Arrabidaea chica chloroform extract modulates estrogen and androgen receptors on luminal breast cancer cells

Abstract

Background: Breast Cancer (BC) is the most common cancer in women worldwide and, although 70% of patients are responsive to selective Estrogen Receptor (ER) modulators such as Tamoxifen (Tam), patients' survival is comprised by resistance to endocrine therapy. Brazilian flora, especially the Amazon biome, is one of the richest global sources of native species with potentially bioactive compounds. Arrabidaea chica is a plant native to the Amazon that has been used in the treatment of different diseases. However, its action on BC remains unclear.

Methods: Herein the biological effects of the chloroform extract of A. chica (CEAC) were evaluated on BC cells and in in vivo model. After confirmation of CEAC antioxidant capacity, cells were treated with CEAC and Tam, alone and with CEAC+Tam. The cell viability was evaluated by MTT and hormone receptor transcripts levels were assessed (ESR1, ESR2 and AR). Finally, anticarcinogenicity of CEAC was recorded in Drosophila melanogaster through Epithelial Tumor Test (ETT).

Results: The study confirmed the antioxidant activity of CEAC. CEAC was selective for MCF-7, downregulating ESR2 and AR transcripts and upregulating ESR2 expression. The modulatory effects of CEAC on ERs did not differ between cells treated with Tam and with CEAC+Tam. Interestingly, previous treatment with CEAC, followed by treatment with Tam promoted a significant decrease in cell viability. The extract also presented anticarcinogenic effect in in vivo assay.

Conclusion: The bioassays on breast tumor cells demonstrated the antiproliferative activity of the extract, which modulated the expression of hormone receptors and sensitized luminal tumor cells to Tam. These results suggest that CEAC could be a complementary treatment for BC.

Keywords: Breast Cancer; Carcinogenicity; Hormone Receptors; Hormonotherapy; Natural Products.

Fig. 1

Antioxidant activity of the chloroform extract of Arrabidaea chica (CEAC) evaluated by the ABTS⁺ method. The tested concentrations of CEAC were 125 μg/mL, 250 μg/mL, 500 μg/mL and 1000 μg/mL. The results are represented relative to Trolox, as reference standard

Fig. 2

Cytotoxic effects of chloroform extract of Arrabidaea chica (CEAC) on human luminal breast cancer cells T-47D, MCF-7. The non-tumorigenic cell line MCF 10A and the triple-negative breast cancer cell MDA-MB231 were included in this study. Cells treated with DMSO (diluent) were used as control. Treatments were performed on the four cell lines with different concentrations of CEAC for 24 (A) and 48 hours (B). Cell viability rates of luminal breast cancer cell lines were also recorded after treatment with CEAC and Tamoxifen (Tam) for 48 hours in T-47D (C) and MCF-7 (D) cell lines. Cells were treated with CEAC and Tam in isolation and combined. Data are presented as mean ± S. D of three independent experiments. Significance was calculated by one-way ANOVA, and Tukey’s post hoc test. # treatments with DMSO differed from all treatments with CEAC, p < 0.0001. a: treatment in MCF-7 differed from treatment in MCF 10A. b: treatment in MCF-7 differed from treatment in T-47D. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 3

Relative levels of hormone receptors transcripts after treatment with the chloroform extract of Arrabidaea chica (CEAC) and Tamoxifen (Tam). Gene expression levels were recorded without treatment in MCF 10A, T-47D, MCF-7 and MDA-MB231 cell lines (A, B, C). T-47D (D, E, F) and MCF-7 (G, H, I) cell lines were treated with 1000 μg /mL of CEAC, Tam (1 μM) and CEAC + Tam (1000 μg /mL, and 1 μM, respectively) for 48 h. The relative expression levels of the genes encoding for Estrogen Receptor alpha (ESR1), Estrogen Receptor beta (ESR2), and Androgen receptor (AR) were quantified by the comparative Cq method. * p <0,05, ** p <0,01, *** p <0,001 e **** p <0,0001

Fig. 4

Cell viability rates of luminal breast cancer cell lines after alternate treatment with chloroform extract of Arrabidaea chica (CEAC) and Tamoxifen (Tam) for 48 hours. T-47D (A) and MCF-7 (B) cell lines were treated with CEAC followed by Tam (1μM) or with Tam (1μM) followed by CEAC (1000 μg/mL). Data are expressed as means ± SD, n = 3. Significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was calculated by one-way ANOVA, and Tukey’s post hoc test. # treatments with DMSO (control) differed from all treatments with CEAC, p < 0.0001

Fig. 5

Effect of chloroform extract of Arrabidaea chica (CEAC) and Tamoxifen (Tam) on tamoxifen-resistant MCF7 cells (MCF-7/TamR). Cell viability of MCF-7/TamR cells after treatment with CEAC (1000 μg/mL), Tam (1μM) or with CEAC + Tamfor 48 hours (A). Relative levels of hormone receptors transcripts after treatment with CEAC, Tam or CEAC + Tam. Gene expression was recorded through Cq method (B). MCF-7/TamR cell lines were also treated with CEAC followed by Tam (1μM) or with Tam (1μM) followed by CEAC (1000 μg/mL) and the viability evaluated by MTT (C). Data are expressed as means ± SD, n = 3. Significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was calculated by one-way ANOVA, and Tukey’s post hoc test. # treatments with DMSO differed from all treatments, p < 0.0001. ESR1: Estrogen Receptor alpha, ESR2: Estrogen Receptor beta (ESR2), and AR: Androgen receptor

Fig. 6

In vivo assays performed with D. melanogaster treated with the chloroform extract of Arrabidaea chica (CEAC). Percentage of survival of D. melanogaster treated with CEAC (A). Treatments were conducted with CEAC (2.5, 5.0, 10.0 and 20.0 μg/μL). Flies, heterozygous for the Warts tumor suppressor gene, were further treated with different concentrations of CEAC (2.5, 5.0 and 10.0 μg/μL) and total tumors were recorded to demonstrate the carcinogenic (B) effect of CEAC. To demonstrate the anticarcinogenic effect of CEAC (C) flies were pre-treated with Doxorubicin (DOX 0.4 mM) and, subsequently, chronic treated for 48h with different concentrations of the extract. The frequency of tumors was analyzed in different segments, and significance was calculated by the Mann-Whitney Test. **Values considered significant when compared to the positive control (DOX 0.4 mM). *Values considered different from the negative control (P < 0.05). NC, negative control (osmosis reverse water). SC, solvent control (Tween 80 1%). PC, positive control (DOX 0.4mM). NC: Negative control (reverse osmosis water); SC: Solvent control (Tween 80 at 1%); PC: Positive control (DOX 0.4 mM)