Preliminary Study Of Mutant Hbv Promoter Regulation Apoptin Expression Induces Apoptosis Of Hepatoma Cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. HCC resists to traditional chemotherapies, and the liver does not tolerate the radiotherapy. Owe to those reason, the clinical therapeutical results of HCC are not satisfactory up to now. The prognosis of HCC is poor and is associated with a high risk of relapse. Therefore, developing new therapeutical approaches are necessary and gene therapy strategies may be new choices. Two questions must be firstly solved for gene therapy of HCC: one is that transgene must target to specific tissue, and the other is selecting most efficient transgene. Precisely targeting therapy can decrease toxicity and increase therapeutic index. One of delicate and efficient methods to achieve targeting is to regulate transgene expression at transcriptional level. Therefore, it is necessary to search for cis-acting elements with tissue- or tumor-specificity. According to epidemiological studies, hepatitis B virus (HBV) infection is usually associated with the development of HCC. Many researches have reported that two domains in the HBV genome had enhancer activity and both enhancers had liver-specific activity and synergistic action. Given these informations, we cloned and constructed a mutated HBV promoter containing two HBV enchancers and HBV core promoter sequences. Transcriptional activity analysis showed this mutated HBV promoter had a higher transcriptional activity in hepatocarcinoma cell lines than positive control, SV40 promoter, especially in 2.2.15 cell line which was integrated by four copies of HBV genome.Many in vitro researches have shown that Apoptin could kill malignant or transformed cells in a tumor cell-specific manner and the nomal cells left intact. Moreover, Apoptin inducing apoptosis is in a p53 and Bcl-2 independent manners which are two main causes for producing resistance of many tumors to traditional chemotherapy and radiotherapy. Tumor cell-specific killing is necessary for successful cancer therapy, so Apoptin becomes our first choice though the molecular mechanism why Apoptin can distinguish tumor cells from normal cells has not been clearly elucidated. We assume that the association of the liver-specificity of the mutated HBV promoter and the tumor-specificity of Apoptin would further increase specific killing to tumor cells, decrease the damadge to normal liver or non-liver cells at the same time and lower the toxic side effect in the application of gene therapy of HCC.In this research, plasmid and adenovirus vectors containing apoptin controlled by the mutated HBV promoter was constructed and their inhibitory effect on hepatocarcinoma cells in vitro was assayed. Results showed that the plasmid vector containing apoptin could efficiently kill hepatocarcinoma cell line HepG2, but the killing effect on 2.2.15 cell line was not obvious. However, Apoptin mediated by adenovirus vector could induce apoptosis of 2.2.15 cell line to some extent, although the efficiency was still not as strong as in HepG2 cell line. These data suggested that it was feasible that Apoptin controlled by mutated HBV promoter mediated by adenovirus vector could be used for targeting gene therapy of hepatocarcinoma, especially which was integrated with HBV genomes.To probe the cause that resulting in the significant difference of Apoptin inducing apoptosis between 2.2.15 and HepG2, we studied from Apoptin's acting mechanism. Firstly, we detected the subcellular localization of Apoptin in 2.2.15 and HepG2. Results showed that the subcellular localization between two cell lines was indeed significantly different, i.e. Apoptin was mainly in nucleus of HepG2 while it mostly localized at cytoplasm of 2.2.15. Because localization at nucleus was necessary for Apoptin's inducing apoptosis in tumor cells, these results further supported the fact that Apoptin induced apoptosis in 2.2.15 and HepG2 by different efficiency. Secondly, it was known that Apoptin utilized intracellular factors to induce apoptosis and the activation of caspase-3 might be necessary for the rapid apoptosis induced by Apoptin. Therefore, the activity of caspase-3 after apoptin was imported into 2.2.15 cell line mediated by adenovirus or plasmid vectors was detected. Results showed that the activity of caspase-3 was indeed increased after apoptin was introduced into 2.2.15. These results suggested Apoptin inducing apoptosis in 2.2.15 invovled the activation of caspase-3. Considering the fact that the efficiency of Apoptin inducing apoptosis in 2.2.15 was relatively lower than in HepG2, one of plausible explains was that Apoptin inducing apoptosis of tumor cells might also involve some important executing factors following the downstream of caspase-3 while these factors were properly scant or absent in 2.2.15. During the caspase-3 activity analysis, we also observed that the effect of cisplatyl as a positive control was stronger in 2.2.15 than in HepG2. The fact as shown by Western blot that the expression of pro-caspase-3 was much higher in 2.2.15 than in HepG2 might be the cause. These data also suggested either more activation of caspase-3 or discovery of the important substrates of caspase-3 might further improve the inhibitory effect of Apoptin on 2.2.15 through combined utlization.The hepatocarcinoma cell line 2.2.15 is derived from HepG2 cells transfected with cloned HBV DNA. The existing of HBV genome might lead to Apoptin's different effect on two cell lines. When we used lamivudine to inhibit the replication of HBV in 2.2.15 cells and then introduced apoptin, the treated 2.2.15 cells were more sensitive to apoptin. The results might provide evidence for the combination of apoptin's gene therapy and anti-HBV therapy.Because HBV infection is a long and complex process, we also detected the global difference of gene expression between 2.2.15 and HepG2 by cDNA microarray. Microarray results suggested that the different expression of many genes including many cytoskeleton genes and genes related to cellular apoptosis in the two cell lines might lead to Apoptin's different effects on them. These data also profited to studies about the mechanism of carcinogenesis by HBV.In summary, we used the strategy of the combination of the liver-specificity of the mutated HBV promoter and the tumor-specificity of Apoptin and constructed the plasmid and adenovirus vector containing apoptin controlled by the mutated HBV promoter successfully. Preliminary researches on inhibiting tumor cells in vitro showed that apoptin controlled by the mutated HBV promoter mediated by adenovirus was able to induce apoptosis of HepG2 cell line and 2.2.15 cell line, so this strategy was feasible to apply for gene therapy of hepatocarcinoma, especially which integrated by HBV genome . Secondly, we for the first time found that the tumor-specific killing activity of Apoptin in hepatocarcinoma could be impacted by HBV infection and the cooperation with anti-HBV reagents would enhance Apoptin's effect on 2.2.15. In addition, changes of factors determining Apoptin's localization at nucleus of tumor cells and the undetermined downstream substrates of caspase-3 might result in the different effects of Apoptin on 2.2.15 and HepG2. Microarray results provided new clues for us to further explore the mechanisms among them.