Anti-metastatic Effects on B16F10 Melanoma Cells of Extracts and Two Prenylated Xanthones Isolated from Maclura amboinensis Bl. Roots

Abstract

Inhibitory effects of Maclura amboinenesis Bl, one plant used traditionally for the treatment of cancers, onmetastatic potential of highly metastatic B16F10 melanoma cells were investigated in vitro. Cell proliferationwas assessed using the MTT colorimetric assay. Details of metastatic capabilities including invasion, migrationand adhesion of B16F10 melanoma cells were examined by Boyden Chamber invasion and migration, scratchmotility and cell attachment assays, respectively. The results demonstrated that n-hexane and chloroformextracts exhibited potent anti-proliferative effects (p<0.01), whereas the methanol and aqueous extracts hadless pronounced effects after 24 h exposure. Bioactivity-guided chromatographic fractionation of both activen-hexane and chloroform extracts led to the isolation of two main prenylated xanthones and characterizationas macluraxanthone and gerontoxanthone-I, respectively, their structures being identified by comparison withthe spectral data. Interestingly, both exhibited potent effective effects. At non-toxic effective doses, n-hexaneand chloroform extracts (10 and 30 μg/ml) as well as macluraxanthone and gerontoxanthone-I (3 and 10μM) significantly inhibited B16F10 cell invasion, to a greater extent than 10 μM doxorubicin, while reducingmigration of cancer cells without cellular cytotoxicity. Moreover, exposure of B16F10 melanoma cells to highconcentrations of chloroform (30 μg/ml) and geratoxanthone-I (20 μM) for 24 h resulted in delayed adhesionand retarded colonization. As insights into mechanisms of action, typical morphological changes of apoptoticcells e.g. membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic bodies and loss ofadhesion as well as cell cycle arrest in the G1 phase with increase of sub-G1 cell proportions, detected by Hoechst33342 staining and flow cytometry were observed, suggesting DNA damage and subsequent apoptotic cell death.Taken together, our findings indicate for the first time that active n-hexane and chloroform extracts as well asmacluraxanthone and gerontoxanthone-I isolated from Maclura amboinensis Bl. roots affect multistep of cancermetastasis processes including proliferation, adhesion, invasion and migration, possibly through induction ofapoptosis of highly metastatic B16F10 melanoma cells. Based on these data, M. amboinensis Bl. represents apotential candidate novel chemopreventive and/or chemotherapeutic agent. Additionally, they also support itsethno-medicinal usage for cancer prevention and/or chemotherapy.