Anticancer study of procupine (hystrix brachyura) bezoar on melanoma and the identification of its active compounds

Abstract

Hystrix brachyura bezoar (PB) has been traditionally used as an alternative medicine to treat cancer. However the anticancer effect of PB is yet to be explored. Therefore this study aims to evaluate PB anticancer potential using metabolomics approach and molecular docking. Four different PBs namely PB-A, PB-B, PB-C and PB-D were procured and used in this study to evaluate the anticancer effect. All PBs were treated with water through ultrasonication-assisted extraction technique to obtain aqueous extracts. The melanoma cells (A375) were chosen for the evaluation of further anticancer effects. A375 cells were subjected to cell proliferation, colony formation, apoptosis, cell cycle arrest, cell migration, cell invasion assays followed by molecular pathways using real time polymerase chain reaction (qPCR) and in vivo antiangiogenesis using zebrafish (Danio rerio) larvae. Additionally, all four PBs were also evaluated for their toxicity effect using zebrafish embryos. The anticancer compounds of PB were putatively identified using liquid chromatography–mass spectrometry (LC-MS) and liquid chromatography–mass spectrometry (LC-MS) based metabolomics approaches. All the compounds identified were then docked to BCL-2, cyclin B/CDK1 complex, VEGF and NM23 crystal structures to predict the ligand-protein interaction. Lastly, the study also developed a validated regression model using Fourier transform infra-red (FT-IR) spectroscopy to predict the anticancer activity of new PB extract to ensure its quality as an effective anticancer agent. All PB extracts exhibited significant cytotoxicity on A375 cells. Further analysis revealed that PB-A, PB-C and PB-D had a good inhibitory effect on cell proliferation, colony formation, cell cycle, cell migration, cell invasion, angiogenesis and apoptosis inducer. PB-A, PB-C and PB-D revealed apoptosis through intrinsic pathway, arresting the cell cycle at G2/M phase by downregulating cyclin B and CDK1. PB-A, PB-C and PB-D at molecular level were showed to suppress NM23, E-cadherin, MMP2 and MMP9. The toxicity assessment showed the morphological developmental defects caused by all four PB extracts such as deformed brain section, contorted backbone with deformities in somites and notochord, deformities in soft tissues (yolk sac, pericardial edema and swim bladder). Additionally, the PB extracts were showed to affect the cardiovascular systems via presence of heart edema, downregulating the heart rate and blood flow. The LC50 values at 96 hpf were <100 µg/mL. Chemical profiling analyses of PB extracts through GC-MS and LC-MS based metabolomics approached identified 4-androsten-4-ol-3,17-dione, acetate cholest-7-en-3-ol, gallic acid, isolongifolol, mangiferin and propafenone as the active principles. The docking results predicted the interaction of the active principles with BCL-2, cyclin B/CDK1 complex, VEGF and NM23 majorly via hydrophobic interaction with protein residues. The docking results showed good binding affinity to the crystal structures of BCL-2, cyclin B/CDK1 complex, VEGF and NM23 for 4-androsten-4-ol-3,17-dione, cholest-7-en-3beta-ol,4,4-dimethyl-, acetate and mangiferin, suggesting the roles of these compounds as the potential anticancer agents. Moreover, the study has also generated a validated statistical model to predict the anticancer activity of new PB extracts. Conclusively, the study has revealed the anticancer activity of Hystrix brachyura bezoar, active principles, in vitro mechanism of action and in vivo toxicity effect using zebrafish model for the first time. Results of this study further support the traditional claims for the use of Hystrix brachyura bezoar as an anticancer agent in Malaysia.