Skip to main content

Phytochemical profile and antiproliferative effect of Ficus crocata extracts on triple-negative breast cancer cells

Abstract

Background

Some species of the Ficus genus show pharmacological activity, including antiproliferative activity, in cell lines of several cancer Types. ficus crocata is distributed in Mexico and used in traditional medicine, as it is believed to possess anti-inflammatory, analgesic, and antioxidant properties. However, as of yet, there are no scientific reports on its biological activity. This study aims to evaluate the phytochemical profile of F. crocata leaf extracts and their effects on breast cancer MDA-MB-231 cells proliferation. Moreover, the study aims to unearth possible mechanisms involved in the decrease of cell proliferation.

Methods

The extracts were obtained by the maceration of leaves with the solvents hexane, dichloromethane, and acetone. The phytochemical profile of the extracts was determined using gas chromatography coupled with mass analysis. Cell proliferation, apoptosis, and cell cycle analysis in MDA-MB-231 cells were determined using a Crystal violet assay, MTT assay, and Annexin-V/PI assay using flow cytometry. The data were analyzed using ANOVA and Dunnett’s test.

Results

The hexane (Hex-EFc), dichloromethane (Dic-EFc), and acetone (Ace-EFc) extracts of F. crocata decreased the proliferation of MDA-MB-231 cells, with Dic-EFc having the strongest effect. Dic-EFc was fractioned and its antiproliferative activity was potentiated, which enhanced its ability to induce apoptosis in MDA-MB-231 cells, as well as increased p53, procaspase-8, and procaspase-3 expression.

Conclusions

This study provides information on the biological activity of F. crocata extracts and suggests their potential use against triple-negative breast cancer.

Peer Review reports

Background

Breast cancer is the most common cancer and the main cause of death for women worldwide. It represents about 12% of all new cancer cases and 25% of all cancers in women [1]. Triple-negative breast cancer (TNBC) represents 10–20% of all breast carcinomas [2] and is characterized by no expression of estrogen receptors (ER) and progesterone receptors (PR), and the lack of overexpression of the HER2 protein. Thus, the tumoral cells do not respond to hormone therapy, and it is considered the subtype with the worst prognosis among breast cancer cases [3,4,5]. Among the different drugs used in cancer, many of these are derived from plants and some are utilized in their natural form or with structural modifications, such as polyphenols (e.g., resveratrol [6], epicathechin [7], and epigallocatechin gallate [8]) and flavonoids (e.g., quercetin [9], silibinin [10], and oncamex [11]), which, in cancer cells, have demonstrated the ability to induce apoptosis [12,13,14,15].

Ficus genus species are employed in traditional medicine for the treatment of asthma, migraine, cough, diarrhea, earache, toothache, scabies, and eye problems [16,17,18,19,20,21]. Several studies have reported that some species of Ficus possess pharmacological activities, such as antioxidant [20, 22,23,24,25,26], antimicrobial [26,27,28,29], antiviral [30,31,32], anti-inflammatory [33,34,35,36], antiparasitic [20, 37], antidiabetic [25, 38,39,40,41,42], antiproliferative [28, 43,44,45,46,47,48,49,50,51,52,53], and cytotoxic activities [32, 53,54,55,56,57]. Extracts of Ficus religiosa have exhibited cytotoxic properties, inducing apoptosis in cervical cancer HeLa cell lines and cell cycle arrest in SiHa cells [45]. Moreover, Ficus fistulosa, Ficus hispida, and Ficus schwarzii have shown antiproliferative activity in human brain glioblastoma (U87MG), lung adenocarcinoma (A549), and colorectal adenocarcinoma (HT-29) cell lines [43]. The biological properties of Ficus species are attributed to the wide range of secondary metabolites identified in the root, stem, leaf, bark, and fruit, which are mostly alkaloids, flavonoids, coumarins, phenols, steroids, terpenoids, and triterpenoids [9, 16, 18, 21, 23,24,25,26, 39, 40, 51, 52, 54, 56, 58, 59]. In Mexico, the presence of 21–40 species of Ficus has been reported, among which 13 species have been identified in southern Mexico, including Ficus crocata [60,61,62]. However, there are no reports on the biological activity of these species of Ficus. This study aimed to evaluate the phytochemical profile of leaf extracts of F. crocata and the effect of the exposure of breast cancer cells MDA-MB-231 to these extracts. Moreover, cell proliferation and the possible mechanisms involved in the decrease of proliferation, such as apoptosis and cell cycle arrest, were investigated.

Methods

Plant material

Leaves of F. crocata were collected from the wild in Petaquillas, Guerrero State, Mexico (latitude: 17.3708, longitude: − 99.5344, altitude: 1160 masl); in accord with Mexican official standard NOM-059-SEMARNAT-2010, there are no restrictions for the collection of this species. The plant was authenticated by Blanca Verónica Juárez-Jaime, M.Sc., and Mauricio Mora-Jarvio, B. Sc, biologists from Herbario Nacional de México (MEXU). A voucher specimen (MEXU-2052) was deposited at the same institute.

Preparation of extracts and fractionation

Leaves of F. crocata (100 g) were dried and ground, and then successively macerated (sequential extraction) with hexane, dichloromethane, and acetone solvents (reactive-grade, 500 mL during 24 h, three times). The macerated material was filtered, and the organic phase was evaporated in a rotary evaporator (Digital Rotary Evaporator Model 410) at 60 °C and 80 rpm. Hexane (1.51% yield; Hex-EFc), and dichloromethane (0.94% yield; Dic-EFc), and acetone (11.75% yield; Ace-EFc) extracts were stored at − 20 °C and protected from light.

Dic-EFc was subjected to fractionation by Open glass Column Chromatography (OCC, ID: 20 mm) packed with 30 g of silica gel (7734, 60 Å, 70–230 mesh; Sigma-Aldrich). The procedure was initiated with the hexane–acetone 9:1 elution system and the polarities were gradually increased with acetone until finally obtaining 149 aliquots (10 mL). Aliquots were analyzed by TLC (silica gel 60, F254, 20 × 20 cm; Merck, Germany) and visualized by UV light at 254 and 365 nm and were combined according to the characteristics observed by TLC in 17 fractions (A1/A17-Dic-EFc). The fractions were monitored by thin layer chromatography (TLC) (Silica gel 60 F254; Merck, Germany) and visualized by ultraviolet (UV) light at 254 nm, then revealed with vanillin-sulfuric acid, vanillin-phosphoric acid, Dragendorff reagent, Liebermann–Burchard reagent, potassium hydroxide reagent (KOH), and acid developer [63].

Gas chromatography coupled with mass analysis (GC-MS)

GC-MS analyses were carried in triplicate out on an Agilent 6890 series gas chromatograph equipped with a mass selective detector 5973 N (USA). The experimental conditions of GC-MS system were as follows [64]: HP-5MS capillary nonpolar column (30 m, ID: 0.20 mm, film thickness: 0.25 μm). The carrier gas was helium at flow rate of 1.0 mL/min. In the gas chromatography part, the temperature program (oven temperature) was 50 °C raised to 230 °C at 2 °C/min and the injection volume was 1 μL. Samples were dissolved in dichloromethane. All results were compared by using NIST/EPA/NIH Mass Spectral library version 1.7a/ChemStation.

