Background
Breast cancer is one of the most aggressive endocrine related cancer which has been considered as common malignancy affecting female worldwide. In spite of numerous therapeutic agents available to treat breast cancer, development of chemoresistance and recurrence of disease is frequently observed day by day [
1]. Although several potent cytotoxic, hormonal and estrogen receptor (ER) targeted agents have been developed for treatment of breast cancer, the disease free survival of the patients remains unsatisfactory [
2,
3]. Moreover, several breast cancer-targeted agents are available for effectively treating ER+ breast cancer [
4,
5]. However, treatment of triple-negative breast cancer (TNBC) patients lack estrogen receptor (ER), progesteron receptor (PR) and human epidermal growth factor receptor 2 (HER2) has been challenging due to heterogeneity and devoid of well-defined molecular targets [
6,
7]. About 20% of breast cancer patients are TNBC and commonly observed in younger patients [
8]. Thus, identification of novel effective and selective agents against TNBC that do not produce considerable side effect is essential at this stage.
Neem (
Azadiracta indica) plant is well-known for its diverse applications in traditional medicine in Indian subcontinent for many years. Various parts of this tree are being used over the years as the home-made remedies for several pathological conditions including hyperglycaemia, ulcer, malaria, cancer and dermatological complications [
9,
10]. Structural diversity in the secondary metabolites of neem plant and more importantly their insecticidal efficacy and pharmacological activities has been explored in last five decades [
11]. Over 150 triterpenoids have been isolated and structurally characterized from neem plant, majority of which belongs to tetranortriterpenoids (limonoids) [
11]. On the basis of structural diversity, neem limonoids can be classified broadly into two groups; (i) basic/ring-intact limonoids possessing 4,4,8-trimethyl-17-furanylsteroidal skeleton (e.g. azadirone, azadiradione, gedunin etc.) and (ii) C-seco limonoids with rearranged framework generated through C-ring opening (e.g. salannin, nimbin, azadirachtin A etc) [
11,
12]. Various neem limonoids including nimbolide, azadirachtin A, gedunin, azadirone and several other ring-intact limonoids have been tested for their cytotoxic potency against various cancer cell lines in vitro [
13‐
17]. However, mode of action and anti-carcinogenic activity of these compounds under in vivo conditions are not well-explored. Our continuous effort to search for potent anti-carcinogenic plant-derived metabolites has prompted us to screen the neem limonoids against breast cancer cell lines and further investigate the molecular mechanism underlying this process. Previous studies have shown that neem-derived epoxyazadiradione limonoid exhibits anti-feedant properties [
18]. Further, it has been shown that epoxyazadiradione acts as anti-inflammatory agent by attenuating macrophage migration inhibitory factor (MIF)-mediated macrophage migration [
19]. Moreover, anti-cancer activity of epoxyazadiradione limonoids is not studied well. We report that epoxyazadiradione acts as anti-cancer agent in both TNBC and ER+ breast cancer models.
Several results revealed that mitochondria play crucial role in apoptosis through reactive oxygen species (ROS), apoptosis inducing factor (AIF) or caspase activation [
20‐
23]. Phosphatidylinositol-3-kinase (PI3K)/Akt, MEK/ERK, GSK and STAT3, FAK and Src-mediated signaling play major role in breast cancer progression [
24‐
27]. However, PI3K/Akt signaling pathway exhibits significant role in various aspect of tumor progression such as cell cycle progression, apoptosis, oncogenic transformation, cytokine production and activation of AP-1 and NF-κB [
28]. Earlier report suggests that several components of PI3K/Akt pathway are dysregulated due to amplification, mutation and translocation more frequently in cancer patients [
29]. This warrants the significant role of PI3K/Akt pathway in cancer specific drug development. Previous studies have shown that epoxyazadiradione inhibits the NF-κB activation and regulates pro-inflammatory cytokine production in RAW 264.7 cells [
19]. Further, studies showed that blocking of PI3K/Akt and MEK signaling pathways are involved in induction of apoptosis and suppression of breast tumor growth [
24,
25,
30].
