Study On The Effect And Mechanism Of Apoptosis On HepG2Cells Induced By TTF-1in Vitro

Aims:To study the inhibitory molecular mechanism of TTF-1on angiogenesis and apoptosis of HepG2cells in vitro. At the same time, our study could promote TTF-1, the herbal extracts, to use as a Chinese medicine on liver cancer.Methods:HepG2cells were treated with TTF-1in vitro, and the cell viability was detected by using MIT assay. Then, the cell morphology changes were observed by phase-contrast microscope and transmission electron microscope. Meanwhile a series of molecular techniques, such as colony formation, wound-healing assay were used to performed the HepG2cells’proliferation, migration. In addition, HepG2cells were treated with TTF-1by flow cytometry, ELISA and hoechst staining to dected the apoptosis rate. For determining the effects and mechanism of apoptosis inhibitor TTF-1on HepG2cells, western blot, immunocytochemistry and immunofluorescence staining were used to detect the location and expression of apoptosis makers, bax、bcl-2、cyc-c、Caspase-3、Caspase-9in the mitochondrial pathway. Transfection and luciferase reporter gene assay was used to test the promote activity inhibiting of NF-κB by TTF-1.Results:1. HepG2cells with TTF-1treatment was suppressed of the cell morphology changes which observed by phase-contrast microscope and transmission electron microscope. The cells treated by TTF-1at50μmol/l for48h showed spindle shapes,and scattered and loose cell-cell interaction.2. TTF-1could effectively inhibit the cell proliferation and migration of HepG2cells in vitro. Meanwhile, MTT assay found that the cell viability was significantly decreased in HepG2cells treated with TTF-1, and showed the dose dependent manner. Moreover, colony-forming assay also indicated that HepG2cells were significantly inhibited by TTF-1. TTF-1could effectively inhibit the HepG2cells via suppressing the motility.3. TTF-1induces the apoptosis of HepG2cells in vitro. Flow cytometry assay revealed that apoptotic rate was significantly increased in the treated cells compare with the control untreated cells. ELISA results showed that DNA fragmentation was higher compared to the negative control and was significantly different. Additionally, Hoechst33342staining showed an even distribution of the stain and round homogeneous nuclei, however, the cells treated by TTF-1showed many apoptotic cells, the nucleus displayed typical changes including reduction of cellular volume, bright staining and condensed or fragmented nuclei.4. The expression of bax, cyt-c, caspase-3/9were significantly increased, the expression of bcl-2was significantly decreased in the HepG2cells treated by TTF-1compared with the control untreated cells. And TTF-1could inhibit the promote activity of NF-κB to induce the apoptosis of HepG2cells.Conclusions:1. TTF-1could effectively suppress the HepG2cells in vitro via inhibiting the cell proliferation, migration by inducing apoptosis.2. TTF-1induced apoptosis of HepG2cells by bax、bcl-2、cyc-c、Caspase-3、 Caspase-9through mitochondrial pathway.3. TTF-1could inhibit the promote activity of NF-κB to suppress HepG2cells in vitro.