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  • 4μ8C br significantly inhibit the viability of TNBC a

    2019-10-21

    
    significantly inhibit the viability of TNBC, a subgroup of breast cancer cell line MDA-MB-231 and another breast cancer 4μ8C SK-BR-3, and induce their ability of apoptosis (Fig. 2). One of the clinical features of TNBC was high metastasis. Metastasis can be diagnosed in 70% of pa-tients, which is a major death cause of breast cancer. According to this, the ability of metastasis of MDA-MB-231 cell line was also measured after being treated with the three drugs. Meanwhile, the metastasis ability of SK-BR-3 cells was also measured. Consistent with the result of the viability assay, the novel compound SM934-Testosterone sup-pressed the metastasis ability of breast cancer cells MDA-MB-231 and SK-BR-3 (Fig. 3). A xenotransplantation model of breast cancer was established in this study to explore the effects of SM934-Testosterone in vivo. As the results shown, SM934-Testosterone had little effects on tumorigenesis in vivo, at least in this model. The water-solubility of SM934-Testosterone was poor, so it could only be suspended by Car-boxymethyl (CMC) and given to mice by intragastrical administration, which would reduce the bioavailability of SM934-Testosterone. It was also analyzed that SM934-Testosterone may metabolize into its pro-drugs which would lead to the effects of SM934-Testosterone in vivo
    Fig. 5. SM934-Testosterone suppressed Cathepsin K expression (A) Quantitative real-time PCR analyzed the expression level of Cathepsin K mRNA in MDA-MB-231 and SK-BR-3 cells treated with different compounds. (B) Western blot was performed to analyze the expression level of Cathepsin K protein in MDA-MB-231 and SK-BR-3 cells treated with different compounds. (C) Quantitative real-time PCR assay showed the expression of Cathepsin K mRNA in MDA-MB-231 and SK-BR-3 cells transfected with Cathepsin K shRNA. (D) Western blot analyzed the protein expression level of Cathepsin K in MDA-MB-231 and SK-BR-3 cells, compared to the psilence group. *P < 0.05; **P < 0.01; ***P < 0.001. Each bar represents the mean ± S.D. of three independent experiments.
    Fig. 6. Regulating the expression of Cathepsin K had effects on the breast cancer cells proliferation, migration and invasion. (A) Colony formation assay showed the proliferation rate of MDA-MB-231 and SK-BR-3 cells with Cathepsin K knockdown. (B) Cell apoptosis was evaluated by Flow cytometry in MDA-MB-231 and SK-BR-3 cells transfected with Cathepsin K shRNA (shRNA) and treated with combination of SM934-Testosterone and Cathepsin K overexpression plasmid (SM934-Tes + CTSK OE). (C) Wound-healing assay showed the migration ability of MDA-MB-231 and SK-BR-3 cells treated with Cathepsin K shRNA and combi-nation of SM934-Testosterone and Cathepsin K overexpression plasmid. (*P < 0.05; **P < 0.01; ***P < 0.001). Scale bar, 200 μm. (D) & (E) Transwell assay showed the migration and invasion ability of MDA-MB-231 and SK-BR-3 cells treated with Cathepsin K shRNA and combination of SM934-Testosterone and Cathepsin K overexpression plasmid. (*P < 0.05; **P < 0.01; ***P < 0.001). Scale bar, 200 μm. Each bar represents the mean ± S.D. of three independent experiments.
    Fig. 7. SM934-Testosterone regulated the expression of Cathepsin K followed by the suppression of Bcl-xL expression. (A) The cascade pathway of Cathepsin K in the apoptosis pathway. CTSK (Cathepsin K) was marked in pink rectangle with its name highlighted in pink also, other entries were all in the black rectangle. (B) Western blot showed the protein expression level of Bcl-xL in MDA-MB-231 and SK-BR-3 cells treated with SM934-Testosterone and Cathepsin K shRNA. (*P < 0.05; **P < 0.01; ***P < 0.001). Each bar represents the mean ± S.D. of three independent experiments.
    similar to the results of SM934 and Testosterone physical mixture (data not shown). According to this, we will optimize the structure of SM934-Testosterone to improve its water-solubility and further explore its ef-fects in vivo in our future study.