The inhibitory effects of butein on cell proliferation and TNF-α-induced CCL2 release in racially different triple negative breast cancer cells

Abstract

Drug resistance is the leading cause of breast cancer-related mortality in women, and triple negative breast cancer (TNBC) is the most aggressive subtype, affecting African American women more aggressively compared to Caucasians women. Of all cancer-related deaths, 15 to 20% are associated with inflammation, where proinflammatory cytokines have been implicated in the tumorigenesis process. The current study investigated the effects of the polyphenolic compound butein (2',3,4,4'-tetrahydroxychalcone) on cell proliferation and survival, as well as its modulatory effect on the release of proinflammatory cytokines in MDA-MB-231 (Caucasian) and MDA-MB-468 (African American) TNBC cell. The results obtained showed that butein decreased cell viability in a time and dose-dependent manner, and after 72-h of treatment, the cell proliferation rate was reduced in both cell lines. In addition, butein was found to have higher potency in MDA-MB-468, exhibiting anti-proliferative effects in lower concentrations. Apoptosis assays demonstrated that butein (50 μM) increased apoptotic cells in MDA MB-468, showing 60% of the analyzed cells in the apoptotic phase, compared to 20% in MDA-MB-231 cells. Additionally, butein downregulated both protein and mRNA expression of the proinflammatory cytokine, CCL2, and IKBKE in TNFα-activated Caucasian cells, but not in African Americans. This study demonstrates butein potential in cancer cell suppression showing a higher cytotoxic, anti-proliferative, and apoptotic effects in African Americans, compared to Caucasians TNBC cells. It also reveals the butein inhibitory effect on CCL2 expression with a possible association with IKBKE downregulation in MDA-MB-231 cells only, indicating that Caucasians and African Americans TNBC cells respond differently to butein treatment. The obtained findings may provide an explanation regarding the poor therapeutic response in African American patients with advanced TNBC.

Fig 1. The effect of butein on cell viability and proliferation in MDA-MB-231 and MDA-MB-468 TNBC cells.

Butein tested concentrations ranged from 0.78–200 μM. All experiments were performed at least 3 times with n = 5 and kept at 5% CO₂ and 37°C. The cytotoxic effect was measured after 24 (A), 48 and 72-h (B), and the anti-proliferative effect after the 72-h treatment period (C). For both assays, cells were also treated with DMSO (<0.1%). For the proliferation assay, Taxol (1 μM) was used as a positive control. The data are presented as the mean ± S.E.M. Statistically significant differences between control vs. treatments were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = p > 0.05.

Fig 2. The apoptotic effect of butein in MDA-MB-231 and MDA-MB-468 TNBC cell lines.

Cells were exposed to butein at concentrations ranging from 12.5–200 μM for 24 h, and control cells were treated with DMSO (< 0.1%). Apoptotic effect was determined by flow cytometry using Annexin V-FITC kit and FACSCalibur Flow cytometer to analyze the percentage of the apoptotic cells compared to the control cells. A and C represent the scatter plots for each one of the cell lines showing the movement of cells from the resting to the apoptotic state, and B and D show the percentage of apoptosis compared to the control group. The results represent the mean ± S.E.M. of two independent studies (n = 3). Statistically significant differences between control vs. treatments were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3. The effect of butein on cytokine expression in TNF-α-activated MDA-MB-231 and MDA-MB-468 TNBC cells (n = 3).

A- Array layout used to assess chemokines/cytokines expression in the cell-free supernatants, showing the cytokines map, and highlighting CCL2 (MCP1), IGFBP-1, IL-6, and positive controls. B and C—Arrays with chemiluminescent spot intensity of supernatants derived from Caucasian breast cancer and African American cells showing cytokine changed expression after treatments. Blots represent the supernatants of 4 treatments: control (cells + DMSO), butein (5 μM), TNF-α (40 ng/ml), and butein (5 μM) + TNF-α (40 ng/ml) after 24-h treatment period.

Fig 4. Normalized protein expression of CCL2, IL-6, and IGFBP1 (A, C, D) in MDA-MB-231 and (B) CCL2 in MDA-MB-468 TNBC cells.

Data represent normalized dot spot intensities from the cytokine arrays calculated based on the positive controls found in the corners of each one of the membranes using RAYBIO^®ANALYSIS software (RayBiotech). Data are expressed as % of control arrays (mean ± S.E.M. n = 3), representing 4 treatments: control (cells + DMSO), butein (5 μM), TNF-α (40 ng/ml), and butein (5 μM) + TNF-α (40 ng/ml). Statistically significant differences between control vs. butein and TNF-α (*)and TNF-α vs. butein + TNF-α (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. **p < 0.01, ****p < 0.0001, ^#p < 0.05, ns = p > 0.05.

Fig 5. ELISA protein expression and mRNA quantification in MDA-MB-231 and MDA-MB-468 TNBC cells.

The effect of butein (5 μM) on CCL2 (MCP1) and IL-6 protein expression in TNF-α stimulated MDA-MB-231 (A,C) and on CCL2 protein expression in TNF-α stimulated MDA-MB-468 cells (B). In D and E, the effect of butein in CCL2 mRNA quantification in Caucasians and African American TNBC cells, respectively. Each data point represents the mean ± S.E.M. of three independent experiments (n = 3), representing 4 treatments: control (cells + DMSO), butein (5 μM), TNF-α (40 ng/ml), and butein (5 μM) + TNF-α (40 ng/ml). Statistically significant differences between control vs. butein and TNF-α (*)and TNF-α vs. butein + TNF-α (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. **p < 0.01, ****p < 0.0001, ^####p <0.001, ns = p > 0.05.

Fig 6. IKBKE mRNA and protein expression quantification in MDA-MB-231 and MDA-MB-468 TNBC cells.

In A and B, the effect of butein (5 uM) in IKBKE normalized mRNA expression in TNF-α stimulated MDA-MB-231 and MDA-MB-468 cells. Each data point represents the mean ± S.E.M. of three independent studies (n = 3), representing 4 treatments: control (cells + DMSO), butein (5 μM), TNF-α (40 ng/ml), and butein (5 μM) + TNF-α (40 ng/ml). In C, electropherogram shows total IKBKE protein expression and the amount of chemiluminescence measured after Caucasian cells were exposed to the different treatments. In D, bands representing the protein expression after the 4 treatments (24 h) for total and phosphorylated IKBKE protein. Statistically significant differences between control vs. butein and TNF-α (*)and TNF-α vs. butein + TNF-α (^#) were evaluated by a one-way ANOVA, followed by Dunnett’s multiple comparison tests. **p < 0.01, ***p < 0.001, ****p < 0.0001, ^#p < 0.05, ns = p > 0.05.

Fig 7. Proposed mechanism of butein effect in MDA-MB-231 and MDA-MB-468 TNBC cells.

The diagram shows butein inhibitory effect in TNF-α-stimulated CCL2 expression at mRNA and protein level, attenuating IKBKE expression as a possible molecular mechanism in Caucasian cells; in addition to a possible signaling pathway for butein apoptotic effects in MDA-MB-231 and MDA-MB-468 TNBC cells.