Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.

Thapsigargin increases apoptotic cell death in human hepatoma BEL-7404 cells

Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL-7404

Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.

Induction of APOBEC3G in human hepatoma cells by interferon-α stimulation

To clarify the role of APOBEC3G （A3G） in cellular defense against hepatitis B virus （HBV）, the expression of A3G in normal human liver and the regulation of the A3G expression in hepatoma cell line （HuH-7） were investigated. Expression level of APOBEC3s mRNA in human liver was determined by RT-PCR. HuH-7 and HepG2 cells were treated with various concentrations of IFN-α（0 U/ml, 100 U/ml, 500 U/ml, 1000 U/ml）for 12 h. The mRNA levels were measured by a quantitative RT-PCR, the results were normalized relative to the specimens without IFN-α stimulation. Total protein of HuH-7 cells treated with various concentrations of IFN-α for 48 h was subjected to Western blot analysis. For reporter gene assay, HuH-7 cells were transfected with the reporter plasmids containing IRF- E sites and its mutants with different lengths. Then the cells were treated with or without 1200 U/ml IFN-α for additional 12 h （ 1000 U/ml） after 24 h of transfection, and the cell lysate was prepared and assayed for lueiferase activity. It was found that normal human liver expressed the rnR_NA of A3G. A3G mRNA expression in HuH-7 and HepG2 cells were up-regulated by IFN-α stimulation in a dose-depen- dent manner. Western blot analysis indicated that A3G protein expression was also enhanced by IFN-α stimulation. Sequence analysis showed the existence of putative sites of IFN regulatory factor element （IRF-E） in 5＇ region of A3G gene upstream the initiation eodon. IFN-α stimulation results in 6- to 8- fold increase in lueiferase activity in cells transfeeted with the plasmid containing IRF-E sites of the 5＇ upstream sequences, whereas luciferase activity did not change in cells transfected with the plasmid containing mutant IRF-E sites or without IRF-E sites. As a conclusion, A3G are expressed in normal human liver. A3G expression was up-regulated by IFN-α stimulation in hepatoma cells and could be involved in host defense mechanisms against HBV. IRF-E site in 5＇ region of APOBEC3G gene upstream the initiation codon plays an important role in this process.