Cell culture and exposure to extracts

MDA-MB-231 cells (ATCC® HTB-26) were cultured in Dulbecco’s Modified Eagle Medium Formula 12 (DMEM/F12) supplemented with 5% Fetal Bovine Serum (FBS), 1% antibiotic (ampicillin/streptomycin), and incubated at 37 °C in a 5% CO2 atmosphere and at 95% humidity. Cells were synchronized with basal medium without FBS for 24 h and exposed to 0–80 μg mL− 1 of F. crocata extracts for 24–48 h. DiMethyl SulfOxide (DMSO) was used as the diluent of extracts (Vehicle). As a positive control, cells were treated with 100 μM Cytarabine (Ara-C), which is known to induce cell death [65]. As a negative control, cells were treated with 5% FBS to induce cell proliferation. All tests were performed in triplicate at three independent times.

Cell growth in monolayer (crystal violet assay)

A total of 15 × 103 cells were plated on 24-well plates (Corning) and cultured in DMEM/F12 medium supplemented with 5% FBS for 24 h. Cells were treated with 0, 5, 10, 20, 40, and 80 μg mL− 1 of F. crocata extracts for 24 and 48 h, fixed after treatment in 4% formaldehyde for 5 min, and the cell morphology was observed using an inverted microscope (EVOS Cell Imaging System; Thermo Scientific). Later, the cells were stained with 0.5% Crystal violet, the excess dye was washed with water and PBS (Phosphate-Buffered Saline) and the stain was extracted with 10% acetic acid. The relative cell density in the monolayer was determined by measuring the optical density (OD) of each well at 600 nm in a biophotometer (Eppendorf Model RS-2312 DH 8.5 mm).

MTT assay

The percentage of viable cells was evaluated using the MTT cell proliferation colorimetric assay (CT02, Millipore Corp., Bedford, MA, USA) according to the manufacturer’s instructions. Briefly, in a 96-well plate, 1 × 104 MDA-MB-231 cells per well were cultured for 24 h with DMEM/F12 medium with 5% FBS and subsequently with basal medium for 24 h to synchronize the cells in the G1 phase and promote a homogeneous cellular response. Then, the treatment with 0–80 μg mL− 1 of F. crocata extracts or fractions was applied for 24 and 48 h. After the treatment, the medium containing the extracts was replaced by fresh basal medium and 100 μL of the MTT reagent was added for 4 h. The formazan crystals were diluted with isopropanol, and the OD of the supernatant was obtained at a wavelength of 540 nm using a biophotometer (Eppendorf Model RS-2312 DH 8.5 mm). The half-maximal inhibitory concentration (IC50) was calculated through the linear eq. (Y = mX + b) using GraphPad prism software v6.0.

Analysis of apoptosis

In order to determine the cell population in apoptosis after the treatment with F. crocata extracts, the Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (556,547; Beckton Dickinson) was used according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were seeded at a density of 2 × 105 cells/well in a six-well plate for 24 h with DMEM and 5% FBS. The cells were synchronized with basal medium for 24 h and subsequently exposed to Dic-EFc (0, 20, 40, and 80 μg mL− 1) or A9, A12, and A13-Dic-EFc fractions (80 μg mL− 1) for 48 h. Cells were collected by trypsinization followed by washing with PBS, and staining with FITC Annexin V and propidium iodide (PI) for 15 min in the dark. They were then immediately analyzed by flow cytometry (FACSCanto II; Beckton Dickinson, USA). The FITC Annexin V- and PI-negative cells were considered viable cells. Cells in early apoptosis were FITC Annexin V-positive and PI-negative, while cells in late apoptosis were both FITC Annexin V- and PI- positive.

Analysis of cell cycle arrest

MDA-MB-231 cells were seeded at a density of 2 × 105 cells/well in a six-well plate and incubated for 24 h in an incubator at 37 °C with a 5% CO2 atmosphere. The cells were synchronized in basal medium for 24 h and subsequently exposed to Dic-EFc (0, 20, 40, and 80 μg mL− 1) or A9, A12, and A13-Dic-EFc fractions (80 μg mL− 1) for 48 h. Cells were collected by trypsinization followed by washing with PBS, centrifugation, and fixing with ethanol. Subsequently, the cells were centrifuged and resuspended in cold PBS. A total of 20 μg mL− 1 RNAsa was added for 30 min and, the cells were stained with Propidium Iodide (PI) in the dark at room temperature for 15 min. The immunofluorescence of PI was analyzed by flow cytometry (FACSCanto II; Beckton Dickinson).

Immunobloting for p53 and caspases

After cell stimulation with the Dic-EFc or A9- Dic-EFc fraction, the cells were solubilized in 0.2 mL of RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA/EGTA, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 1.5 mM MgCl2, 0.1% SDS, 100 mM NaF, 1 mM sodium orthovanadate and 1 mM PMSF). Whole cell lysates (20 μg) were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated in 5% non-fat dried milk in PBS for 1 h at room temperature (RT), and incubated with the primary antibodies anti-p53 (Sc-126, SC Biotech, 1:2000 dilution), anti-caspase-3 (which also recognizes other caspases in according to cell type) (H-277, sc-7148, 1:2000 dilution), and anti-GAPDH (AB clonal AC001, 1:5000 dilution) at 4 °C overnight. Later, the membranes were incubated for 2 h at RT with secondary anti-mouse or anti-rabbit (1:5000 dilution, MERCK Millipore), and revealed with a chemiluminescent substrate from Bio-Rad (Hercules, CA, USA).

Statistical analysis

Data analysis was performed using the GraphPad Prism version 7.0 statistical software. The data were shown as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used with Dunnett’s test. A statistically significant difference was considered when p < 0.05.

Results

Phytochemical analysis

The GC-MS analysis of Hex-EFc, Dic-EFc and Ace-EFc extracts revelated the existence of 19, 10 and 6 phytochemical compounds, respectively (Table 1). The most prevailing compounds were the terpenes lup-20(29)-en-3-ol acetate (28.39%) and lupeol (18.61%) in Hex-EFc; lupeol (27.86%), the fatty acid 10,13,13-trimethyl-11-tetradecen-1-ol acetate (14.80%), and lup-20 (29)-en-3-ol acetate (10.90%) in Dic-EFc; and β-sitosterol (42.36%) and stigmastan-3,5-dien (23.87%) in Ace-EFc. Compounds identification was established based on the peak area (%), and retention time (RT) (Table 1). The chemical structures of the molecules identified are expressed in Figs. S1, S2, and S3.

Table 1 The chemical composition of extracts from F. crocata using GC-MS.

Leaf extracts of F. crocata decreased the proliferation of MDA-MB-231 cells

To evaluate the antiproliferative activity of F. crocata extracts on MDA-MB-231 breast cancer cells, crystal violet and MTT assays were performed. It was observed that Hex-EFc, Dic-EFc, and Ace-EFc decreased the number of cells in the monolayer in a concentration- and time-dependent manner. At 24 h of treatment, only 40 and 80 μg mL− 1 concentrations decreased the cell density compared to the untreated control cells (p < 0.001). The effect of extracts was greater at 48 h, showing a lower cell density in the monolayer at all of the tested concentrations and, decreasing the number of cells as the concentration increased (p < 0.001) (Fig. 1). In addition, Dic-EFc and Ace-EFc induced morphologic changes in MDA-MB-231 cells, such as a decrease in cell size, a rounded shape, and the formation of intracellular vacuoles suggestive of apoptosis (Fig. 2). These cellular changes were evident at 48 h with 5-, 10-, and 20-μg mL− 1 concentrations, mainly with Dic-EFc. Figure 2 shows the effect of the extracts at 20 μg mL− 1 after 48 h of treatment.

