Studies On Autophagic Cell Death Induced By TIPs In Human Hepatoma Cells HepG2

AIM:To investigate the mechanism of proliferation inhibition of TIP-6 (7-(4-methoxyphenyl)-5,Sa-diphenyl-1,2,3,7,8,Sa-hexahydroimidazo[1,2-a]pyridine) in human hepatoma cells HepG2 and human normal hepatocyte L02.METHODS:After treatment with varying concentration of TIP-6, Typan Blue Assay and MTT assay was used to determine the proliferation rate treated-cells.The IC50 of cells was figured out.The morphology of cells was observed by the phase contrast microscope.The cell cycle was determined by Flow cytometry(FCM) analysis in treated-cells.Autophagy and autophagic cell death were detected after acridine orange(AO) staining under fluorescent microscopy.The submicrostructure was observed by transmission electron microscope.Apoptosis was analyzed by Annexin V/7-AAD,DAPI staining and DNA agarose gel electrophoresis assays.The expression of nuclear factor-kappa B(NF-κB) was detected by cellular immunochemistry.RESULTS:We found that TIP-6 can induce different cell line cytotoxity. While TIP-6 causes 50%inhibition in HepgG2 at 138.6μmol/ml for 24 hrs,88.6μmol/ml for 48 hrs,52.5μmol/ml for 72 hrs,same inhibition in L02 was caused at 142.5μtmol/ml for 24 hrs,93.3μmol/ml for 48 hrs,59.1μmol/ml for 72 hrs.The result based on Typan Blue Assay shown that TIP-6 can suppress the proliferation of HepG2 cells in a dose- and time- dependent manner.TIP-6 has stronger inhibition in HepG2 than in L02 cells.After treatment with varying concentration of TIP-6 for 24, 48,72 hrs,more vacuoles in cells were found by phase contrast microscope.FCM showed that cells were blocked in G2/M phase.Annexin V/7-AAD and DAPI staining and DNA agarose gel electrophoresis assays indicated that TIP-6 can't inhibit proliferation followed by inducing apoptosis in cells.The of AO staining and transmission electron microscope showed that TIP-6 can cause autophagic cell death in HepG2 cells.Moreover,we found that the expression level of NF-κB increased by TIP-6 treatment.CONCLUSION:TIP-6 inhibited cell proliferation,as cells were blocked in G2/M phase.TIP-6 can inhibit proliferation of cells by causing autophagic cell death in HepG2 and L02 cells.The NF-κB pathway may be involved in these processes.