Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells
Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells
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DOI：
10.1002/jbt.23184
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发表时间：
2022-08
影响因子：
3.6
通讯作者：
Toshiyuki Nakamura;Chiharu Tsutsui;Y. Okuda;Naomi Abe-Kanoh;Saori Okazawa;S. Munemasa;Y. Murata;Y. Kato;Yoshimasa Nakamura
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文献类型：
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作者：
Toshiyuki Nakamura;Chiharu Tsutsui;Y. Okuda;Naomi Abe-Kanoh;Saori Okazawa;S. Munemasa;Y. Murata;Y. Kato;Yoshimasa Nakamura
异硫氰酸苄酯(BITC)是从十字花科蔬菜中提取的一种有机硫化合物，在多种人类癌细胞中具有抗增殖作用。在这项研究中，我们评估了BITC作为一种潜在的破骨细胞生成抑制剂，并探讨了其潜在的机制。5μM时，破骨细胞样分化的RAW264.7细胞活力显著降低，与破骨细胞分化的主要生物标志物，如抗酒石酸酸性磷酸酶活性和活化T细胞核因子基因表达下调一致。BITC及其代谢产物对正常RAW264.7细胞的增殖均有抑制作用，提示BITC在体内摄取代谢后具有抗破骨细胞生成的作用，可能是通过抗增殖作用实现的。BITC及其代谢产物也能促进DNA片段化和caspase-3活性，但较高浓度的BITC有抑制这种作用的趋势。当细胞与其代谢产物一起处理细胞时，BITC通过降解为游离形式而在细胞内积聚。利用蛋白质分解/高效液相色谱技术进行的定量实验表明，细胞内BITC-赖氨酸硫脲的量也随时间的增加而增加，这表明BITC处理的细胞中确实发生了细胞蛋白的赖氨酸修饰。在细胞蛋白中，被切割的caspase-3被确定为BITC赖氨酸修饰的潜在靶点。综上所述，BITC从其代谢物中解离出来，以及它的游离形式可能调节破骨细胞的形成，可能是通过蛋白质修饰抑制细胞增殖。
Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 μM significantly decreased the viability of the osteoclast‐like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate‐resistant acid phosphatase activity and nuclear factor of activated T‐cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti‐osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase‐3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC‐lysine thiourea in the cells was also increased in a time‐dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase‐3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.