| Summary: | 碩士 === 國立陽明大學 === 傳統醫藥研究所 === 102 === Backgrounds: Andrographolide is the major component in the Chinese herb Andropgraphis paniculata and known to exhibit anti-cancer activity. We recently found that andrographolide can suppress the invasion activity of a highly metastatic melanoma cell B16F10 but the molecular targets underlying this phenomenon are unclear. Serine hydrolases have been found to play roles in cancer progression and metastasis, we thus applied the chemical proteomics approach to identify the serine hydrolases associated with andrographolide-mediated suppression on invasion of B16F10. Monoacylglycerol lipase (MAGL), among the candidate serine hydrolases whose activity were suppressed by andrographolide, is previously found to be elevated in aggressive cancer and associated with tumor metastasis. Consistently, we found the mRNA expression of MAGL was suppressed after andrographolide treatment. In this study, we further investigate whether MAGL is a novel molecular target of andrographolide during cancer cell migration and examine the regulatory mechanism of MAGL by andrographolide.
Materials and Methods: The fluorescence-labeled chemical probe, FP-benzyl-BODIPY, was used to monitor the activity of serine hydrolases. JZL184, a chemical compound known to inhibit the activity of MAGL, was used to validate the signal derived from MAGL activity and to determine whether suppression of MAGL activity could reduce the migration activity of B16F10. In addition, a MAGL-overexpressing B16F10 cell line was established to examine whether MAGL overexpression can abrogate andrographolide-induced suppression of B16F10 migration. Furthermore, Western blot and reporter assays were applied to examine whether Erk, Akt, PPARs or NF-B signaling pathways are participated in the MAGL downregulation triggered by andrographolide. Moreover, quantitative RT-PCR was used to validate the roles of these signaling pathways on MAGL mRNA expression. Moreover, the effects of several andrographolide analogues on MAGL activity and cellular migration in B16F10 cells were also measured.
Results: After andrographolide treatment, both the activity and the expression of MAGL were reduced. Either andrographolide or JZL184 could reduce the MAGL migration activity of B16F10. Overexpression of MAGL abolished the andrographolide-mediated inhibition on the migration activity of B16F10 cells. Consistently, andrographolide could also reduce the activity of MAGL in human melanoma cell A2058. Moreover, NF-B and PPAR activity were suppressed in andrographolide-treated B16F10 cells and the inhibitors for NF-B and PPAR , Bay11-7082 and GW9662 respectively, could efficiently decrease the activity and expression of MAGL. In contrast, andrographolide induced Erk activation in Bl6F10 cells while inhibition of Erk signaling enhanced MAGL activity and expression. Among the andrographolide derivatives we tested, NCTU-030, NCTU-322 and Andro-NBD also exhibited inhibition on MAGL activity and cellular migration in B16F10 cells.
Conclusions: MAGL identified from chemical proteomics is the target suppressed by andrographolide treatment. In addition, MAGL downregulation is a key step in andrographolide-suppressed migration of melanoma cells. Moreover, NF-B, Erk and PPAR signaling pathways are involved in andrographolide-mediated downregulation of MAGL. Notably, several active andrographolide derivatives showing significant inhibition on MAGL activity and melanoma cell migration were identified. In the future, the regulatory mechanism of MAGL expression by andrographolide and the application potential of andrographolide on melanoma treatment merit further investigation.
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