Biological Characters Of Hepatocellular Carcinoma Transfected With X Gene

Hepatocellular carcinoma (HCC), one of the most common human malignancies ofdigestive system, ranks as the second death rate in cities and the first in countryside inour country. The incidence rate has risen in recent years. The worldwide incidence ofHCC is approximately 260 thousand cases a year and 42.5% of them in China. Themortality rate of HCC in China is 20.4/100,000. The diagnosis of HCC is usually toolate. Usually no potentially curative therapy is possible in the patients with unresectabletumor or recurrence after surgery. It is estimated that 90% of HCC is etiologically associated with HBV in China.Individuals who are chronic carriers may suffer from HCC with 5 ~ 30-fold higher riskthan persons without HBV. HBV DNA contains genes which encode four kinds ofprotein. They are HBsAg, HBcAg, HBeAg and HBX. Increasing evidences indicate thatthe HBV excoded x antigen (HBxAg) contributes to the development of HCC. Most ofinvestigations have concentrated on its carcinogenesis. X gene encodes 16.5 kDamultifunctional protein termed pX or HBX, which is a transcriptional activator that mayplay an important role in HBV-associated hepatocarcinogenesis. However, thecharacters of HCC cells transfected with HBx are largely unknown. The effects of HBxor HBX on expression of oncogene, cell growth, cellular signal transduction moleculesare still unclear. Our previous investigation found that somatostatin analogue could significantly 8inhibit HCC growth in vitro or in vivo in nude mice. However, the results of thistherapy in patients with HCC were quite different. Like most of the cellular models forhepatocarcinoma therapeutic studies, we didn't consider the effects of X gene and Xprotein on HCC. Cyclooxygenase-2 (COX-2) is a hot topic in the field of tumorinvestigation because it has been considered as related with the development of tumor.What happened with COX-2 and with cellular proliferation when HCC cells transfectedwith X gene was interesting for us. Lamivudine, a nucleoside analogue, is an inhibitor of HBV DNA polymerase. Ithas been applied extensively for treatment of hepatitis B. It is worth to be discussedwhether or not lamivudine inhibit the replication, transcription and expression of Xgene and play synergistic action in liver cancer treatment. Therefore, investigation on the biological characters of HCC transfected with Xgene would be benefit for HCC therapy.AIMSTo investigate:1. Whether or not lamivudine could inhibit the replication, transcription and expression of X gene and be used in the experiment for X gene as a tool?2. Whether or not HBV X gene or X protein could affect the growth of HCC cell?3. Whether or not HBV X gene or X protein could affect the expression of oncogene ras in HCC cells?4. Whether or not HBV X gene or X protein had an effect on COX-2 expression in HCC cells? And if selective COX-2 inhibitors could arrest the proliferation of HCC cells transfected with X gene? And how about its mechanism.5. Whether or not somatostatin analogue octreotide could still inhibit the growth of HCC transfected with X gene and how about its mechanism. 9METHODS1. The proliferation of HepG2 HCC cells was measured by H-thymidine incorporation 3 into DNA.2. Immunocytochemistry was used to detect the expression of PCNA,H-Ras and COX-2 in tumor cells.3. Annexin-V labeled assay for detecting the early stage of apoptotic cells.4. TUNEL in situ assay for detecting the late stage of apoptotic cells.5. The replication of transfected X gene was measured by polymerase chain reaction. The mRNA of transfected X gene was analyzed by reverse transcriptase polymerase chain reaction.6. The expression of X protein was detected by biosensor.7. The extracellular signal-regulated kinase (ERK-1/ERK-2),somatostatin receptor (...