Regulation Of COX - 2 On Metastasis And Chemoradiotherapy Of Breast Cancer And Its Mechanism

Part I COX-2promotes breast cancer metastasis through PI3K/AKT signaling pathwayPurpose:Breast cancer is the sixth cause leading to women’s death in China, as result of distant recurrence and metastasis before or after surgery and standard treatment. In this study, we tried to demonstrate the relationship between cyclooxygenase-2(COX-2) and breast cancer metastasis.Methods:COX-2expressions were detected by reverse transcription-PCR in normal breast epithelial cell line and9human breast cancer cell lines. The effects of COX-2on the proliferation, migration and invasion in vitro were tested by CCK8, scratch-wound assay and transwell using the COX-2-overexpressing cells (MCF7-COX-2, SKBR3-COX-2, MDA-MB-231-COX-2) and COX-2-silencing cells (MDA-MB-231-COX-2i, MDA-MB-231HM-COX-2i, MDA-MB-231BO-COX-2i) which were established by stable transfection. We established an orthotopic xenograft tumor model in BALB/c nude mice using the above6cell lines and explored the influence of COX-2overexpression or knockdown on tumor growth and metastasis in vivo. Protein expression in cell lines was examined by Western blot.Results:COX-2expression is higher in high-metastatic cell lines than in low-metastatic cell lines. Overexpression of COX-2promotes cell proliferation, migration, invasion and tumor formation and metastasis, but silencing of COX-2inhibits those activities both in vitro and in vivo. Mechanism studies revealed that COX-2suppresses AKT1to promote breast cancer metastasis by regulating E-cardherin and p-catenin.Conclusion:Our results suggest that COX-2controls breast cancer metastasis through regulating AKT1. Thus, antagonists of COX-2may be used to treat breast cancer patients particularly with high metastasis. Part II COX-2promotes breast cancer cell chemoresistance via inhibiting p53-mediated cellular pro-apoptosisPurpose:A major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. However, the mechanisms underlying this drug resistance are unknown. In this study, we investigated the role of COX-2in breast cancer chemoresistance.Methods:The function role of COX-2was investigated by using a chemo-resistant breast cancer cell line MDA-MB-231Gem and its parental cell line MDA-MB-231cells before or after COX-2was silenced by a specific shRNA. The IC50and apoptosis were measured by MTT and flow cytometery. Protein expression was analyzed by Western blot.Results:Under the same dose treatment of Gemcitabine, overexpression of COX-2promotes chemoresistance, leading to decreased apoptotic cells and increased IC50. However, silencing of COX-2inhibits these activities. COX-2is upregulated in MDA-MB-231Gem cells compared with in MDA-MB-231cells, and silencing of COX-2expression by shRNA in MDA-MB-231Gem cells increases the number of apoptotic cells and decreases the IC50, leading to the enhanced chemosensitivity. Mechanism studies revealed that silencing of COX-2activates p53phosphorylation (Serine15) and E2F1expression, but inhibits MDM2, Bcl-xL and Bcl-2, leading to decreased anti-apoptosis.Conclusions:Our data suggests that inhibition of COX-2could reduce chemoresistance of breast cancer cells by activating p53-mediated apoptosis signal pathway, and may be considered as potential thereapeutic targets against chemoresistant breast cancer.Part III COX-2promotes breast cancer cell radioresistance via p38/MAPK-mediated cellular anti-apoptosis and invasivenessPurpose:Radioresistance is one of the major barriers to improve the survival rate of breast cancer patients. COX-2is usually overexpressed in highly invasive and metastatic breast cancer, which may indicate an association with breast cancer radioresistance. Methods:The function role of COX-2was investigated by using a radioresistant breast cancer cell line MDA-MB-231RR10and its parental cell line MDA-MB-231cells before or after COX-2was silenced by a specific shRNA. The cell proliferation, migration, invasion, colony formation and apoptosis were measured by CCK-8, scratch-wound, transwell, clone formation assay, and flow cytometery. Protein and mRNA expression were analyzed by Western blot and quantitative reverse transcription-polymerase chain reaction.Results:COX-2is upregulated in MDA-MB-231RR10cells compared with in MDA-MB-231cells, and silencing of COX-2expression by shRNA in MDA-MB-231RR10cells decreases the expression of Bcl-2and Bcl-XL, but increases the pro-apoptotic protein BAK, leading to the increased apoptosis following treatment with gamma-irradiation in comparison with those in control cells. Silencing of COX-2also increases the expression of β-catenin and E-cadherin, two anti-invasion proteins, resulting in reduced cell migration and invasion tested by transwell chambers and wound healing assays. Further study demonstrated that COX-2-induced radioresistance is negatively regulated through the phosphorylation of p38at Tyr182, and that the phosphorylation of p38induced by TNF-alpha reduces the expression of Bcl-2, BCL-XL, but increases β-catenin and E-cadherin, leading to the decreased invasiveness of cells.Conclusions:Our data suggests that COX-2, p38, Bcl-2, Bcl-XL, β-catenin, and E-cadherin may be considered as potential thereapeutic targets against radioresistant breast cancer.