Proteomics analysis demonstrating rosmarinic acid suppresses cell growth by blocking the glycolytic pathway in human HepG2 cells
Authors: Hu Yan
Lu Li
Xiao-Bin Li
Yang Yang
Ya-Jiao Wang
Zhan-Jun Ma
Xu Jing-Wen
Xue-Xi Wang
An-Cheng Shi
Lu Yu-Bao
Journal: Biomedicine & Pharmacotherapy (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Apoptosis; Glycolytic pathway; HepG2 cells; Proteomics; Rosmarinic acid.
Affiliations: The Second Clinical School, Lanzhou University, Lanzhou, Gansu, 730000, China., School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, China., Clinical College of Hebei Medical University, Shijiazhuang, Hebei, 050031, China., School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou, 730000, China., School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou, 730000, China. Electronic address: .

Abstract:  Rosmarinic acid (RA), isolated from herbal balm mint plants, has demonstrated potent anti-tumor properties against liver cancer. However, the precise underlying mechanisms remain unclear. This study aimed to investigate the molecular mechanisms of RA in HepG2 cells. RA anti-tumor activity was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and Hoechst 33258 staining. Apoptosis and the cell cycle distribution were evaluated by flow cytometry. A proteomics approach was used to identify differentially expressed proteins following RA treatment in HepG2 cells, and quantitative reverse transcription-quantitative polymerase chain reaction was used to validate the results. Bioinformatics analysis was also implemented to further understand the identified proteins, and western blotting was used to analyze the associated proteins. Our results suggested that RA treatment significantly inhibits the viability of HepG2 cells. The MTT and LDH assays indicated dose-dependent decreases in cell proliferation following RA treatment. Hoechst 33258 staining and flow cytometry analysis showed that RA exhibits an apoptosis-inducing effect and induces cell cycle arrest in G1. The proteomics analysis successfully identified 16 differentially expressed proteins. Bioinformatics analysis indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to inactivation of the glycolytic pathway. Further western blotting analysis showed that RA could downregulate the expression of glucose transporter-1 and hexokinase-2, leading to the suppression of glucose consumption and generation of lactate and ATP. Taken together, our study found that RA exhibits significant cytotoxic effects by inhibiting cell proliferation and inducing apoptosis and cell cycle arrest, possibly by blocking the glycolytic pathway in human HepG2 cells. 

Retracted: No

PubPeer comments:
 Two images in Figure 3 appear to overlap, with different dimensions. Roughly indicated with magenta boxes. *** ![file] , 
 #1 Dear Dr. Biskrensis: Thank you very much for your comment. First of all, I sincerely apologize to you. I carefully checked the original data with the first author, and we found that some mistakes of the Hoechst 33258 staining pictures of Figure. 3 in this paper. Unfortunately, we found that the picture of 6.25 u03bcg ml group and the picture of 25 u03bcg ml group were repeated when they were processed with PowerPoint. Besides, we also found that the pictures of 12.5 and 50 u03bcg ml group had little size errors based on our original results. Unfortunately, we didnu2019t realize this mistake in time. We are very sorry for our mistake and carelessness. Thus, we re-corrected our pictures, the correct images of Hoechst 33258 staining were attached here. We have contacted Biomedicine Pharmacotherapy journal to correct this figure. We highly appreciate your work hard for the scientific research environment. Meanwhile, thank you again very much for your concern on the figure. I m looking forward to hearing from you soon. We will strive to do our best. Thank you again for your hard work. Yous sincerely, Xuexi Wang ![file] ,