The 3-fold increase in luminescence intensity (Figure ?(Figure8D)8D) of pGL3-FASN when compared to that of pGL3 suggests the involvement of OPG in the upregulation of FASN gene transcription. Celecoxib and C75 induces cell apoptosis via caspase 9/3 dependent pathway Since SUM149PT and SUM1315MO2 cells have upregulated FASN and COX-2 pathways, we screened the effects of FASN inhibitor C75 and COX-2 inhibitor celecoxib or both in combination on the breast cancer cell lines using caspase 9 and caspase 3/7 assays. upregulation of the COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells. Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and fatty acid synthase (FASN). FASN is a multifunctional enzyme involved in lipid biosynthesis. Immunofluorescence staining revealed the Rabbit Polyclonal to CEP78 co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be crosstalk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Interestingly, knocking down OPG by CRISPR/Cas9 gene editing in breast cancer cells decreased FASN expression at the protein level. Here, we CCT245737 identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 and COX-2 inhibitor celecoxib individually decreased the number of lipid bodies/cell, downregulated phosphorylation of ERK, GSK3, and induced apoptosis by caspase-3/7 and caspase-9 activation. But a more efficient and effective decrease in lipid bodies/cell and survival kinase signaling was observed upon combining the drug treatments for the aggressive cancer cells. Collectively, the novel biological crosstalk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic target to treat highly invasive breast cancer. observations, we analyzed breast tissue sections from invasive breast cancer patients for the presence of OPG, FASN and PGE2 by immunofluorescence staining (Figure ?(Figure6A).6A). Abundant OPG, FASN and PGE2 expression were detected in breast cancer tissue sections (Figure ?(Figure6A).6A). There was consistent elevated expression of FASN throughout the breast cancer tissue samples (Figure ?(Figure6A).6A). However, OPG and PGE2 were expressed in a spatial-temporal mutually exclusive manner (Figure ?(Figure6A).6A). However, little significant staining of OPG, FASN and PGE2 was observed in control breast tissue sections (Figure ?(Figure6B).6B). Collectively these results for the first time show the co-existence of OPG, PGE2 and FASN in breast cancer tissues when compared to the control breast tissues sections. Open in a separate window Figure 6 OPG, FASN and PGE2 CCT245737 expression is significantly elevated in patient invasive breast cancer tissueA. Breast cancer tissue samples were analyzed by immunofluorescence and confocal microscopy staining for OPG (red), FASN (orange) and PGE2 (green). Nuclei were counterstained with DAPI. Magnification is 60X. B. Normal control breast tissue samples were analyzed by immunofluorescence and confocal microscopy staining for OPG (red), FASN (orange) and PGE2 (green). Nuclei were counterstained with DAPI. Magnification is 60X. We also performed RT-PCR for profiling the gene expression of OPG, CCT245737 CCT245737 FASN, COX-2 and mPGES-1 in inflammatory breast cancer patient samples. Almost ~ 15 and ~6 fold higher expression of COX-2 and mPGES-1 was observed in IBC samples when compared to control normal human mammary tissue samples(Figure ?samples(Figure7).7). Remarkably very high gene expression of OPG and FASN was observed in IBC samples in comparison to normal healthy samples (Figure ?(Figure77). Open in a separate window Figure 7 Upregulation of COX-2, mPGES-1, FASN and OPG expression in patient inflammatory breast cancer tissuecDNAs isolated from patient inflammatory breast cancer tissue and normal healthy mammary tissue samples were subjected to quantitative real time PCR analysis for the gene expression of COX-2, mPGES-1, FASN and OPG using their specific primers. Each point represents the average +/? SD from three independent experiments Effect of OPG on COX-2 promoter transcription regulation via NF-B regulatory element It is well known that COX-2 promoter activity is regulated in other systems by several transcription factors, including NF-kB, nuclear factor IL-6, AP-1, CRE,.