Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro
Authors: Xiu-guo Han
Qi-ming Fan
Ting-yu Wang
Wei Yan
Yu-gang Wang
Ting-ting Tang
Han Qiao
An Qin
Journal: Acta Pharmacologica Sinica (2015)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Zoledronic acid; Plumbagin; Breast cancer; Synergistic cytotoxicity; Apoptosis
Affiliations: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China., Shanghai Key Laboratory of Orthopaedic Implants, Department of Pharmacy, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China., Shanghai Key Laboratory of Orthopaedic Implants, Wendeng Zhenggu Hospital of Shandong Province, Wendeng 264400, China.

Abstract:  Aim: Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro. Methods: Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined. Results: PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 μmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2. Conclusion: Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis. 

Retracted: No

PubPeer comments:
 Figure 3A: ![file] , 
 Figure 3C: Can you find any more? ![file] , 
 See image manipulation in another paper from this group: Suppressive Effects of Plumbagin on Invasion and Migration of Breast Cancer Cells via the Inhibition of STAT3 Signaling and Down-regulation of Inflammatory Cytokine Expressions Bone Research (2013) - 3 Comments pubmed: 26273514 doi: 10.4248 br201304007 issn: 2095-4700 issn: 2095-6231 https: pubpeer.com publications FA4C59EE1DA2B7FB8439EBB9AB85DC , 
 #2 Fig 4C. ![file] Figures 4A + C: ![file] , 
 I have attempted to locate email addresses for the authors. I was able to locate information on last author Ting-ting Tang (Professor) who appears to be the [Director](http: www.ortholab-snph.com faculty.asp) of the Shanghai Key Laboratory of Orthopaedic Implants, and [President of International Chinese Musculoskeletal Research Society (ICMRS)](http: www.ortholab-snph.com bulletin_view.asp?id=45). I also found a biography [here](http: www.ortholab-snph.com 3Dworkshop-zc-01.asp) which includes 2 email addresses. I added institutional address to the author email of this thread. , 
 Thank you so much for your comments. Actually, we have already found these problems in our APS publication. Immediately, we started our self-examination process. We found that this mistake was due to the carelessness when we tried to compare different apoptosis and transwell rates between various treatments. Specifically, when we pasted a small new image to produce a large figure, we failed to notice that we did not actually selected a new image, and instead we pasted the same previous image. This is the careless mistake we will definitely avoid next time. We have already send our correction and raw data to the APS editors, and passed the editorial investigation. We are informed that we are neat the correction stage. Thank you so much for your serious comments again. Your thoughtful understanding is highly appreciated. Best regards, Han Qiao , 
 There is also duplication between the bottom left in 4A and bottom right in 4C, which means that all of the bottom row of 4C are part of the same image as the bottom left of 4A. ![file] , 
 Thank you so much for your serious comments. Actually, we have already found these problems in our APS publication. Immediately, we started our self-examination process. We found that this mistake was due to the carelessness when we compared different apoptosis and transwell rates between varying treatments. Specifically, when we pasted a small new image to produce a large figure, we failed to notice that we did not actually select a new image, and instead we pasted the same previous image. This is the careless mistake we will definitely avoid next time. We have already send our correction and raw data to the APS editors, and passed the editorial investigation. We are informed that we are currently at the correction stage. Thank you so much for your serious comments again. Your thoughtful understanding is highly appreciated. Best regards, Han Qiao , 
 #7 You are amazing! But do you buy the author s words? Even if he could provide raw data which I don t believe he can, how did this manuscript pass through the peer review and editor? , 
 #7  There is also duplication between the bottom left in 4A and bottom right in 4C Many thanks. That right-hand image was nagging at me, because the scale is slightly different from other panels in 4C, but I couldn t quite focus on the overlap. , 
 #10 See another image manipulation from the same author and same group in anther very new paper: Plumbagin Ameliorates Collagen-Induced Arthritis by Regulating Treg Th17 Cell Imbalances and Suppressing Osteoclastogenesis Frontiers in Immunology (2019) - 0 Comments doi: 10.3389 fimmu.2018.03102 issn: 1664-3224 pubmed: 30671063 , 
 Just to complete the record, there is a further small area of overlap in two of the panels in the middle rows of Fig 4 A C:![file] , 
 The authors regretted to find the mis-representative images in Fig. 3a, c and Fig. 4a, c when re-read our previously published article Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro in the journal of Acta Pharmacologica Sinica. The first mistake occurred due to the careless compilation when the authors tried to show the synergistic effect against tumor during figure presentation process. The second mistake occurred because the authors carelessly mixed up representative pictures of different magnification during microscopic observation of breast cancer cells after drugs treatments. The right Fig. 3a, c and Fig. 4a, c were provided below. Despite that this correction does not affect the results and conclusions of the aforementioned paper, all the authors still consent on the correction of this negligence. We apologize to the Editor and the readership of the journal for any inconvenience caused. Your thoughtful understanding is highly appreciated. ,