The second E3 lig ase, SCFB TrCP, was discovered to only recognize Mcl 1 that has been phosphorylated by GSK3 at Ser159. This interaction between SCFB TrCP and Mcl 1 is facili tated by phosphorylation of the same selleck Y-27632 serine and threo nine residues that have been identified previously as potential sites of recognition by the X linked ubiquitin specific peptidase 9, a deubiquitinase. Hence, it is possible that SCFB TrCP and USP9X compete for Mcl 1 binding. USP9X binds Mcl 1 protein and removes the Lys 48 linked polyubiquitin chains that normally mark it for proteasomal degradation. Mcl 1 ubiquitination is thus offset by the activities of USP9X and it has been reported that increased USP9X expres sion correlates with increased Mcl 1 protein levels and a poor prognosis in lymphoma patients.
The silencing of USP9X using siRNAs increases the sensitivity of CML cells, to imatinib and other apoptotic stimuli. The deubiquitination activities of USP9X can be inhibited by WP1130, a partially selective DUB inhibitor. It has been demonstrated in this regard that a reduction in the Mcl 1 levels in WP1130 treated cancer cells parallels the inhibition of USP9X activity. In our current study, we further tested the hypothesis that Mcl 1 and Bcl xL are both overexpressed in colon and lung cancers. Our analysis reveals that the overex pression of both of these anti apoptotic proteins causes resistance to chemotherapeutic agents. In addition, the blocking of USP9X activities using a small molecule in hibitor decreases Mcl 1 expression by promoting its degradation and thus sensitizes tumor cells to che motherapeutic agents.
Methods Cell culture I45, REN, A549, H1299 and H23 as well as DLD 1 and HCT116 were purchased from the American Type Culture Collection. DLD 1, H1299, H23, I45 and REN were cultured in 10% fetal bovine serum supplemented RPMI 1640 medium. A549 cells were cultured in 10% FBS supplemented F12 medium. HCT 116 cells were cul tured in McCoys 5A medium containing 10% fetal bovine serum. Authentication of these cell lines was per formed by the ATCC. Reagents Cycloheximide, 5 FU, Taxol, PS341, WP1130 and ABT 737 were obtained from Selleck Chemicals. The HDAC inhibitor SAHA was purchased from Biovision. The rabbit anti human USP9X poly clonal antibody used was obtained from Bethyl Laboratories. Rabbit antibodies against Bcl xL and Mcl 1 were purchased from Santa Cruz Biotechnology Inc.
Mouse monoclonal anti B actin was obtained from Sigma. The siRNA transfection reagents, and siR NAs targeting Bcl xL, Mcl 1 and a control scrambled siRNA, were obtained Cilengitide from Ambion Biotechnology, Inc. Apoptosis assay After various treatments, cancer cells were stained for Annexin V using a FITC Annexin V staining kit and then measured with BD FACSCanto II Flow cytometry.