Effect And Mechanism Of Celecoxib On The Proliferation, Invasion And Sensitivity To Chemotherapy Drug

Ovarian cancer is one of the most common life-threatening malignant tumors among women and it is the first leading cause of death from gynecologic cancers. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, majority of cases have progressed to an advanced stage at the time of primary diagnosis. Although most patients will undergo an attempt at surgical debulking followed by 6 cycles of chemotherapy with a platinum-based regimen, the 5-year survival remains 25-30%[1] and 85-90% patients will develope tumor recurrence or metastasis. Cisplatin as the critical component in the chemotherapy regimens, its usage has been limited because of its cumulative toxicities, especially the toxicity to the kidney and the intrinsic or acquired resistance to cisplatin in some patients[2; 3]. In recent years, the interests of combining cisplatin and new active chemotherapeutic agents to treat ovarian cancer have arisen for promoting the effectiveness and reducing the side effect of cisplatin.Cyclooxygenase (COX) is an important speed-limited enzyme in the conversion of arachidonic acid to prostaglandins. Two isoenzymes of COX, COX-1 and COX-2, have been idendified. Overexpression of COX-2 is known to be closely associated with tumor growth[4-8], metastasis[9-11] and chemotherapy resistance[12; 13] in several kinds of human tumors. The selective inhibitor of COX-2 has been shown to suppress tumor growth, invastion and metastasis in solid tumors, and increase the cytotoxicity of chemotherapy drugs to tumor cells[14-18]. It has been found that COX-2 is stably expressed in ovarian cancer tissue, but not in normal tissues[19; 20]. Although there are several reports about COX-2 inhibitors used in ovarian cancer, the results are debatable and the mechanism is unclear. There is no report about the combination of celecoxib with chemotherapy drug in ovarian cancer cells nowaday.Objective1. To investigate the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell proliferation in human ovarian cancer cell line SKOV3.2. To investigate the effect and mechanism of Celecoxib on cell sensitivity to Cisplatin in human ovarian cancer cell line SKOV3.3. To investigate the effect and mechanism of Celecoxib on cell ivasion in human ovarian cancer cell line SKOV3.MethodsPart One the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell proliferation in human ovarian cancer cell line SKOV3The proliferation assay and flow cytometry were carried out to investigate the effect of Celecoxib on cell proliferation and apoptosis in ovarian cancer cell line SKOV3. As a comparison, the same work was done in SKOV3 cells in which the COX-2 expression was inhibited by COX-2 siRNA. Western blot was carried out to assess the changes of the expression of related proteins (Caspase-9, Caspase-3, PARP, Bcl-2, Bax, Cyclin D1).Part Two the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell sensitivity to Cisplatin in human ovarian cancer cell line SKOV3.The proliferation assay was carried out to assess the enhancement effect of 10μM Celecoxib (clinical available) on cell sensitivity to Cisplain. The SKOV3/COX-2i cell, in which the COX-2 protein expression was inhibited by COX-2 siRNA, was used as the COX-2 negtive control to observe whether the COX-2 expression is dependent in the enhancement effect of Celecoxib on cell sensitivity to Cisplain. Western blot was carried out to assess the changes of the expression of related proteins (Caspase-9, Caspase-3, PARP, Bcl-2, Bax, P-gp).Part Three the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell invasion in human ovarian cancer cell line SKOV3.The cell adhesion assay, transwell chamber and scratch wound assay were carried out to detect the abilities of adhesion, invasion and migration in SKOV3. The activity of MMP-2 and MMP-9 were examined by Zymography, the protein expression of COX-2, MMP-2, MMP-9 and E-cadherin was analyzed by Western blot. The same work was done in SKOV3/COX-2i to investigate the potential role of COX-2 in the changes caused by Celecoxib.Reults1. The cell growth was inhibited obviously in SKOV3 cells