Ginsenoside 20(S)‑Rg3 inhibits the Warburg effect through STAT3 pathways in ovarian cancer cells
Authors: Jie Li
Xu Li
Zhongxue Ye
Ting Liu
Wei Chen
Le Zhao
Huilian Hou
Journal: International Journal of Oncology (2015)
Discipline: CLINICAL MEDICINE
Keywords: Cancer cells; aerobic glycolysis; Warburg effect; STAT3 signaling; ginsenoside 20(S)-Rg3
Affiliations: Center for Translational Medicine, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China., Center for Laboratory Medicine, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China., Department of Pathology, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.

Abstract:  Cancer cells prefer to metabolize glucose through aerobic glycolysis, known as the Warburg effect. It plays a crucial role in proliferation and progression of cancer cells. However, the complete mechanism remains elusive. In recent studies, the signal transducer and activator of transcription 3 (STAT3) signaling has been discovered to have roles in cancer‑associated changes in metabolism. In this study, we find that the ginsenoside 20(S)‑Rg3, a pharmacologically active component of the traditional Chinese herb Panax ginseng, inhibits glycolysis in ovarian cancer cells by regulating hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2). We also show that 20(S)‑Rg3 regulates HK2 through downregulation of p‑STAT3 (Tyr705). Furthermore, overexpression of STAT3 in ovarian cancer cells weakened the suppression of Warburg effect induced by 20(S)‑Rg3. Importantly, 20(S)‑Rg3 treatment represses HK2 expression in nude mouse xenograft models of ovarian cancer. Taken together, our results show that 20(S)‑Rg3 inhibits the Warburg effect by targeting STAT3/HK2 pathway in ovarian cancer cells, highlighting the potentiality of 20(S)‑Rg3 to be used as a therapeutic agent for ovarian cancer. 

Retracted: No

PubPeer comments:
 Concern about Figure 3A. A sharp transition is visible between lanes 1 and 2 in the GAPDH panel and the 2nd FLAG panel. In addition, within each of the panels, background areas appear to be visible multiple times. Highlighted with rounded boxes of the same color. Image made darker to enhance details. ![file] , 
 Figure 3D also appears to show some repetitive elements. Image made darker to enhance details. ![file] , 
 Figure 2J also appear to have some repetitive features. Image made darker to enhance details. ![file] , 
 Update on Figure 3D after Tjibbe Donker (through Twitter) spotted an additional potential similar-looking area in the Palm-STAT3 blot. Here is the new version. ![file] , 
 #1 - #4 : Note that the paper includes  uncropped scans in the Supplementary Information. (Often they are only slightly-less cropped than in the main figures.) They might be useful for further analysis of these issues; a quick inspection suggests that several of the irregularities noted above are indeed also evident in the  uncropped Supplementary images. , 
 Nothing convincing here to indicate the bands are duplicated. Where is the evidence for splicing? , 
 indeed strange stuff are happening in the SI too ? ![file] , 
 #7 RE: * indeed strange stuff are happening in the SI too ? * What is your point? It is not at all clear. The features you have circled are likely to be markings on the film, made with pen, to show the location of the MW markers (which are not otherwise visible on the film, but only on the membrane). This is standard and common practice known to anyone with experience w WB techniques. By contrast, the implications of your blue and red circles are unclear: Are you surprised that they don t look like bands? Are you suggesting that you think they are related in the two images? Something else?? In general, posted comments should contain enough context and explanation so that your meaning is clear. See here: https: pubpeer.com static faq#6 , 
 #6  Nothing convincing here to indicate the bands are duplicated. Where is the evidence for splicing? In, for instance Fig 3a, how well would you expect the faint upper bands in lanes 1 and 3 to cancel out? ![file] No-one seems to have alleged anything as crude as **splicing**. , 
 #7 SI for Fig 3d: ![file] Am I right to deduce, from the different felt-tip marks, that these were separate groups of lanes on the same gel? , 
