Chemical proteomics study of the anti-metastasis activity of andrographolide on B16 melanoma cells

• Main Authors: Ting-Yun Huang, 黃庭筠
• Other Authors: Chao-Hsiung Lin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/33991252848652125984

碩士 === 國立陽明大學 === 生物藥學研究所 === 100 === Serine hydrolase family, estimated as 1% of human genome, is one of the largest and diverse enzyme classes in mammalian proteome, which participates in essential physiological functions. Besides, the activity levels of several serine hydrolases have been recently reported to correlate with tumor progression or migration. However, more than half members of this enzyme family remain unclear for their substrates and physiological function. Therefore, systematic identification of disease-related serine hydrolases is very important. Andrographolide, a major chemical ingredient of Chinese herb Andrographis panicμlata, was previously shown to exert its anticancer activity through cell growth inhibition, apoptosis induction, antiangiogenesis and metastasis inhibition. In previous mechanistic studies, andrographolide suppressed cell invasion of colorectal cancer cells (CT26 and HT29) via inhibiting MMP2 activity and ERK expression. However, whether the activity of serine hydrolase was involved in andrographolide-mediated inhibition of cancer cell migration has not been reported. Therefore, my thesis study intended to investigate whether serine hydrolases participate in the anti-migration activity of andrographolide. A chemical proteomics approach was applied to analyze serine hydrolases and to identify alteration of activity profiles of serine hydrolases in two mouse melanoma cells, B16F1 and B16F10 which differ in migration ability, upon andrographolide treatment. The biotin-tagged probes further used to isolate serine hydrolases whose activities were affected by andrographolide, which woμld increase our understanding towards the mechanism and involved pathways of andrographolide-induced suppression of cancer cell migration. Through collaboration with Professor Chung-Ming Sun from Dept. of Applied Chemistry in NCTU and Professor Lee-Chiang Lo from Dept. of Chemistry in NTU, two types of serine hydrolase probes carrying either biotin or fluorophore tags, namely FP-edo-biotin, FP-edo-fluorescein and FP-benzyl-biotin, FP-benzyl-BODIPY, were prepared. I first used the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis to examine the probe efficiency at diverse reaction concentration and time. Moreover, the fluorescent probes were used to monitor alteration of the fluorescence-based activity profiles of serine hydrolases in melanoma cells, B16F1 and B16F10. A fluorescence band was observed to be correlated with difference of cell migration between melanoma cells. Interestingly, this signal was suppressed upon andrographolide-treatment. Finally, analyses of mass spectrometry and Western blot demonstrated that such fluorescence change was likely resμlted from activity alteration of monoacylglycerol lipase (MAGL). In summary, my study applied the chemical proteomics to discover that andrographolide coμld inhibit the activity of MAGL in melanoma cell, providing a putative mechanism for the anti-migration activity of andrographolide.