In this context, we report the potential anti-cancer activities of neem-derived limonoid epoxyazadiradione under in vitro and in vivo conditions. It is noteworthy that out of ten major limonoids, epoxyazadiradione is highly potent cytotoxic agent. It induces apoptosis in both TNBC and ER+ breast cancer cells through mitochondrial-dependent caspase 3 and 9 activation. We have also shown that epoxyazadiradione induces apoptosis through ROS and AIF independent manner. Our findings suggest that it significantly attenuates breast cancer cell viability, migration and angiogenesis. It inhibits PI3K/Akt-mediated AP-1 activation and suppresses the expression of MMP-9, Cox2, OPN and VEGF leading to attenuation of breast tumor growth, angiogenesis and metastasis. Taken together, our study demonstrates that epoxyazadiradione may act as a potential therapeutic agent for control of TNBC and ER+ breast cancers.
Methods
Isolation and purification of neem limonoids
Ten major neem limonoid compounds (
1: Epoxyazadiradione;
2: Azadiradione;
3: 17β-hydroxyazadiradione;
4: Gedunin;
5: Nimbin
6: 6-Deacetylnimbin;
7: Salannin;
8: 3-Deacetylsalannin;
9: Azadirachtin A;
10: Azadirachtin B) were extracted and purified from
Azadirachta indica as described earlier [
12,
19]. Drugs were solubilized in DMSO and DMSO was used as vehicle control.
Cell cultures and transfection
Human breast cancer cells, MDA-MB-231 and MCF-7 and normal human breast epithelial cells, MCF-10A were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured as per standard conditions. pcDNA6-HA-Akt1 was transiently transfected in MDA-MB-231 cells using Dharmafect-1 (Dharmacon International) as per manufacturer’s instructions.
MTT assay
To determine the cytotoxic effect of neem-derived limonoids, MTT assay was performed as described [
24]. Briefly, MDA-MB-231 and MCF-7 (1 × 10
4 cells/well) cells were plated in 96-well flat-bottom microplate. Further, cells were treated with all ten neem-derived limonoids independently at 100 μM and 200 μM for 24 h. MTT was added into each well and incubated at 37 °C for 4 h. After incubation, formazan crystals were dissolved with isopropanol and optical density of formazan solution, as a measure of cell viability was observed using a microplate reader at 570 nm (Thermo Scientific). In separate experiments, MDA-MB-231, MCF-7 and MCF-10A cells were independently treated with epoxyazadiradione (0–200 μM) in time-dependent manner and cytotoxic effect was determined by MTT assay as described above. In other experiments, MDA-MB-231 cells were pre-treated with Caspase 9 inhibitor-I (Calbiochem) or ROS scavenger agents, catalase (CAT) or N-acetyl-cysteine (NAC) (Sigma) independently for 1 h and further incubated with epoxyazadiradione (150 μM) for 24 h and MTT assay was performed.
Annexin V/propidium iodide staining
MDA-MB-231 cells were treated with/without epoxyazadiradione (0–150 μM) for 24 h and stained with annexin V-FITC followed by propidium iodide (PI) and apoptosis was studied using apoptosis detection kit (BD Pharmingen) according to the manufacturer’s instructions. Stained cells were analyzed by FACSCalibur cytometer (BD Biosciences). In separate experiments, the effect of epoxyazadiradione on cell-cycle analysis was studied using PI staining as described [
24]. Briefly, MCF-7 cells were treated with epoxyazadiradione (0–150 μM) for 24 h, stained with PI and analyzed on FACSCalibur cytometer. The cell cycle distribution was analyzed using CellQuest software (BD Immunocytometry System).
Immunofluorescence study
Cells were grown on cover slips, treated in absence or presence of epoxyazadiradione with increasing concentrations (0–150 μM) for 24 h and immunofluorescence analysis was performed as described [
31]. MDA-MB-231 or MCF-7 cells were fixed with 2% paraformaldehyde, blocked with 10% FBS and incubated with anti-c-Jun, anti-c-Fos or anti-AIF (Santa Cruz Biotechnology) antibody for overnight followed by fluorescence conjugated Cy2 or Cy3 (Calbiochem) specific antibody. To study the actin cytoskeleton reorganization, epoxyazadiradione treated MDA-MB-231 or MCF-7 cells were stained with FITC conjugated phalloidin (Sigma). Nuclei were stained with DAPI and analyzed under confocal microscope (Zeiss).
