According to the World Health Organization (WHO) [1
], Breast Cancer (BC) is the most common malignant tumor among women. An increase from 14 million in 2012 to 22 million new cases is estimated by 2022. By 2035, the number of deaths is expected to grow by 70% [2
]. BC is a disease that encompasses numerous entities, with peculiar biological and behavioral characteristics favored by a complex molecular microenvironment [3
Malignancy results from regulatory imbalances involving pathways closely related to growth and proliferation [4
]. In this context, receptors for estrogen and androgenic hormones are important proteins in cancer progression and play key roles in deciding the appropriate treatments [8
Estrogen hormone binds to receptors on the nucleus membrane and regulates the expression of genes associated with survival, proliferation, and differentiation of mammary cells [9
]. The estrogen receptor alpha (ESR1) is the main receptor responsible for these events, thus hormone therapy is considered when tumors display this receptor. However, therapies using the ESR1 as a target may cause some patients to become more resistant [8
]. The ESR1 protein forms a homodimer or a heterodimer with the protein ESR2 (estrogen receptor beta) and is responsible for regulating gene function [11
gene encodes for the androgen receptor, which is a transcription factor activated by a steroid hormone [14
]. Structurally related to ESR1, this protein is expressed in 80% of breast tumors, of which 55% are ESR1-positive and 35% are classified as triple-negative tumors (negative for ESR, progesterone receptor, and HER2 receptor). In recent years, 18 AR
) have been described and characterized, especially focusing on their role in disease progression [16
]. In prostate cancer, AR-V7
is involved in cancer cell growth in the absence of androgens, which represents a highly advanced form of the disease [14
]. From a clinical perspective, AR
can be a favorable prognostic indicator [20
], but its role in BC needs a deeper understanding [21
In light of the regulatory role of ESR and AR, agents able to modulate these receptors’ gene expression emerges as a fundamental strategy for tumor aggressiveness control, and could potentially be used as new therapies.
Brazil has approximately 25% of the world’s biodiversity, providing great opportunities for the development of cancer drugs and therapies [25
]. Different natural products present antitumor properties, endorsing the importance of scientific studies that elucidate their mode of action [26
Among these diverse plants, the extracts from the species Azadirachta indica
A. Juss., commonly known as “neem”, have been used for the treatment of inflammation, viral infections, hypertension, and displays insecticidal, nematicide, and fungicidal properties [30
]. Although the bioactive compounds present in neem are found in different tissues of this plant, those from their seeds and leaves are more concentrated, accessible, and easily obtained by water or organic solvents extraction methods, such as those that use hydrocarbons, alcohols, ketones, or ethers [34
Considering that natural phytochemicals contain phenolic compounds with antimetastatic activity [36
], A. indica
should be investigated in cancer research, since phenolic compounds were found in this species [40
]. Balasenthil et al. (1999) [41
] demonstrated that neem leaves extract administered to hamsters with oral carcinoma promoted tumor suppression by modulating lipid peroxidation, antioxidant action, and detoxification. Leaves of this species are also capable of activating an immune response [42
]. It has also been reported that flavones isolated from neem flowers have antimutagenic effects by inhibition of the enzymatic activation of heterocyclic amines [43
In this study, we hypothesized that ethanolic extracts from Azadirachta indica leaves (EENL) obtained by dichloromethane (DCM) or ethyl acetate (EA) extraction could modulate the expression of estrogen and androgen receptors, thus promoting molecular changes that would hinder the mammary tumor activity. Therefore, our goal was to evaluate the cytotoxic and mutagenic effects of the extracts and their effect on the expression of genes coding for the hormonal receptors in the lineages MCF 10A (non-tumorigenic), MCF7 (ESR + BC), and MDA-MB-231 (triple-negative BC [TNBC]).
2.1. Bioactive Compounds and Antiproliferative Effects of EENL
Total phenols of DCM and EA extracts were calculated according to the standard curve of gallic acid equivalents (GAE) subjected to a linear regression. Concentrations of this bioactive compound were 40.415 ± 0.566 mg GAE/g and 45.200 ± 0.569 mg GAE/g for EA and DCM, respectively (P < 0.01). Antiproliferative activity of EENL extracts was further investigated in three breast lineages (MCF 10A, MCF7, and MDA-MB-231) through MTT assay.
