Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways
Authors: Vishal Chandra
Rituraj Konwar
Murli Manohar
Bal Gangadhar Roy
Sandeep Kumar
Utsab Debnath
Yenamandra S Prabhakar
Karan Singh Saini
Kanchan Hajela
Anila Dwivedi
Ruchi Saxena
Kaling Megu
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: epidermal growth factor receptor (EGFR); breast cancer; benzopyran compound; ER-negative; anti-proliferative
Affiliations: Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.

Abstract:  Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors. 

Retracted: Yes

PubPeer comments:
 This paper was reported to PLOS ONE in 2015, but it s been 4 years without action, so let s discuss this one here. I combined all the findings into one screenshot to better appreciate the number of possible similarities, all found within the same paper. Dashed colored lines indicate a mirrored image. Similarities were found in the following figures: * Figure 5: 2 panels within the same figure look very similar (shown with red boxes) * Figure 6: 2 sets of 2 panels within the same figure look very similar (shown with orange and purple boxes) * Figure 7 8 9: 3 panels look very similar, although the one in Fig. 8 might be in mirror image (shown with blue boxes) * Figure 9: 2 set of panels appear to show an area of overlap (shown with green boxes) ![file] , 
 #1 Figure 5 and one set from fig 6 nuclear b actin is not similar. See carefully one by one. , 
 They look much too similar to be different to me. Appears the possible 6A-6D duplications have been stretched slightly. The features at the center-bottom of the third- and fourth-from-left bands are dead giveaways, comparing the lavender boxes. May be a difference comparing the first (from left) bands in the orange boxes, but the 2nd and 3rd bands are extremely similar. Red boxes and blue boxes are pretty obvious dupes imo, 2nd and 3rd bands very distinctive and easy to identify. , 
 I agree with  much too similar to be different . ![file] Dr Bik has indicated a difference in vertical scale between the  beta-actin nuclear beta-actin pair from Fig 6. , 
 We are grateful to pubpeer reviewers for reading the manuscript and pointing out these errors. We did not know that there were any such reports earlier in 2015. We deeply regret these inadvertent errors that resulted from mistakes in image assembly and blot scans. Given that each figure has many similar bands, we sincerely apologize for not looking at the final figures carefully. We have replaced the representative blots in the new figures wherever it was required. These changes do not alter the overall result of the paper. We will also get in touch with the journal for publishing an erratum. Fig 5B: The p-Akt and p-NF-u03baB blots do look similar here. In the new figure we have replaced the p-NF-kB band with the correct representative band. https: pubpeer.com storage image-1559904373726.jpg Fig 6A: The nuclear actin band in Fig 6A is actually different from actin band in Fig 6D though they look very similar as pointed out. We do realize that cytosolic b-actin has been wrongly placed and replaced with the right figure. The bands in figure 6D are the correct bands. https: pubpeer.com storage image-1559904487678.jpg Fig 7B: The cdk4 band does look similar to the CTGF band. We have replaced it with another representative image. https: pubpeer.com storage image-1559904539280.jpg Fig 8: The cleaved PARP band is the correct representation of the effect of compound 85 287 on MDA-MB 231 cells and remains unchanged. Fig 9A: The control image in Fig 9A has been replaced with the correct image. https: pubpeer.com storage image-1559904599882.jpg Fig 9C: The CTGF band has been replaced with the correct representative image. https: pubpeer.com storage image-1559904639541.jpg Best, Ruchi , 
 We are grateful to pubpeer reviewers for reading the manuscript and pointing out these errors. We did not know that there were any such reports earlier in 2015. We deeply regret these inadvertent errors that resulted from mistakes in image assembly and blot scans. After the errors were pointed out, we checked our data records and were able to retrieve the correct representative images. Given that each figure has many similar bands, we sincerely apologize for not looking at the final figures carefully. We have replaced the representative blots in the new figures wherever it was required. These changes do not alter the overall result of the paper. We will also get in touch with the journal for publishing an erratum. Fig 5B: The p-Akt and p-NF-u03baB blots do look similar here. In the new figure we have replaced the p-NF-kB band with the correct representative band. https: pubpeer.com storage image-1559904373726.jpg Fig 6A: The nuclear actin band in Fig 6A is actually different from actin band in Fig 6D though they look very similar as pointed out. We do realize that cytosolic b-actin has been wrongly placed and replaced with the right figure. The bands in figure 6D are the correct bands. https: pubpeer.com storage image-1559904487678.jpg Fig 7B: The cdk4 band does look similar to