 #10 RE: * Am I right to deduce, from the different felt-tip marks, that these were separate groups of lanes on the same gel? * I don t think so. My assumption would be that these two are **meant** to represent the same four lanes, but probed with two different antibodies (along with two additional antibodies, for a total of four, in the final figure). , 
 I don t believe the Figure 3D example is evidence of anything strange. The blot is showing total STAT3 and palmitoylated STAT3, so the two panels should come from the same blot and will have the same markers, as shown. Unless, I am missing something, it looks legitimate. , 
 #12 RE: * ...so the two panels should come from the same blot... * No, in fact the samples are different: the STAT3 panel is Input, whereas the Palm-STAT3 panel is a streptavidin pulldown (following covalent modification of the sample). So it is not a single blot probed with two different antibodies. Presumably you are responding to comment #7. I am still unsure of the point of that comment, unless it is meant to suggest that the pen markings are surprisingly similar, given that these should be distinct blots. , 
 Here s a close up view of the Flag Stat3 bands in Figure 3A pointed out by Elizabeth Bik. The areas around the left 2 bands and the bands themselves match pixel for pixel. https: imgur.com Zpctb6S https: i.imgur.com Zpctb6S.jpg https: imgur.com B99VWTz https: i.imgur.com B99VWTz.jpg , 
 #4 ZDHHC19 panel, lane 2 and lane 4 are duplicates. Also, the background seems to be heavily manipulated. , 
 #13 Yes, you are correct that I was replying to #7. Also, you correctly pointed out that they are indeed different samples. I missed that it was a pulldown panel and input panel in the original figure. Thanks for pointing that out. , 
 #7 I am just pointing out that the left side pen marks are pixel-wise identical in form. To my knowledge it should not be the case. , 
 From the SI for Fig 3d: the noise in the two HA bands looks very similar: ![file] , 
 #9 OK I see the issue with the upper bands on 3a now. The upper bands themselves in lanes 1 and 3, which is what I was initially focusing on, do seem different but the surrounding background is almost certainly duplicated. Duplication of 3 bright vertical markings and pixel spot are the most apparent to me. Lane 1: ![file] Lane 3: ![file] If there is manipulation, and the case is valid, then it has been done in a highly sophisticated manner. I suppose if anyone is going to try and pull this off with a Nature paper then it would be. Apologies to the original poster! She has a very good eye for this! , 
 #1 Dear Dr. Bik- I am the corresponding author of this paper. I appreciate your bringing this up to our attention. I was first in shock to see these concerns. To ensure the viewers and the community, I have immediately started investigation of these issues, and have contacted our institution for their advice. We will carry out a thorough investigation and figure out the details of these issues. The particular experiment in Fig3a has been repeated multiple times. Other gel blots are available to examine as well. I appreciate the posts and all the other contributors for their comments. Sincerely, Xu , 
 Please don t provide  other gel blots . Provide the original film with high resolution and without any cutting. , 
 Dear Dr. Xu Wu, Thank you so much for your fast response. I very much appreciate that you take this very seriously, and I realize how much of a shock this must have been for you, your lab, and your institition. The  uncropped films provided in the supplemental materials also appear to contain at least some of these similarities, so those might not be able to provide an answer for what happened here. I look forward to hear from you once you have more information to share. Kind regards, Elisabeth Bik. , 
 #22 Dear Dr. Bik- I am still in shock, but have taken this very very seriously and honestly. Upon learning the concerns, I have immediately reported to my department and our institutional officer, and will thoroughly investigate. Thanks for mentioning the SI Fig1 for the uncropped images. It will also be examined. I am currently checking the data from a co-author who provided the gel scans and the assembled western blot figures. We apologize if this will take some time to finish. But please trust me that I want to find out what happened from the bottom of my heart. Best regards, Xu , 
