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Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G1 cell cycle arrest and apoptosis: A quantitative proteomic study.

ABSTRACT: Objective:The present study aimed to investigate the molecular events in alisol B 23-acetate (ABA) cytotoxic activity against a liver cancer cell line. Methods:First, we employed a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) to identify the different proteins expressed in HepG2 liver cancer cells upon exposure to ABA. Next, bioinformatics analyses through DAVID and STRING on-line tools were used to predict the pathways involved. Finally, we applied functional validation including cell cycle analysis and Western blotting for apoptosis and mTOR pathway-related proteins to confirm the bioinformatics predictions. Results:We identified 330 different proteins with the SILAC-based quantitative proteomics approach. The bioinformatics analysis and the functional validation revealed that the mTOR pathway, ribosome biogenesis, cell cycle, and apoptosis pathways were differentially regulated by ABA. G1 cell cycle arrest, apoptosis and mTOR inhibition were confirmed. Conclusions:ABA, a potential mTOR inhibitor, induces the disruption of ribosomal biogenesis. It also affects the mTOR-MRP axis to cause G1 cell cycle arrest and finally leads to cancer cell apoptosis.

SUBMITTER: Xia J

PROVIDER: S-EPMC6513739 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G<sub>1</sub> cell cycle arrest and apoptosis: A quantitative proteomic study.

Xia Ji J Luo Qiang Q Huang Shengbin S Jiang Fuquan F Wang Lin L Wang Guanghui G Xie Jingjing J Liu Jie J Xu Yang Y

Chinese journal of cancer research = Chung-kuo yen cheng yen chiu 20190401 2

<h4>Objective</h4>The present study aimed to investigate the molecular events in alisol B 23-acetate (ABA) cytotoxic activity against a liver cancer cell line.<h4>Methods</h4>First, we employed a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) to identify the different proteins expressed in HepG2 liver cancer cells upon exposure to ABA. Next, bioinformatics analyses through DAVID and STRING on-line tools were used to predict the pathways i ...[more]

PMID: 31156308

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Project description:Introduction: Alisol B 23-acetate (AB23A), a major bioactive constituent in the Chinese herb Zexie (Rhizoma Alismatis), has been found with multiple pharmacological activities. AB23A can be readily hydrolyzed to alisol B in mammals, but the hydrolytic pathways of AB23A in humans and the key enzymes responsible for AB23A hydrolysis are still unrevealed. This study aims to reveal the metabolic organs and the crucial enzymes responsible for AB23A hydrolysis in human biological systems, as well as to decipher the impact of AB23A hydrolysis on its biological effects. Methods: The hydrolytic pathways of AB23A in human plasma and tissue preparations were carefully investigated by using Q-Exactive quadrupole-Orbitrap mass spectrometer and LC-UV, while the key enzymes responsible for AB23A hydrolysis were studied via performing a set of assays including reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses. Finally, the agonist effects of both AB23A and its hydrolytic metabolite(s) on FXR were tested at the cellular level. Results: AB23A could be readily hydrolyzed to form alisol B in human plasma, intestinal and hepatic preparations, while human butyrylcholinesterase (hBchE) and human carboxylesterases played key roles in AB23A hydrolysis in human plasma and tissue preparations, respectively. It was also found that human serum albumin (hSA) could catalyze AB23A hydrolysis, while multiple lysine residues of hSA were covalently modified by AB23A, suggesting that hSA catalyzed AB23A hydrolysis via its pseudo-esterase activity. Biological tests revealed that both AB23A and alisol B exhibited similar FXR agonist effects, indicating AB23A hydrolysis did not affect its FXR agonist effect. Discussion: This study deciphers the hydrolytic pathways of AB23A in human biological systems, which is very helpful for deep understanding of the metabolic rates of AB23A in humans, and useful for developing novel prodrugs of alisol B with desirable pharmacokinetic behaviors.

Project description:IntroductionEmerging severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 causes a global health disaster and pandemic. Seeking effective anti-pan-CoVs drugs benefit critical illness patients of coronavirus disease 2019 (COVID-19) but also may play a role in emerging CoVs of the future.ObjectivesThis study tested the hypothesis that alisol B 23-acetate could be a viral entry inhibitor and would have proinflammatory inhibition for COVID-19 treatment.MethodsSARS-CoV-2 and its variants infected several cell lines were applied to evaluate the anti-CoVs activities of alisol B 23-aceate in vitro. The effects of alisol B 23-acetate on in vivo models were assessed by using SARS-CoV-2 and its variants challenged hamster and human angiotensin-converting enzyme 2 (ACE2) transgenic mice. The target of alisol B 23-acetate to ACE2 was analyzed using hydrogen/deuterium exchange (HDX) mass spectrometry (MS).ResultsAlisol B 23-acetate had inhibitory effects on different species of coronavirus. By using HDX-MS, we found that alisol B 23-acetate had inhibition potency toward ACE2. In vivo experiments showed that alisol B 23-acetate treatment remarkably decreased viral copy, reduced CD4+ T lymphocytes and CD11b+ macrophages infiltration and ameliorated lung damages in the hamster model. In Omicron variant infected human ACE2 transgenic mice, alisol B 23-acetate effectively alleviated viral load in nasal turbinate and reduced proinflammatory cytokines interleukin 17 (IL17) and interferon γ (IFNγ) in peripheral blood. The prophylactic treatment of alisol B 23-acetate by intranasal administration significantly attenuated Omicron viral load in the hamster lung tissues. Moreover, alisol B 23-acetate treatment remarkably inhibited proinflammatory responses through mitigating the secretions of IFNγ and IL17 in the cultured human and mice lymphocytes in vitro.ConclusionAlisol B 23-acetate could be a promising therapeutic agent for COVID-19 treatment and its underlying mechanisms might be attributed to viral entry inhibition and anti-inflammatory activities.

Dataset Information

Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G1 cell cycle arrest and apoptosis: A quantitative proteomic study.

Publications

Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G<sub>1</sub> cell cycle arrest and apoptosis: A quantitative proteomic study.

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