Chan Yau Sang, Xia Lixin, Ng Tzi Bun
State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University, School of Medicine, Shenzhen University, Nanhai Ave 3688, Shenzhen, Guangdong 518060, PR China.
School of Biomedical Sciences, Lo Kwee Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Int J Biol Macromol. 2016 Apr;85:335-45. doi: 10.1016/j.ijbiomac.2015.12.094. Epub 2016 Jan 6.
A 60-kDa glucosamine binding lectin, white kidney bean lectin (WKBL), was purified from Phaseolus vulgaris cv. white kidney beans, by application of anion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, and FPLC-size exclusion on Superdex 75. The anti-proliferative activity of WKBL on HONE1 cells and HepG2 cells was stronger than the activity on MCF7 cells and WRL68 cells (IC50 values for a 48-h treatment with WKBL on HONE1 cells: 18.8 μM; HepG2 cells: 19.7 μM; MCF7 cells: 26.9 μM; and WRL68 cells: >80 μM). The activity could be reduced by addition of glucosamine, which occupies the binding sites of WKBL, indicating that carbohydrate binding is crucial for the activity. Annexin V-FITC and PI staining, JC-1 staining and Hoechst 33342 staining revealed that apoptosis was induced on WKBL-treated HONE1 cells and HepG2 cells, but not as obviously on MCF7 cells. Cell cycle analysis also showed a slight cell cycle arrest on HONE1 cells after WKBL treatment. Western blotting suggested that WKBL induced apoptosis of HONE1 cells occurred through the extrinsic apoptosis pathway, with detection of increased level of active caspase 3, 8 and 9.
一种60 kDa的氨基葡萄糖结合凝集素，即白芸豆凝集素（WKBL），通过在Q-Sepharose上进行阴离子交换色谱、在Affi-gel蓝凝胶上进行亲和色谱以及在Superdex 75上进行FPLC尺寸排阻，从菜豆品种白芸豆中纯化得到。WKBL对HONE1细胞和HepG2细胞的抗增殖活性强于对MCF7细胞和WRL68细胞的活性（用WKBL处理HONE1细胞48小时的IC50值：18.8 μM；HepG2细胞：19.7 μM；MCF7细胞：26.9 μM；WRL68细胞：>80 μM）。添加占据WKBL结合位点的氨基葡萄糖可降低该活性，表明碳水化合物结合对该活性至关重要。膜联蛋白V-FITC和PI染色、JC-1染色以及Hoechst 33342染色显示，WKBL处理的HONE1细胞和HepG2细胞诱导了凋亡，但对MCF7细胞的诱导不明显。细胞周期分析还表明，WKBL处理后HONE1细胞出现轻微的细胞周期阻滞。蛋白质免疫印迹表明，WKBL诱导HONE1细胞凋亡是通过外源性凋亡途径发生的，检测到活性半胱天冬酶3、8和9的水平升高。
