Biotechnology And Mass Spectrometry-based Anti-cancer Mechanism Study Of Celastrol And Its Analogue

Celastrol, a quinine methide triterpene extracted from a Chinese medicine(Trypterygium wilfordii Hook F.), has the potential to become an new drug for treatingchronic inflammatory, autoimmune disease, neurodegenerative disorders and cancer withpromising prospects. Nowdays, although investigations focusing on celastrol’s effects onspecifc cellular pathways have revealed a number of targets in a diverse array of in vitromodels, there is still lack of a thorough insight into its anticancer effects from a globalview. Meanwhile, the chemical instability and low aqueous solubility and oralbioavailability of celastrol restrict its further therapeutic applications. Therefore, it will benecessary to improve celastrol water solubility and bioavailability while still retaining itsactivities in order to develop new celastrol-based therapeutics. In current study, theanticancer mechanisms of celastrol and its analogue NST-001were elucidated using anion-trap gas chromatography–mass spectrometry based metabolomics combined withmultivariate statistical analysis, and the direct target of NST-001was investigated usingDARTS (Drug affinity responsive target stability) coupled with MALDI-TOF/TOF MS(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry).Subsequently, the proposed direct molecular target of NST-001, namely fatty acidsynthase, was validated by molecular docking.1. Effects of celastrol on human cervical cancer cells as revealed by ion-trap gaschromatography–mass spectrometry based metabolic proflingCell culture metabolomics has been a powerful method to study metabolic profles incell line after drug treatment, which can be used for discovery of drug targets andinvestigation of drug effects. We analyzed the metabolic modifcations induced bycelastrol treatment in human cervical cancer cells, using an ion-trap gaschromatography–mass spectrometry based metabolomics combined with multivariatestatistical analysis, which allows simultaneous screening of multiple characteristicmetabolic pathways related to celastrol treatment. Three representativeapoptosis-inducing cytotoxic agents, namely cisplatin, doxorubicin hydrochloride andpaclitaxel, were selected as positive control drugs to validate reasonableness andaccuracy of our metabolomics investigation on celastrol. Anti-proliferation and apoptoticeffects of celastrol were demonstrated by CCK-8assay, Annexin-V/PI staining method,mitochondrial membrane potential (ΔΨm) assay and caspase-3assay. Several signifcant metabolites involved in energy, amino acid and nucleic acid metabolism in HeLa cellsinduced by celastrol and positive drugs were reported. Our method is proved to beeffective and robust to provide new evidence of pharmacological mechanism of celastrol.The metabolic alterations induced by drug treatment showed the impaired physiologicalactivity of HeLa cells, which also indicated anti-proliferative and apoptotic effects ofcelastrol and these positive drugs. GC/MS-based metabolomics approach applied to cellculture could give valuable information on the systemic effects of celastrol in vitro andhelp us to further study its anticancer mechanism.2. The novel water-soluble celastrol analogue NST-001without conjugatedketone displays similar anticancer activity in human cervical cancer cellsAnti-proliferation and apoptotic effects of celastrol analogue NST-001weredemonstrated by CCK-8assay, Annexin-V/PI staining method, cell cycle analysis,mitochondrial membrane potential (ΔΨm) assay and caspase-3assay and western blotting.NST-001can inhibit the growth of HeLa cells in a time-and dose-dependent manner, andinduce G2cell cycle arrest and apoptosis in HeLa cells. And then, the anticancer effectswere investigated using our validated cell culture metablomics, which the results showedthat the NST-001treatment induced low level of citrate cycle, high level of glycolysis anddown-regulated amino acid metabolism and protein biosynthesis of HeLa cells. All thesefindings suggested the apoptosis state of HeLa cells after NST-001treatment.3. Identification of direct target of NST-001in HeLa cells using DARTS coupledwith MALDI-TOF/TOF MSSmall-molecule target identification is a vital and daunting task for drug study.Identification of drug target is essential for elucidating pharmacological activity andmechanism of action of drug. Although investigations focusing on celastrol’s effects onspecific cellular pathways have revealed a number of targets in a diverse array of in vitromodels, the direct targets remain elusive. In current study, the proposed direct target ofNST-001, namely fatty acid synthase (FAS), was identified using DARTS couple withMALDI-TOF/TOF MS. The FAS inhibitor drug Orlistat, which has been approved by theUS Food and Drug, can inhibit the growth of prostate tumor xenografts throughinteraction with the thioesterase (TE) domain in FAS. Based on these findings, theproposed direct molecular target FAS was validated by molecular docking of NST-001and celastrol to TE domain, which suggested that NST-001was a better FASN inhibitorthan celastrol. In this study, referring to methods and ideas of systems biology，biotechnologycoupled with mass spectrometry method was employed to investigate anti-cancermechanism of celastrol and its analogue NST-001, which provided an important approachfor active constituent in traditional Chinese medicine. A robust, accurate and fast GC-MSbased cell culture metabolomics was successfully developed, which also demonstratedthe mechanism of action of celastrol in cell culture metabolomics. And then, the directtarget of NST-001was successfully identified by DARTS coupled withMALDI-TOF/TOF MS, and validated by molecular docking.