Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells
Authors: Anna Kawiak
Ewa Lojkowska
Journal: PLoS ONE (2016)
Discipline: Multidisciplinary
Keywords: PI3K signaling pathway; ramentaceone; breast cancer cells; apoptosis; PI3K/Akt inhibition
Affiliations: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland., Laboratory of Human Physiology, Faculty of Health Sciences with Subfaculty of Nursing, Medical University of Gdansk, Tuwima 15, 80-210, Gdansk, Poland.

Abstract:  The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer. 

Retracted: No

PubPeer comments:
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 #3  Figure 7: The left and right blot panels show unexpected similarities, in particular in the background of the blots. Here dramatized by twiddling the image contrast and darkness. ![file] There are some curious features even within the background of the Bak slice. , 
 Figure 3. All six panels of blots brought together, with contrast adjustments to focus on the features of the backgrounds. ![file] Lots of unexpected similarities. , 
 Figure 6C (just the blots): ![file] Increased contrast reveals unexpected features in common across the three bands of the BT474 half, and across the SKBR3 half. ![file] Below, I have added the BT474 Akt band from Figure 3A, after rotation through 180 deg. It seems to the the same. I have also added the SKBR3 beta-actin band from Figure 3C. , 
  Fig 8. The role of Akt inhibition in ramentaceone-mediated apoptosis induction. (A) Silencing of Akt by siRNA in BT474 and SKBR3 cells. Cells were transiently transfected with Akt siRNA and 24 after transfection, Akt silencing was confirmed with Western blot analysis. ![file] , 
 2019 expression of concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0211655 After publication of this article [1], concerns were raised about results reported in Figs 2, 5, 7, and 8: Fig 2B: It was raised that the same image appears to be presented in the 0 and 0.5 u03bcM panels for BT474, and that there are several areas of similarity in the 0.5 and 1 u03bcM panels for SKBR3. Fig 5: Similarities were noted between several regions of the MCF-7 panels, and between several regions of the MDA-MB-231 panels. Similarities were noted between the BT474u201310 panel of Fig 5 and the BT474 u2013Akt siRNA 5 u03bcM ramentaceone panel of Fig 8B. Similarities were noted between the SKBR3u201310 panel of Fig 5 and the SKBR3 u2013ctrl siRNA 5u03bcM ramentaceone panel of Fig 8B. Similarities were noted between the background pixelation patterns of Bax, Bak, Bcl-2, and u03b2-actin panels for the BT474 and SKBR3 experiments in Fig 7. Fig 8B: Similarities were noted between regions of three BT474 panels (ctrl siRNA non-treated, cntrl siRNA 5 u03bcM ramentaceone, Akt siRNA non-treated), and between regions of three SKBR3 panels (cntrl siRNA non-treated, Akt siRNA non-treated, Akt siRNA 5 u03bcM ramentaceone). The PLOS ONE Editors have notified the University of Gdansk and Medical University of Gdansk of these concerns and issue this Expression of Concern so readers are aware of the issues whilst they are investigated. Reference 1. Kawiak A, Lojkowska E (2016) Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K Akt Signaling in Breast Cancer Cells. PLoS ONE 11(2): e0147718. https: doi.org 10.1371 journal.pone.0147718 pmid:26840401 , 
 #9 SO the editors have taken heed of Dr Bik s critique from two years ago, but have not yet noticed the problems in Figs 3, 6C, 8A. , 
 2019 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0226703 After publication of this article [1], concerns were raised about results reported in Figs 2, 3, 5, 6, 7 and 8, as detailed below. In light of some of these, an Expression of Concern was issued by the PLOS ONE Editors on January 29, 2019 [2]. Since, the authors stated that a third-party company conducted the colony assay, western blot, and flow cytometry experiments reported in the article, but that initial colony assay and western blot replicates were done in the authorsu2019 laboratory. According to the authors, the results reported in Figs 2, 3, 5, 6, 7, 8 were provided by the third-party company, and the authors do not have the underlying data supporting these figures. The authors did not provide data from the original experiments or comments to address the specific concerns about Figs 2, 3, 5, 6, 7 and 8, but the company provided clarifications and supporting files. Specific concerns include: In Fig 2B, similarities were noted between the 0 and 0.5 u03bcM panels for BT474, and between the 0.5 and 1 u03bcM panels for SKBR3. A representative of the third-party company clarified that the BT474 0.5 u03bcM image was mistakenly included as also representing the BT474 0 u03bcM condition. Furthermore, an image showing the plate from the SKBR3 0.5 u03bcM experiment after an additional wash step was errantly reported as representing the SKBR3 1 u03bcM experiment. Original images were provided to support the Fig 2B results. Several concerns were raised about similarities in FACS plots: u25cb. Fig 5: Similarities were noted between several regions of the MCF-7 panels, and separately between several regions of the MDA-MB-231 panels. u25cb. Similarities were noted between regions of the BT474u201310 panel of Fig 5 and the BT474 u2013Akt siRNA 5 u03bcM ramentaceone panel of Fig 8B. u25cb. Similarities were noted between regions of the SKBR3u201310 panel of Fig 5 and the SKBR3 u2013ctrl siRNA 5u03bcM ramentaceone panel of Fig 8B. u25cb. Fig 8B: Similarities were noted between regions of three BT474 panels (ctrl siRNA non-treated, cntrl siRNA 5 u03bcM ramentaceone, Akt siRNA non-treated), and between regions of three SKBR3 panels (cntrl siRNA non-treated, Akt siRNA non-treated, Akt siRNA 5 u03bcM ramentaceone). The company representative commented that raw data files do not include overlaying cells. Supporting files were provided by the company but have not undergone a full editorial review in light of the other issues raised in this case. As such, the concerns about the above FACS plots have not been verified. When adjusted for brightness and contrast, similarities between western blot background pixilation patterns were noted between BT474 and SKBR3, results shown in Fig 7 (Bax, Bak, Bcl-2, and u03b2-actin panels), and between several additional western blot panels in Figs 3, 6C and 8A. The company representative clarified that in assembling the panels, the respective bands were cropped and placed onto background templates, i.e. the background image in each figure panel does not represent the background as apparent on the original data. Common templates were used and thus there is overlap in background between panels. The company representative provided some files in support of the western blot results, but these files did not resolve the concerns about how data were reported or clarify the validity of the results. The western blot concerns also have implications for the validity of the quantitative data reported in Figs 3, 6 and 8. The contributions of the third-party company were not disclosed in the published article. Hence, the listed author contributions do not fully and accurately describe who conducted the reported experiments. In response to these issues, the authors provided replication data in support of the results in question. Nevertheless, in light of the confirmed concerns about western blot data reporting and substantial undisclosed contributions by a third-party company to the reported work, the PLOS ONE Editors retract this article. AK and EL did not agree with retraction. References 1.Kawiak A, Lojkowska E (2016) Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K Akt Signaling in Breast Cancer Cells. PLoS ONE 11(2): e0147718. https: doi.org 10.1371 journal.pone.0147718 2.The PLOS ONE Editors (2019) Expression of Concern: Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K Akt Signaling in Breast Cancer Cells. PLoS ONE 14(1): e0211655. https: doi.org 10.1371 journal.pone.0211655 pmid:30695056 , 
 #11  The company representative clarified that in assembling the panels, the respective bands were cropped and placed onto background templates, i.e. the background image in each figure panel does not represent the background as apparent on the original data. Common templates were used and thus there is overlap in background between panels.  That is some clarification. The name of the company that follows this  background template policy would be helpful. ,