Effects And Mechanisms Of COX-2 And Its Selective Inhibitors On HBV Related HCC

[ Background ] Hepatocellular carcinoma (HCC) is one of the most common cancers in our country, and severely harmed the health of people. Epidemiological and molecular studies revealed that persistent chronic infection of hepatitis B virus (HBV) is a major cause of HCC. HBV is a small DNA virus and its genomic DNA contains four partially overlapping open reading frames, named S, C, P and X. Among the proteins translated from those open reading frames, the X-gene product (HBx),which is encoded by the smallest ORF, might play an important role in the development of HCC. HBx is important for HBV viral replication. HBx is a trans-activation that regulated the expression of many genes. HBx binds to and functionally inactivates the tumor suppressor, p53. Moreover, HBx regulates many cytoplasmic signal transduction pathways including the ras-raf-MEK-mitogen-activated protein kinase cascade. However, the mechanisms mentioned above can only partially explain the role of HBx in HCC. Some of the mechanisms involved in the HBx-associated hepatocarcinogenesis remain unknown. COX-2, an isoform of cyclooxygenase, has been associated with carcinogenesis of many types of tumors including gastrointestinal tumors, and NSAIDs can reduce the incidence of colorectal cancer and several other cancers by selectively blocking the function of COX-2. However, the role of COX-2 in HBV related HCC and the relationship between HBx and COX-2 are unknown, nor the efficacy of selective COX-2 inhibitors on HBV associated liver cancer. Toaddress this issue, we observed the expression of HBx and COX-2 in adjacent tumor tissues (precancerous tissues) and HCC tissues, established the HCC cell stably transfected with HBx gene, and also analyzed the effects of COX-2 selective inhibitors on HBx positive liver cancer cells, hoping to give some clues on the development, progression and treatment of HBV associated HCC.[ Aims ] 1. To examine the expression and relationship of HBx and COX-2 in HCC tissues and pericancerous tissues. 2. To detect the expression and function of COX-2 in HBx stably transfected cells. 3. To analyze the effect and mechanisms of COX-2 selective inhibitors on HBx stably transfected cells.[ Methods ] 1 .Immunohistochemistry was perfomed to examine the expression of HBx and COX-2 in human HCC tissues and precancerous tissues. 2. PCDNA3/HBx was transfected into HepG2 cells by using Lipofectamine, and individual clones were isolated with G418 and verified by western blot.3. Western blot and RT-PCR were performed to examine the protein and mRNA expression of COX-1 and COX-2 in HepG2-X and HepG2-PC cells, and ELISA was used to evaluate the level of PGE2 in cell medium. 4. Trypan blue exclusion was performed to detect the growth of both cells. 5. Trypan blue exclusion was performed to examine the inhibitory effects of COX-2 selective inhibitor (celecoxib), COX-2 nonselective inhibitor (indomethin) and COX-1 selective inhibitor (SC560) on cell growth of both transfectants.6.IC5o of celecoxib and indomethin in these two transfectants were measured by colony formation assay.7.Light microscope and electron microscope were used to observe the morphology of cells with or without treatment of celecoxib. 8.Cell apoptosis and the cell cycle distributions in these two transfectants with or without treatment of celecoxib were analyzed by FACS.9.RT-PCR and Western blot were used to detect the apoptosis associated proteins,including Akt, raf, p53, NF-kB, Bcl-2, Bax, Fas/FasL, cytochrome C, caspase-9, caspase-8, caspase-6, caspase-3, and cell cycle related moleculars, including p21~wafl , p27kipl, CyclinA, CyclinB, CyclinDl, CyclinD2, CycinD3, CycinE, Cdkl, CDK2, CDK4, CDK6.