the CTGF band. We have replaced it with another representative image. https: pubpeer.com storage image-1559904539280.jpg Fig 8: The cleaved PARP band is the correct representation of the effect of compound 85 287 on MDA-MB 231 cells and remains unchanged. Fig 9A: The control image in Fig 9A has been replaced with the correct image. https: pubpeer.com storage image-1559904599882.jpg Fig 9C: The CTGF band has been replaced with the correct representative image. https: pubpeer.com storage image-1559904639541.jpg , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0231282 After this article [1] was published, concerns were raised about several of the reported results. In Fig 2A, there is a vertical discontinuity in the background between the NC (negative control) lane and the Control lane, and the NC lane area does not appear to contain verifiable image data. The authors commented that NC data are included in the figure and that the lack of apparent image data is due to having no background signal in this lane. The original data needed to confirm these results have not been provided. Similarities were noted between the following: p-NFu03baB and p-FOXO-3a panels in Fig 5A, especially lanes 1 and 5 of each panel: The authors stated that these are different panels. MDA-MB-231 u03b2-actin panels in Figs 5A and 6D: Authors commented that the results in the MDA-MB-231 panels of these figures are derived from the same blot, and so the same representative u03b2 -actin blot was reported in both figures. p-Akt and p-NFu03baB panels in Fig 5B: Authors noted this image was duplicated in error, they provided an updated Fig 5B in which the p-NFu03baB panel is replaced. Fig 6A Nuclear u03b2-actin and Fig 6D Primary breast adenocarcinoma cells u03b2-actin: The authors stated that these are different bands. Fig 6A Cytosolic u03b2-actin and Fig 6D Primary breast adenocarcinoma cells Rb: The authors provided an updated version of Fig 6A in which the Cytosolic u03b2-actin panel was replaced. Fig 7B cdk4, Fig 8B Cleaved PARP (horizontally flipped), and Fig 9C CTGF: The authors offered replacement panels for the Fig 7B cdk4 and Fig 9C CTGF panels, in each case noting the replacement panel is u201cthe correct representative imageu201d. They noted that the cleaved PARP data are correct in the published figure. In Fig 9A, the right side of the Control panel appears to overlap with the left side of the EGF panel. The authors noted this was due to an error in figure preparation and they provided an updated version of Fig 9A in which the control image is replaced. Fig 9C does not include a loading control blot for the western blot experiment examining CTGF levels in conditioned media. The authors noted that they did not know of an appropriate loading control for proteins in the conditioned media at the time the experiment was conducted, and that equal amounts of protein were loaded in each lane. In light of this issue, the quantification data presented in the bar graph in Fig 9C are not normalized across lanes or replicates, which raises questions about the validity of the statistical analyses. The original data supporting the above results are not available, and so we have been unable to resolve the concerns about the published figures. In addition, concerns were raised about the mouse tumor sizes reported in Fig 10. In response to questions about these experiments the authors commented that to prevent autoregression of tumors they initiated treatment dosing with vehicle or compound after allowing tumors to grow for 14 days; at this timepoint the tumors had attained sizes of approximately 2000 mm3 as is shown in Fig 10C. They then treated the mice as described in the Materials and Methods section until animals in the control group reached 20 mm in one dimension (approximately 4000 mm3), at which point animals were euthanized. They also considered humane endpoints based on assessments of tumor size with respect to the percent reduction in body weight, impairment of mobility, food and water intake, and other behavioral observations. All animals in the experiment were sacrificed when the experiment was terminated on Day 30. A member of PLOS ONEu2019s Animal Research Advisory Group reviewed the article and the authorsu2019 comments and confirmed that the tumor sizes exceed community standards for humane endpoint limits in mouse tumor studies. The advisor noted that the tumor results present a significant animal welfare and ethical concern. In light of the above concerns, the PLOS ONE Editors retract this article. The editors regret that these concerns were not addressed at the time of the original review process. AD notified the journal that the authors do not agree with retraction. RS, VC, YSP confirmed they do not agree with retraction. The other authors either could not be reached or did not respond directly. Reference 1.Saxena R, Chandra V, Manohar M, Hajela K, Debnath U, Prabhakar YS, et al. (2013) Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K Akt FOXO and MEK Erk AP-1 Pathways. PLoS ONE 8(6): e66246. https: doi.org 10.1371 journal.pone.0066246 ,