2-methoxyestradiol sensitizes tamoxifen-resistant MCF-7 breast cancer cells via downregulating HIF-1α

Abstract

The clinical studies for breast cancer (BC) are now assessing the efficacy of 2-Methoxyestradiol (2-ME), a naturally occurring derivative of estradiol. Our study aimed to explore the potential of combining the 2-ME and tamoxifen (TAM) on sensitization of TAM-resistant cells using LCC2 the TAM-resistant cells as a model and comparing the results to the sensitive cells MCF-7. Sulphorhodamine-B (SRB) assay is used to examine the 2-ME chemo-sensitizing impact on the cytotoxicity of TAM on LCC2 cells. Colorimetric assay kits were used to assess the level of the apoptosis-related markers caspases 3, Bcl2, and Bax in cell lysate. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was measured using western blotting. Total cholesterol and triglyceride (TG) levels were examined colorimetrically, using the BIOLABO kit. The use of 2-ME enhanced the cytotoxic effects of TAM and effectively reversed TAM resistance. This was achieved by inhibiting the expression of HIF-1α, while concurrently increasing the levels of apoptotic marker caspase-3, as well as the pro-apoptotic protein Bax. Additionally, there was a reduction in the levels of Bcl2, an anti-apoptotic protein. Furthermore, a reduction in TG and cholesterol levels was noted. Our findings show that HIF-1α plays an important role in TAM resistance and that suppression of HIF-1α by 2-ME-mediated sensitization of BC-resistant cells to TAM. Therefore, the concurrent administration of TAM/2-ME might potentially serve as a viable therapeutic approach to address TAM resistance and enhance the overall therapy efficacy for patients with BC.

Keywords: 2-Methoxyestradiol; Breast cancer; HIF-1α; LCC2; Tamoxifen-resistance.

Fig. 1

TAM and 2-ME suppress MCF-7 and LCC2 cell viability in a concentration-dependent manner. TAM reduces the viability of MCF-7 A and LCC2 B cells in a dose-dependent way. After a period of 48 h of being subjected to various TAM concentrations (2.5–15 μM for MCF-7 cells and 6.25–100 μM for LCC2 cells), the viability of the MCF-7 and LCC2 BC cell lines was assessed using the SRB test technique. The results, which were presented in relation to the control cells, were summarized as means ± SD obtained from three separate experiments, each of which was done in triplicate. The TAM IC50 was determined in both cells. Similarly, 2-ME decreases MCF-7 C and LCC2 D cell viability in a dose-dependent way. After 48 h of exposure to different 2-ME concentrations (0.625–10 μM for LCC2 and MCF-7 cells), the vitality of both cells was assessed using the SRB test technique. The results were presented as the mean ± SD derived from 3 consecutive experiments, each carried out in triplicate, and were given relative to the control cells. The 2-ME IC50 was detected in both cells

Fig. 2

2-ME improves TAM’s cytotoxic impacts on MCF-7 and LCC2 cells. Effect of 2ME alone and in combination with TAM on dose–response in MCF-7 cells A. Exemplary SRB test demonstrating the interaction between two doses of TAM (2.5 μM and 5 μM) and various amounts of 2-ME in MCF-7 cells. Each line depicts the viability of cells following treatment with 2ME alone (●), with 2.5 μM (■), or with 5 μM (▲) of TAM. The concentration of 2ME is given on the -axis. Cell viability was assessed by contrasting it to the survival of cells in untreated (negative control) cultures, which was standardized to 1. Results are shown as mean ± SD from three separate trials. The 2ME IC50 alone and when coupled with TAM were calculated. B: In LCC2 cells, the dose–response correlation between TAM alone and coupled with 2ME. Typical SRB test demonstrating a relationship between two different concentrations of TAM citrate (17.5 μM and 35 μM) and different doses of 2ME in LCC2 cells. The 2ME amount is shown on the x-axis, and each line shows the survival of cells following treatment with 2ME alone (●), 17.5 μM (■), or 35 μM (▲) of TAM. The survival rate of cells in the negative control cultures, which was adjusted to 1, was used to compare cell viability. Results are shown as mean ± SD from three separate trials. The IC50 of 2ME singly and in combination with TAM were calculated

Fig. 3

Combined treatment of TAM/2-ME decreases hypoxia-inducible factor alpha (HIF-1α) protein expression level in both MCF-7 and LCC2 cells. 2-ME/TAM co-treatment decreases HIF-1α expression in both MCF-7 and LCC2. Western blot analysis showed that the combination of 2-ME and TAM inhibited HIF-1α in both MCF-7 and LCC2 cells were treated with 2-ME (2.5 M, 1.25 µM, respectively) and TAM (2.5, 35 µM, respectively) for 48 h. Numbers under the bands represent the densitometric analysis of the western band compared to β-actin control

Fig. 4

Co-treatment with TAM and 2-ME increases the level of apoptosis-related markers in MCF-7 and LCC2 cells. In MCF-7 and LCC2 CELLS, 2-ME/TAM concurrent treatment led to a rise in caspase 3, Bax, and a reduction in Bcl2. Two different concentrations of 2-ME and TAM were applied to MCF-7 and LCC2 cells, respectively, during a 48-h period. By using an ELISA, the levels of caspase 3 A, Bax B, and Bcl2 C in each cell were determined. The findings were presented as mean ± SD derived from 3 separate studies. “a” Significantly distinct from MCF-7 control, “b” Significantly distinguished from MCF-7 treated with 2-ME. At P 0.05, the following comparisons were found to be significant: “c” Notably different from MCF-7 treated with TAM, “d” Remarkably different from LCC2 control cells, “e” Significantly distinctive from LCC2 treated with 2-ME, and “f” Statistically different from LCC2 treated with TAM

Fig. 5

TAM/2-ME combination therapy reduced the amount of cholesterol and triglycerides (TG) per 100 mg of protein in MCF-7 and LCC2 cells. Following a combination with TAM and 2-ME, MCF-7 and LCC2 cells showed increased TG and cholesterol content (mg/100 mg protein). MCF-7 and LCC2 cells were exposed to 2-ME (5 μM and 2.5 μM, respectively) and TAM (5 μM and 17.5 μM, respectively) for 48 h. TG (B) and cholesterol (A) concentrations in MCF-7 and LCC2 cells that had undergone different treatments were measured using spectroscopic measurement The results of three separate studies (n = three) are computed as mean ± SD. Employing one-way ANOVA and Tukey’s post hoc multiple comparison tests, the statistical significance was evaluated. Statistically different from MCF-7 controls in “a” and “b” Statistically different from MCF-7 treated with 2-ME, “d” Significantly different from LCC2 control cells, “e” Significantly different from LCC2 treated with 2-ME and “f” Significantly different from LCC2 treated with TAM, at P < 0.05