A Study On Cyclooxygenase-2 Protein Expression And Angiogenesis Mechanisms During Experimental Rat Liver Carcinogenesis

OBJECTIVE: Primary hepatocellular carcinoma (HCC) is one of the most common tumors in the world. The prognosis of HCC is generally poor, and the 5-year survival rate is limited to 25%~49% after surgery. Tumor angiogenesis is a necessary condition for the growth, invasiveness and metastasis of tumor. Cyclooxygenase (COX) is a rate-limiting enzyme involved in the conversion of arachidonic acid to prostaglandins. Two isoenzymes of COX have been identified: COX-1 and COX-2. Recent studies have shown that COX-2 involved in a variety of human malignancies including hepatocellular carcinoma. However, the relationship between COX-2 expression and neovascularization in patients with hepatocellular carcinoma is not clear. The aim of this study is to develop animal model of liver cancer, observe the dynamic changes of expression of COX-2 protein and its association with tumor angiogenesis during experimental rat liver carcinogenesis, explore the mechanisms of COX-2 promoting tumor angiogenesis. At the same time, we have investigated the inhibitory effects of rofecoxib, a selective inhibitor, in the early stage of liver cancer formation and intended to explore new methods of liver cancer therapy.METHODS: A total of 86 male Wistar rats were randomly divided into three groups: control group, inducing group and interferential group. Except the controlgroup all rats were fed with the water that contained 0.01% diethylnitrosamine (DEN), a chemical carcinogen-inducing drug. 14 weeks later, the water was changed to normal water. The rats in inducing group were killed respectively in the 6th week, 12th week and 18th week, while the rats in interferential group were also fed with rofecoxib, a selective COX-2 inhibitor, at a dose of 30mg.kg-l.day-l in the beginning and were killed in the 6th week and 9th week respectively. The rats in control group received normal diet and were killed according to experimental design. The tissue samples of all rats were collected on the experimental design.The expression of COX-2mRNA was assayed by reverse transcriptate polymerase chain reaction (RT-PCR) and COX-2 protein by immunohistochemical stain. We also examined microvessel density (MVD), vascular endothelial growth factor (VEGF) and VEGF receptor 2(VEGFR2, KDR / flk-1), matrix metalloproteinase 2 (MMP-2) , Hypoxia-inducible factor 1 alpha (HIF-1 a ) and proliferating cell nuclear antigen (PCNA) as well as Bcl-2 in interferential group by immunohistochemistry. The level of prostaglandinE2 in serum was assessed by Enzyme-linked immunosorbent assay (ELISA). All data were analyzed by statistic methods.RESULTS: At the 18th week of experiment, the model of experimental liver cancer was confirmed by HE stains. The incidence of the experimental liver cancer was 100%, and specimens showed a development from liver inflammation to cirrhosis and cancer respectively on the 6th, 12th and 18th week.Inducing group: RT-PCR and immunohistochemical stain showed expression of COX-2 emerged at early stage of the experiment, and became more and more obvious according to the course of experiment. At the stage of cancer, the expression was the most obvious but no expression of COX-2 was observed in normal control group;the difference was significant between the two groups (p<0.05). Because the expression of MVD was insignificant between early stage of cancer and control group, the difference was not significant (p >0.05). However, the expression at stage of cirrhosis increased and reached to the tiptop at stage of cancer. Positive correlationbetween COX-2 and MVD was established (r=0.788, p <0.01) . Correlation between MVD and VEGF(r=0.8587,p<0.01), that between MVD and MMP-2( r=0.6837, p<0.01), and that between MVD and HIF-la(r=0.7031,p<0.01) were also significant.VEGF is now thought to be one of the most important factors regulating angiogenesis which combines with its receptors, especially KDR / flk-1, on vascular endothelial cells and induces mitosis and migration of EC intensely. The expression of VEGF and KDR emerged at early stage of experiment and became more and more obvious in the stage of cirrhosis and reached its climax in the stage of cancer. Difference among the groups was significant (p <0.01). COX-2 was able to act on gene of VEGF through PPAR8 and upregulate expression of VEGFmRNA and protein.MMP-2 is a member of a large family of endogenous proteases that degrade various components of the extracellular matrix. MMP2 may make EC deform, proliferate and transform through degenerating ECM.Expression of MMP-2 was similar to VEGF during the experiment. Difference among the groups was also significant (p <0.01) . COX-2 upregulated expression of MMPs, led to imbalance of MMPs