Effects of Curcumin and Estrogen Receptor Alpha in Luminal Breast Cancer Cells

Abstract

This work examined the potential benefit of curcumin in breast cancer patients as a supplementary drug in ER-positive cancers. The results indicated that in the MCF-7 human breast cancer cell line, E2 and curcumin decreased cell proliferation and the colony-forming capacity and down-regulated protein expression as well as important molecules associated with cell proliferation, such as PCNA and estrogen receptor alpha; genes associated with the epithelial-mesenchymal transition, such as β-catenin, Vimentin, and E-cadherin; and molecules associated with apoptosis. Clinical studies in bioinformatics have indicated a positive correlation between ESR1 and either CCND1 or BCL2 gene expression in all breast cancer patients. Thus, curcumin could become a potential natural adjuvant treatment for patients with estrogen receptor alpha-positive breast cancer and those with resistance or a poor response to endocrine therapy since the reactivation of estrogen receptor alpha is inevitable.

Keywords: 17ß-estradiol; antiestrogens; breast cancer; cancer therapy; curcumin; estrogen receptor alpha; estrogens.

Figure 1

(A) The effect of 25 μM curcumin (CUR) for 48 h on cell viability in the MCF-7 human breast cancer cell line, an estrogen receptor-positive cell line. (B) 17ß-estradiol (E2) at 1 × 10⁻⁷ for 48 h. The viability was measured using (C) an MTT assay and (D) crystal violet staining, and DMSO was used as a control (Ct). (E) The effect of CUR, E2, and a combination of both on anchorage-independent growth in the MCF-7 cell line, measured using colony formation assays in semisolid agar for 31 days. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (A–D). The data are expressed as the mean ± average with standard deviations (**: p < 0.01; ***: p < 0.001).

Figure 2

Effect of CUR, E2, and combination of both on proliferating nuclear antigen (PCNA) protein expression in MCF-7 cell line using (A) Western blot (β-actin served as control for loading) and (B) graph that represents PCNA protein expression according to relative peroxidase intensity in MCF-7 cell line, and its corresponding representative peroxidase images of immunocytochemistry analysis of PCNA (sc-56, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in MCF-7 cells treated with E2, CUR, or both combined for 48 h. Pictures were captured at 40× magnification using an Olympus CX31 optical microscope. Data are expressed as mean ± average with standard deviations (*: p < 0.05).

Figure 3

(A) The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) on ERα gene (ESR1) expression levels in the MCF-7 cell line. The cells were treated with DMSO as the control (Ct), E2, CUR, and CUR+E2 combined for 48 h. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls. (B) The effect of E2 and CUR, both alone and combined, on the ERα protein expression, with a Western blot and representative graphs, in MCF-7 cells treated for 96 h. β-actin was the loading control. (C) This graph represents the ERα protein expression according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images for the immunocytochemistry of ERα (D6R2W, sourced from Cell Signaling, CA, USA) in MCF-7 cells. The images were taken with 40× magnification using an Olympus CX31 optical microscope. The data are expressed as the average with the standard deviation (*: p < 0.05; **: p < 0.01).

Figure 4

(A) The impact of curcumin (CUR) by itself and when combined with 17β-estradiol (E2) on the protein expression of Bcl-2 and Bax in the MCF-7 cell line, using a Western blot analysis and graphs for 48 h. DMSO was used as the control (Ct) and β-actin as the loading control. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05). (B) The graphs represent Bcl-2 and Bax protein expressions according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images of the immunocytochemistry of Bcl-2 and Bax (sc-492 and sc-526, respectively, both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in the same cell line. The pictures were captured at 40× magnification using an Olympus CX31 light microscope.

Figure 5

The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) for 48 h on the levels of the cathepsin D gene (CTSD), the epidermal growth factor receptor gene (EGFR), the cyclin D1 gene (CCND1), and the BCL2 apoptosis regulator gene in MCF-7 cells, with DMSO as the control (Ct). The data are expressed as the average with the standard deviation. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05; **: p < 0.01).