 #23 Dr. Wu, Your willingness to investigate these matters seriously and thoroughly is appreciated. As you undergo the process of considering the possible origins and explanations for the figure irregularities noted above, you might want to be aware of some similar issues that Dr. Bik has uncovered in earlier papers from the same first author (which you may or may not already be aware of). See below: * https: pubpeer.com publications A8349A81F02D0407AF7A6ACC7C6D9E * https: pubpeer.com publications 69204B1B2D6819E4592A1A8D09D2A4 * https: pubpeer.com publications 66EC0AE87809DB7DDAEB5883C22995 , 
 After I notified our institution officers, the band similarity issues are now handled by MGH. All inquiries should be directed to MGH research compliance officer. We re-examined results, including results from multiple repeated but same experiments for Fig 2J, 3a and 3d. The results are reproducible. The scientific conclusions remains valid and unchanged. , 
 Thanks a lot for reading the paper! Yes, these results are exactly what we have stated in the paper. C682 is the major site of modification, and C712 might be also involved. 1) APE assay is quite different from metabolic labeling using chemical probes. It is less sensitive. C682S lost most of the signal, and C712S has a much weaker upper band (suggesting the C712S mutation decreases palmitoylation). 2) Alk-C16 labeling and click methods are very sensitive, and also tend to have higher background signal. in Alk-C16 experiment, C682S has a much weaker signal (but not totally gone). C712S has a weaker labeling than WT, but stronger than C682S. 3) Given C682S is the major site, we only included C682S in the IL-6 stimulation assay. The data can not conclude that C682 is the only site regulated by IL-6. But it is a major site. , 
 In Fig. 1f, Streptavidin Pulldown show both C687 C712 for palmitoylation, but in Extended Data Fig. 1m, STAT3-PEG show that only C687 was labelled. Also, in Extended Data Fig. 2b, IL6 only increase C687 s palmitoylation. So, there may be only one site (C687) for palmitoylation. The above is my opinion, just for reference. ![file] ![file] ![file] , 
 #27 I m surprised you replied so soonuff0cthanks for your reply. I agree with your explanation. Your good work has provided me some new research ideas. Thank you again. , 
 I am surprised that Palm-STAT3 shows such a clear band on gel. Lipid-modified proteins should show smear like band pattern. Even glycosylation-modified proteins will never show clean band on gel. Also I am surprised that reviewer let this pass without requesting the most important and fundamental experiment using 3[H]-palmitic acid labeling to validate Palm-STAT3 in cells. , 
 #29 RE: * I am surprised that Palm-STAT3 shows such a clear band on gel. Lipid-modified proteins should show smear like band pattern. * It s not clear why you expect this, and you provide no citations to support your claim. Here are some examples that contradict your suggestion that smeariness is a standard or necessary consequence of palmitoylation: Fig 4A here: http: www.jbc.org content 290 49 29259 F4.expansion.html Fig 1B here (bCdc42 lanes labelled OD): https: mcb.asm.org content mcb 33 7 1417.full.pdf Fig 1D here (pan-Ras blot w palmitoylated [4A, H, N] and non-palmitoylated [4B] forms of Ras): https: www.pnas.org content 112 3 779 Fig 6A here: http: www.jbc.org content 285 49 38104 F6.expansion.html , 
 June 17, 2020 Retraction Notice available at: https: www.nature.com articles s41586-020-2414-6 * We, the authors, are retracting this Letter owing to anomalies in Figs. 2j, 3a and 3d. We and the Nature editors reviewed the data and found that these anomalies were present in the digital scans of the original western blot films, which could not be located for further examination. Although repeat experiments at the time of writing the paper have reproduced the data, the anomalies undermine confidence in the published Letter. We therefore believe that the most appropriate course of action is to retract this Letter in its entirety. We thank the readers who brought the issues to our attention, and apologize to the scientific community. We remain confident that the key findings of the Letter are valid and reproducible. All authors agree with the Retraction. * ,