and its natural antagonist TIMPs and increased malignance of tumor cell.PGE2 is one of the main products of arachidonic acid catalyzed by C0X2, PGE2 interacts with its receptor Eps, acts on PKC or PKA message system, promotes expression of VEGF. PGE2 is also able to dilate blood vessel directly, increase vessel osmosis, accelerate plasma protein exosmosis, strengthen reaction for EC and promote the growth of blood vessel. Meanwhile, when C0X2 induces growth of PGE2, PGE2 is able to feedback positively expression of C0X2 and further strengthen effect of COX-2. Concentration of PGE2 in serum in the experiment indicated that it was lower in normal rats and became more and more obvious during the inducing process. The change of PGE2 and COX-2 was consistent in the experiment.Hypoxia-inducible factor 1 alpha (HIF-la) is a gene that regulates a critical reaction in the body under the hypoxia. HIF-la is expressed during the stage of cirrhosis and reached its climax in HCC. In contrast, the expression of HIF-la innormal tissue and early inducing stage was not found. This illuminated that hypoxia induced the expression of HIF-la. HIF-la interacted with VEGF and promoted the expression of VEGF. In this process HIF-la induced directly or indirectly expression of COX-2 which may be through some mechanisms of conduction. There was a kind of cooperative function and a significant correlation between HIF-la and COX-2 (r =0.7791, p<0.01) or between HIF-la and MVD(r=0.7031, p<0.0l).COX-2 itself in the process of promoting growth of tumor and angiogenesis was modulated by various factors. First, multifunctional nuclear factor kappaB (NF-kB) modulated COX-2 gene and promoted its expression. Secondly, activation of PPAR 5 was able to promote expression of COX-2. Moreover, various vascular growth factors in accepting the modulation of COX-2 could form positive feedback for COX-2. PGE2 and VEGF were able to farther increase the function of COX-2 by upregulation of expression of COX-2. Furthermore, MMP-2, HIF-la also increased the effect of COX-2 and promoted all together the growth, invasiveness, metastasis and angiogenesis of tumor.In conclusion, we have shown that the selective COX-2 inhibitor intervened and slowered the process of rat experimental liver cancer and presented the effect in prevention and treatment of tumor.Interferential group: Expression of COX-2 in interferential group was far lower than that in inducing group at stage 6th (p<0.01) and 9th (p<0.05) week of the experiment, while expression of MVD in two groups was low and difference was not significant at two stages of the experiment. PCNA is one of the indicators of tumor cell proliferating ability and malignant degree. Its expression is lower in interferential group than that in inducing group at the 9th week (p<0.05), but was no significant meaning in 6th week (p>0.05). Bcl-2 is the most important anti-apoptosis gene in the body;it can refrain various tissue cells from apoptosis and promote tumor growth. Its expression was also lower in interferential group than that in inducing group at the 9th week (p<0.05) and 6th week (p<0.01).In summary, we have shown that the selective COX-2 inhibitor intervenes and slowers the process of rat experimental liver cancer though the interferentialexperiment of rat liver cancer in the early stage. There is an effect in prevention and treatment of tumors. The interferential effect in the early stage of tumor formation mainly bases on the inhibition of proliferation and induction of apoptosis, but there is little effect on tumor angiogenesis. This is maybe also the main mechanism of inhibiting tumor growth of NSAIDs and worthy of continuous study. Meanwhile, this studies also offers a valuable reference for prevention of hepatocellular carcinoma.CONCLUSIONS:1.COX-2 involves in DEN-induced experimental rat hepatocellular carcinogensis.2.There was a positive correlation between expression of COX-2 protein and MVD count. COX-2 produced PGE2 and upregulated the expressions of "VTXjT^ KDR and MMP-2. HIF-la induced directly or indirectly expression of COX-2, perhaps through some mechanisms of conduction. There was cooperative function between HIF-la and COX-2. All the factors promoted tumor growth and angiogenesis3.Rofecoxib, a selective COX-2 inhibitor, significantly suppressed DEN-induced formation of experimental rat liver caner through inhibiting COX-2. The COX-2 inhibitor in the early stages of tumor formation led to a marked growth inhibition of tumor mainly based on the inhibition of proliferation and inducement of apoptosis, but it was little effect on tumor angiogenesis.4.There is great scientific and clinical significance in continuing the experiments of COX-2 inhibitors on inhibiting tumors and clinical studies and exploring new safe selective COX-2 inhibitors.