Fig. 1
figure1

Effect of extracts of Ficus crocata on the growth in a monolayer of MDA-MB-231 cells. Crystal violet assay; a Hex-EFc: hexane extract of F. crocata. b Dic-EFc: dichloromethane extract of F. crocata. c Ace-EFc: acetone extract of F. crocata. V: vehicle, DMSO. (−): negative control, 5% FBS. (+): positive control, 100 μM Ara-C (cytarabine); One-way ANOVA, Dunnett’s test: *p < 0.05, **p < 0.01 and ***p < 0.001 versus V

Fig. 2
figure2

Morphologic changes in MDA-MB-231 cells exposed to F. crocata extracts. The images are representative of cells treated for 48 h with FBS 5% (a), V: Vehicle, DMSO (b), 100 μM Ara-C (cytosine β-D-arabinofuranoside) (c), and 20 μg mL− 1 of Hex-EFc (d), Dic-EFc (e), and Ace-EFc (f). The treatment induced a decrease in cell size (blue arrow), rounded shape (white arrow), and the formation of intracellular vacuoles (black arrow). 20× magnification

In order to validate the data observed with the Crystal violet assay, the effect of the extracts on cell proliferation was determined by MTT assay in order to consider only metabolically viable cells. After 24 h of treatment, a significant decrease in cell proliferation was observed with ≥5 μg mL− 1 of Dic-EFc, ≥10 μg mL− 1 of Ace-EFc, and ≥ 20 μg mL− 1 of Hex-EFc compared to untreated control cells. After prolonging exposure to extracts for 48 h, Hex-EFc, Dic-EFc, and Ace-EFc decreased cell proliferation at all tested concentrations compared to control cells (p < 0.001). However, this effect was more evident with Dic-EFc, which decreased the percentage of viable cells by more than 50% with 10–80 μg mL− 1 (p < 0.001) (Fig. 3). Antiproliferative activity at 48 h was greater for Dic-EFc (IC50: 32.43 μg mL− 1), followed by Ace-EFc (IC50: 78.49 μg mL− 1), and Hex-EFc (IC50: 164.05 μg mL− 1) (Fig. S4). In addition, the effect of DIC-EFc on viability of MCF-10A non-tumor cells was preliminary evaluated, and only 320 μg mL− 1 of extract showed cytotoxic effect at 48 h of treatment (Fig. S5).

Fig. 3
figure3

Effect of leaf extracts of F. crocata on MDA-MB-231 cell viability. MTT assay; V: vehicle, DMSO. (−): negative control, 5% FBS. (+): positive control, 100 μM Ara-C (cytarabine); a Hex-EFc: hexane extract of F. crocata. b Dic-EFc: dichloromethane extract of F. crocata.c Ace-EFc: acetone extract of F. crocata. One-way ANOVA, Dunnett’s test: **p < 0.05, **p < 0.01 and ***p < 0.001 versus V

Considering that Dic-EFc exhibited the best effect in decreasing cell proliferation (IC50: 32.43 μg mL− 1), 17 fractions of Dic-EFc were obtained. The fractions demonstrated the presence of cardiotonic glycosides and anthraquinones (Table S1). Considering the phytochemical profile, three fractions (A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc) were selected to evaluate their effect on the proliferation of MDA-MB-231 cells. The A9-Dic-EFc fraction exhibited the presence of anthraquinones, triterpenoids, lignans, essential oils, phenylpropanoids, cucurbitacins, steroids, and saponins. The A12-Dic-EFc fraction revealed only the presence of anthraquinones, while the A13-Dic-EFc fraction contained lignans, anthraquinones, and cardiotonic glucosides (Table S1). Exposure to ≥40 μg mL− 1 of A9-Dic-EFc for 24 h decreased the number of viable cells by around 70% (p < 0.001) and this effect was maintained at 48 h of exposure. On the other hand, after 24 h of exposure to > 40 μg mL− 1 of A12-Dic-EFc, the cell proliferation was decreased by around 50%, and > 20 μg mL− 1 of A13-Dic-EFc decreased cell proliferation by around 60%. However, at 48 h, both fractions maintained their antiproliferative activity at a concentration of only 80 μg mL− 1 (Fig. 4). Low concentrations of the A9-Dic-EFc fraction (5–20 μg mL− 1) exhibited no effect on the proliferation of MDA-MB-231 cells, while low concentrations of A-12- and A13-Dic-EFc fractions (5–10 μg mL− 1) appeared to stimulate cell proliferation after 24 h (A12- and A13-Dec-EFc) or 48 h of exposure (A12-Dec-EFc) (Fig. 4 b and c). Considering the above, the A9-Dic-EFc fraction showed a better effect for decreasing cell proliferation and this observation could be explained by differences in the phytochemical profile of A9-Dic-EFc with respect to the A12- and A13-Dic-EFc fractions described previously (Table S1). Antiproliferative activity against MDA-MB-231 cells after 48 h of exposure was greatest for the A9-Dic-EFc fraction (IC50: 39.89 μg mL− 1), followed by the A13-Dic-EFc fraction (IC50: 80.49 μg mL− 1) and the A12-Dic-EFc fraction (IC50: 126.76 μg mL− 1) (Fig. S4).

Fig. 4
figure4

Effect of A9-, A12-, and A13-Dic-EFc fractions on MDA-MB-231 cells viability. MTT assay. a A9-Dic-EFc fraction, (b) A12-Dic-EFc fraction, (c) A13-Dic-EFc fraction. V: vehicle, DMSO. V: vehicle, DMSO. (−): negative control, 5% FBS. (+): positive control, 100 μM Ara-C (cytarabine); One-way ANOVA, Dunnett’s test: *p < 0.05, **p < 0.01 and ***p < 0.001 versus V

The dichloromethane extract of F. crocata induced apoptosis and cell cycle arrest in MDA-MB-231 cells

To determine possible mechanisms to reduce the proliferation of MDA-MB-231 cells exposed to the dichloromethane extract of F. crocata, an apoptosis assay and cell cycle analysis were performed. Only the concentrations of Dic-EFc (20–80 μg mL− 1) and its fractions (80 μg mL− 1) that reduced around 50% or more of cell proliferation were analyzed. It was observed that the Dic-EFc treatment induced apoptosis in 4.3 and 7.7% of the cell population with 20 μg mL− 1 and 40 μg mL− 1, respectively, while 80 μg mL− 1 increased the apoptotic population to 19.3% (4.3 and 15% in early and late apoptosis, respectively). Interestingly, the fractionation of Dic-EFc components enhanced the ability of the extract to induce apoptosis in MDA-MB-231 cells. It was observed that A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc at 80 μg mL− 1 all induced apoptosis. The A9-Dic-EFc fraction showed the highest percentage of apoptotic cells with 53% (30.7% in early apoptosis and 22.3% in late apoptosis), followed by the A12-Dic-EFc and A13-Dic-EFc fractions, with 51.9 and 48.8%, respectively (Fig. 5). Moreover, a decrease was observed in the cell population in the S and G2/M phases of the cell cycle, and an increase during the sub-G0 phase was noted after treatment with Dic-EFc and its fractions A9, A12, and A13. A highlighted effect was observed with fraction A9, with more than 50% of the cell population occurring during the sub-G0 phase (Fig. 6). This high percentage in the sub-G0 phase was consistent with the apoptosis results observed with the Annexin/PI assay.