TUNEL assay
To analyze the DNA fragmentation in response to epoxyazadiradione, TUNEL assay was conducted using APO-DIRECT™ Kit (BD Pharmingen) in MDA-MB-231 cells as per manufacturer’s instructions. Images were captured using fluorescence microscope (Leica).
Determination of ROS production
To measure the effect of epoxyazadiradione on intracellular ROS production, MDA-MB-231 or MCF-7 cells were independently treated with increasing concentrations of epoxyazadiradione (0–150 μM) for 24 h. These cells were then stained with dihydroethidine (DHE) (Molecular Probes) for 20 min at 37 °C and analyzed on FACSCanto cytometer (BD Biosciences).
Measurement of mitochondrial membrane potential (∆ψm)
To examine the effect of epoxyazadiradione on mitochondrial membrane potential which is a crucial event in caspase-mediated apoptosis [
32], MDA-MB-231 or MCF-7 cells were independently treated with epoxyazadiradione at different doses (0–150 μM) and stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide (JC-1) (Molecular Probes) at 37 °C for 20 min and washed. The JC-1 aggregates, (healthy cells with functional mitochondria) and monomers, (apoptotic or unhealthy cells with collapsed mitochondria) were measured on FACSCanto cytometer (BD Biosciences). In separate experiments, MDA-MB-231 cells were treated with either perifosine or epoxyazadiradione and stained with JC-1. In another experiment, Akt1 overexpressed MDA-MB-231 cells were treated with epoxyazadiradione and stained with JC-1 and analyzed as described above.
Immunoblot analysis
MDA-MB-231 or MCF-7 cells were treated with epoxyazadiradione (0–150 μM) for 24 h, lysed in lysis buffer and lysates containing equal amount of total proteins (40 μg) were resolved by SDS-PAGE and blotted onto nitrocellulose membranes as described [
33]. The levels of apoptosis specific molecules such as Bax, Bad, Bcl2 (Santa Cruz Biotechnology), PARP, cleaved Caspase 9 and cleaved Caspase 3 (Cell Signaling Technology), metastasis and angiogenesis specific molecules such as Cox2, Flk1, VEGF (Santa Cruz Biotechnology) and cell signaling molecules such as OPN (Abcam), PI 3 kinase (p85 subunit) and p-Akt (Cell Signaling Technology), c-Jun and c-Fos (Santa Cruz Biotechnology) were analyzed using their specific antibodies. Actin was used as a loading control. All details of antibodies used are described in Additional file
1: Table S1.
Wound and Transwell migration assays
To check the effect of epoxyazadiradione on breast cancer cell migration, wound and Transwell migration assays were performed as described [
34]. Briefly, MDA-MB-231 cells were grown in monolayer and synchronized in serum free medium for 24 h and pretreated with caspase inhibitor (Sigma) for 1 h to avoid apoptosis induced by epoxyazadiradione and migration assay was performed. Wound with uniform size was made using sterile tip and the cells were treated with epoxyazadiradione at concentrations of 0–20 μM. Wound photographs were captured at
T = 0 and
T = 12 h using phase contrast microscope (Nikon), distance migrated was measured and analyzed (Image-Pro plus software) and represented as bar graph (Sigma Plot 10.0 software). In another experiments, to examine the involvement of PI3K/Akt on cell migration, MDA-MB-231 cells were either treated with perifosine (Akt inhibitor) [
35] or epoxyazadiradione or transfected with pcDNA6-HA-Akt1 and then treated with epoxyazadiradione and wound assay was performed as describe above.
In separate experiments, cell migration assay was performed using Transwell Boyden chamber (Corning) at above conditions as described earlier [
36]. Briefly, MDA-MB-231 cells (1 × 10
5) were pretreated with epoxyazadiradione or perifosine or transfected with pcDNA6-HA-Akt1 followed by treatment with epoxyazadiradione and used in the upper portion of Boyden chamber. In the lower chamber, 5% FBS was used as chemoattractant. Cells were incubated further at 37 °C for 12 h, the migrated cells to the lower surface of the Transwell membrane were fixed with 4% paraformaldehyde for 10 min and stained with 5% Crystal Violet in 25% methanol for 10 min and washed. Migrated cells were photographed at five high power fields (hpf) under inverted microscope at magnifications of 10X (Nikon), counted, analyzed statistically and represented graphically (Sigma Plot 10.0 software).
