Mechanism Of TTF1-NP Induced Human Liver Cancer HEPG-2Cell Apoptosis By Ers Signal Pathway

Objective:Explore the mechanism on TTF1-NP induced liver cancer cell(HepG-2, Hep3B) and nomal liver cell (ChangLiver) apoptosis through ER stress pathway.Method:Observe the proliferation inhibition of different time (24h,48h,72h)and different dose (25,50,100μmol/L) in HepG-2, Hep3B and ChangLiver cells by TTF1-NP. To observe the morphological changes on TTF1-NP induced HepG-2cell apoptosis by HE, Hoechst and AO/EB staining; To detect HepG-2cell apoptosis by Flow cytometry (FCM) apoptosis analysis; To observe the expression of ERS key protein GRP78, CHOP, JNK, p-JNK and Caspase-4by Western Blot and Immunohistochemistry method.Result:1. The MTT assay TTFl-NP-inhibited cell growth results:TTF1-NP in different time (24h,48h,72h) and dose (25,50,100μmol/L) intervention3kinds of cell, the inhibitive effect of cell growth is different and showed a certain degree of dose and time dependent manner, the proliferation inhibition in HepG-2cell is most obvious, then Hep3B, the inhibition in ChangLiver cells was not significant.2. Morphological test results:(1) Optical microscope test results (HE staining), the HepG-2cell in control group was in nomal cell morphology. With the drug dose increasing, the number of cell decrease obviously, morphological changed, become smaller and circular, the nucleus pycnosis and hyperchromatic.(2) Fluorescence microscope test results, AO/EB staining show that, the HepG-2cell in control group was almost all green, dosing group cells dyed red; Hoechst staining show that, the HepG-2cell in control group was in blue uniform, dosing group cells could be seen blue fluorescence in nucleus.3. Flow cytometry (FCM) apoptosis analysis result:the survival rate detected by flow cytometry (FCM) in control group was91.34%, the apoptosis rate was just4.92%. The apoptosis rate increased gradually in dosing group with the increase of drug concentration. In100μmol/L dose group the apoptosis rate was93.79%, and the survival rate is just4.17%.4. The ERS key protein test results:Western Blot analysis result show that, the protein (GRP78, JNK, Caspase-4) expression in control group was very low, the protein of CHOP don’t exprss, with the drug dose increasing, all the protein express obviously incloud CHOP. Immunohistochemistry testing results was basically the same with Western Blot.5. The ERS inhibitors4-PBA test results show that:There is no significant change in the expression in GRP78, p-JNK, Caspase-4in Con and Con-4-PBA group. However, There is significant change in the expression in TTF1-NP and TTF1-NP-4-PBA group.Conclusion:TTF1-NP induced human liver cancer HepG-2cell apoptosis; Endoplasmic reticulum stres pathway is one of the mechanism that TTF1-NP induced HepG-2cell apoptosis.