Fig. 5
figure5

Dic-EFc and the A9-, A12-, and A13-Dic-EFc fractions induced apoptosis in MDA-MB-231 cells. Representative pictograms of cells treated as indicated for 48 h. Q3: viable cells. Q4: Annexin V-positive (early-apoptotic cells). Q2: Annexin V/PI double-positive (late-apoptotic cells). Q1: dead cells (no apoptosis). Vehicle: cells treated with 1% DMSO

Fig. 6
figure6

Effect of treatment with Dic-EFc and the A9-, A12-, and A13-Dic-EFc fractions on the cell cycle of MDA-MB-231 cells. Representative pictograms of cells treated as indicated for 48 h. Vehicle: cells treated with 1% DMSO

Exposure to A12-Dic-EFc and A13-Dic-EFc fractions caused an accumulation of G1 phase cells of 62 and 53%, respectively, suggesting that metabolites in these fractions induce a G1-phase cell cycle arrest.

The dichloromethane extract of F. crocata increased the p53, procaspase-8 and procaspase-3 expression

MDA-MB-231 cells were treated with 0–80 μg mL− 1 of Dic-EFc and 80 μg mL− 1 of the A9-Dic-EFc fraction to determine changes in expression of proteins such as p53, procaspase-8, and procaspases-3, which are associated with apoptosis and the cell cycle (Fig. 7). It was observed that the Dic-EFc and A9 fraction increased p53 expression at 24 h of treatment (Fig. 7b), although it markedly decreased p53 expression at 48 h (Fig. 7f). The increase in p53 expression could be associated with the diminution of cell number in the G1 and S phases of the cell cycle. With respect to caspase expression, in Western blot assays, we observed bands around 32 kDa and 55 kDa, which correspond to procaspase-3 and procaspase-8, respectively (Fig. 7a and e). Dic-EFc increased procaspase-8 expression at 48 h of treatment only at the 80 μg mL− 1 concentration (Fig. 7g). Minor concentrations (20–40 μg mL− 1) of Dic-EFc increased procaspase-3 expression at 24 h and 48 h of treatment (Fig. 7d and h). On the other hand, the A9-Dic-EFc fraction increased procaspase-8 expression at 24 h and 48 h of treatment (Fig. 7c and g), and procaspase-3 expression at 48 h (Fig. 7h). It is possible that accumulated procaspase-3 and procaspase-8 are being processed into active forms—i.e., caspase 8 and caspase 3, respectively— which contribute to the progression of apoptosis induced mainly by treatment with the A9-Dic-EFc fraction in MDA-MB-231 cells.

Fig. 7
figure7

Dic-EFc and the A9-Dic-EFc fraction increased p53, procaspase-8 and procaspase-3 expression. Top panel: 24 h of treatment; Bottom panel: 48 h of treatment. Representative western blot of p53, procaspase-8, procaspase-3 and GAPDH, after 24 h (a) and 48 h (e) of treatment with Dic-EFc or A9-Dic-EFc fraction. (b, c and d) Densitometric analysis of (a). V, vehicle: cells treated with diluent of extract, 1% DMSO. (f, g and h) Densitometric analysis of (e). One-way ANOVA, Dunnett’s test: *p < 0.05; **p < 0.01 and ***p < 0.001 versus V

Discussion

TNBC represents 15–20% of breast cancer cases and is related to poor prognosis, metastasis, and death. This type of breast tumor does not respond to conventional chemotherapy because the cells do not express ER and PR and lack overexpression of the HER2 protein, thus representing the sub-type with worst prognosis among breast cancer cases [66]. Current research is focused on the chemical compounds of plants and their effect on cellular models of cancer [14]. It has been observed that natural products obtained from plants exhibit antiproliferative, antimigratory, and anti-invasive have effects on cancer cells and could be used as adjuvant therapies in cancer [12,13,14,15]. Studies conducted in species of the Ficus genus have revealed the greatest antiproliferative potential in cervical, hepatic, leukemia, lung, and colon-cancer cell lines [43, 45, 47, 50]. It has been described that this activity is due to the presence of compounds such as alkaloids, flavonoids, coumarins, phenols, steroids, terpenoids, and triterpenoids [16, 18, 21, 23,24,25,26, 39, 40, 51, 52, 54, 56, 58, 59]. In Mexico, Ficus species are distributed throughout the country [60, 61, 67], yet there have been no studies, to our knowledge, on the chemical composition or biological properties of the Ficus species distributed in Mexico. In this study, we analyzed the phytochemical profile of leaf extracts of F. crocata and their effect on the cell proliferation of TNBC MDA-MB-231 cells.

This is the first study, to our knowledge, to evaluate the cytotoxic activity of leaf extracts of F. crocata. We observed that Hex-EFc, Dic-EFc, and Ace-EFc decreased the proliferation of MDA-MB-231 cells, with Ace-EFc and mainly Dic-EFc being the most active. An interesting observation in this study was that Dic-EFc did not show a cytotoxic effect on MCF-10A non-tumor cells at the same concentrations that affected the viability of MDA-MB-231 cells (5–80 μg mL− 1), which supports the possible use of extracts of F. crocata as an alternative therapy against breast tumors. However, this observation must be analyzed further. These observations are in agreement with other studies, which reported a decrease of cell viability in the breast cancer cell lines T47D and MDA-MB-231 exposed to the leaf extracts of Ficus septica Burm. and Ficus carica [56, 68].

To separate compounds into groups based on their polarity, Dic-EFc was fractionated and we observed that the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions exerted a greater effect in terms of decreasing the viability of MDA-MB-231 cells. A9-Dic-EFc was the most effective of the fractions, as compared to Dic-EFc. This effect suggests that isolation of compounds increased their biological activity. The phytochemical composition of Dic-EFc and the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions revealed the presence of alkaloids, coumarins, lignans, anthraquinones, phenols, terpenoids, and triterpenoids. The presence of these compounds has already been reported for the Ficus genus, and it has been demonstrated that these compounds possess biological activity [16, 46, 56, 59].

We observed that the exposure of MDA-MB-231 cells to F. crocata extracts induced morphological changes such as a decrease in cell size, a rounded shape, and the formation of intracellular vacuoles. In a concentration-dependent manner, Dic-EFc and A9-Dic-EFc decreased the cell population in the S and G2/M phases of the cell cycle and increased the cell population in the sub-G0, which was consistent with the increase of apoptotic cells that could explain the morphologic changes in the cells and the decrease in cell viability. The apoptotic effect of Dic-EFc was potentiated with the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions, inducing apoptosis in around 50% of the cell population. In this regard, we observed that Dic-EFc and the A9-Dic-EFc fraction increased p53 expression at 24 h of treatment, which could be associated with apoptosis and cell cycle arrest, but curiously, p53 expression markedly decreased after of 48 h with 80 μg mL− 1 of Dic-EFc and A9-Dic-EFc. This increase and a subsequent decrease in the expression of p53 has also been observed in another study with HCT116 cells treated with betulinic acid (BA), in which the treatment also induced apoptosis, suggesting that other proteins could be involved in BA-induced apoptosis in addition to p53 [69]. Moreover, in breast cancer cell lines MCF-7 and MDA-MB-231, it was observed that Vernonia amygdalina (VA) extracts induced apoptosis and cell cycle arrest in a p53-independent manner. Interestingly, these authors observed that VA increased p53 expression in a time-dependent manner in MCF-7 cells, whereas in MDA-MB-231, p53 expression decreased after 48 h [70]. We observed a similar event in our study. On the other hand, the A9-Dic-EFc fraction increased the expression of procaspase-8 and procaspase-3, which are initiator and executioner caspases, respectively, involved in the extrinsic apoptosis pathway [71]. These proteins could be processed in their active form and induce apoptosis, which would explain the increase in the apoptotic cell population induced mainly by the A9-Dic-EFc fraction in MDA-MB-231 cells. Dic-EFc also increased procaspase-8 expression at 48 h of treatment at an 80 μg mL− 1 concentration, while 20–40 μg mL− 1 concentrations increased procaspase-3 expression, which could be associated with the slight increase in the apoptotic cell population under these treatment conditions. It is probable that the apoptosis induced by the extracts of F. crocata and its fractions in MDA-MB-231 cells is through an p53-independent pathway, such as PUMA protein for example, which is known to be a powerful apoptosis inducer associated or not with p53 [72]. However, this hypothesis should still be tested.