To examine the effect of epoxyazadiradione on angiogenesis, tube formation assay was performed with HUVECs as described [
34]. Briefly, HUVECs (Lonza) were seeded (1 × 10
4) onto Matrigel pre-coated 96-well plate and treated with epoxyazadiradione (0–20 μM) and used for tube formation assay. After 8 h, tube like structures were observed and photographed using a phase contrast microscope (Nikon).
Zymography
To examine the effect of epoxyazadiradione on MMP-9 activity, gelatinolytic assay was performed as described previously [
37]. Briefly, MDA-MB-231 cells were treated with epoxyazadiradione (0–150 μM) for 24 h in basal medium. Conditioned medium (CM) was collected, dialyzed, lyophilized and CM containing equal amount of total proteins was loaded on gelatin gel and gelatinolytic activity of MMP-9 was studied.
Electrophoretic mobility shift assay (EMSA)
To determine the role of epoxyazadiradione on AP-1-DNA binding, EMSA was performed as described earlier [
38,
39]. Briefly, MDA-MB-231 cells were treated with different concentration of epoxyazadiradione (0–150 μM). After 24 h, cells were washed and nuclear extracts were prepared and incubated with γ-
32P-labeled double-stranded oligonucleotide containing AP-1 consensus sequence (5’-CGC TTG ATG ACT CAG CCG GAA-3′) in binding buffer (100 mM Tris-HCl, 500 mM NaCl, 10 mM DTT, 50% Glycerol) with 1 mg/ml BSA and 1 μg sonicated salmon sperm DNA for 30 min at room temperature. AP-1-DNA complex were resolved on native gel electrophoresis (8%). The gel was dried and exposed to an X-ray film overnight at −80 °C for autoradiography.
Tumor xenograft and IVIS analysis
All mice experiments were performed according to the institutional guidelines, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of National Centre for Cell Science (NCCS), Pune, India. MDA-MB-231-Luc (2 × 10
6) cells were mixed with Matrigel (1:1) (BD Biosciences) and administered orthotopically into mammary fat pad of 6-week old female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Once tumor formed, mice were randomly divided into three groups. Further, two doses of epoxyazadiradione (25 mg/Kg and 100 mg/Kg body wt) was injected intraperitoneally (i.p.) twice a week into these mice. Tumors length and breadth were measured twice a week using Vernier Calipers. Tumor volumes were calculated by using the formula, V = π/6 [(l x b)
3/2]. In vivo bioluminescence imaging was conducted using Living Image acquisition and analysis software on a cryogenically cooled In Vivo Imaging System (IVIS) (Xenogen Corp.) as described earlier [
40]. At the end of experiments, mice were sacrificed and tumor samples were removed, photographed, weighed and fixed in formalin. Further, these tumor sections were stained with H & E and analyzed by immunohistochemistry using anti-VEGF antibody.
Statistical analysis
The data were expressed as mean ± SEM using Sigma Plot 10.0 software. The levels of significance were calculated using unpaired Student’s t test or a one-way ANOVA test. A ‘p’ value less than 0.05 (p < 0.05) was considered as statistically significant.
Discussion
Despite of several drugs available for the treatment of breast cancer, emerging drug resistance leads to high mortality is observed in many cases. Hence, identification of novel and selective anti-cancer agents which exhibit potent anti-cancer activity and less side effects is essential for the treatment of TNBC and ER+ breast cancer.
In this study, we have screened the anti-cancer properties of 10 major neem-derived limonoids and found that epoxyazadiradione exhibits most potent anti-cancer activity. It induces cell death in both TNBC and ER+ breast cancer cells through attenuation of PI3K/Akt-mediated mitochondrial depolarization and induction of caspase-dependent apoptosis. Further, attenuation of PI3K/Akt pathway by epoxyazadiradione leads to inhibition of c-Jun and c-Fos expression and AP-1-DNA binding. Epoxyazadiradione also inhibits the important hallmarks of cancer such as cell proliferation, migration and angiogenesis that is probably due to inhibition of OPN, VEGF, Cox2 and MMP-9 expression and activation. Taken together, epoxyazadiradione suppresses cell migration, angiogenesis and breast tumor growth through downregulation of PI3K/Akt-mediated mitochondrial depolarization and induction of caspase-dependent apoptosis and blocking of AP-1 activation and expression of pro-angiogenesis and metastasis genes (Fig.
