Experimental Research Of TTF1-NP Induced Autophagy In Human Liver Cancer HepG-2 Cell

Objective:To observe the autophagy of HepG-2 cell induced by TTF1-NP and explore the possible mechanism.Method:The viability of HepG-2 cells that were treated by TTF1-NP with different dose (15-240 μM) in different time (12 h,24 h,48 h) was measured by MTT assay. Autophagosomes and autolysosomes were identified by transmission electron microscopy. Western Blot were performed to analyze the protein level of LC3-II, Beclin-1, and proteins in PI3K/Akt/mTOR signal pathway (Akt, p-Akt, mTOR and p-mTOR) and in MAPK/JNK signal pathway (p-JNK) induced by TTF1-NP with different dose (80 μM,160 μM,240 μM) and different time (24 h,48 h,72 h).Result:1. After treatment of TTF1-NP, the growth of HepG-2 cells were significantly inhibited in dose-time dependent manner.2. According to transmission electron microscopy results, autophagosomes with double-membrane and autolysosomes were found in TEM pictures. This finding suggested that TTF1-NP induced autophagy.3. Western Blot results show that the protein level of LC3-Ⅱ, Beclin-1 were significantly increased in TTF1-NP treatment groups compared to control group (P<0.01).4. Western Blotting demonstrated that TTF1-NP inhibited the phosphorylation of Akt and the phosphorylation of the downstream proteins, such as mTOR. With the increasing of dose and interval of TTF1-NP, the expression of p-Akt and p-mTOR reduced. After treatment with insulin, the level of two proteins increased compared to control group (P>0.01). TTF1-NP inverted the increasing by down-regulated expression of p-Akt and p-mTOR. LC3-II was up-regulated after TTF1-NP treatment. There was significantly change of the expression between TTF1-NP and TTF1-NP+insulin group.5. MAPK/JNK signal pathway was tested by Western Blotting, and the results show that the expression of p-JNK increased with treatment of TTF1-NP in different dose and times. After treatment with SP600125, the expression of p-JNK and LC3-Ⅱin TTF1-NP+SP600125 group decreased compared to TTF1-NP group, but significantly increased when compared with control group.Conclusion:TTF1-NP may induce autophagy in HepG-2 cell and the mechanism may be related to the inhibition of PI3K/Akt/mTOR and the activation of MAPK/JNK signal pathway.