Our observations are consistent with those of other studies. Zhang et al. (2018) reported that F. carica extracts induce apoptosis and cell cycle arrest at the S phase in MDA-MB-231 cells, increasing the expression of genes that promote apoptosis and the regulation of the cell cycle, such as BAX, TP53, and TP21 [68]. Similarly, in a study by Choudhari et al., the authors reported that the aqueous extract of Ficus religiosa induces cell cycle arrest in the G1 phase in SiHa cells, accompanied by an increase in the expression of p53, p21, and pRb proteins. Moreover, in HeLa cells, the extract induces apoptosis through an increase in intracellular Ca2+, leading to the loss of mitochondrial membrane potential, the release of cytochrome-C, and an increase in caspase-3 expression [47]. It has also been reported that the flavonoid quercetin induces apoptosis in HeLa cells, promoting the accumulation of reactive oxygen species (ROS), upregulating proapoptotic proteins such as cytochrome C, Apaf-1, and caspases, and downregulating the antiapoptotic proteins Bcl-2 and survivin [73, 74].

The primary compound in Dic-EFc was lupeol, a triterpene also present in Ace-EFc and Hex-EFc, although in smaller proportions (Table 1). Triterpenes were only present in the A9-Dic-EFc fraction, which suggests that lupeol was only present in A9-Dic-EFc and not in the A12- and A13-Dic-EFc fractions. It has been reported that lupeol induces apoptosis and cell cycle arrest in several cancer cell lines. For example, in pancreatic cancer cells, lupeol induces apoptosis by decreasing the levels of p-AKT and p-ERK, as well as cell cycle arrest in the G0/G1 phase, by upregulating P21 and P27 and downregulating cyclin D1 proteins [75]. A similar effect was observed in osteosarcoma cells MNNG/HOS and MG-63 [76]. In gallbladder carcinoma GBC-SD cells, lupeol was shown to induce apoptosis and inhibit invasion by downregulating the activity of p-EGFR and MMP-9 [77]. In non-small cell lung cancer cells, it was observed that lupeol inhibits the phosphorylation of EGFR by binding to its tyrosine kinase domain and reducing STAT3 phosphorylation, which contributes to the induction of apoptosis [78]. In the hepatocarcinoma cell lines SMMC7721 and HepG2 as well as lung carcinoma A427 cells, lupeol was shown to induce low expression of the antiapoptotic protein Bcl-2 and upregulate the proapoptotic protein BAX [79, 80]. Considering the previously noted observations, it is possible that the metabolites present in F. crocata extracts, such as lupeol, probably in synergy with other metabolites, induce cell cycle arrest and apoptosis in MDA-MB-231 cells, altering the expression of cell cycle regulatory proteins such as p53, p21, p27, and cyclins, as well as the expression of anti- and proapoptotic proteins, such as caspases, PUMA, Bax, and Bak. However, this hypothesis still needs to be further analyzed.

One limitation of this study is that the assays were executed in only one breast cancer cell line. It would be important to evaluate the effect of F. crocata extracts on a larger number of tumor and non-tumor cell lines. Furthermore, the effect of pure metabolites isolated from F. crocata extracts, such as lupeol, Stigmastan, and others, has not, to our knowledge, been tested. This effect will be evaluated in a subsequent study. Our observations comprise, to our knowledge, a first approach to investigating the possible use of extracts of F. crocata as alternative or complementary therapy against cancer. Nonetheless, their cytotoxicity and molecular mechanisms must be analyzed in more study models.

Conclusions

The leaf extract of F. crocata decreases the proliferation capacity of MDA-MB-231 triple-negative breast cancer cells, decreasing the S and G2/M cell cycle phases, and inducing apoptosis possibly through an p53-independent pathway. By fractionating the extract, the antiproliferative activity against MDA-MB-231 cells can be further potentiated. These findings provide information on the biological activity of F. crocata extracts and suggest their potential use as an alternative or complementary therapy against triple-negative breast cancer. Nevertheless, more studies are required to determine the components responsible for this biological activity, as well as the molecular and cellular mechanisms involved.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

Hex-EFc:

Hexane extract of F. crocata

Dic-EFc:

Dichloromethane extract of F. crocata

Ace-EFc:

Acetone extract of F. crocata

GC-MS:

Gas chromatography coupled with mass analysis

TNBC:

Triple-negative breast cancer

ER:

Estrogen receptors

PR:

Progesterone receptors

OCC:

Open glass Column Chromatography

DMEM:

Dulbecco’s Modified Eagle Medium

FBS:

Fetal bovine serum

DMSO:

Dimethyl Sulfoxide

Ara-C:

Cytarabine, cytosine β-D-arabinofuranoside

PBS:

Phosphate-Buffered Saline

RIPA:

Radioimmunoprecipitation assay buffer

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

EDTA:

Ethylene-diamine-tetraacetic acid

EGTA:

Ethylene glycol tetraacetic acid

OD:

Optical density

IC50 :

Half-maximal inhibitory concentration

PI:

Propidium iodide

RT:

Room temperature

SD:

Standard deviation

ANOVA:

One-way analysis of variance

References

  1. 1.

    Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–86. https://doi.org/10.1002/ijc.29210.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Badve S, Dabbs DJ, Schnitt SJ, Baehner FL, Decker T, Eusebi V, Fox SB, Ichihara S, Jacquemier J, Lakhani SR, Palacios J, Rakha EA, Richardson AL, Schmitt FC, Tan PH, Tse GM, Weigelt B, Ellis IO, Reis-Filho JS. Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod Pathol. 2011;24:157–67. https://doi.org/10.1038/modpathol.2010.200.

    Article  Google Scholar 

  3. 3.

    Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J, Shi B. Breast cancer intrinsic subtype classification, clinical use and future trends. Am J Cancer Res. 2015;5:2929–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Kalimutho M, Parsons K, Mittal D, Lopez JA, Srihari S, Khanna KK. Targeted therapies for triple-negative breast Cancer: combating a stubborn disease. Trends Pharmacol Sci. 2015;36:822–46. https://doi.org/10.1016/j.tips.2015.08.009.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Ramos AYD, Marmón TER, Crespo GC, Junco SB, Valiente MW. Breast cancer, its epidemiological characterization. Rev. Ciencias Médicas. 2015;19:619–29.

    Google Scholar 

  6. 6.

    Poschner S, Maier-Salamon A, Thalhammer T, Jäger W. Resveratrol and other dietary polyphenols are inhibitors of estrogen metabolism in human breast cancer cells. J Steroid Biochem Mol Biol. 2019;190:11–8. https://doi.org/10.1016/j.jsbmb.2019.03.001.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Shay J, Elbaz HA, Lee I, Zielske SP, Malek MH, Hüttemann M. Molecular mechanisms and therapeutic effects of (−)-epicatechin and other polyphenols in cancer, inflammation, diabetes, and neurodegeneration. Oxidative Med Cell Longev. 2015:181260–13. https://doi.org/10.1155/2015/181260.

  8. 8.

