Studies On Celecoxib-induced Hepatocarcinoma Cell Apoptosis And Its Molecular Mechanism

A new generation of non-steroidal anti-inflammatory drugs (NSAIDs), have been described that selectively targets the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2). This isoform is expressed at sites of inflammation, which has led to the speculation that its inhibition could provide all the benefits of current NSAIDs but without their major side-effects on the gastrointestinal system (which are due to inhibition of COX-1). The first specific COX-2 inhibitor, namely celecoxib, oral agents and U.S. Food and Drug Administration approved, has been shown preclinically and clinically to has efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis. Recent studies have shown increased levels of COX-2 in a variety of human malignancies, including hepatocellular carcinoma (HCC), but so far it is unknow whether COX-2 contributes to the malignant growth and whether inhibition of COX-2 function modifies the malignant potential of tumors. Substantial evidence indicates that celecoxib displays anti-tumor effect by sensitizing cancer cells to apoptosis and reduces the number of adenomatous colorectal polyps in patients with familial adenomatous polypsis. However, the research or the effects of celecoxib on HCC have just begun and the exact anti-tumor mechanisms are not well known. Therefore, in this article, we studied the biological effects of celecoxib on human three HCC cell lines,such as HepG2, BEL-7402 , SMMC-7721 in vitro, in order to investigate thepotential mechanisms of celecoxib on HCC and to provide a theoreticalevidence for searching an new agent for the treatment of HCC in the future.Objectives:1 .To detect the expression of COX-2 mRNA and protein in three human HCCcell lines : HepG2 , BEL-7402 and SMMC-7721. 2.To study the biological effects of inhibiting proliferation and inducingapoptosis by celecoxib, selective COX-2 inhibitor. 3.To investigate the potential mechanisms of the effects of celecoxib on thethree HCC cells lines. Methods: 1.Cell culture: Three human HCC cell lines HepG2 , BEL-7402 andSMMC-7721 were cultured as research models in vitro. 2.In situ hybridization was used to evaluate the expression of COX-2 mRNA.Western blot analyses and immunohistochemistry were used to detect theexpression of COX-2 protein. 3.Morphological changes were observed under the light and invertedmicroscope. 4.After treated with 6.25-100 mol/L celecoxib for 12-48 hours, the effects ofcelecoxib on the proliferation of the three HCC cell lines were measured bymeans of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide(MTT)-assays.5.The expression of proliferating cell nuclear antigen (PCNA) and thephosphorylation of Akt Thr were detected by immunohistochemistry. 6.Apoptotic index (AI) was detected by Flow Cytometry (FCM) and terminaldeoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)method. 7. Western blot analyses were used to detect the phosphorylation of Akt Thr308and activation of caspase-9 and caspase-3. 8.The results of TUNEL and immunohistochemistry were analysed byComputer Image Analysis System: Leica Qwin. Result:1. COX-2 mRNA and protein can be detected in the three HCC cell lines by in situ hybridization and Western blot. COX-2 protein was located in cytoplasm in those positive cells.2. Celecoxib could significantly inhibit the proliferation of the three HCC cell lines in a dose and time dependent manner. Typical apoptotic morphology, especially apoptotic bodies, was observed after treatment with celecoxib.3. After treatment with celecoxib, the histogramic analysis of DNA contents revealed the appearance of hypodiploid DNA peak immerged before G1 phase.4. The apoptotic index measured by TUNEL increased gradually with adding the concentrations of celecoxib and acting time. With 50u,mol/L celecoxib treatment for 24 hours, the apoptotic index of BEL-7402, SMMC-7721 andHepG2 were 26.40 3.05%, 3...