br were not Moreover Cathepsin
were not. Moreover, Cathepsin K has been reported to be highly ex-pressed in breast cancer, and was related to the migration and invasion ability of breast cancer cells (Brubaker et al., 2010).
According to this, Glide docking was further conducted in Maestro program of Schrödinger to observe the binding position between Cathepsin K and SM934-Testosterone. Comparing Fig. 4B and C, the 2D protein-ligand interaction diagrams revealed that the SM934-Testos-terone could form a good hydrogen bond with GLY 66 and ASN 161 alike 7AS, but 7AS can form Pi-Pi stacking with TYR 67. Fig. 4F de-monstrated the 3D protein-ligand interaction of SM934-Testosterone.
protein-ligand interaction surface status. We could find that SM934-Testosterone was well wrapped in the active pocket. SM934 and Tes-tosterone + linker were also docked with Cathepsin K, respectively. As shown in Fig. 4D&E, SM934 and Testosterone did not bind well to Cathepsin K compared to SM934-Testosterone. The docking results of SM934 showed no hydrogen bonding interaction, probably because the space volume of SM934 was not large enough to occupy this binding pocket on the periphery of Cathepsin K. Testosterone + linker is mainly a nitrogen Fasudil on Linker that forms a hydrogen bond interaction with ASP 61 and GLY64, and an anthracene ring moiety can form a hydro-phobic interaction.
3.5. SM934-testosterone suppressed cathepsin K expression
Based on the docking result of SM934-Testosterone and Cathepsin K, we further measured whether SM934-Testosterone could suppress the expression of Cathepsin K in breast cancer cells MDA-MB-231 and SK-BR-3. The data of quantitative real-time PCR analysis revealed that the level of Cathepsin K mRNA was suppressed in cells treated with SM934-Testosterone compared to other groups of cells (Fig. 5A). Meanwhile, the protein level of Cathepsin K in breast cancer cells treated with SM934-Testosterone was also detected, and the results showed that the expression level of Cathepsin K protein was diminished in cells treated with SM934-Testosterone (Fig. 5B).
3.6. SM934-testosterone inhibited the breast cancer cells proliferation, migration and invasion via regulating the expression of cathepsin K To further ascertain the role of Cathepsin K expression in SM934-Testosterone mediated breast cancer cells MDA-MB-231 and SK-BR-3 suppressive activity, the shRNA of Cathepsin K was constructed. As is shown in Fig. 5C–D, the mRNA and protein levels of Cathepsin K were significantly knocked down in MDA-MB-231 and SK-BR-3 cells trans-fected with Cathepsin K shRNA. After verifying the efficiency of the Cathepsin K expression knock-down plasmid, colony formation assay was performed. Colony numbers in MDA-MB-231 and SK-BR-3 cells transfected with Cathepsin K shRNA significantly reduced (Fig. 6A), which indicated that down-regulation of Cathepsin K suppressed cell proliferation in breast cancer cells. Moreover, down-regulation of Ca-thepsin K triggered cell apoptosis in MDA-MB-231 and SK-BR-3 cells, while rescuing the low-expression of Cathepsin K caused by SM934-Testosterone using Cathepsin K overexpression plasmid showed the opposite results compared with the cells treated with SM934-
Testosterone (Fig. 6B). Furthermore, wound healing assay and trans-well assays showed that down-regulation of Cathepsin K suppressed both invasion and migration in breast cancer cells MDA-MB-231 while the metastasis ability of cells treated with the combination of Cathepsin K overexpression plasmid and SM934-Testosterone was rescued com-pared to the cells treated with SM934-Testosterone. (Fig. 6C–D). The experiments on SK-BR-3 also showed the same results (Fig. 6C–E). In summary, regulating the expression of Cathepsin K by applying shRNA or overexpression plasmid could affect the proliferation and metastasis ability of breast cancer cells MDA-MB-231 and SK-BR-3. According to this, it is concluded that SM934-Testosterone could inhibit the pro-liferation, migration and invasion ability of breast cancer cells via regulating the expression of Cathepsin K.