Yap Hui-Yeng Y, Fung Shin-Yee, Ng Szu-Ting, Tan Chon-Seng, Tan Nget-Hong
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
J Ethnopharmacol. 2015 Nov 4;174:437-51. doi: 10.1016/j.jep.2015.08.042. Epub 2015 Aug 28.
The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has been traditionally used as a complementary and alternative medicine for cancer treatment by the local communities of Southeast Asia. Despite the continuous research interest in its antiproliferative activity, the identity of the bioactive compound(s) responsible has yet to be determined. This study aims to bridge the gap in existing research literature by using proteomics approach for investigation of the nature of the anticancer substance of L. rhinocerotis.
To elucidate the proteome of L. rhinocerotis TM02 sclerotium by protein mass spectrometry and to further isolate and identify the cytotoxic component(s) bearing anticancer potential.
The proteome of L. rhinocerotis sclerotium was analyzed by label-free quantitative shotgun proteomics, using 1D-SDS-PAGE coupled with nano-ESI-LC-MS/MS based on the availability of its genome-sequence database. The cytotoxicity of L. rhinocerotis sclerotial extracts against human breast adenocarcinoma cells (MCF7) were assessed by MTT cytotoxicity assay prior to successive purification steps by a combination of gel filtration chromatography, ammonium sulfate precipitation, and anion exchange chromatography. Bioactive compound(s) in the extracts was identified by shotgun proteomics and N-terminal protein sequencing.
Several proteins with interesting biological activities including lectins, fungal immunomodulatory proteins, and several antioxidant proteins were identified from the proteome of L. rhinocerotis. A cytotoxic protein fraction (termed F5) which was partially purified from its sclerotial cold water extract F5 shows two distinct bands of 31 and 36 kDa in reducing SDS-PAGE and exhibited potent selective cytotoxicity against MCF7 cells with IC50 value of 3.00 ± 1.01 μg/ml. Both bands were identified to be serine protease by LC-MS/MS analysis. Phenylmethylsulfonyl fluoride, a specific serine protease inhibitor, inhibited both the proteolytic activity and cytotoxicity of F5, suggesting that the cytotoxicity of F5 is related to its protease activity.
This study provides the first comprehensive and semi-quantitative profiling of the proteome of L. rhinocerotis sclerotium. Further investigation into its selective cytotoxicity shows that a serine protease-like protein, termed F5, may be targeted for new anticancer agent development.
虎乳灵芝（Lignosus rhinocerotis (Cooke) Ryvarden）的菌核传统上被东南亚当地社区用作癌症治疗的补充和替代药物。尽管对其抗增殖活性的研究兴趣持续存在，但负责的生物活性化合物的身份尚未确定。本研究旨在通过蛋白质组学方法研究虎乳灵芝抗癌物质的性质，以弥合现有研究文献中的差距。
通过蛋白质质谱法阐明虎乳灵芝TM02菌核的蛋白质组，并进一步分离和鉴定具有抗癌潜力的细胞毒性成分。
基于其基因组序列数据库的可用性，采用无标记定量鸟枪法蛋白质组学分析虎乳灵芝菌核的蛋白质组，使用一维SDS-PAGE结合纳米电喷雾液相色谱-串联质谱（nano-ESI-LC-MS/MS）。在通过凝胶过滤色谱、硫酸铵沉淀和阴离子交换色谱的连续纯化步骤之前，通过MTT细胞毒性试验评估虎乳灵芝菌核提取物对人乳腺腺癌细胞（MCF7）的细胞毒性。提取物中的生物活性化合物通过鸟枪法蛋白质组学和N端蛋白质测序进行鉴定。
从虎乳灵芝的蛋白质组中鉴定出几种具有有趣生物活性的蛋白质，包括凝集素、真菌免疫调节蛋白和几种抗氧化蛋白。从其菌核冷水提取物F5中部分纯化得到的细胞毒性蛋白组分（称为F5）在还原SDS-PAGE中显示出两条明显的条带，分子量分别为31 kDa和36 kDa，并对MCF7细胞表现出强大的选择性细胞毒性，IC50值为3.00±1.01μg/ml。通过LC-MS/MS分析确定这两条带均为丝氨酸蛋白酶。苯甲基磺酰氟，一种特异性丝氨酸蛋白酶抑制剂，抑制了F5的蛋白水解活性和细胞毒性，表明F5的细胞毒性与其蛋白酶活性有关。
本研究首次提供了虎乳灵芝菌核蛋白质组的全面和半定量分析。对其选择性细胞毒性的进一步研究表明，一种称为F5的丝氨酸蛋白酶样蛋白可能是开发新型抗癌药物的靶点。
