Curcumol enhances the anti-tumor effects of metformin via suppressing epithelial-mesenchymal transition in triple-negative breast cancer
Authors: Gangyue Wang
Yi Dong
Heng Liu
Journal: Annals of Translational Medicine (2020)
Discipline: CLINICAL MEDICINE
Keywords: Triple-negative breast cancer (TNBC); curcumol; epithelial–mesenchymal transition; metformin.
Affiliations: Department of Breast, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.

Abstract:  Background: Triple-negative breast cancer (TNBC) is a severe disease with a high mortality rate. Metformin has been found to possess anti-tumor properties. Curcumol, an active ingredient extracted from curcuma, exerts the protective effect in TNBC cells through inducing apoptosis. However, the effects of curcumol combined with metformin on the treatment of TNBC have yet to be fully established. Methods: TNBC cells MDA-MB-231 and MDA-MB-468 cells were used in the study. TNBC cells were treated with curcumol and metformin alone or treated with curcumol combined with metformin. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The levels of proteins were measured using Western blot. Wound healing assay and Transwell invasion assays were used to determine cell migration and invasion ability, respectively. A xenograft model was established to investigate the tumor growth ability. Immunohistochemistry was performed to determine the expression of Ki-67 and Vascular endothelial growth factor (VEGF). Results: In the study, the administration of curcumol alone had no significant effects on the TNBC cells. However, the anti-proliferation, anti-metastasis, and anti-epithelial-mesenchymal transition (EMT) effects of metformin were enhanced by the addition of curcumol. Further, curcumol reversed TNBC cell proliferation, migration, invasion, and EMT induced by rucaparib, and enhanced the effect of metformin on rucaparib-induced TNBC cells. The combination of curcumol and metformin also suppressed tumor growth, EMT marker expression, and the activation of Wnt2/β-Catenin signaling during in vivo experiments. Conclusions: The combination of curcumol and metformin enhances the anti-tumor effects of metformin on TNBC via inhibiting EMT. Curcumol combined with metformin may hold promise as a therapeutic strategy for TNBC. 

Retracted: No

PubPeer comments:
 Figure 1 and 2. * Red boxes: Lanes 3 and 4 of the bottom blot in Figure 1C look similar to lanes 1 and 2 of the bottom blot in Figure 2B. Note the presence of two dots between the lanes. * Blue boxes: In the top panel of Figure 2B, lane 3 (30 min) and 5 (8h) appear to look similar. The 4th (4h) lane appears to contain a rectangle that has a lighter background than the other lanes. ![file] , 
 Figure 2A. * Orange boxes: The 0h timepoints for both treatments appear to look similar. This might be acceptable, but is not really good practice to have 1 control serve multiple experiments, unless perhaps if they were all performed at the same time machine conditions. Could the authors please clarify? * Aqua boxes: One cell in the bottom 30 min panel looks similar to a cell in the bottom 4h panel. Since every sample was taken after a certain amount of time of incubation, and then fixed and used for microscopy, this does not appear to be a simple time series. Could the authors please clarify? ![file] Clarification by the authors would be greatly appreciated. The methods state: *In the next day, the coverslips were washed twice in PBS and then the cells were incubated in Hepes buffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM Hepes, 10 mM glucose, pH 7.2) and exposed to different stimuli for the indicated lengths of time at 37 u25e6 C. Then the cells were fixed with 3.7% paraformaldehyde. Fluorescent images were collected using a confocal laser scanning microscope (TCS SP2 from Leica Microsystem; 488 nm argon excitation, 515 nm long pass barrier filters).* , 
 Figures 1, 2, and 3. An update on comment #1. * Red boxes: Lanes 3 and 4 of the bottom blot in Figure 1C look similar to lanes 1 and 2 of the bottom blot in Figure 2B. Note the presence of two dots between the lanes. * Blue boxes: In the top panel of Figure 2B, lane 3 (30 min) and 5 (8h) appear to look similar. The 4th (4h) lane appears to contain a rectangle that has a lighter background than the other lanes. * Green boxes: Lanes 2-5 of the bottom blot in Figure 2B appear to look similar to the bottom blot of Figure 3, top. Since Figures 1C and 2B also show an overlap (marked in red) that overlap is also partially with Figure 3. ![file] , 
 To top it off with a cherry, there are some regions of similarity within and between flow cytometry panels in Figure 6E. Boxes of the same color highlight groups of dots that look similar. ![file] ,