    Losada-Echeberría M, Herranz-López M, Micol V, Barrajón-Catalán E. Polyphenols as promising drugs against main breast cancer signatures. Antioxidants. 2017;6(4):88. https://doi.org/10.3390/antiox6040088.

    CAS  Article  PubMed Central  Google Scholar 

  9. 9.

    Srivastava S, Somasagara RR, Hegde M, Nishana M, Tadi SK, Srivastava M, Raghavan SC. Quercetin, a natural flavonoid interacts with DNA, arrests cell cycle and causes tumor regression by activating mitochondrial pathway of apoptosis. Sci Rep. 2016;6(1):24049. https://doi.org/10.1038/srep24049.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Jiang K, Wang W, Jin X, Wang Z, Ji Z, Meng G. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol Rep. 2015;33(6):2711–8. https://doi.org/10.3892/or.2015.3915.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Martínez-Pérez C, Ward C, Turnbull AK, Mullen P, Cook G, Meehan J, Langdon SP. Antitumour activity of the novel flavonoid oncamex in preclinical breast cancer models. Br J Cancer. 2016;114(8):905–16. https://doi.org/10.1038/bjc.2016.6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Amin A, Gali-Muhtasib H, Ocker M, Schneider-Stock R. Overview of major classes of plant-derived anticancer drugs. Int J Biomed Sci. 2009;5:1–11.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Greenwell M, Rahman PK. Medicinal Plants: Their Use in Anticancer Treatment. Int J Pharm Sci Res. 2015;6:4103–12. https://doi.org/10.13040/IJPSR.0975-8232.6 (10).4103–12.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Seca AML, Pinto D. Plant secondary metabolites as anticancer agents: successes in clinical trials and therapeutic application. Int J Mol Sci. 2018;19(1):E263. https://doi.org/10.3390/ijms19010263.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Weaver BA. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell. 2014;25:2677–81. https://doi.org/10.1091/mbc E14-04-0916.

    Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Mawa S, Husain K, Jantan I. Ficus carica L. (Moraceae): Phytochemistry, traditional uses and biological activities. Evid Based Complement Alternat Med. 2013;2013:974256. https://doi.org/10.1155/2013/974256.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Bouasla A, Bouasla I. Ethnobotanical survey of medicinal plants in northeastern of Algeria. Phytomedicine. 2017;36:68–81. https://doi.org/10.1016/j.phymed.2017.09.007.

    Article  PubMed  Google Scholar 

  18. 18.

    Chee-Yan C, Sulong SY. A review on the phytochemicals, ethnomedicine uses and pharmacology of Ficus species. Curr Traditl Med. 2016;2(1):3–17. https://doi.org/10.2174/2215083802999160722152307.

    CAS  Article  Google Scholar 

  19. 19.

    Neamsuvan O, Komonhiran P, Boonming K. Medicinal plants used for hypertension treatment by folk healers in Songkhla province, Thailand. J Ethnopharmacol. 2018;214:58–70. https://doi.org/10.1016/j.jep.2017.11.032.

    Article  PubMed  Google Scholar 

  20. 20.

    Ahmed AF, Mueen AK, Abedin M, Karim A. Traditional uses and pharmacological potential of Ficus exasperata Vahl. Sys Rev Pharm. 2012;3(1):15–23.

    CAS  Article  Google Scholar 

  21. 21.

    Shiksharthi AR, Mittal S. Ficus racemosa: Phytochemistry, traditional uses and pharmacological properties: a review. Int J Adv Pharm Res. 2011;4:6–15.

    Google Scholar 

  22. 22.

    Abusufyan S, Ibrahim M, Mohib K. Comparative in vitro antidiabetic and antioxidant activity of various extracts of Ficus species. Pharm J. 2018;10(2):349–54. https://doi.org/10.5530/pj.2018.2.59.

    CAS  Article  Google Scholar 

  23. 23.

    Devanesan AA, Zipora TG, Smilin BA, Deviram G, Thilagar S. Phytochemical and pharmacological status of indigenous medicinal plant Pedalium murex L.-a review. Biomed Pharmacother. 2018;103:1456–63. https://doi.org/10.1016/j.biopha.2018.04.177.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Sirisha N, Sreenivasulu M, Sangeeta K, Chetty CM. Antioxidant properties of Ficus species – a review. IntJ PharmTech Res. 2010;2(4):2174–82.

    Google Scholar 

  25. 25.

    Taviano MF, Rashed K, Filocamo A, Cacciola F, Dugo P, Mondello L, Bisignano C, Acquaviva R, D'Arrigo M and Miceli N. Phenolic profile and biological properties of the leaves of Ficus vasta Forssk. (Moraceae) growing in Egypt. BMC Complement Altern Med 2018; 18: 161. DOI: https://doi.org/10.1186/s12906-018-2210-0.

  26. 26.

    Ajaib M, Almas M, Khan KM, Perveen S, Shah S. Phytochemical screening, antimicrobial and antioxidant activities of Ficus natalensis. J Chem Soc Pak. 2016;38(2):345–51.

    CAS  Google Scholar 

  27. 27.

    Bhawana R, Kaur J, Pal Vig A, Arora S, Kaur R. Evaluation of antibacterial potential of Ficus species. J Pharm Sci Res. 2018;10(5):1251–5.

    CAS  Google Scholar 

  28. 28.

    Yessoufou K, Elansary HO, Mahmoud Eman A, Skalicka-Wòzniak K. Antifungal, antibacterial and anticancer activities of Ficus drupacea L. stem bark extract and biologically active isolated compounds. Ind Crop Prod. 2015;74:752–8. https://doi.org/10.1016/j.indcrop.2015.06.011.

    CAS  Article  Google Scholar 

  29. 29.

    Azam S, Ishtiag M, Magbool M, Mushtag W, Mehmood H, Azam A, Shahman M. Antimicrobioal potential of leaves of four ficus species from district Bhimber Azad Kashmir, Pakistan. Pak J Bot. 2017;49:211–20.

    CAS  Google Scholar 

  30. 30.

    Cagno V, Civra A, Kumar R, Pradhan S, Donalisio M, Sinha BN, Ghosh M, Lembo D. Ficus religiosa L. bark extracts inhibit human rhinovirus and respiratory syncytial virus infection in vitro. J Ethnopharmacol. 2015;176:252–7. https://doi.org/10.1016/j.jep.2015.10.042.

    Article  PubMed  Google Scholar 

  31. 31.

    Ghosh M, Civra A, Ritta M, Cagno V, Mavuduru SG, Awasthi P, Lembo D, Donalisio M. Ficus religiosa L. bark extracts inhibit infection by herpes simplex virus type 2 in vitro. Arch Virol. 2016;161(2):3509–14. https://doi.org/10.1007/s00705-016-3032-3.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Lazreg Aref H, Gaaliche B, Fekih A, Mars M, Aouni M, Pierre Chaumon J, Said K. In vitro cytotoxic and antiviral activities of Ficus carica latex extracts. Nat Prod Res. 2011;25(3):310–9. https://doi.org/10.1080/14786419.2010.528758.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Anasane PD, Chaturvedi A. Evaluation of anti-inflammatory effects if Ficus hispida L. leaves extract against carageenan induced paw edema in rats. J Pharm Sci Res. 2017;9(4):364–7.

    CAS  Google Scholar 

  34. 34.

    Ghori SS, Khan M, Tabassum R. Anti-inflammatory activity of Ficus dalhousiae miq roots ethanolic extract in wistar albino rats. Asian J Pharm Clin Res. 2015;8(1):117–9.

    Google Scholar 

  35. 35.

