Inhibition of Cancer Stem-like Cells by Curcumin and Other Polyphenol Derivatives in MDA-MB-231 TNBC Cells

Abstract

Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers and is highly aggressive. Despite an initial positive response to chemotherapy, most patients experience rapid disease progression leading to relapse and metastasis. This is attributed to the presence of breast cancer stem cells (BCSCs) within the tumor, which are characterized by self-renewal, pluripotency, and resistance mechanisms. Targeting BCSCs has become critical as conventional therapies fail to eradicate them due to a lack of specific targets. Curcumin, a polyphenol derived from turmeric (Curcuma longa), exhibits anticancer effects against breast cancer cells and BCSCs. The use of curcumin derivatives has been suggested as an approach to overcome the bioavailability and solubility problems of curcumin in humans, thereby increasing its anticancer effects. The aim of this study was to evaluate the cellular and molecular effects of six synthetic compounds derived from the natural polyphenol epigallocatechin gallate (EGCG) (TL1, TL2) and curcumin derivatives (TL3, TL4, TL5, and TL6) on a TNBC mesenchymal stem-like cell line. The activity of the compounds against BCSCs was also determined by a mammosphere inhibition assay and studying different BCSC markers by Western blotting. Finally, a drug combination assay was performed with the most promising compounds to evaluate their potential synergistic effects with the chemotherapeutic agents doxorubicin, cisplatin, and paclitaxel. The results showed that compounds exhibited specific cytotoxicity against the TNBC cell line and BCSCs. Interestingly, the combination of the curcumin derivative TL3 with doxorubicin and cisplatin displayed a synergistic effect in TNBC cells.

Keywords: cancer stem cells; curcumin; polyphenols; triple-negative breast cancer.

Figure 1

Structure of G28, curcumin, and curcumin analogues 1 and 2.

Figure 2

Structures of polyphenols TL1–TL6.

Scheme 1

Synthetic strategy for the preparation of polyphenols TL1–TL6.

Figure 3

Effect of curcumin and TL3–TL6 on breast cancer stem cells (BCSCs) in MDA-MB-231 cells evaluated using a mammosphere formation assay after treatment with concentrations equivalent to their IC₃₀ for 7 days. (A) Mammosphere Formation Index (MFI) for each compound. Significant differences in MFI compared to curcumin treatment are indicated as * (p < 0.05) and ** (p < 0.01). (B) Mammosphere Inhibition Index (MFIn) for each compound.

Figure 4

Notch1, Shh, and β-catenin protein expression analysis of MDA-MB-231 cells treated with curcumin and polyphenols TL3–TL6 for 72 h. GAPDH was used as a loading control. Quantified Western blotting results obtained using ImageLab software (Image Lab Software for Mac Version 6.1). Expression ratios are relativized with GAPDH and control group (untreated cells). Significant differences are indicated as * (p < 0.05) and *** (p < 0.001). See Western blots in Supplementary Figure S3A.

Figure 5

CD24, CD44, and ALDH1 protein expression analysis of MDA-MB-231 cells treated with curcumin and polyphenols TL3–TL6 for 72 h. GAPDH was used as a loading control. Quantified Western blotting results obtained using ImageLab software (Image Lab Software for Mac Version 6.1). Expression ratios are relativized with GAPDH and control group (untreated cells). Significant differences are indicated as * (p < 0.05) and ** (p < 0.01). See Western blots in Supplementary Figure S3B.

Figure 6

Combinatorial treatment between TL3 or TL4 and chemotherapeutic agents at concentrations equivalent to their IC₃₀ (Doxo: doxorubicin; Pacli: paclitaxel; and Cispla: cisplatin) in MDA-MB-231 cells. (A) Combination index (CI) from treatments with TL3 or TL4 and chemotherapeutic agents. Data are expressed as mean ± SD from three independent experiments and are based on the Chou and Talalay method. The dotted line indicates additive effect (CI approximately equal to 1). CI > 1 is indicative of an antagonistic effect and CI < 1 a synergistic effect. Significant differences are indicated as * (p < 0.05). (B) Dose–response curves of TL3 (from 0.1 to 8 µM) or TL4 (from 0.4 to 2.5 µM) alone and in combination with chemotherapeutic agents at concentrations equivalent to their IC₃₀ for 72 h. Results shown are mean ± SE from at least three independent experiments.