actin monoclonal antibody was purchased from Sigma Aldrich Chemical Co. The polyclonal antibody to TIMP 2 was bought from R D Systems. Phospho extracellular signal regulated kinase 12, ERK12, phospho p38, p38, phospho c Jun N terminal kinase, and JNK antibodies have been pur chased from Cell Signaling. JS K and JS 43 126, a JS K analog that does not release NO, had been ready as previously described. Stock solutions of JS K and JS 43 126 had been ready in dimethylsul foxide and had been stored at 20 C. The structures of JS K and JS 43 126 are presented in Figure 1. Cell lines and culture conditions The human MDA MB 231 breast cancer cell line was obtained from American Type Cell Culture. The MDA MB 231 cell line is an estrogen independent, highly met astatic human breast cancer cell line.
Breast cancer generally metastasizes for the skeletal technique. MDA MB 231F10 is usually a bone metastatic derivative of MDA MB 231 cells chosen in vivo by repeated intracardiac injections selleckchem Paclitaxel with the MDA MB 231 cells into female nude mice till no micrometastases were detected histologically in any organs other than bone. The F10 cell line was kindly provided by Dr Toshiyuki Yoneda. Breast cancer also commonly metastasizes to lymph nodes. Elevated COX two expression in invasive breast tumor is associ ated with lymph node metastasis. MCF 7COX two cells are estrogen dependent MCF 7 cells stably transfected with plasmids encoding the human COX two gene. The parental MCF 7 cells are poorly invasive but the MCF 7COX 2 cells are highly invasive.
The MDA MB 231 and F10 cell lines had been cultured in DMEM F12 supplemented with 5% heat inactivated FBS at 37 C under 5% carbon dioxide in a humidified incubator. MCF 7COX 2 cells were constantly cultured in DMEMF12 medium containing 5% FBS and 500g ml antibiotic G418. Western blot analysis Protein lysates from untreated exponentially developing MDA MB 231, selleck chemical F10, and MCF 7COX two breast cancer cells were loaded onto 15% polyacrylamide gels to decide the expression of GST and GST. The MDA MB 231 cells, F10 cells, and MCF 7COX two cells were plated in T25 flasks in five ml DMEMF12 medium supplemented with 5% FBS. The next day, cells had been treated with JS K for 24 hours. Protein lysates were loaded onto 12% polyacrylamide gels to establish the activity and expression of ERK12, p38, and JNK mitogen activated protein kinases. Proteins were electro phoresed and electrotransferred as described previously.
Membranes were incubated with the suitable antibodies. actin was used as a loading manage. Protein bands were vis ualized by enhanced chemiluminescence. Pictures had been scanned and quantified by an Alpha Innotech densitometer employing the Alpha Imager application system. Nitric oxide assay The MDA MB 231 cells, F10 cells, and MCF 7COX 2 cells had been plated in T25 flasks in 5 ml DMEMF12 medium supplemented with 5% FBS.