EENL–EA did not reduce the proliferation of breast cancer cell lines (Figure 1
A) after 24 h of treatment. The non-tumorigenic lineage was more sensitive to the EA extract at 0.0078125 μg/mL, 0.125 μg/mL, 0.25 μg/mL, and 1.0 μg/mL. In addition, the viability of MCF7 increased after treatment at 0.0078125 μg/mL up until 0.25 μg/mL for 48 h (Figure 1
B). However, in the highest concentration, MCF7 viability decreased compared to MCF 10A (P
The treatment with EENL–DCM extract for 24 h reduced the proliferation of MCF7 compared to MCF 10A (P
< 0.05) at 0.015625 μg/mL (Figure 1
C). After 48 h (Figure 1
D), DCM extract inhibited the proliferation of the triple-negative tumor cell line (MDA-MB-231), with extracts’ concentrations ranging from 0.0625 to 0.25 μg/mL, when compared to MCF7 lineage. Compared to MCF 10A, the ESR + tumor cell line (MCF7) showed a reduction in viability at a concentration of 1.0 μg/mL. Only at the concentration 0.03125 μg/mL and 0.0625 were there significant differences (P
< 0.001) between the non-tumorigenic lineage (MCF 10A) and the triple-negative cells’ (MDA-MB-231) viability.
Based on these results, the concentrations that showed antiproliferative effect on tumor cell lines, but did not decrease MCF 10A viability, were chosen for molecular assays. For EENL–EA extract we used 1.0 µg/mL (P
< 0.001), and for EENL–DCM extract 0.03125 µg/mL (P
< 0.0001) after 48 h of treatment, as demonstrated in the time-course Figure 2
2.2. Transcriptional Profile of Hormone Receptors after Treatment with EENL
Gene expression of ESR1
in breast cells was evaluated before and after 48 h of treatment with 1.0 μg/mL of EENL–EA extract (Figure 3
expression increased 2.85-fold (P
< 0.01) in treated MDA-MB-231 cells after 48 h (Figure 3
H). The remaining genes in MDA-MB-231, and all genes in MCF7, did not display statistically significant differences in gene expression levels. Although not significant, the mean AR
relative gene expression levels were higher in 48 h-treated MDA-MB-231 cells (31.74, 2.03, 7.07, and 4.37-fold, respectively).
No significant gene expression changes were found in MCF7 cells treated with EENL–DCM extract at 0.03125 μg/mL for 48 h (Figure 4
). However, the TNBC cell line MDA-MB-231 had a 28.41-fold increase on the expression of AR-V7
upon treatment (P
< 0.05) (Figure 4
2.3. In Vivo Experiments with Drosophila Melanogaster
The results showed the carcinogenic action of EENL isolate at the concentrations 0.03125%, 0.0625%, and 0.125%, and the modulating effect of the EENL on the carcinogenic action of doxorubicin (DXR at 0.4 mM). DXR may intercalate on DNA and induce formation of DNA adducts at active promoter sites, increasing torsional stress and enhancing nucleosome turnover. Furthermore, it may trap topoisomerase II at breakage sites, causing double strand breaks. Enhanced nucleosome turnover might increase the exposure of DNA to reactive oxygen species (ROS) resulting in DNA damage and cell death [44
]. The frequency of the tumor clone per segment of Drosophila melanogaster
is demonstrated in Table 1
and Figure 5
The study design included a negative control, which was flies with mutated gene; and DXR as a positive control. For the negative control, the frequency of 0.02 of tumors per fly were observed, and this discrete tumor induction occurs due to the genetic predisposition of the test organism. On the other hand, the positive control induced a frequency of 0.46 of tumors per fly, proving that the organism lineage responded to tumor induction.
Larvae that were exposed to the EENL isolate, at concentrations of 0.03125%, 0.0625%, and 0.125% displayed frequencies of 0.21, 0.16, and 0.20 tumors per fly, respectively. Compared to the negative control, a statistically significant increase in tumor induction confirmed the carcinogenic effect of the extract on D. melanogaster. When 0.03125%, 0.0625%, and 0.125% of EENL were applied together with doxorubicin at 0.4 mM, the frequencies of tumors per fly were 5.43, 10.24, and 0.69, respectively. These results demonstrate that EENL with DXR increased tumor frequency at the lowest concentrations, when compared to the positive control. However, in the highest concentration used (0.125%), even when associated with DXR 0.4 mM, the tumor frequency decreased.
