The Effect And Mechanisms Of The Selective Cyclooxygenase-2 Inhibitor (Celecoxib) Combined With Radiation And Epirubicin On Breast Cancer Cells

Objective To investigate the expression of cyclooxygenase-2 and its clinical and prognostic relevance in breast cancer, and determine the correlation between COX-2 and HER-2/neu, or VEGF expression.Methods Paraffin imbedding tissues taken from 60 patients with breast cancer were detected for COX-2, HER-2/neu and VEGF ptoteins by immunohistochemistry. Results A strong cytoplasmic signal for COX-2 was observed in 23 of breast cancer samplts, the strong positive rate for COX-2 was 38.3%.Statistic analysis (Chi-sguare test) showed that an increased COX-2 expression was significantly associated with lymph node metastasis (P=0.024) and higher TNM stage (P＜0.005). However, the results could not demonstrate a significant relationship between COX-2 expression and patient' s age, tumor size, pathological type, ER or PR expression (P＞0.05). A significant relationship was observed between elevated COX-2 expression and increased expression of HER-2/neu, and VEGF. There was significant difference in TNM stage of the patients with coexpression of COX-2 and HER-2/neu(later stage), and with single protein expression(earlier stage). Cox-2 overexpression in breast cancer was related negatively to the patient's overall survival.Conclusions An elevated COX-2 expression in breast cancer tissue is significantly associated with more aggressive growth of breast cancer and poor prognosis. COX-2 expression of is significantly associated with HER-2/neu and VEGF expression. Coexpression of COX-2 and HER-2/neu might be a useful prognositic indicator. Objective To investigate the radiosensitivity enhancement and underlying mechanisms of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on human cancer cells expressing differential COX-2 levels. Methods Two human cancer cells were studied: A549 cell lines, human lung adenocarcinoma, exhibited a high constitutive COX-2 protein expression level and the MCF-7 cells, human breast carcinoma,manifested low expression of the COX-2 protein. COX-2 expression was measured by RT-PCR.MTT reduction assay was used to evaluate the inhibitory rate of the two cell lines and clonogenic assay was used for radiation survival experiment.The cell cycle distribution and apoptosis index (AI) were analysed by flow cytometry. Results The IC50 of celecoxib dealing with MCF-7 tumor cells in 30 hours was 43.7±1.12μmol/l and A549 tumor cells in 24 hours was 37.5±0.80μmol/l. Celecoxib's irradiation-enhanced cell inhibition was observed in COX-2-expressing A549 cells but the same inhibition was not observed in the MCF-7 cells after the same treatment. Cell cycle analysis showed that four group cells have the different cell cycle distribution. In celecoxib-treated group, the cell population in G₀/G₁ phase have increased; in irradiation group, most cells were in the G₂/M phase. There were no significant apoptosis found in A549 and MCF-7 cells treated by 10～80μmol/1 celecoxib after 6 hours but non-apoptotic cell death was noted when 160μmol/l celecoxib was added. In the two tumor lines, the AI of irradiation+ celecoxib group was no significantly different from that in the irradiation alone group. Conclusion Celecoxib significantly inhibited the growth and proliferation of A549 and MCF-7 tumor cells, this effect was time and dose dependent but seemed to be non-COX-2 expression-dependent. Celecoxib leaded to accumulation of tumor cells in G₀/G₁ phase. Celecoxib could not induce apoptosis nor enhance radiation induced apoptosis in the two lines. Celecoxib significantly enhanced the proliferation inhibition by irradiation on A549 tumor cells probably via COX-2 inhibition ,G₀/G₁ arrest and other mechanisms, however detailed mechanisms remain to be investigated. Object: to investigated the effect of the selective cyclooxygenase -2(cox-2) inhibitor Celecoxib on the breast cancer cell line MCF-7, and explore the possible mechanism. Methods: MCF-7 cells were divided into control(C) group, Celecoxib(Ce) treatment group, epirubicin(E) treatment group and Celecoxib plus epirubicin(Ce+E) intervention group respectively.MTT assay was used to study the cell growth inhibition,Hochest 33258 staining and flow cytometry assay were employ -yed for apoptosis assessment, RT-PCR technique for cox-2 mRNA expression, and Western blot analysis for expression of bcl-2/bax,caspase-3 and STAT3,STAT5,STAT5b proteins. Result: Both Celecoxib and epirubicin in given dose could inhibit the proliferation of MCF-7 in the ways with dose-dependent and time-dependent.The inhibition was markedly increased with the combination of two agents(Ce+E). The inhibitory rates were 0.877±0.023 vs 0.503±0.018 in Ce+E treated cells and E treated alone(p＜0.05). The sensitivity enhancement ratio was 1.74.The highest percentage(44.56±5.14%) of apoptotic cells was also noted in Ce+E settings, although some apoptotic phenomenon was seen in the Ce or E treated cells by hochest 33258 staining and flow cytometry. The Westem blot results showed that bcl-2 expression was decreased in the Ce or E treated cells, and marked decrease was noted in Ce+E settings,however no alteration of bax expression was observed. In the mean time, caspase-3 was activated and the more evident activation was also noted in the Ce+E settings. On the other hand, Celecoxib did not result in the alteration of STAT1, STAT3,STAT5 expression,but epirubicin induced a mild decrease. Futhermore a significant STATs expression happened down-regnlation was noticed as cells treated with epirubicin and Celecoxib. Conclusion: Celecoxib and epirubicin can inhibit the proliferation and induce apoptosis of MCF-7 cells that express a lower level of cox-2, both separately and in combination.The most prominent effects was noted in the Ce+E settings,which suggests that Celecoxib coordinates with epirubicin in growth inhibition and apoptosis. These effect may be mediated by down-regulation of bcl-2, STAT1,STAT3,STAT5b,p-STAT5 and activation of caspase-3.Celecoxib could be used as a sensitizor of epirubicin for cancer therapy.