Basic Study
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World J Gastroenterol. Jun 14, 2022; 28(22): 2437-2456
Published online Jun 14, 2022. doi: 10.3748/wjg.v28.i22.2437
18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells
Ling Yuan, Yi Yang, Xia Li, Xin Zhou, Yu-Hua Du, Wen-Jing Liu, Lei Zhang, Lei Yu, Ting-Ting Ma, Jia-Xin Li, Yan Chen, Yi Nan
Ling Yuan, Yi Yang, Xia Li, Yu-Hua Du, Ting-Ting Ma, Jia-Xin Li, College of Pharmacy, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Xin Zhou, Yan Chen, Yi Nan, Traditional Chinese Medicine College, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Wen-Jing Liu, Lei Zhang, Yan Chen, Yi Nan, Key Laboratory of Hui Ethnic Medicine Modernization of Ministry of Education, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Lei Yu, Department of Infectious Diseases, The Fourth Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China
Author contributions: Yuan L carried out most of the in vivo studies, analyzed the data, and wrote the manuscript; Yang Y carried out most of the in vitro experiments, wrote the manuscript, and carried out the chart-making work; Li X, Zhou X, Du YH, and Li JX were responsible for the proteomic analysis and PPI network; Ma TT performed parts of the in vivo and in vitro experiments, and conducted statistical analysis of the data; Yu L supervised the process of research and provided clinical guidance; Liu WJ and Zhang L conducted molecular biological experiments; all authors have read and approved the manuscript; Nan Y provided the conceptual and technical guidance as well as revised the manuscript critically for important intellectual content; Chen Y designed the study and revised the manuscript.
Supported by Ningxia Natural Science Foundation, No. 2020AAC03130; and Ningxia Medical University Project, No. XM2020005.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of Ningxia Medical University (No. 2020-071 and No. 2021-018).
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Ningxia Medical University (IACUC protocol number: 2019-083).
Conflict-of-interest statement: All authors declare no financial or commercial conflict of interest for this article.
Data sharing statement: All data generated or analyzed during this study are included in this paper, and further inquiries can be directed to the corresponding author (E-mail: 20080011@nxmu.edu.cn).
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Yi Nan, MD, PhD, Professor, Key Laboratory of Hui Ethnic Medicine Modernization of Ministry of Education, Ningxia Medical University, No. 1160 Shengli Street, Yinchuan 750004, Ningxia Hui Autonomous Region, China. 20080011@nxmu.edu.cn
Received: December 8, 2021
Peer-review started: December 8, 2021
First decision: March 11, 2022
Revised: March 24, 2022
Accepted: April 27, 2022
Article in press: April 27, 2022
Published online: June 14, 2022
Processing time: 184 Days and 2.1 Hours
Abstract
BACKGROUND

Gastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets.

AIM

To explore the mechanism by which 18β-glycyrrhetinic acid (18β-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells.

METHODS

Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18β-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells.

RESULTS

The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18β-GRA intervention compared with control. We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18β-GRA concentration.

CONCLUSION

18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

Keywords: Gastric carcinoma; 18β-glycyrrhetinic acid; Mitochondrial ribosomal protein L35; Proliferation; Invasion; Apoptosis

Core Tip: 18β-glycyrrhetinic acid (18β-GRA) is a pentacyclic triterpene derivative extracted from the natural medicine licorice. Our results showed that 18β-GRA could inhibit gastric carcinoma (GC) cell proliferation, migration, invasion, and tumor formation, induce GC cell apoptosis, and arrest the cell cycle. Tandem mass tag analysis revealed that the expression of mitochondrial ribosomal protein L35 (MRPL35) was significantly decreased after 18β-GRA intervention in GC cells, which was confirmed by Western blot results. These data indicate that 18β-GRA inhibits the proliferation/migration and promotes apoptosis of GC cells by down-regulating MRPL35 expression, suggesting that MRPL35 is a therapeutic target for GC.