Figure 6

The effect of curcumin (CUR) alone and in the presence of 17β-estradiol (E2) on (A) the β-catenin, Vimentin, and E-cadherin protein expression in MCF-7 cells treated for 48 h. Western blot analysis: DMSO was used as a control (Ct) and β-actin was used as the loading control. Comparisons between all the treated groups were made using an ANOVA and then Dunnett’s test to indicate statistical differences among the groups and the controls (*: p < 0.05). (B) Graphs that represent ß-catenin, Vimentin, and E-cadherin protein expressions according to the relative peroxidase intensity in the MCF-7 cell line and the corresponding representative peroxidase images of the immunocytochemistry of β-catenin, Vimentin, and E-cadherin (sc-1496, sc-7557, and sc-8426, respectively; provided by Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in the same cell line. The images were taken with 40× magnification in an Olympus CX31 optical microscope.

Figure 7

(A) The heatmap table shows the correlation between the estrogen receptor alpha gene (ESR1) and the cathepsin D (CTSD), epidermal growth factor receptor (EGFR), cyclin D1 (CCND1), and BCL2 apoptosis regulator (BCL2) gene expression levels in invasive breast carcinoma (BRCA) subtypes. The red color indicates a statistically significant positive correlation (Spearman’s, p < 0.05), the blue color indicates a statistically significant negative correlation (Spearman’s, p < 0.05), and gray denotes a non-significant result. (B) The scatter plots represent the significant (Spearman’s, p < 0.05) correlations between the expression of ESR1 with a purity adjustment (on the left) and the expression levels of the CTSD, EGFR, CCND1, and BCL2 genes (on the right) in patients with breast cancer. The correlation figures for each examination are indicated in red on the right side (adjusted partial Spearman’s rho value as the degree of their correlation). The expression level was estimated using TIMER2.0 (accessed on 17 June 2022) in breast cancer subtypes [20].

Figure 8

Variations in gene expression levels between tumor and normal tissues in invasive breast carcinoma across different subtypes. The box diagrams illustrate the (A) cathepsin D (CTSD), (B) epidermal growth factor receptor (EGFR), (C) cyclin D1 (CCND1), and (D) BCL2 apoptosis regulator (BCL2) gene expression levels in tumors compared to normal tissues (using the Wilcoxon rank-sum test, ***: p < 0.001). These levels were determined using TIMER2.0 (accessed on 17 June 2022) in patients with invasive breast carcinoma [20]. 1: Tumor (n = 1093), 2: Normal (n = 112), 3: Basal.Tumor (n = 190), 4: Her2.Tumor (n = 82), 5: Luminal A.Tumor (n = 564), 6: Luminal B.Tumor (n = 217).

Figure 9

Box plot representations of the transcript expressions for the following genes: (A) CTSD, or the cathepsin D gene; (B) EGFR, or the epidermal growth factor receptor gene; (C) CCND1, or the cyclin D1 gene; and (D) BCL2, or the BCL2 apoptosis regulator gene, all within the context of invasive breast carcinoma. The studied cohort was TCGA breast cancer (BRCA), with 782 subjects, and it was stratified by nature2012 based on the estrogen receptor status. A one-way ANOVA test was used for the statistical analysis, with a p-value less than 0.05 considered significant. The information was sourced from the raw data obtained via the UCSC Xena functional genomics explorer from the University of California, Santa Cruz (https://xena.ucsc.edu/), accessed on 20 August 2022 [21].

Figure 10

Survival in breast cancer patients. (A) The table depicts the normalized coefficient of the cathepsin D gene (CTSD), the epidermal growth factor receptor gene (EGFR), the cyclin D1 gene (CCND1), and the BCL2 apoptosis regulator gene (BCL2) in the Cox model, adjusted by clinical factors such as the stage in invasive breast carcinoma subtypes. The blue color indicates a statistically significant decreased risk (Z-score, p < 0.05) and gray denotes a non-significant result. (B) The Kaplan–Meier curve of the significant genes such as BCL2 in Luminal B patients. These levels were determined using TIMER2.0 (accessed on 10 July 2024) in patients with invasive breast carcinoma, reference number [20].