The Intrabody Targeting COX-2 Inhibits Growth And Metastasis Of Breast Cancer Cells

Breast cancer is a common malignant tumor in women with the highest mortality.Although early diagnosis and comprehensive treatment?including surgery,adjuvant chemoradiotherapy,endocrine therapy,immunotherapy,and targeted therapy?have been performed and good therapeutic effects have been obtained in a certain extent.However,since brast cancer cells are susceptible to drug resistance and high metastasis,breast cancer tends to recur afterwards.Therefore,exploring more safe and effective strategies for the treatment of breast cancer has important scientific and practical value.Cyclooxygenase-2?COX-2?is a key rate-limiting enzyme that catalyzes the production of prostaglandins?PGs?by arachidonic acid?AA?.A number of studies have shown that COX-2 is overexpressed in brast cancer cells and participates in the occurrence and development of breat cancer.For example,COX-2 is mainly involved in the proliferation and apoptosis of breast cancer cells,immunosuppression,induction of various carcinogens,and lead to tumorigenesis and metastasis by promotion of tumor angiogenic factors,and so on.So COX-2 has become a potential target for the treatment of breast cancer.Intrabody?or intracellular antibody?technology,which allows antibodies to be expressed in cells by means of genetic engineering,can achieve targeted interference and block the process of transcription,translation,processing,and modification of intracellular antigen proteins.Therefore,in our previous studies,an anti-COX-2intrabody Intra-OX-1 was successfully constructed.The results have shown that the intrabody against COX-2?Intra-OX-1?can target COX-2 and inhibits HepG₂ cell growth and proliferation,promotes cell apotosis.Intra-OX-1 shows a good anti-tumor effect.But it is unclear whether it can effectively inhibit the proliferation and migration of breast cancer cells.In this study,we investigated the inhibitory effects of Intra-OX-1 on the breast cancer cell growth and migration.The details are as follows:The expression of COX-2 in breast cancer cells was detected by immunofluorescence and it was overexpressed and mainly distributed in the cytoplasm of MCF-7 cells.The anti-COX-2 intrabody was transfected into MCF-7breast cancer cells.Then the expression levels of the fusion proteins were analyzed by RT-PCR,indirect immunofluorescence and westrn blot.The results demonstrated that Intra-OX-1 was efficiently expressed in MCF-7 cells and retained in endoplasmic reticulum.The binding activity between Intra-OX-1 and endogenous COX-2 is measured by co-IP.The results showed that Intra-OX-1were found to be able to bind specifically to COX-2 in MCF-7 cells.It was found that Intra-OX-1 can significantly inhibit the proliferation of MCF-7 cells and the colony formation of cells by MTT and colony formation experiments.Intra-OX-1 also can obviously induce the apoptosis of MCF-7 cells by flow cytometry detection.In the"wound healing"assay,the Intra-OX-1 group significantly inhibited the migration of MCF-7 cells.Transwell experiments showed that the Intra-OX-1 expression significantly inhibited the invasion of MCF-7 cells in vitro.Finally,using real-time fluorescence quantitative PCR to detect the expression of m RNA involved in tumorigenesis and development in breast cancer cells stably expressing Intra-OX-1.The results showed that Intra-OX-1 can down-regulate the expression of EGFR,VEGF,CDK1,CDK2,Bcl-2,c-Met mRNA,and up-regulate the transcription of p21.This study lays the foundation for breast cancer treatment with intrabody targeting COX-2.