Departament of Natural Products, Institute of Pharmaceuticals Technology, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, Rio de Janeiro, 21045-900, Brazil.
Laboratory of Bioprospection and Applied Ethnopharmacology, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
Protein J. 2024 Apr;43(2):333-350. doi: 10.1007/s10930-023-10175-9. Epub 2024 Feb 12.
A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.
从绿豆（Cajanus cajan）新鲜叶片中通过亲和层析部分纯化了一种新型胰蛋白酶抑制剂（TIC）。SDS-PAGE 显示一条约 15 kDa 的带，通过酶谱法显示出高表达的胰蛋白酶抑制剂活性。TIC 对胰蛋白酶具有高亲和力（Ki = 1.617 μM），是该丝氨酸蛋白酶的竞争性抑制剂。TIC 在 70°C 下处理 24 小时后、在不同 pH 值下处理 1 小时后以及在β-巯基乙醇浓度增加的情况下，其活性保持不变，表现出高结构稳定性。然而，TIC 的活性在存在氧化剂的情况下受到影响。为了研究 TIC 对分泌性丝氨酸蛋白酶的影响以及对细胞培养生长曲线的影响，SK-MEL-28 转移性人黑色素瘤细胞系和 CaCo-2 结肠腺癌细胞系在补充有 DMEM 的条件下生长，细胞外部分经过盐析和亲和层析以获得新的分泌性丝氨酸蛋白酶。TIC 几乎完全抑制了这些丝氨酸蛋白酶的活性（96%至 89%），并分别降低了黑色素瘤和结肠腺癌细胞的生长（48%和 77%）。此外，这是首次从绿豆叶片中分离和鉴定胰蛋白酶抑制剂，并且从 SK-MEL-28 和 CaCo-2 癌细胞系中分离和部分鉴定了癌症分泌性丝氨酸蛋白酶。此外，TIC 被证明是一种有效的肿瘤蛋白酶抑制剂，可影响细胞生长，并且可能是黑色素瘤和结肠腺癌化学疗法中潜在的候选药物。
