The Effects Of Celecoxib Combined With Tea Poly-Phenols On The Proliferation And Apoptosis Of Breast Cancer Cell And Its Possible Mechanisms

Objective:To investigate the effects of celecoxib combined with tea polyphenols on the proliferation and apoptosis of triple-negative breast cancer cell MDA-MB-231and to explore the possible mechanisms of apoptosis induced by celecoxib combined with tea polyphenols.Methods:MTT assay was adopted to detect the effects of celecoxib, tea polyphenols and celecoxib combined with tea polyphenols on the proliferation of breast cancer cell MDA-MB-231. The changes of morphology of the cancer cells were observed under inverted fluorescent contrast phase microscope. Hoechst33258staining was used to observe the morphologic changes. Cell apoptosis was detected by Annexin V-FITC/PI double staining. The mRNA expression of Bcl-2and Bax was determined by Real-time PCR. Western Blot was taken to detect the expression of proteins including Bcl-2, Bax, caspase-3and caspase-9which are related to apoptosis.Results:1. MTT assay showed that celecoxib and tea polyphenols inhibited the proliferation of MDA-MB-231cells (P<0.05). The cell proliferation inhibition rate of celecoxib (10μmol/L) combined with tea polyphenols (6.25、12.5、25、50、100μg/mL) was higher than that of the single drug groups with significant difference (P<0.05).2. The shrinking cells, the variation of the refractive index and the increased cell fragments could be observed in both single drug groups or combination groups of celecoxib (10μmol/L) and tea polyphenols (50、100μg/mL) under an inverted microscope. The number of normal cells and the morphological changes of combination groups were significantly different compared with the single drug groups.3. Typical apoptotic morphology such as shrunken of nuclei and chromatin con-densation could be observed when the cells were treated with celecoxib (10μmol/L) and tea polyphenols (50、100μg/mL) alone or in combination under fluorescence microscope. The apoptotic morphological changes were more significant in combination groups compared with single drug groups.4. Flow cytometry showed that celecoxib (10μmol/L) and tea polyphenols (50,100μg/mL) alone and in combination can induce apoptosis, and the rate of apoptosis of combination groups was significantly higher than that of single drug groups(P<0.05).5. The results of Real-time PCR showed that celecoxib (10μmol/L) and tea polyphenols (100μg/mL) decreased the expression of mRNA of Bcl-2and increased the expression of Bax mRNA, and the two drugs showed a synergistic effect.6. The results of Western Blot showed that the celecoxib (10μmol/L) and tea polyphenols (100μg/mL) alone and in combination can significantly reduce the expression of anti-apoptotic protein Bcl-2and increase the expression of protein Bax. They can also decrease the expression of apoptosis-related protein including cleaved caspase-3, and cleased caspase-9which are the active forms of protein caspase-3and caspase-9. The differences between combination groups and single drug groups were statistically significant (P<0.05).Conclusions:1. Celecoxib and tea polyphenols alone and in combination can inhibit the proliferation of breast cancer cell MDA-MB-231. The inhibition rate of proliferation in combined groups was significantly higher than that of single drug groups. The result showed that the two drugs using together can enhance the effects of single drugs to inhibit the cell proliferation.2. Celecoxib and tea polyphenols alone and in combination could induce cell apoptosis, and the two drugs showed a synergistic effect.3. The mechanisms of apoptosis induced by celecoxib combined with tea polyphenols may be that they acted on the targets Bcl-2and Bax, down-regulated the gene of Bcl-2and up-regulated the gene of Bax. The changes actived the caspase-9and caspase-3, thereby triggered cascade reaction of downstream and induced apoptosis through mitochondrial pathway.