A Study Of Correlation Between Cyclooxygenase-2 Expression And Akt Activation In Promotion Liver Cancer Cell Growth

Objective: Cyclooxygenase-2 (COX-2)-controlled prostaglandin (PG) metabolism has recently been implicated in the cell proliferation and pathogenesis of some cancers. However, the biological role and molecular mechanism of COX-2-mediated PGE2 in the control of liver cancer growth have not been established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth control of liver cancer cells and the correlation between COX-2 and Akt cell signaling system.Methods: 12 specimens of human HCC and 4 cholangiocarcinoma samples were taken for COX-2 and phospho-Akt(Thr308) immunohistochemistry detections. 5 human liver cancer cell lines including 2 human hepatocellular carcinoma (HCC) cell lines ( Hep3B and HepG2) and 3 cholangiocarcinoma cell lines ( HuCCTl, SG231 and CCLP1) were taken for following experiments: COX-2 overexpression transfection and COX-2 antisense transfection, exogenous PGE2 treatment, selective COX-2 inhibitor (celecoxib) treatment, caspase inhibitor (Z-VAD-fmk) treatment, PI3K inhibitor (LY294002) treatment, cell viability and growth rate (WST-1) assay, cell apoptosis detections including: cell morphological changes, apoptotic nuclear characters, plasma membrane alteration ( by method of light and fluorescence microscopy observation, flow cytometry ) and caspase protease activity measurements, immunocytochemistry assay for detecting COX-2, phospho-Akt ( Thr308 ) and phospho-Akt ( Ser473 ) expressions and their correlation, Western blot analyses for detecting of COX-2 expression, Akt cellsignaling pathway activity and apoptosis associated protein and enzyme changes after COX-2 transfection or COX-2 inhibitor treatments , etc.Results: Our experiments showed that 1) High level of COX-2 expressionand Akt(Thr308 ) phosphorylation was observed in human liver cancer tissues, the average staining intensity for COX-2 expression in HCC is 2.2, which is significantly higher than that in nontumor liver tissue(0.99), and the averagestaining intensity for phospho-Akt(Thr308 ) is 2.1 in HCC versus 0.79 in nontumor liver tissue. 2) Positive correlation and co-location between phospho-Akt(Thr308) and COX-2 was observed both in liver cancer tissues and cultured HCC cells. 3) Human liver cancer cells transfected with PCDNA3-COX-2 vector showed increased PGE2 production (about 15% increase) and cell growth rate (about 25.8-30.4% increase) when compared with PCDNA3 control transfection cells, while the HuCCTl cell, which is a human COX-2 highly expressed cholangiocarcinoma cell line, when transfected with COX-2 antisense plasmids, showed decreased PGE2 production (about 18% decrease) and cell viability (about 20% decrease). 4) Human HCC Hep3B cells transfected with PCDNA3-COX-2 vector showed enhanced phosphorylation of Akt (Thr308) but the level of phospho-Akt(Ser473) remained unchanged. 5) Liver cancer cells treated with COX-2 inhibitor celecoxib showed remarkable decreasing of cell viability, significant reduction of Akt phosphorylation (both Thr308 and Ser473), markedmorphological and biochemical characteristics of apoptosis: the cells became shrunken, round, and detached from the dish. Fluorescence microscopy following Pi-staining showed marked chromatin condensation, fragmentation and attachment to nuclear envelope. Annexin-V and PI double labeling flowcytometry analysis showed a result of 25-55% of cell apoptosis inducement ratio after celecoxib treatment compared to 5-8% of control cells. Western blot analysis revealed that celecoxib induced activation of caspase-9and caspase-3 with concomitant release of cytochrome C. The results of colometric caspase protease activity assay showed that after celecoxib treatment, about 2 to 7-fold increased caspase-9 protease activity and about 8 to 13-fold increased caspase-9 activity were observed in liver cancer cells and these effects can be partially blocked by intruduction of Z-VAD-fmk, a kind of caspase inhibitor. 6) Inhibition of Akt activation by PI3-kinase inhibitor LY294002 significantly decreased...