The association between phenolic compounds in plants and antioxidant activity is due to phenolic hydroxyl groups that have a strong free radical scavenger activity [45
]. Abdelhady et al. (2015) [40
], during a phytochemical search for active substances, demonstrated that several phenolic compounds are present in Azadirachta indica.
In fact, natural phytochemicals contain phenolic compounds with the ability to prevent cancer metastasis [36
]. Several studies have shown that different flavonoids and polyphenols exert an anti-metastatic effect [37
]. Here we evaluated the cellular effects of ethanolic extracts of neem in breast cell lines. The presence of phenolic compounds in the EENL–DCM and EENL–EA demonstrate their potential antiproliferative activity, which may reduce tumor aggressiveness. The different cellular responses, evaluated by the MTT reduction, after treatments with EENL extracts obtained using DCM and EA, indicated this behavior. For the assays with Drosophila melanogaster
, the EENL extracts induced higher tumors at a higher concentration when applied with DXR, probably due to the toxic effect of EENL together with the chemotherapeutic DXR, inducing cell death, which makes the appearance of tumors unfeasible.
According to Paul et al. (2011) [51
], the major secondary metabolites present in the Azadirachta indica
leaves are nimbolide, vilasinin, nimbinene, 6-deacetyl nimbinene, nimbandiol, nimocinol, β-sitosterol, β-sitosterol-β-d
-glicoside, neem leaf glycoprotein, quercetin, glycoside of quercetin, glycoside of kaemferol, quercetin-3-galactoside (hyperin), and rutin. Nimbolide is the most abundant tetranortriterpenoid isolated from leaves of A. indica
, showing apoptotic and antiproliferative activity acting in the following pathways: (1) oxidative stress and caspase activation; (2) reduction of the expression of anti-apoptotic proteins (Bcl-xl; Bcl-2) and increasing the expression of pro-apoptotic proteins (Bax, Bad, Bid, cytochrome c); (3) activation of the tumor suppressor p53; (4) activation of the extrinsic apoptosis pathway; (5) inhibition of IGF-1; (6) reducing levels of cyclin-dependent kinase (CDKs) and cyclins, promoting cell cycle arrest; and (7) inhibition of NFκB and its pro-tumorigenesis pathway [52
]. The cytotoxicity profile verified in the MTT assays may also be attributed to the probable induction of apoptosis by the EENL. MCF7 cells were more sensitive to high concentrations of EENL. This behavior was previously observed in prostate cancer, in which hormone-responsive cells (LNCaP) were more sensitive to treatment with this extract [55
]. Therefore, analysis of the gene expression, especially of hormone receptor genes, may indicate molecular patterns involved in EENL modulation in breast tumor cells.
The results of Aleskandarany et al. (2016) [56
] have shown that there is an association between the expression of AR
and good prognosis in BC. Comparing AR
expression in HER+, TNBC, and luminal tumors, they observed that luminal BC presented a higher receptor expression. However, AR is an oncogene in triple-negative tumors by “replacing” the estrogen receptor and stimulating tumor growth [57
]. After treatment with EENL we observed a decrease of the AR
transcripts in the MDA-MB-231 line. This effect, therefore, strongly suggests a possible action of EENL in triple-negative tumors and patients treated with this phenolic compound would have a better prognosis considering the behavior of AR signaling in TNBC [58
After treatment with EENL–EA and EENL–DCM we detected an increase in AR-V7
transcripts in MDA-M-231. The AR-V7
gene is expressed in primary BC and in breast tumor cell lines, which can promote growth and mediate resistance in androgen deprivation therapies (ADT) in BC subsets [59
]. In this context, the expression of AR
and its variants emerge as a new strategy for BC treatment.
The effects of AR
are directly linked to the ESR
]. ESR+ breast tumor cells, such as MCF7, have the growth stimulated by androgens and inhibited by the antiandrogens [64
]. AR antagonizes the ESR growth by: (1) directly inhibiting ESR target genes; (2) competing with ESR for binding in the estrogen responsive elements (ERE); (3) sequestering transcriptional factors (TFs); and (4) inducing apoptosis by direct negative regulation of cyclin D1
gene expression [62
Taken together, our results demonstrate a differential effect of EENL in breast tumor cell lines (MCF7 and MDA-MB-231), specially modulating nuclear receptor expression. The behavior of AR-V7 in the MDA-MB-231 tumor cell line indicates new pathways involved in tumor biology, especially as a therapeutic target. Further studies are needed to better understand the role of these compounds in the modulation of such receptors.