    Panday DR, Rauniar GP. Effect of root-extracts of Ficus benghalensis (banyan) in memory, anxiety, muscle co-ordination and seizure in animal models. BMC Complement Altern Med. 2016;16:429. https://doi.org/10.1186/s12906-016-1413-5.

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Yang Y, Zheng K, Mei W, Wang Y, Yu C, Yu B, Deng S, Hu J. Anti-inflammatory and proresolution activities of bergapten isolated from the roots of Ficus hirta in an in vivo zebrafish model. Biochem Biophys Res Commun. 2018;496(2):763–9. https://doi.org/10.1016/j.bbrc.2018.01.071.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Desrivot J, Waikedre J, Cabalion P, Herrenknecht C, Bories C, Hocquemiller R, Fournet A. Antiparasitic activity of some new Caledonian medicinal plants. J Ethnopharmacol. 2007;112(1):7–12.

    Article  Google Scholar 

  38. 38.

    Deepa P, Sowndhararajan K, Kim S, Park SJ. A role of Ficus species in the management of diabetes mellitus: a review. J Ethnopharmacol. 2018;215:210–32. https://doi.org/10.1016/j.jep.2017.12.045.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Olaokun OO, McGaw LJ, Janse VI, Eloff JN, Naidoo V. Antidiabetic activity of the ethyl acetate fraction of Ficus lutea (Moraceae) leaf extract: comparison of an in vitro assay with an in vivo obese mouse model. BMC Complement Altern Med. 2016;16:110. https://doi.org/10.1186/s12906-016-1087-z.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Solanki ND, Bhavsar SK. Evaluation of phytochemical profile and antidiabetic activity of Ficus racemosa (Linn.) stem bark in rats. Indian Drugs. 2017;54(1):49–54.

    Google Scholar 

  41. 41.

    Stephen IS, Christudas S, Antony S, Duraipandiyan V, Naif Abdullah AD, Ignacimuthu S. Protective effects of Ficus carica leaves on glucose and lipids levels, carbohydrate metabolism enzymes and beta-cells in type 2 diabetic rats. Pharm Biol. 2017;55(1):1074–81. https://doi.org/10.1080/13880209.2017.1279671.

    Article  Google Scholar 

  42. 42.

    Yahaya N, Mohd Dom NS, Adam Z and Hamid M. Insulinotropic Activity of Standardized Methanolic Extracts of Ficus deltoidea from Seven Varieties. Evid Based Complement Alternat Med 2018; 2018: 3769874. DOI: https://doi.org/10.1155/2018/3769874.

  43. 43.

    Abubakar IB, Lim KH, Loh HS. Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells. Nat Prod Res. 2015;29(22):2137–40. https://doi.org/10.1080/14786419.2014.991927.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Raheel R, Saddique Z, Iram M, Afzal S. In vitro antimitotic, antiproliferative and antioxidant activity of stem bark extracts of Ficus benghalensis L. S Afr J Bot. 2017;11:248–57. https://doi.org/10.1016/j.sajb.2017.03.037.

    Article  Google Scholar 

  45. 45.

    Larbie C, Appiah-Opong R, Acheampong F, Tuffour I, Uto T, Yeboah GA, Abboah-Offei O, Tagoe DNK, Inkabi SE. Anti-proliferative effect of Ficus pumila Linn. On human leukemic cell lines. Int J Basic Clin Pharmacol. 2015;4:257–61.

    Google Scholar 

  46. 46.

    Parveen M, Malla AM, Alam M, Ahmad F, Silva PS, Silva MR. Two new phenolic compounds from Ficus rumphii and their antiproliferative activity. Nat Prod Res. 2014;28:646–52. https://doi.org/10.1080/14786419.2014.891201.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Choudhari AS, Suryavanshi SA, Kaul-Ghanekar R. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 positive) and apoptosis in HeLa (HPV-18 positive). PLoS One. 2013;8:e70127. https://doi.org/10.1371/journal.pone.0070127.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Hanafi MMM, Afzan A, Yaakob H, Aziz R, Sarmidi MR, Wolfender JL and Prieto JM. In Vitro Pro-apoptotic and Anti-migratory Effects of Ficus deltoidea L. Plant Extracts on the Human Prostate Cancer Cell Lines PC3. Front Pharmacol 2017; 8: 895. DOI: https://doi.org/10.3389/fphar.2017.00895.

  49. 49.

    Bafor EE, McKenna J, Rowan EG, Edrada-Ebel R. Characterisation of the antiproliferative constituents and activity of Ficus exasperata (Vahl) on ovarian cancer cells -a preliminary investigation. Nat Prod Res. 2017;31(18):2164–8. https://doi.org/10.1080/14786419.2016.1277348.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Santiago LA, Mayor AB. Antiproliferative and apoptotic effects of ficus pseudopalma Blanco (Moraceae) against hepatocarcinoma (hepg2). Asian J Pharm Clin Res. 2015;8(2):257–61.

    Google Scholar 

  51. 51.

    Yap VA, Qazzaz ME, Raja VJ, Bradshaw TD, Loh HS, Sim KS, Yong KT, Low YY, Lim KH. Fistulopsines a and B antiproliferative septicine-type alkaloids from Ficus fistulosa. Phytochem Lett. 2016;15:136–41. https://doi.org/10.1016/j.phytol.2015.12.007.

    CAS  Article  Google Scholar 

  52. 52.

    Din A, Uddin G, Hussain N, Khan I, Shad AA, Choudhary M. Bioassay-guided isolation of new antitumor agent from Ficus faveolata (wall. Ex Miq.). J Cancer Sci Ther. 2013;5:404–8. https://doi.org/10.4172/1948-5956.1000233.

    CAS  Article  Google Scholar 

  53. 53.

    Sathiyamoorthy J, Sudhakar N. In vitro cytotoxicity and apoptotic assay in HT-29 cell line using Ficus hispida Linn: leaves extract. Pharmacogn Mag. 2018;13(4):S756–61. https://doi.org/10.4103/pm.pm_319_17.

    Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Al-Khdhairawi AAQ, Krishnan P, Mai CW, Chung FF, Leong CO, Yong KT, Chong KW, Low YY, Kam TS, Lim KH. A Bis-benzopyrroloisoquinoline alkaloid incorporating a cyclobutane core and a chlorophenanthroindolizidine alkaloid with cytotoxic activity from Ficus fistulosa var. tengerensis. J Nat Prod. 2017;80(10):2734–40. https://doi.org/10.1021/acs.jnatprod.7b00500.

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Wangkheirakpam SD, Wadawale A, Leishangthem SS, Gurumayum JS, Laitonjam WS. Cytotoxic triterpenoids from Ficus pomifera wall. Indian J Chem. 2015;54B:676–81.

    CAS  Google Scholar 

  56. 56.

    Nugroho AE, Akbar FF. Wiyani a and Sudarsono. Cytotoxic effect and constituent profile of alkaloid fractions from Ethanolic extract of Ficus septica Burm. f. Leaves on T47D breast Cancer cells. Asian Pac J Cancer Prev. 2015;16:7337–42. https://doi.org/10.7314/apjcp.2015.16.16.7337.

    Article  PubMed  Google Scholar 

  57. 57.

    Ogunlaja OO, Moodley R, Singh M, Baijnath H, Jonnalagadda SB. Cytotoxic activity of the bioactive principles from Ficus burtt-davyi. J Environ Sci Health B. 2018;53(4):261–75. https://doi.org/10.1080/03601234.2017.1410385.

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    El-Rafie HM, Sleem AA. Phytochemical studies of Ficus Binnendijkii leaf extracts: fractionation and bioactivities of its petroleum ether extract. J Pharmacogn Phytochem. 2016;8(10):1742–50.

