Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration
Authors: Ying-Fang Song
Hong-Jie Chen
Shou-Sen Wang
Zhao-Cong Zheng
Zheng Liu
Jing-Fang Hong
Journal: Molecular Medicine Reports (2016)
Discipline: CLINICAL MEDICINE
Keywords: anticancer effects; apoptotic effects; taraxerol acetate; U87 human glioblastoma cells; cell cycle
Affiliations: Department of Neurosurgery, Fuzhou General Hospital of Nanjing Military Command, Dongfang Hospital, Xiamen University, Fuzhou, Fujian 350025, P.R. China., Department of Pulmonary and Critical Care Medicine, Fuzhou General Hospital of Nanjing Military Command, Dongfang Hospital, Xiamen University, Fuzhou, Fujian 350025, P.R. China.

Abstract:  The aim of the present study was to investigate the in vitro and in vivo anticancer and apoptotic effects of taraxerol acetate in U87 human glioblastoma cells. The effects on cell cycle phase distribution, cell cycle-associated proteins, autophagy, DNA fragmentation and cell migration were assessed. Cell viability was determined using the MTT assay, and phase contrast and fluorescence microscopy was utilized to determine the viability and apoptotic morphological features of the U87 cells. Flow cytometry using propidium iodide and Annexin V-fluorescein isothiocyanate demonstrated the effect of taraxerol acetate on the cell cycle phase distribution and apoptosis induction. Western blot analysis was performed to investigate the effect of the taraxerol acetate on cell cycle‑associated proteins and autophagy‑linked LC3B‑II proteins. The results demonstrated that taraxerol acetate induced dose‑ and time‑dependent cytotoxic effects in the U87 cells. Apoptotic induction following taraxerol acetate treatment was observed and the percentage of apoptotic cells increased from 7.3% in the control cells, to 16.1, 44.1 and 76.7% in the 10, 50 and 150 µM taraxerol acetate‑treated cells, respectively. Furthermore, taraxerol acetate treatment led to sub‑G1 cell cycle arrest with a corresponding decrease in the number of S‑phase cells. DNA fragments were observed as a result of the gel electrophoresis experiment following taraxerol acetate treatment. To investigate the inhibitory effects of taraxerol acetate on the migration of U87 cell, a wound healing assay was conducted. The number of cells that migrated to the scratched area decreased significantly following treatment with taraxerol acetate. In addition, taraxerol acetate inhibited tumor growth in a mouse xenograft model. Administration of 0.25 and 0.75 µg/g taraxerol acetate reduced the tumor weight from 1.2 g in the phosphate‑buffered saline (PBS)‑treated group (control) to 0.81 and 0.42 g, respectively. Similarly, 0.25 and 0.75 µg/g taraxerol acetate injection reduced the tumor volume from 1.3 cm3 in the PBS-treated group (control) to 0.67 and 0.25 cm3, respectively. 

Retracted: Yes

PubPeer comments:
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 Fig 10. ![file] For comparison, Fig 7 from [Liu et al] . ![file] , 
 *[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 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 . ![file] , 
 *[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 . *[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 . ![file] , 
  Figure 7. Inhibitory effects of taraxerol acetate on the migration of U87 cells. (A) Scratch at 0 h, prior to any treatment; (B) Control at 48 h; cells treated with (C) 50 and (D) 150 u00b5M taraxerol acetate at 48 h . ![file] The repeated cell clumps were evidently not a source of concern for the reviewers. , 
 #2 Fig 10 again: ![] Original source seems to be Fig 3 from [Shi et al (2014)] .  Scutellarein suppresses tumor growth in a mouse xenograft model. (A) Images of tumors removed from the mice in each group . ![file] , 
 Retracted [20 April 2021] .  Following the publication of the above paper, a concerned reader drew to the Editor s attention that several figures (Figs. 3, 4, 7 and 10) contained apparent anomalies, including repeated patternings of data within the same figure panels. Furthermore, Fig. 3 contained data that bore striking similarities to data published in Fig. 6 in another paper published in *Molecular Medicine Reports*, which has now been retracted [Zhu Yu2011Y, Huang Hu2011Y and Wu Yu2011L: 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. Mol Med Rep 12: 5012u20115018, 2015]. After having conducted an independent investigation in the Editorial Office, the Editor of *Molecular Medicine Reports* has determined that the above paper 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.  ,