AKT and TrkB proteins by Western blot to clarify the role of LF in the anoikis-resistant of NPC cells.Results: After suspension culture of HNE1 and HONE1 cells for 48 hours,the survival rate of cells were stable,the anoikis-resistant NPC cells were constructed successfully.Compared with parental nasopharyngeal carcinoma cells,a reduced percentage of anoikis-resistant NPC cells was detected at G0/G1 phase(P< 0.001),an increased percentage of cells in S phase(P< 0.001),the apoptosis rate was also significantly reduced(P<0.001).The survival rate of anoikis-resistant NPC cells was significantly increased(P < 0.001),and the number of cells passing through the basal glue was increased(P < 0.001).The expressions of VEGF,MMP9 and Vimentin in anoikis-resistant NPC cells were increased(P< 0.001,P<0.001,P< 0.001),while E-cadherin were decreased(P< 0.001).After transfection of LF overexpressed or interfered plasmids into cells,thesurvival rate and invasion ability of HNE1 cells which transfected with LTF were significantly lower than control group(P< 0.001,P< 0.001).Flow cytometry showed that LTF could significantly increased the cell proportion of G0/G1 phase(P< 0.001)and reduced the cell proportion of S phase(P< 0.001)along with increasing the apoptosis rate(P< 0.001).In contrast,the survival rate and invasion ability of HONE1 cells which transfected with LTF shRNA were significantly higher than control group(P< 0.001,P< 0.001),LTF shRNA could significantly reduced the cell proportion of G0/G1 phase(P< 0.001)and increased the cell proportion of S phase(P< 0.001)along with decreasing the apoptosis rate(P< 0.001).Western blot indicated that the expression of AKT and TrkB in anoikis-resistant NPC cells were significantly increased(P< 0.001,P<0.001),meanwhile,the expression of AKT and TrkB in control cells were higher than overexpression cells and lower than knockdown cells(P<0.001,P< 0.001).Conclusion: The study shows that anoikis-resistant NPC cells has strong ability of proliferation,invasion and anti-apoptosis.LF can inhibit the anoikis-resistant NPC cells,and further inhibit the metastasis of NPC.The related molecular mechanism is related to AKT signaling pathway.