Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells
Authors: Yue-Yong Zhu
Hong-Yan Huang
Yin-Lian Wu
Journal: Molecular Medicine Reports (2015)
Discipline: CLINICAL MEDICINE
Keywords: Hepatocellular carcinoma; oleanolic acid; anticancer activity; HepG2 cells; cell viability
Affiliations: Liver Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, P.R. China., Department of Gastroenterology, Taizhou Municipal Hospital, Taizhou, Zhejiang 318000, P.R. China.

Abstract:  Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‚Ä?23 DNA‚Äëbinding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‚Äëdependent, as well as time‚Äëdependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 ¬µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‚ÄëG1 (apoptotic) phase of the cell cycle, in a dose‚Äëdependent manner. Staining with Annexin V‚Äëfluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‚Äëdependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‚Äëdependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. 

Retracted: Yes

PubPeer comments:
 Fig 8A.  Effect of oleanolic acid on the mitochondrial u0394u03a8m of HepG2 cells. Cells treated with oleanolic acid for 48 h were incubated with rhodamine-123 and measured using flow cytometry. The percentages of cells in the right section of the histogram indicate the number of u0394u03a8m-collapsed cells.  Appears to be the original of Fig 6 of [Zhao et al (2015)] .  , 
 *[right]* Fig 3 from [Zhu et al (2015)] . Reworked to become *[left]* Fig 5 from [Zhang et al (2015)] .  , 
 #1 Another appearance of *[left]* Fig 8A: it was reworked to become *[right]* Fig 8 from [Jia et al (2015)] .  , 
 *[above]* Figure 6 from [Zhu, Huang Wu (2015)] .  Morphological observation following acridine orange and ethidium bromide staining (magnification, x400). HepG2 cells were treated (A) without and (B) with oleanolic acid at 5 u00b5M, (C) 25 u00b5M and (D) 50 u00b5M for 48 h . *[below]* Figure 3 from [Hong et al (2016)] .  Visualization of apoptotic morphological changes induced by taraxerol acetate in U87 glioblastoma cells using acridine orange ethidium bromide staining. (A) Untreated cells (control) and cells treated with (B) 10, (C) 50 and (D) 150 u00b5M taraxerol. The orange arrows indicate apoptotic cells.  , 
 *[above]* Figure 3 from [Zhu, Huang Wu (2015)] .  Analysis of apoptosis using flow cytometry in HepG2 cells. Cells were treated with various concentrations of oleanolic acid. (A-D) represent treatment with 0, 5, 25 and 50 u00b5M oleanolic acid, respectively, for 48 h. Binding of Annexin V fluorescein isothiocyanate to phosphatidyl serine was determined by flow cytometry. The percentage of apoptotic cells determined by the number of Annexin V propidium iodide-positive cells is shown in the scattered plot . *[below]* Figure 4 from [Hong et al (2016)] .  Effect of taraxerol acetate on U87 cell apoptosis. Cells were treated with (A) 0, (B) 10, (C) 50 and (D) 150 u00b5M taraxerol acetate for 48 h. The Annexin Vu2011fluorescein isothiocyanateu2011propidium iodide assay and flow cytometry were utilized to quantify apoptosis. The quadrants, Q1u20114 represent necrotic, late apoptotic, viable and early apoptotic cells, respectively .  , 
 Retracted [13 April 2021] .  Following the publication of the above paper, a concerned reader drew to the Editor s attention that several figures (principally, Figs. 3, 6 and 8) contained data that bore striking similarities to data published in other papers, some of which had been published around the same time and written by different authors based at different research institutions. After having conducted an independent investigation in the Editorial Office, the Editor of *Molecular Medicine Reports* has determined that this article should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. ,