Therefore, Hec1 emerges as an excellent target for treating cancer clinically. Small molecules targeting the Hec1 Nek2 pathway was first discovered by Drs. Chen in the laboratory of Dr. W. H. Lee using the inducible reverse yeast two hybrid screening of a library of 24,000 compounds. A series of compounds was designed based on this pub lished initial hit molecule as the starting template to optimize the potency for drug development. The original template with micromolar in vitro potency was improved to low nanomolar potency, enabling possible clinical utility of the Hec1 targeted compound. This study explores the features and potential of the improved anticancer agent targeting Hec1, TAI 1, for preclinical development and clinical utility.
The in vitro and in recommended reading vivo biological activity, mechanism of action, toxicity and safety, and transla tional implications are investigated. Methods Cell lines Development Center for Biotechnology, New Taipei City, Taiwan, MDA MB 453, T47D, ZR 75 1, ZR 75 30, MDA MB 361, Hs578T, NCI H520, Hep3B, PLC PRF 5 were from Bioresource Collection and Research Center, Hsinchu, Taiwan. Cell lines were maintained in complete 10% fetal bovine serum and physiologic glucose in DME. Studies conducted using cell lines RPMI8226, MOLT 4, and N87, drug resistant cell lines MES SA Dx5, NCI ADR RES, and K562R were from and tested by Xenobiotic Laboratories, Plainsboro, NJ, USA. In vitro potency assay Cells were seeded in 96 well plates, incubated for 24 hours, compounds added and incubated for 96 hours. All testing points were tested in triplicate wells.
Cell viability was determined by MTS assay using CellTiter 96 Aqueous Non radioactive Cell Proliferation Assay system according to manu facturers instructions {over at this website| selleck|selleckchem|selleck chemicals|LDC000067 ic50 with MTS and PMS. Data retrieved from spectropho tometer were processed in Excel and GraphPad Prism 5 to calculate the concentration exhibiting 50% growth inhibition. All data represented the results of triplicate experiments. Immunoblot and co immunoprecipitation analysis Western blotting and co immunoprecipitation were done as described previously. Primary antibodies used, mouse anti Nek2 and mouse anti Mcl 1, rabbit anti Hec1, mouse anti actin, mouse anti P84 and mouse anti RB, rabbit anti Cleaved Caspase3, rabbit anti Cleaved PARP, rabbit anti XIAP, and mouse anti P53, mouse anti Bcl 2, mouse anti Tubulin.
For co immunoprecipitation, cells were lysed in buffer, 250 mM NaCl, 5 mM EDTA, 0. 1% Triton X 100, 1 mM PMSF, 50 mM NaF, and protease inhibitor cocktail for 1 hour then incubated with anti Nek2 antibody or IgG as control for 4 hours at 4 C, collected by protein G agarose beads and processed for immunoblotting. Immunofluorescent staining and microscopy For quantification of mitotic abnormalities, cells were grown on Lab Tek II Chamber Slides, washed with PBS buffer before fixation with 4% paraformalde hyde.
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