Recently, however,

Recently, however, during systemic delivery of siRNA com bined with a special nanoparticle successfully knocked down a target gene in melanoma in a clinical trial. The use of such a technique to attempt specific knock down of SON in pancreatic cancer cells in a clinical model is worth trying Inhibitors,Modulators,Libraries and is an issue to be resolved in a future study. The results of this study also suggest that develop ment of a molecule oriented chemical substance against SON as therapy for pancreatic cancer is warranted. Conclusion This study indicates that SON is overexpressed and plays a critical role in the proliferation, survival, and tumorigenicity Inhibitors,Modulators,Libraries of pancreatic cancer cells, suggesting that SON is a novel therapeutic molecular target for pancre atic cancer.

Methods Cell culture Human pancreatic cancer cell lines, MIA PaCa 2 and PCI 35, and Inhibitors,Modulators,Libraries the human embryonic kidney cell line 293 were obtained and cultured as previously described. The immortalized human pancreatic duct epithelial cell line, HPDE, was kindly provided by Dr. MS Tsao and cultured as previously described. Transfection of siRNA and cell proliferation assay siRNAs targeting each downstream MAPK associated molecule were custom designed and manufactured. Cells were seeded at 5 103 cellswell in Inhibitors,Modulators,Libraries 96 well plates with 100 uL of appropriate culture medium and incubated at 37 C with 5% CO2 for 24 hours. Then, the medium was replaced with OPTI MEM, and the cells were transfected with siRNA at 10 nM with Oligofectamine according to the manufacturers recommendations. After 4 hours of incu bation, the transfection reagent was replaced with the ap propriate culture medium.

A colorimetric cell proliferation assay3 2,5 diphenyltetrazolium bromide assaywas performed daily for 5 days as previously described. Colony formation assay with shRNA vectors pSUPER vector was used for the construction Inhibitors,Modulators,Libraries of vectors expressing shRNAs by cloning the oligonucleotides o serve as a control harboring a nonspecific sequence against the human genome according to the manufac turers instructions. MIA PaCa 2 and PCI 35 cells were seeded at 1 105 cellswell in enzalutamide mechanism of action 6 well plates and incubated for 24 hours at 37 C with 5% CO2. The shRNA SON vec tor or shRNA Nons vector were transfected into the cells with LipofectamineTM reagent accord ing to the manufacturers recommendations. The cells were dissociated with trypsin 48 hours after transfection and reseeded in three 10 cm tissue culture dishes, con taining the appropriate culture medium supplemented with 10% FBS and G418 at 400 ugmL for PCI 35 and 500 ugmL for MIA PaCa 2. After 3 weeks, the cells were fixed with 10% formalin solution and stained with hematoxylin. The number of colonies was assessed with the COLONY program.

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