Yoo Im Sonia, Ramalho Bonturi Camila, Miti Nakahata Adriana, Ryuichi Nakaie Clóvis, Pott Arnildo, Pott Vali Joana, Vilela Oliva Maria Luiza
Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), 04044-020 São Paulo, SP, Brazil.
Laboratório de Genômica e Biologia Molecular, Centro Internacional de Pesquisa (CIPE) do A.C.Camargo Cancer Center, 01508-010 São Paulo, SP, Brazil.
Plants (Basel). 2021 Mar 23;10(3):602. doi: 10.3390/plants10030602.
Metastasis, the primary cause of death from malignant tumors, is facilitated by multiple protease-mediated processes. Thus, effort has been invested in the development of protease inhibitors to prevent metastasis. Here, we investigated the effects of protease inhibitors including the recombinant inhibitors rBbKI (serine protease inhibitor) and rBbCI (serine and cysteine inhibitor) derived from native inhibitors identified in seeds, and EcTI (serine and metalloprotease inhibitor) isolated from the seeds of on the mouse fibrosarcoma model (lineage L929). rBbKI inhibited 80% of cell viability of L929 cells after 48 h, while EcTI showed similar efficacy after 72 h. Both inhibitors acted in a dose and time-dependent manner. Conversely, rBbCI did not significantly affect the viability of L929 cells. Confocal microscopy revealed the binding of rBbKI and EcTI to the L929 cell surface. rBbKI inhibited approximately 63% of L929 adhesion to fibronectin, in contrast with EcTI and rBbCI, which did not significantly interfere with adhesion. None of the inhibitors interfered with the L929 cell cycle phases. The synthetic peptide RPGLPVRFESPL-NH, based on the BbKI reactive site, inhibited 45% of the cellular viability of L929, becoming a promising protease inhibitor due to its ease of synthesis.
转移是恶性肿瘤致死的主要原因，多种蛋白酶介导的过程会促进转移。因此，人们致力于开发蛋白酶抑制剂以防止转移。在此，我们研究了蛋白酶抑制剂的作用，包括从种子中鉴定出的天然抑制剂衍生而来的重组抑制剂rBbKI（丝氨酸蛋白酶抑制剂）和rBbCI（丝氨酸和半胱氨酸抑制剂），以及从 种子中分离出的EcTI（丝氨酸和金属蛋白酶抑制剂）对小鼠纤维肉瘤模型（谱系L929）的影响。rBbKI在48小时后抑制了80%的L929细胞活力，而EcTI在72小时后显示出类似的效果。两种抑制剂均呈剂量和时间依赖性作用。相反，rBbCI对L929细胞的活力没有显著影响。共聚焦显微镜显示rBbKI和EcTI与L929细胞表面结合。与EcTI和rBbCI不同，rBbKI抑制了约63%的L929细胞与纤连蛋白的黏附，EcTI和rBbCI对黏附没有显著干扰。这些抑制剂均未干扰L929细胞周期阶段。基于BbKI反应位点的合成肽RPGLPVRFESPL-NH2抑制了45%的L929细胞活力，因其易于合成而成为一种有前景的蛋白酶抑制剂。
