Enhancing Tamoxifen Therapy with α-Mangostin: Synergistic Antiproliferative Effects on Breast Cancer Cells and Potential Reduced Endometrial Impact

Abstract

Breast cancer is the most prevalent neoplasia among women worldwide. For the estrogen receptor-positive (ER+) phenotype, tamoxifen is the standard hormonal therapy; however, it carries the risk of promoting endometrial carcinoma. Hence, we aimed to evaluate the antiproliferative effect of the phytochemical α-mangostin (AM) as a co-adjuvant alongside tamoxifen on breast cancer cells to improve its efficacy while reducing its adverse effects on endometrium. For this, ER+ breast cancer cells (MCF-7 and T-47D) and endometrial cells (N30) were treated with AM, 4-hydroxytamoxifen (4-OH-TMX), and their combination. Cell proliferation was evaluated using sulforhodamine B assay, and the pharmacological interaction was determined through the combination index and the dose reduction index calculation. The genes KCNH1, CCDN1, MKI67, and BIRC5 were amplified by real-time PCR as indicators of oncogenesis, cell cycle progression, cell proliferation, and apoptosis, respectively. Additionally, genes involved in ER signaling were analyzed. In breast cancer cells, the combination of AM with 4-OH-TMX showed a synergistic antiproliferative effect and favorable dose reduction. AM and 4-OH-TMX decreased KCNH1, CCND1, and BIRC5 gene expression. In endometrial cells, AM decreased MKI-67 gene expression, while it reverted the 4-OH-TMX-dependent CCND1 upregulation. This study establishes the benefits of incorporating AM as a co-adjuvant for first-line ER+ breast cancer therapy.

Keywords: KCNH1; breast cancer; combination index; endometrium cells; synergism; tamoxifen; α-mangostin.

Figure 1

Antiproliferative effect of AM and 4-OH-TMX on MCF-7 and T-47D breast cancer cell lines. The cells were treated with increased concentrations of (a) α-mangostin (AM) and (b) 4-hydroxy-tamoxifen (4-OH-TMX) for 6 days, followed by the analysis of proliferation by the sulforhodamine B (SRB) assay. As depicted, 4-OH-TMX and AM inhibited MCF-7 and T-47D cell proliferation in a concentration-dependent manner. Results are the mean ± SEM of at least four independent experiments. The data from the vehicle-treated cells were normalized to 100%. * p < 0.001 vs. vehicle.

Figure 2

Antiproliferative effect of 4-OH-TMX and AM in combination. (a) The antiproliferative effect of 4-OH-tamoxifen (4-OH-TMX) and α-mangostin (AM) was evaluated at their respective inhibitory concentrations at 20% (IC₂₀) and 50% (IC₅₀) in MCF-7 and (b) T-47D cell lines. For further details on inhibitory concentrations, refer to Table 1. After 6 days of treatment, the cell proliferation was evaluated by the sulforhodamine B (SRB) assay. Results are shown as the mean ± SEM of at least five independent experiments. Data from vehicle-treated cells were normalized to 100% and are depicted as the first bars in each graphic. The letters above the bars indicate significant statistical differences (p ˂ 0.05) among the treatment groups, assuming that bars lacking a shared letter are considered significantly statistically different. These differences were determined by a one-way analysis of variance, followed by the post-hoc Holm–Sidak method for multiple comparisons.

Figure 3

Combination index values as a function of the fraction affected in ER+ breast cancer cells. The combination index and the fraction affected were determined by combining 4−OH−tamoxifen (4−OH−TMX) and α-mangostin (AM) at their respective inhibitory concentrations at 20% (IC₂₀) and 50% (IC₅₀) in MCF−7 (black symbols) and T−47D (white symbols) cell lines. For further details on inhibitory concentrations, refer to Table 1. Symbols below, on, or above the horizontal line (which indicates a value of 1) represent synergism, addition, or antagonism, respectively. n ≥ 5 independent experiments.

Figure 4

4-OH-TMX and AM decrease KCNH1 and CCND1 gene expression in breast cancer cells. MCF-7 (a,c) and T-47D (b,d) cell lines were treated with 4-OH-tamoxifen (4-OH-TMX) and α-mangostin (AM), alone and in combination, at their respective inhibitory concentrations at 50% (IC₅₀). For further details on inhibitory concentrations, refer to Table 1. The results are shown as the mean ± SEM of relative gene expression of KCNH1 (a,b) and CCND1 (c,d) after normalizing against the housekeeping gene ribosomal protein (RPL32). The data from the treatments were normalized to the vehicle, to which the value of 1 was arbitrarily given.Results from vehicle treatments are represented by the first bars of each graph. * p ˂ 0.05 vs. vehicle, n ≥ 4 independent experiments.

Figure 5

Effect of 4-OH-TMX and AM upon BIRC5 gene expression in breast cancer cells. MCF-7 (a) and T-47D (b) cell lines were treated with 4-OH-tamoxifen (4-OH-TMX) or α-mangostin (AM), alone and in combination, at their respective inhibitory concentrations at 50% (IC₅₀). For further details on inhibitory concentrations, refer to Table 1. The results are shown as the mean ± SEM of BIRC5 relative gene expression after normalizing against the housekeeping gene ribosomal protein (RPL32). The data from the treatments were normalized to the vehicle, to which the value of 1 was arbitrarily given, and are represented by the first bars of each graphic. * p ˂ 0.05 vs. vehicle, n ≥ 3 independent experiments.

Figure 6

Effect 4-OH-TMX alone and combined with AM on CYP19A1, PGR, and PRL expression in MCF-7 cells. The effect of the inhibitory concentrations at 50% (IC₅₀) of α-mangostin (AM, IC₅₀ = 3.53 µM) and 4-hydroxy-tamoxifen (4-OH-TMX, IC₅₀ = 2.44 µM) was evaluated upon the gene expression of (a) CYP19A1, (b) progesterone receptor (PGR), and (c) prolactin (PRL), which are involved in the synthesis of estrogens and ER signaling. The results are shown as the mean ± SEM of relative gene expression after normalizing against the ribosomal protein (RPL32) used as a housekeeping gene. The data from the treatments were normalized to the vehicle, to which the value of 1 was arbitrarily given, and are represented by the first bars of each graph. * p ˂ 0.05 vs. vehicle, n ≥ 4 independent experiments.

Figure 7

Effect of 4-OH-TMX or AM alone and combined on the gene expression of MKI67 and CCND1 in N30 cells. The concentrations required to inhibit MCF-7 cell proliferation in 50% (IC₅₀) by α-mangostin (AM, IC₅₀ = 3.53 µM) and 4-hydroxy-tamoxifen (4-OH-TMX, IC₅₀ = 2.44 µM) were used as a reference to assess their impact on the gene expression of (a) MKI-67 and (b) CCND1 in N30 cells. The results are the mean ± SEM of relative gene expression after normalizing against the RPL32 gene used as a housekeeping gene. The normalized values of the vehicle-treated cells are represented by the first bars of each graph. * p ˂ 0.05 vs. vehicle and ** p ˂ 0.05 vs. 4-OH-TMX; n ≥ 5 independent experiments with three replicates each one.