[Results] l.In cancer samples, COX-2 was detected in 73(73%), and HBx in 65(65%). COX-2 and HBx were almost exclusively cytoplasmic in the cancer cells, except for HBx with occasional nuclear staining. The correlation coefficient of HBx and COX-2 expression in cancer tissues was evaluated by spearman correlation analysis according to the total score of immunohistochemistry from every case that combines staining intensity with positive cell percentage. Correlation coefficient between HBx and COX-2 was 0.511(p<0.01). When analyzed according to differentiation grade of each case of cancer sample, both HBx and COX-2 were found to have significant correlation with HCC differentiation status (rs= -0.538, rs= -0.72, P<0.01 respectively), which is the higher differentiation of HCC, the higher expression of HBx and COX-2. Importantly, in HBx positive HCC tissues, 60(88 %) with the COX-2 expression, compared to 16(50 %) in HBx negative tissues, there is notable difference between them too (P<0.0l). In precancerous tissue of HCC, cytoplasmic COX-2 was present in 80(80 %) and HBx in 74(74 %). The close correlation was also found in these two markers according to the immunoreactive score. Correlation coefficient between HBx and COX-2 was 0.607(P<0.01).2.PcDNA3-HBx plasmid was stably transfected into HepG2 cells. Western blot and RT-PCR analysis confirmed that HBx was stably expressed in HBx transfected cells (HepG2-X), whereas no signal could be detected from cells transfected with empty vector (HepG2-PC). 3.COX-2 expression at protein and mRNA level were elevated in HepG2-X cells compared with HepG2-PC cells, whereas there was no difference of COX-1 expression in both cells. The PGE2 level in the cell supernatant of HepG2-X cell was 25.1% higher than that of control cell.4.HepG2-X cell grew faster than HepG2-PC cell.5.Celecoxib and indomethin suppressed the growth of both cells in a dose-dependent manner and celecoxib also in a time-dependent manner, while SC560 inhibited cell growth in a constant manner. HepG2-X cell was significantly susceptible to celecoxib and indomethin when compared with HepG2-PC cells. The IC50S for celecoxib and indomethin were 40.54 uM and 185.57uM in HepG2-X celland 55.76 uM and 341.56uM in HepG2-PC cell, respectively. 6. After 72 hours of treatment with celecoxib, morphological changes of apoptosis were found in HepG2-X and HepG2-PC by light microscope. Cells firstly became sparse, rounded, and then detached from the dishes. Under electron microscope, the early manifestations of apoptosis including cell exfoliation, and condensation of chromatin in the edge of the cells, as well as the typical manifestations of apoptosis including the concentration of cytoplasm and nucleus could be also observed in both cells. These effects were more evident with HepG2-X cell. Flow cytometry showed that celecoxib induced apoptosis in a dose-dependent manner in both cells, and HepG2-X cells also showed more apoptosis. After treatment with 50 and 70umol/L celecoxib, the percentages of apoptosis cells of HepG2-X cell were significantly higher than of HepG2-PC cell, respectively. 7. Flow cytometry showed that celecoxib caused a concentration-dependent decrease in the number of cells in the S and G2/M phase in both HepG2-X and HepG2-PC cells, but without significant differences between these two cell clones. 8. After 50 uM celecoxib treatments for 72h, both HepG2-X and HepG2-PC cells showed decreasing level of COX-2 expression, and this is obvious in HepG2-X cell. In addition, celecoxib inhibited PGE2 production in all cell lines, and this effect was also significant in HepG2-X cell. Exposure of both cells to 1 to 5 p.g/L PGE2 consistently rescued the celecoxib-mediated effect on cell viability in a dose-dependent manner. 9. Both p~473Ser Akt and raf were suppressed after treatment with celecoxib, while the p53 was induced. However, NF-kB was not affected. And all these altered proteins were changed dose-dependently and the extent of the change was prominent in HepG2-X cells. 10.The release of cytochrome C was increased, and activation of caspase-9, caspase-3 and caspase-6 were also observed after treatment with celecoxib, while the cleaved caspase-8 was not detectable. Fas ligand was also induced while its receptor, Fas was unchanged. And there was no alteration with Bcl-2 and Bax expression. 11. CyclinA, CyclinDl, CDK1 and CDK2 expressions were dose-dependent decreased while the expressions of p21~Wafl and p27~Kipl were...