Ceramide Induced Autophagy Through The Akt1/Foxo3a Pathway In Ovarian Cancer Cells

Objective:This study was to investigate C₂-ceramide-induced autophagy through Akt1/Foxo3a signaling pathway and its mechanisms in ovarian cancer cells.Methods:Use of MTT method to study the rate of inhibition of ovarian cancer cells in vitro treated with different concentrations of C₂-ceramide ( 0μmol/L ,12.5μmol/L , 25μmol/L, 50μmol/L, 100μmol/L) . The rate of early apoptosis of cancer cells was analysed by Flow cytometry , when the different concentrations of C₂-ceramide were added into the culture medium. The autophagy induced at 25μmol/L C₂-ceramide in ovarian cancer A2780 cells was observed by transmission electron microscropy.Real time quantitative PCR was used to analyse the expression of Beclin1, LC3 in ovarian cancer cells when a concentration of 25μmol/L C₂-ceramide was used . The method of western- blot was used to analyse the expression of Beclin1,LC3, p-Akt1 , Foxo3a at protein lever in ovarian cancer cells when treated with 25μmol/L C₂-ceramide.Results:The rate of inhibition of cell proliferation increased with the concentration of C₂-ceramide elevated, the IC50 value was 55.719μmol/L; The rate of early apoptosis of the ovarian cancer cells depended on the concentrations of C₂-ceramide. But, for the concentration of C₂-ceramide less than 25μmol/L, there was almost no initiation of ovarian cancer cells apoptosis. Transmission electron microscopy showed autophagic vacuoles of A2780 cells treated with C₂-ceramide. With the supplement of C₂-ceramide, the mRNA and protein expression of Beclin1 and LC3 in the ovarian cancer cells were increased. LC3 had two subunits, LC3 I and LC3 II .The expressions of LC3 I and LC3 II protein were increased. However , there was a greater increase in LC3II than LC3I. P-Akt1 (ser473) protein expression level decreased with the addition of C₂-ceramide, meanwhile the Fox3a protein expression level was increased.Conclusions:1. C₂-ceramide could induce ovarian cancer cells autophagic death through Akt1 /Foxo3a signaling pathway.2. C₂-ceramide could activate autophagy- related genes Beclin1 and LC3 expression.