7h).
Neem contains several limonoids (triterpenoids) that showed a considerable research interest in recent years. Several reports showed that it has potent anti-oxidant, anti-proliferative, anti-inflammatory and insecticide effects [
19,
50]. Kikuchi et al. have isolated 35 neem limonoids including 15 azadiradione type and evaluated their cytotoxic activity against different cancer cell lines [
17]. Previous data showed that neem limonoids azadirachtin and nimbolide induce mitochondria-mediated apoptosis in human cervical cancer, HeLa cells [
14]. However, we found that azadirachtin is less cytotoxic in breast cancer cells suggesting that activity of these limonoids are cancer cell type specific. It has been also shown that neem oil limonoids induce p53 independent apoptosis and autophagy [
51]. In our study, we have comparatively evaluated the cytotoxic activity of 10 major neem limonoids in TNBC and ER+ breast cancer cells. We found that epoxyazadiradione, a derivative of azadiradione exhibits most potent cytotoxic activity among 10 different limonoids. Epoxyazadiradione shares the same structural scaffold of azadiradione but differ in that it has an epoxide group instead of alkenyl group (Fig.
1a).
During apoptosis, disturbance of mitochondria homeostasis is linked with cancer progression [
52]. While apoptosis, the expression of Bax and Bad are upregulated whereas Bcl2 expression is dowregulated which further activate mitochondria-mediated apoptotic pathway which in turn release cytochrome C, followed by caspase 9 and 3 activation leading to PARP cleavage [
30,
53]. Moreover, apart from Caspase-dependent apoptosis, ROS are known to play crucial role in apoptosis. There are several anti-cancer drugs like taxol and etoposide induce apoptosis through upregulation of intracellular ROS [
54‐
56]. In addition to this, several studies demonstrate that mitochondrial apoptosis inducing factor (AIF) which translocate to nucleus upon apoptotic signals and induce chromatin condensation and fragmentation, play an important role in programmed cell death [
57]. In agreement with these results, our findings demonstrate that epoxyazadiradione induce apoptosis in both TNBC and ER+ breast cancer cells through disturbance of mitochondrial membrane potential and activation of Caspase 9 and 3-mediated PARP cleavage. However, epoxyazadiradione does not affect either of the intracellular ROS level or translocation of AIF into the nucleus.
Various signaling molecules and cytokines such as OPN, VEGF, Flk1, Cox2 and MMP-9 play an important role during process of tumor angiogenesis and metastasis [
37,
58‐
60]. At the time of metastasis, tumor cell secretes mettallomatrix proteins (MMPs) which help in the degradation of extracellular matrix (ECM) that allows tumor cells to invade into the surrounding tissues [
61]. Targeting tumor angiogenesis is an important therapeutic aspect in the regulation of tumor progression [
62]. Therefore, controlling tumor angiogenesis may provide prolonged survival of cancer patients. Our results further revealed that epoxyazadiradione attenuates breast cancer cell migration and endothelial cell tube formation. Moreover, our data showed that epoxyazadiradione did not have any role on the migration potential of MDA-MB-231 cells significantly in the presence of mitomycin C, a cell cycle blocker indicating that the observed migratory effect is not due to proliferation. Further, it inhibits the expression and activation of pro-angiogenic and pro-metastatic molecules like OPN, VEGF, Flk1, Cox2 and MMP-9. Thus epoxyazdiradione effectively inhibits the various hallmarks associated with aggressive breast cancer growth.
It has been shown that PI3K/Akt pathway is generally active in most of the cancer types. Constitutive activation of PI3K/Akt pathway plays a crucial role in cell growth, survival, migration and invasion [
63]. Further, this pathway protects the cancer cells against apoptosis [
28,
30,
48]. Our findings demonstrate that epoxyazadiradione attenuates PI3K/Akt pathway. Further, using selective Akt inhibitor, perifosine or overexpression of Akt1 demonstrates that it regulates breast cancer cell migration, angiogenesis and induces apoptosis through PI3K/Akt pathway. Next, our results also showed that epoxyazadiradione downregulates the AP-1-DNA binding in these cells. Our in vitro findings also supported by in vivo data using NOD/SCID mice where epoxyazadiradione showed significant reduction in breast tumor growth and angiogenesis.