Effects Of Celecoxib On Cell Proliferation And Apoptosis In Human Colorectal Cancer Cell Line HT-29 And Molecular Mechanisms Involved

Background Colorectal cancer is one of the most common malignancies of the gastrointestinal track,the incidence of which has been increased in china. This disease is often detected at a late stage when treatment is costly and clinical outcome is poor. Development of better prevention and therapeutic strategies is critical to improve the prognosis for patients with colorectal cancer. Non-steroidal anti-inflammatory drugs (NSAIDs) represent one class of chemopreventive agents, which have activity against colorectal cancer. Nemerous epidemiologic,animal,and clinical studies have shown that NSAIDs reduce the risk of colorectal cancer. One known target for this class of drugs is the cyclooxygenase enzyme. Cyclooxygenases (COXs) are key rate-limiting enzymes that mediate the production of prostaglandins from arachidonic acid. Two isoforms of cyclooxygenase, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), have been identified. COX-1 is constitutively expressed in various types of cells and plays important roles in homeostasis. COX-2 is usually absent under basal conditions, but is inducible by various cytokines and growth factors and mitogens. Overexpression of COX-2 has been well demonstrated in different malignancies,including colorectal, esophageal, gastric, liver, lung, prostate, and breast cancers. These findings suggest that COX-2 may play an important role in carcinogenesis. It is thought that inhibition of COX-2 activity by NSAIDs as the antineoplastic mechanism of this class of drugs and gastrointestinal complications of using NSAIDs attribute to the inhibition of COX-1.Tranditional NSAIDs inhibit COX-1 so that a range of normal physiologic functions are affected,which frequently results in untoward gastrointestinal side effects such as ulceration and bleeding . This limits their clinical prolonged use in human and has lead to the development of more selective COX inhibitors. For this reason, development of selective COX-2 inhibitors was promoted. However,the efficacy and the possible cellular and molecular mechanisms involved of treating established colorectal carcinomas with the selective COX-2 inhibitors has not been thoroughly evaluated. In this study, HT-29 human colorectal cancer cells were treated with various concentrations of Celecoxib, the aim of which is to investigate whether Celecoxib can inhibit proliferation, induce apoptosis in HT-29 cells and to explore the molecular mechanisms involved. ChapterⅠ Effects of Celecoxib on cell proliferation in human colorectal cancer cell line HT-29 and molecular mechanisms invovled Objectives To explore the effect of Celecoxib on cell proliferation of human colorectal cancer cell line HT-29 and the probable mechanisms involved. Methods MTT assay and flow cytometry (FCM) were used to quantify the influence of Celecoxib in the proliferation of HT-29 cells. The expressions of CDK2,CDK4 , P21WAF1/CIP1 proteins were observed by Western blot analysis. Results 1. MTT assay showed : Celecoxib decreased the cell survival rates of HT-29 cells in a concentration -dependent manner from 0 to 120 μM. 2. FCM showed : Celecoxib increased the proportion of cells in G0/G1 phase, whereas decreased the proportion of cells in the S and G2/M phase in a concentration -dependent manner from 0 to 120 μM in HT-29 cells. 3. Western blot analysis showed : the expressions of CDK2, CDK4 proteins were down-regulated, whereas the expression of P21WAF1/CIP1 was up-regulated in a concentration -dependent manner from 0 to 120 μM inHT-29 cells. Conclusions Celecoxib inhibited cell proliferation in HT-29 cells,which may be associated with down-regulation of the expressions of CDK2,CDK4 proteins, up-regulation of the expression of P21WAF1/CIP1 protein, and alteration cell cycle progression. ChapterⅡ Effects of Celecoxib on cell apoptosis in human colorectal cancer cell line HT-29 and molecular mechanisms invovled Objectives To explore the effect of Celecoxib on cell apoptosis of human colorectal cancer cell line HT-29 and the probable mechanisms involved. Methods Fluorescence staining was used to quantify the influence of Celecoxib in the apoptosis of HT-29 cells. The expressions of Cytochrome C in cytosol ,Caspase-9, PARP proteins were observed by Western blot analysis. Results 1. FCM showed : Celecoxib increased the apoptotic rates of HT-29 cells in a concentration -dependent manner from 0 to 120 μM. 2. Fluorescence staining showed : Celecoxib induced apoptosis in HT-29 cells. Typical morphological feature of apoptotic cell included cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptosis bodies. Apoptotic index increased in a concentration -dependent manner from 0 to 120 μM. 3. Western blot analysis showed : The expression of Cytochrome C protein in cytosol was up-regulated, whereas the expressions of pro-caspase-9 and pro-PARP proteins were down-regulated in a concentration -dependent manner from 0 to 120 μM in HT-29 cells.. After 30μM Celecoxib incubated with HT-29 cell for 48hrs, it began to activate caspase-9 and consequently trigger PARP cleavage. The effects served as a concentration -dependent fashion from 0 to 120 μM .Conclusions Celecoxib induced cell apoptosis in HT-29 cells, which may be associated with up-regulation of the expressions of Cytochrome C protein in cytosol, activation caspase-9 and PARP.