used to dock the SM testosterone into protein
used to dock the SM-934_testosterone into protein 5TDI. Five optimal poses of SM934-Testosterone were output.
2.11. Pathway enrichment
KEGG Mapper, as one of the most trendy pathway analysis tools currently, was used to retrospect the biological function of Cathepsin K in the context of the whole organism as complicated system. In KEGG Mapper webpage, we input “CTSK” in the box of “Enter objects” column and click “Exec” button to start search. Then the pathway search results appeared in a new page. European Journal of Pharmacology 858 (2019) 172382
2.12. The establishment of breast cancer xenotransplantation model and analysis of tumors
In the breast cancer subcutaneous xenograft model, 6-week-old BALB/c nude mice were randomly divided into five groups and there were five mice per group. Approximately 5 × 106 MDA-MB-231 Chloramphenicol in 120 μl of physiological saline were implanted into the subcutaneous tissues of the mice's right armpits. Then, the tumor volumes were measured with calipers every two days, and the volumes of tumors were calculated by the following formula: volume = length × width2/2. After the tumors' volumes reached to 100 mm3, different groups of the mice were given SM934, Testosterone, the combination of SM934 and Testosterone, and SM934-Testosterone by intragastrical administration every day. The control group was treated with physiological saline. After 25 days, the mice were killed and tumors were collected. The weight and volumes of different groups' tumors were measured and analyzed.
2.13. Statistical analysis
All experiments were performed at least three times, and all values are presented as the mean ± standard deviation (S.D.). All statistical analyses were conducted using GraphPad Prism software. Student's t-tests were employed for analyzing the differences among individual groups. The significance of difference was described as *P < 0.05; **P < 0.01; ***P < 0.001. P-value < 0.05 were considered sig-nificant.
3.1. New compound SM934-Testosterone inhibited the survival of breast cancer cells MDA-MB-231 and SK-BR-3
In our former study, the toxicity of SM934-Testosterone was ex-amined on gastric cancer cells and prostate cancer cell and the new compound showed no effect on these cancer cells. Here MDA-MB-231 and SK-BR-3 cells were treated with gradually-increased concentrations of SM934 - Testosterone for 24 h, 48 h, and 72 h. The results of the MTT viability assay in Fig. 2A showed that SM934-Testosterone dose and time-dependently inhibited the survival rate of MDA-MB-231 cells and SK-BR-3 cells. MTT assays also showed that the pro-drugs of SM934-Testosterone including SM934 and Testosterone and their combination nearly had no effect on the survival rate of MDA-MB-231 and SK-BR-3 (Fig. 2B). Human normal liver cells L02 were also treated with gradu-ally-increased concentrations of SM934 - Testosterone for 48 h, and the results showed that the novel compound had little toxicity on human normal liver cells (Fig. 2C). All the IC50 ± S.D. of the above MTT
Fig. 1. (A) Chemical structure of SM934. (B) Chemical structure of Testosterone. (C) Chemical structure of the newly synthesized anticancer agent (SM934-Testosterone).
assays were summarized in Fig. 2D.
3.2. SM934-testosterone promoted apoptosis of breast cancer cells but had nearly no effect on tumorigenesis in vivo
Cell apoptosis analysis was exerted to investigate whether SM934-Testosterone inhibited cell proliferation by promoting apoptosis in breast cancer cells MDA-MB-231 and SK-BR-3. The cells were treated with SM934, Testosterone, the combination of SM934 and Testosterone, and the new compound SM934-Testosterone for 48 h, respectively. As shown in Fig. 2E–F, SM934-Testosterone markedly induced apoptosis of breast cancer cells both of MDA-MB-231 and SK-BR-3. This finding elucidated that the inhibition of cell proliferation was partially owing to the induction of cell apoptosis by SM934-Tes-tosterone.