Amentoflavone suppresses cell proliferation and induces cell death through triggering autophagy-dependent ferroptosis in human glioma
Authors: Ning Li
Hui Li
Mingdi Liu
Hui Peng
Yan Chen
Haijing Wang
Jing Wang
Ningning Wang
Zhaohui Wang
Yu Zhang
Yihong Li
Journal: Life Sciences (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Amentoflavone; Autophagy; Ferroptosis; Glioma.
Affiliations: The Affiliated Qingdao Hiser Hospital of Qingdao University, Qingdao, Shandong, China., The Affiliated Qingdao Hiser Hospital of Qingdao University, Qingdao, Shandong, China. Electronic address: .

Abstract:  Aims: Glioma is the most common type of malignant tumor of the nervous system, and aggressiveness and recurrence are major obstacles for treatment. This study is designed to explore the effects of amentoflavone (AF) on glioma, and to investigate the underlying mechanism of the anti-cancer activities of AF. Methods: Cell morphology was recorded under microscopy. Cell viability and cell death ratio were determined by CCK-8 assay and lactate dehydrogenase (LDH) release assay, respectively. Cell cycle progression was assessed by flow cytometry. The levels of iron, MDA (malondialdehyde), lipid ROS, and GSH (reduced glutathione) were assessed by ELISA kit. The cycle-related proteins, ferroptosis-related protein, autophagy-related protein, and the phosphorylation of AMPK, mTOR and p70S6K were analyzed by western blotting. The autophagic flux was observed by transfecting cells with mRFP-GFP-LC3 plasmids. The xenograft murine models were established to analyze the effects of amentoflavone in vivo. The immunohistochemistry assay was performed to analyze the expression of LC3B, Beclin1, ATG5, ATG7, and ferritin heavy chain (FTH). Results: Our results showed that AF treatment led to reduction in cell viability and cell death. In addition, AF was found to block cell cycle progression in a dose-dependent manner in vitro. Following treatment with AF, the intracellular levels of iron, MDA, and lipid OS were increased, and the levels of GSH and the mitochondrial membrane potential were reduced. In addition, our results showed that AF promoted the autophagic by regulating autophagy-relevant proteins. Our results also showed that the autophagy-induction by AF was associated with regulation of AMPK/mTOR signaling. Mechanistically, the inhibition effects of AF on glioma cell were reversed by DFO, ferreostatin-1 as well as upregulation of FTH. Meanwhile, the FTH levels were increased by compound C and knockdown of ATG7. Moreover, both autophagy inhibitor Baf A1 and knockdown of ATG7 were able to compromising AF-induce ferroptosis and cell death. In vivo, the tumor growth was suppressed by AF in a dose-dependent manner. The level of MDA in the tumor tissue was increased while the level of GSH in tumor tissue was decreased by AF in a dose-dependent manner. Furthermore, the expression of LC3B, Beclin1, ATG5, ATG7 were increased, and the expression of FTH were decreased by AF in a dose-dependent manner in vivo. Conclusion These results demonstrate that AF triggered ferroptosis in autophagy-dependent manner. Our results suggest that AF has the potential to be considered as a novel treatment agent in glioma. 

Retracted: No

PubPeer comments:
 This paper is one of a set of 121 papers that all contain images from the same library of about 100 photos and plots. This is unexpected, because each paper has different authors and different affiliations, with little to no overlap, and describes different cancer types and molecules. It would be very hard to explain how authors from different labs would all obtain the same photo. Therefore, the papers might have all be derived from the same source, such as a paper mill that sells ready-to-be-published papers. This  Stock Photo paper set has been described in more detail on my blog post, https: scienceintegritydigest.com 2020 07 05 the-stock-photo-paper-mill and in a Wall Street Journal article by Eva Xiao (currently behind a paywall): https: www.wsj.com articles chinese-research-papers-raise-doubts-fueling-global-questions-about-scientific-integrity-11593939600 The vast majority (113 121) of the papers were published in the same journal, European Review for Medical and Pharmacological Sciences (ERMPS). A link to the full set of papers can be found here: https: docs.google.com spreadsheets d 172RWS18w1lTNJ9Nnj80fuXXTqTm4fGkoK0sdYyvKE3g edit?usp=sharing All papers have been reported to the journal editors on July 2, 2020. , 
 Article withdrawn. The retraction notice is not linked to the original article, but is found here: https: www.europeanreview.org article 22197 It reads:  *Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, u201cLncRNA AB073614 promotes tumor migration and invasion by repressing CDKN1A in non-small cell lung cancer, by W.-D. Zhao, B.-X. Zhang, X.-H. Cui, J. Zhang, N. Du, Y.-F. Zhang, published in Eur Rev Med Pharmacol Sci 2019; 23 (13): 5815-5822u2013DOI: 10.26355 eurrev_201907_18320u2013PMID: 31298333u201d has been withdrawn. * , 
 Even without the stock photos, I would have expected the reviewers to pay more attention to Fig 3A B. ![file] ,