and Immunoblotting Protein extraction, SDS PAGE separation of proteins and Western blot analysis were performed as described previously. Cells were lyzed in an M PER mammalian cell protein extraction buffer supplemented with Na3VO4 and protease inhibitor cocktail and followed by freeze Chrysin 480-40-0 and thaw three times. After being kept on ice for 40 min, the extracts were centrifuged at 15,000g for 15 min 4. The supernatant was designated as the cell lysate. The complex formation of uPAR with other signaling molecules was determined by immunoprecipitation according to the methods described by Nykjaer et al with some modifications. Cell lysate was incubated with corresponding antibodies followed by incubation of protein A/G beads. The immunoprecipitates were subjected to SDS PAGE under non reduced conditions, and immunoblot analysis was performed as described below.
Separately, the immunoprecipitated complex or the cell lysate containing equal amounts of protein were solubilized in Laemmli,s sample buffer and were subjected to SDS PAGE. Separated proteins were then transferred onto nitrocellulose Cryptotanshinone 35825-57-1 membranes. Membranes were blocked with 5% nonfat dry milk in Tris buffered saline containing 0.05% Tween 20 and then probed with antibodies as indicated. Immunoblots were visualized by an enhanced chemiluminescence kit and analyzed by densitometry. Data were obtained from three independent experiments. Immunofluorescence Microscopy Cells grown on coverslips were treated as indicated in the figure 3 legend. Cells were fixed and processed as described. Cells were stained with anti uPAR and anti EGFR antibodies in 0.
1% BSA/PBS, or with vehicle alone. After washing and blocking, secondary antibody in 0.1% BSA/PBS containing DAPI was added. Standard epifluorescence was captured with an Axioskop epifluorescence photomicroscope. Statistical Analysis Statistical analyses were performed by One Way Analysis Of Variance and all pairwise multiple comparison procedures. Results were considered significant when P0.05. The result presented as mean SEM. RESULTS HKa and D5 inhibit migration and invasion of prostate cancer cell Growth factors induce uPAR internalization by initially activating pro uPA followed by complex formation with PAI 1 and interaction of the ternary complex uPAR/uPA/PAI 1 with a member of the LDL receptor like family.
During cell migration, uPAR is redistributed to focal adhesions at the leading edge either by lateral movement or by internalization and recycling of the receptor. We previously showed that binding of HKa or D5 to uPAR could prevent the process of uPAR internalization and inhibit endothelial cell migration. We postulated that HKa and D5 also would inhibit the migration of tumor cells expressing high levels of uPAR. We evaluated the inhibitory potential of HKa and D5 on a human prostate tumor cell line, DU 145, which expresses high levels of uPAR Liu et al. Page 4 Oncogene. Author manuscript, available in PMC 2010 April 28. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript. In fig. 1, bFGF induced cell migration was significantly decreased to 242.4% by HKa while D5 inhibition on cell migration at 33.3, 100 and 300 nM was 360.6, 413.4 and 505.7%, respectively. The inhibition of cell migration by HKa is significantly greater than D5 . uPA is synthesized as a 55 kDa single chain proenzyme and converted into the two cha