Wilkinson Richard D A, Young Andrew, Burden Roberta E, Williams Rich, Scott Christopher J
Molecular Therapeutics, School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom.
Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom.
Mol Cancer. 2016 Apr 21;15:29. doi: 10.1186/s12943-016-0513-7.
Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.
Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.
We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.
In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.
组织蛋白酶S已被证明与多种恶性肿瘤有关，基因敲除研究表明其在肿瘤侵袭和新生血管生成中起关键作用。因此，组织蛋白酶S抑制剂的应用可能在癌症治疗中具有临床应用价值。在本研究中，我们将一种最初开发用于靶向炎症性疾病中组织蛋白酶S的细胞渗透性二肽基腈抑制剂应用于体外和体内肿瘤模型。
通过使用重组组织蛋白酶蛋白酶检测荧光底物周转率来验证组织蛋白酶S的选择性。进行完整的动力学分析并计算真实的Ki值。使用Transwell迁移试验在体外对小鼠MC38和人MCF7细胞系进行肿瘤侵袭的消除实验。使用原代人脐静脉内皮细胞（HUVEC）评估对内皮管形成的影响。分别使用在C57BL/6和BALB/c小鼠中传代的细胞评估抑制剂在体内对MC38和MCF7肿瘤进展的影响。随后对MCF7肿瘤进行增殖（Ki67）和凋亡（TUNEL）的免疫组织化学染色。
我们证实该抑制剂能够选择性地靶向组织蛋白酶S，而不是其家族成员K、V、L和B。该抑制剂还显著降低了MC38和MCF7细胞的侵袭，此外，在体外显著减少了HUVEC内皮管的形成。体内分析表明，该化合物可显著减小小鼠MC38同基因模型和MCF7异种移植模型中的肿瘤体积。对MCF7肿瘤的免疫组织化学分析显示，组织蛋白酶S抑制剂治疗显著降低了增殖并增加了凋亡。
总之，这些结果突出了使用体外和体内肿瘤模型对这种腈类组织蛋白酶S抑制剂的特性描述，展示了一种可用于进一步剖析组织蛋白酶S在癌症进展中的作用且可能具有治疗潜力的化合物。