with high concentration of Celecoxib(50μM) at 24h, 48h and 72h, or in SKOV3/COX-2i in which the COX-2 protein expression was inhibited by COX-2 siRNA, but not in SKOV3 cells with low concentration of Celecoxib(2μM,10μM). Compared with the control and SKOV3 with Celecoxib 10μM , the SKOV3 with 50μM Celecoxib at 24h and SKOV3/COX-2i were retarded in G0/G1 phase and percentage of them in G0/G1 phase increased significantly and G1 subpeak before G1 phase peak(apoptosis peak) was shown. Compared with the control and SKOV3 with 10μM Celecoxib, the early apoptosis cells ( Annexin V(+)PI(-) ) and late apoptosis cells ( Annexin V(+)PI(+) ) were increased significantly. Western blot results showed that the expression of cell cycle protein Cyclin D1 was decreased and the amount of the active subunit of caspase-9 , caspase-3 and the cleaved subunit of PARP was increased, however, neither Bcl-2 nor Bax. Compared with the control, no significant changes of above-mentioned proteins were observed in SKOV3 with 10μM Celecoxib.2. Our data indicated that a remarkable increase of growth inhibition was observed in SKOV3 with the combination of 10μM Celecoxib with 10μM Cisplatin(p<0.05), nevertheless the treatment with 10μM Celecoxib or 10μM Cisplatin alone had little effect to induce growth inhibition(p>0.05). The same result was shown in SKOV3/COX-2i in which the COX-2 expression was inhibited specifically by COX-2 siRNA. In combination treatment groups, Western blot results showed that the amount of the active subunit of caspase-9 , caspase-3 and the cleaved subunit of PARP was increased, however, neither Bcl-2 nor Bax. Compared with the control, no significant changes of above-mentioned proteins were observed in SKOV3 with 10μM Cisplatin. Western blot results showed that MDR1 gene coded P-gp protein was hardly expressed in SKOV3 and SKOV3 with the combination of 10μM Celecoxib with 10μM Cisplatin for 24h, but increased in SKOV3 with 10μM Cisplatin for 24h.3. Treated with 10μM Celecoxib for 24 hours, cell adhesive assay, transwell chamber assay and scratch wound assay indicated that the adhesive, invasive and migratory ability of SKOV3 cells was inhibited significantly(P<0.05). Zymography results showed the descended activity of MMP-2 and MMP-9 and Western blot results showed the unchanged protein expression of COX-2 and decreased protein expression of MMP-2, MMP-9 and E-cadherin in SKOV3 cells treated with 10μM Celecoxib for 24 hours. But the same changes were not observed in SKOV3/COX-2i .Conclusions1. High concentration of COX-2 selective inhibitor Celecoxib (50μM) could inhibit the cell proliferation and increase the cell apoptosis of human ovarian cancer cell SKOV3 by inhibiting the cell cycle protein Cyclin D1 and activating the "Caspase/PARP" pathway. Specifical inhibition of COX-2 by COX-2 siRNA could inhibit the cell proliferation and increase the cell apoptosis of SKOV3, but the effect was no as good as Celecoxib (50μM). The effect mentioned above of Celecoxib might be through both COX-2 dependent and independent way, but Bcl-2 independent way.2. The combination of low concentration of Celecoxib (10μM) with Cisplatin (10μM) could enhance the sensitivity of SKOV3 to Cisplatin by inhibiting the expression of MDR1 gene coded P-gp increased by Cisplatin and activating the "Caspase/PARP" pathway. But the sensitivity of SKOV3 to Cisplatin could not be enhanced by specifical inhibition of COX-2 by COX-2 siRNA. The enhancement of sensitivity of SKOV3 to Cisplatin by low concentration of Celecoxib (10μM) might be COX-2 independent.3. Low concentration of Celecoxib (10μM) could increase the adhension and inhibit invasion and migration of SKOV3 by increasing the expression of E-cadherin and inhibiting the expression and activation of MMP-2 and MMP-9. Specifical inhibition of COX-2 by COX-2 siRNA could not cause the changes mentioned above. The inhibition of invasion of SKOV3 caused by low Celecoxib might be COX-2 independent.4. Celecoxib has a positive antitumor effect and the effective concentration of 10μM is clinical available when it is used as NSAIDs. It might be a new chemotherapy adjunctive drug and has a potential clinical prospect.