Ethanolic Extracts from Azadirachta indica Leaves Modulate Transcriptional Levels of Hormone Receptor Variant in Breast Cancer Cell Lines

Abstract

Breast Cancer (BC) encompasses numerous entities with different biological and behavioral characteristics, favored by tumor molecular complexity. Azadirachta indica (neem) presents phenolic compounds, indicating its potential as an antineoplastic compound. The present study aimed to evaluate the cellular response of MCF10, MCF7, and MDA-MB-231 breast cell lines to ethanolic extracts of neem leaves (EENL) obtained by dichloromethane (DCM) and ethyl acetate (EA) solvent. Extracts’ antiproliferative activities were evaluated against MCF 10A, MCF7, and MDA-MB-231 for 24 and 48 h using MTT assay. ESR1, ESR2, AR, AR-V1, AR-V4, and AR-V7 transcripts were quantified through qPCR for 0.03125 μg/mL of DCM and 1.0 μg/mL for EA for 48 h. The EENL was tested on Drosophila melanogaster as a sole treatment and then also together with doxorubicin. Antiproliferative effect on tumor cell lines without affecting MCF 10A were 1.0 µg/mL (P < 0.001) for EA, and 0.03125 µg/mL (P < 0.0001) for DCM, both after 48 h. Transcriptional levels of AR-V7 increased after treatment. In vivo assays demonstrated that EENL induced fewer tumors at a higher concentration with doxorubicin (DXR). The behavior of AR-V7 in the MDA-MB-231 tumor lineage indicates new pathways involved in tumor biology and this may have therapeutic value for cancer.

Keywords: Azadirachta indica; androgen receptor variant-7; breast cancer; ethanolic extracts; neem.

Figure 1

Effect of Ethanolic Extract of Neem Leaves (EENL) prepared from A. indica on breast cells (MCF 10A, MCF7, and MDA-MB-231) proliferation. (A,B) Treatment with EENL obtained with Ethyl Acetate (EENL–EA) extract for 24 and 48 h, respectively. (C,D) Treatment with EENL extract obtained using dichloromethane (EENL–DCM) for 24 and 48 h respectively. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Figure 2

Time-course response to Ethanolic Extract of Neem Leaves (EENL). Treatment with 1.0 μg/mL of EENL obtained with ethyl acetate (EENL–EA) (A), and EENL obtained with dichloromethane (EENL–DCM) (B).

Figure 3

Relative expression of hormone receptors transcripts after treatment with Ethanolic Extract of Neem Leaves (EENL). Cell lines were treated with 1.0 µg/mL of EENL obtained with ethyl acetate (EENL–EA) for 48 h. Relative gene expression levels were evaluated for AR (A), AR-V1, AR-V4 (B), AR-V7 (C), ESR1 and ESR2 (D) in MCF7 cell line. In MDA-MB-231 the gene expression levels were evaluated for AR (E), AR-V1 (F), AR-V4 (G), AR-V7 (H), ESR1 and ESR2 (I). The analyses were performed using the comparative Cq calibrated with data from MCF 10A and ** p < 0.01.

Figure 4

Relative expression of hormone receptors genes after treatment with Ethanolic Extract of Neem Leaves (EENL). Cell lines were treated with 0.03125 µg/mL of EENL obtained using dichloromethane (EENL–DCM) for 48 h. Relative gene expression levels were evaluated for AR (A), AR-V1, AR-V4, and AR-V7 (B), ESR1 and ESR2 (C) in MCF7 cell line. In MDA-MB-231 the relative gene expression levels were evaluated for AR and AR-V1 (D), AR-V4 (E), AR-V7 (F), ESR1 and ESR2 (G). The analyses were performed using the comparative Cq calibrated with data from MCF10 * P < 0.05.

Figure 5

Frequency of tumors in Drosophila melanogaster. Drosophila melanogaster heterozygous for the warts tumor suppressor gene were treated with different concentrations of isolated Ethanolic Extract from Neem Leaves (EENL) (0.03125%, 0.0625%, and 0.125%) and associated with doxorubicin (DXR) (0.03125%, 0.625%, and 0.125%). The frequency of tumors was analyzed in different segments (eyes, head, wings, body, legs, and halteres). Statistical analysis was according to Mann–Whitney U-test with a significance level of P < 0.05. + value considered statistically different from the negative control (P < 0.05) and ** value considered statistically different from the positive control (DXR 0.4 mM) (P < 0.05).