Fig 2: The TXA2 pathway is required for maintenance of malignant characteristics of MCF-7, T47D, and SK-BR-3 breast cancer cells. a Knockdown of TBXAS1 inhibits anchorage-independent growth of human breast cancer cells. b Knockdown of TBXA2R inhibits anchorage-independent growth of human breast cancer cells. Knockdown of TBXAS1 or TBXA2R in breast cancer cells was analyzed by Western blot. Anchorage-independent cell growth was evaluated by colony formation in soft-agar. Data are presented as mean values ± S.E.M. from three independent experiments. The asterisks (***) indicate a significant (p < 0.001) decrease compared to the Mock group. c Effect of ectopic expression of TBXAS1 on anchorage-independent cell growth and invasion. At 50–60% confluence, 4T1 cells were transiently transfected with either an empty vector (pcDNA3.1) or a TBXAS1 plasmid (pcDNA3.1-V5-TBXAS1). After 24 h, G418 (1200 ng/mL) was added for selection of stable subclones. After 3 week, the stable clones obtained were verified by Western blot. Anchorage-independent cell growth was evaluated by colony formation in soft-agar. Cell invasion was determined by transwell assay. Data are presented as mean values ± S.E.M. from three independent experiments. The asterisks indicate a significant difference compared with vector group (**p < 0.01; ***p < 0.001). d Effect of NSAIDs on cell invasion. 4T1 cells were plated in a Matrigel-coated upper chamber with or without NSAIDs in the medium. After incubation for 24 h, non-migrated cells on the upper surface of the filter were wiped out, and cells on the lower surface of the membrane were stained with crystal violet and counted under a light microscope. The asterisks indicate a significant difference compared with the control group (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig 3: Aspirin reduces CRC risk in FAP patients by targeting the TXA2 pathway. (A) Effects of regular aspirin use on circulating PG biosynthesis in FAP patients. 1 = healthy subjects (n = 16); 2 = FAP patients, aspirin nonusers (n = 24); and 3 = FAP patients, aspirin users (n = 14). FAP patients who reported taking two or more standard (325 mg) aspirin tablets per week were classified as regular aspirin users and those taking less aspirin were defined as aspirin nonusers. Data are presented as means ± S.D. The asterisks (***) indicate a significant (p < 0.001) decrease in circulating TXA2 levels associated with aspirin intake. (B) Effects of regular aspirin use on the expression patterns of TBXA2R, TBXAS1 and Ki-67 in FAP patients. Original magnification: 200 ×. Immunostaining intensities are defined in “Materials and Methods.” (C) Effects of aspirin treatment on TBXA2R and TBXAS1 protein expression in colon cancer cells. HT29 and HCT116 colon cancer cells were treated with aspirin for 72 h. After treatment, cell lysates were subjected to Western blot analysis as described in “Materials and Methods”.

Fig 4: Pathophysiological role of the TXA2 pathway in CRC. (A) Immunohistochemical staining of TBXA2R, TBXAS1 or mPGES-1 in biopsy samples, which included normal colonic mucosa, polyps, adenomas, and adenocarcinomas. For antibody-negative controls, the primary antibodies were substituted with normal rabbit serum. Original magnification: 200 ×. (B) The TXA2 pathway is associated with tumorigenic properties in human colorectal cancer cells. Knockdown of TBXA2R or TBXAS1 in HT29 or HCT116 colon cancer cells was analyzed by Western blot (upper panels). Mock and knockdown HT29 and HCT116 colon cancer cells were then subjected to anchorage-independent growth assays (lower bar graphs) as described in “Materials and Methods”. The asterisks (***) indicate a significant (p < 0.001) decrease in colony formation by knockdown HT29 or HCT116 colon cancer cells.

Fig 5: Chemoprevention of HER2-positive breast cancer by targeting theTXA2 pathway. a TBXAS1 is up-regulated in HER2-driven mammary tumorigenesis in an MMTV-Her2/neu transgenic mouse mammary tumor model. Immunohistochemical staining of TBXAS1 in mouse mammary tumors or normal mammary tissues. Original magnification: 200×. b Switching off TXA2 biosynthesis attenuates HER2-driven breast tumorigenesis. MMTV-HER2/neu transgenic mice were treated with EPA for a total of 60 weeks. c Effects of EPA intake on circulating TXA2 and PGE2 levels. d Effects of EPA intake on TBXAS1 or TBXA2R in the MMTV-neu mammary glands and tumors. Because the FVB background of MMTV-Her2/neu transgenic mice, matched non-transgenic FVB mice were used as a baseline to minimize the genetic differences. Production of serum PGs was measured by ELISA as described in “Materials and Methods”. Data are presented as mean values ± S.E.M. The asterisks indicate a significant difference among groups (*p < 0.05, ***p < 0.001)