Mehdad A, Xavier Reis Giselle, Souza A A, Barbosa Jarg, Ventura M M, de Freitas S M
Laboratory of Molecular Biophysics, Institute of Biological Sciences, University of Brasilia , Brasilia, Brazil.
Faculty of Medicine, Department of Molecular Pathology, University of Brasilia , Brasilia, Brazil.
Cell Death Discov. 2016 Mar 21;2:15067. doi: 10.1038/cddiscovery.2015.67. eCollection 2016.
Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman-Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.
蛋白酶体抑制剂正作为一类新型化学预防剂崭露头角，并在乳腺癌治疗中作为潜在的药理学工具而变得极为重要。对蛋白酶及其特异性抑制剂在人体内所起作用的进一步了解为预防和治疗干预提供了新的且具有挑战性的机遇。在本研究中，我们证明了来自豇豆种子的鲍曼-伯克蛋白酶抑制剂，即黑眼豆胰蛋白酶/糜蛋白酶抑制剂（BTCI），通过抑制20S蛋白酶体的活性有效抑制人乳腺腺癌细胞的活力。BTCI对一组乳腺癌细胞具有负向生长效应，伴随着细胞周期G2/M期的细胞生长抑制作用以及细胞凋亡增加，这可通过亚G1期细胞数量增加以及膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）染色观察到。相比之下，BTCI对正常乳腺上皮细胞无细胞毒性作用。此外，BTCI处理的细胞中细胞内活性氧（ROS）水平升高以及线粒体膜电位变化表明线粒体损伤是导致凋亡过程的关键细胞事件。与未处理细胞相比，BTCI处理的肿瘤细胞中半胱天冬酶活性更高，这表明BTCI以半胱天冬酶依赖性方式诱导细胞凋亡。BTCI影响非侵袭性和侵袭性乳腺癌细胞系中NF-κB靶基因的表达，在侵袭性细胞中的作用尤为明显。在两种细胞系中还观察到白细胞介素-8（IL-8）表达增加。综上所述，这些结果表明BTCI在蛋白酶体抑制后通过ROS诱导的线粒体损伤促进细胞凋亡。这些发现突出了BTCI在乳腺癌治疗中的药理学潜力和益处。
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