Curcumol enhances the anti-tumor effects of metformin via suppressing epithelial-mesenchymal transition in triple-negative breast cancer

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.

Keywords: Triple-negative breast cancer (TNBC); curcumol; epithelial–mesenchymal transition; metformin.

Figure 1

Curcumol enhanced the anti-proliferative effect of metformin in TNBC cells. (A) Cell viability was detected by CCK-8 assay. Three different types of cells MDA-MB-231, HCC1806, and MDA-MB-468 were treated with different concentrations of curcumol (0, 1, 5, 25, 50, 100, 200, and 400 µM). (B) After MDA-MB-231 and HCC1806 cells were treated with curcumol 50 µM, metformin 10 µM, or a combination of both, their viability was detected by CCK-8 assay. Each experiment was performed three times. **, P<0.01 versus the control group; ^##, P<0.01 versus the metformin group. TNBC, triple-negative breast cancer.

Figure 2

Curcumol enhanced the effect of metformin in promoting TNBC cell apoptosis. MDA-MB-231 and HCC1806 cells were treated with curcumol 50 µM, metformin 10 µM, or a combination of both. (A,B) Cell apoptosis was determined by TUNEL assay. (C) The expression levels of cleaved caspase-3, Ki67, and PCNA were detected by western blot. (D,E) Quantitative analysis of related proteins was performed. Each experiment was performed three times. **, P<0.01 versus the control group; ^##, P<0.01 versus the metformin group. Scale bar: 40 µm. TNBC, triple-negative breast cancer.

Figure 3

Curcumol enhanced the anti-metastatic and anti-EMT effects of metformin in TNBC cells. (A,B) Cell invasion of MDA-MB-231 cells and HCC1806 cells was detected by Transwell assay. (C,D) Cell migration was detected by wound healing assay in MDA-MB-231 cells and HCC1806 cells. (E,F) The related proteins in MDA-MB-231 and HCC 18006 cells were detected by western blot. (G,H) Quantitative analysis of related proteins in both MDA-MB-231 and HCC 18006 cells was carried out. Each experiment was performed three times. **, P<0.01 versus the control group; ^##, P<0.01 versus the metformin group. Scale bar: 40 µm. EMT, epithelial-mesenchymal transition; TNBC, triple-negative breast cancer.

Figure 4

Curcumol reversed proliferation, migration, invasion and EMT induced by rucaparib, and enhanced the effects of metformin on TNBC cells. Cells were divided into 8 groups: the control, curcumol (50 µM), metformin (10 µM), curcumol (50 µM) + metformin (10 µM), rucaparib (5 µM), curcumol (50 µM) + rucaparib (5 µM), metformin (10 µM) + rucaparib (5 µM), and curcumol (50 µM) + metformin (10 µM) + rucaparib (5 µM) groups. (A) Cell viability was examined by CCK-8 assay. (B) Cell apoptosis was detected by TUNEL assay, respectively. (C) Transwell assay was carried out to detect cell invasion. (D) Cell migration was detected by wound healing assay. (E) The related proteins E-cadherin, N-cadherin, and Twist1, were detected by western blot. Each experiment was performed three times. *, compared with the control group; ^#, compared with the Ruca 5 group. **, P<0.01; ^#, P<0.05; ^##, P<0.01. Scale bar: 40 µm. EMT, epithelial-mesenchymal transition; TNBC, triple-negative breast cancer.

Figure 5

Curcumol enhanced the anti-growth and anti-EMT effects of metformin in vivo. The in vivo experiments were carried out via injecting MDA-MB-231 cells into nude mice. The tumor growth factors were detected every 7 days. On day 28 after injection, the mice were sacrificed. (A) The representative xenografts tumor pictures of nude mice co-treated with 50 curcumol and metformin. (B) Tumor volume was determined after nude mice were treated with the combination of curcumol and metformin. (C,D) The expression of Ki67 and VEGF in the mouse tumor tissues was determined by immunohistochemistry. (E) The immunohistochemistry results for Ki67 and VEGF were analyzed. (F) The expression levels of related proteins in tumor tissues were detected by western blot. (G,H) The results of western blot were quantitatively analyzed. Each experiment was performed three times. **, P<0.01 versus the control group; ^##, P<0.01 versus the metformin group. Scale bar: 50 µm. EMT, epithelial-mesenchymal transition.