    Google Scholar 

  59. 59.

    Ahmad S, Rao H, Akhtar M, Ahmad I, Munawarr MH, Iqbal Z. and Nisar-ur-Rahman. Phytochemical composition and pharmacological prospectus of Ficus bengalensis Linn. (Moraceae)- a review. J Med Plant Res. 2011;5(28):6393–400. https://doi.org/10.5897/JMPR11.455.

    CAS  Article  Google Scholar 

  60. 60.

    González-Castañeda N, Cornejo-Tenorio G, Ibarra-Manríquez G. El género Ficus (Moraceae) en la provincia biogeográfica de la depresión del balsas. México Bol Soc Bot Méx. 2010;87:105–24.

    Google Scholar 

  61. 61.

    Ibarra-Manríquez G, Cornejo-Tenorio G, González-Castañeda N, Piedra-Malagón EM, Luna A. El género Ficus L. (Moraceae) en México. Bot Sci. 2012;90:389–452.

    Article  Google Scholar 

  62. 62.

    Somashekhar MNN, Mahesh AR. Botanical study of four Ficus species of family Moraceae: a review. Int J Univ Pharm Bio Sci. 2013;2:558–70.

    Google Scholar 

  63. 63.

    Wagner H, Bladt S. Plant Drug Analysis. A Thin Layer Chromatography Atlas, vol. 60. 2nd edicion ed. New York: J. Springer-Verlag Berling Heidelberg; 1997. p. 428–30.

    Google Scholar 

  64. 64.

    Bunawan H, Amin NM, Bunawan SN, Baharum SN, Mohd Noor N. Ficus deltoidea jack: A review on its phytochemical and pharmacological importance. Evid Based Complement Alternat Med. 2014;2014:902734. https://doi.org/10.1155/2014/902734.

    Article  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Hewish MSA, Elliott R, Cunningham D, Lord CJ, Ashworth A. Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair deficient tumour cells by enhancing levels of intracellular oxidative stress. Br J Cancer. 2013;108:983–92. https://doi.org/10.1038/bjc.2013.3.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Diana A, Franzese E, Centonze S, Carlino F, Corte CMD, Ventriglia J, Petrillo A, De Vita F, Alfano R, Ciardiello F, Orditura M. Triple-negative breast cancers: systematic review of the literature on molecular and clinical features with a focus on treatment with innovative drugs. Curr Oncol Rep. 2018;20(10):76. https://doi.org/10.1007/s11912-018-0726-6.

    CAS  Article  PubMed  Google Scholar 

  67. 67.

    Duran-Ramírez CA, Fonseca-Juárez RM, Ibarra-Marquez G. Estudio florístico de Ficus (Moraceae) en el estado de Guerrero, México. Rev Mex Biodiversidad. 2010;81:239–62.

    Google Scholar 

  68. 68.

    Zhang Y, Wan Y, Huo B, Li B, Jin Y, Hu X. Extracts and components of Ficus carica leaves suppress survival, cell cycle, and migration of triple-negative breast cancer MDA-MB-231 cells. Onco Targets Ther. 2018;11:4377–86. https://doi.org/10.2147/OTT.S171601.

    Article  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Wang S, Wang K, Zhang C, Zhang W, Xu Q, Wang Y, Zhang Y, Li Y, Zhang Y, Zhu H, Song F, Lei Y, Bu Y. Overaccumulation of p53-mediated autophagy protects against betulinic acid-induced apoptotic cell death in colorectal cancer cells. Cell Death Dis. 2017;8:e3087. https://doi.org/10.1038/cddis.2017.485.

    Article  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Wong FC, Woo CC, Hsu A, Tan BKH. The anti-Cancer activities of Vernonia amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent and p53-independent pathways. PLoS One. 2013;8(10):e78021. https://doi.org/10.1371/journal.pone.0078021.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Morgan CW, Julien O, Unger EK, Shah NM, Wells JA. Turning ON Caspases with genetics and small molecules. Methods Enzymol. 2014;544:179–213. https://doi.org/10.1016/B978-0-12-417158-9.00008-X.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Yu J, Zhang L. PUMA, a potent killer with or without p53. Oncogene. 2008;27(Suppl 1):S71–83. https://doi.org/10.1038/onc.2009.45.

  73. 73.

    Vidya Priyadarsini R, Senthil Murugan R, Maitreyi S, Ramalingam K, Karunagaran D, Nagini S. The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-kappaB inhibition. Eur J Pharmacol. 2010;649:84–91. https://doi.org/10.1016/j.ejphar.2010.09.020.

  74. 74.

    Bishayee K, Ghosh S, Mukherjee A, Sadhukhan R, Mondal J, Khuda-Bukhsh AR. Quercetin induces cytochrome-c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug-DNA interaction. Cell Prolif. 2013;46:153–63. https://doi.org/10.1111/cpr.12017.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Liu Y, Bi T, Wang G, Dai W, Wu G, Qian L, Gao Q, Shen G. Lupeol inhibits proliferation and induces apoptosis of human pancreatic cancer PCNA-1 cells through AKT/ERK pathways. Naunyn Schmiedeberg's Arch Pharmacol. 2015;388(3):295–304. https://doi.org/10.1007/s00210-014-1071-4.

  76. 76.

    Liu Y, Bi T, Dai W, Wang G, Qian L, Shen G, Gao Q. Lupeol induces apoptosis and cell cycle arrest of human osteosarcoma cells through PI3K/AKT/mTOR pathway. Technol Cancer Res Treat. 2016;15(6):NP16–24. https://doi.org/10.1177/1533034615609014.

  77. 77.

    Liu Y, Bi T, Shen G, Li Z, Wu G, Wang Z, Qian L, Gao Q. Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway. Cytotechnology. 2016;68(1):123–33. https://doi.org/10.1007/s10616-014-9763-7.

  78. 78.

    Min TR, Park HJ, Ha KT, Chi GY, Choi YH, Park SH. Suppression of EGFR/STAT3 activity by lupeol contributes to the induction of the apoptosis of human non-small cell lung cancer cells. Int J Oncol. 2019;55(1):320–30. https://doi.org/10.3892/ijo.2019.4799.

  79. 79.

    Shen X, Cui X, Cui H, Jin Y, Jin W, Sun H. Geraniol and lupeol inhibit growth and promote apoptosis in human hepatocarcinoma cells through the MAPK signaling pathway. J Cell Biochem. 2019;120(4):5033–41. https://doi.org/10.1002/jcb.27779.

  80. 80.

    He W, Li X, Xia S. Lupeol triterpene exhibits potent antitumor effects in A427 human lung carcinoma cells via mitochondrial mediated apoptosis, ROS generation, loss of mitochondrial membrane potential and downregulation of m-TOR/PI3Ksol; AKT signalling pathway. J Buon. 2018;23(3):635–40.

Download references

Acknowledgements

The authors wish to thank QBP Natividad Sales, QBP Karina Melquiades, and QBP Gabriela García for their technical assistance in the project, and the INHA Botanical Garden at Cuernavaca, Morelos, particularly Biol. Margarita Avilés, B.Sc. Biology, and Macrina Fuentes, B.Sc. Biology.

Funding

The materials and reagents necessary for this research were acquired with the support of Universidad Autónoma de Guerrero through SEMILLA 2018 grant. C.A.S.-V. received a postgraduate Master fellowship from the National Council of Science and Technology (CONACYT).

Author information

Affiliations

Authors

Contributions

CASV and PAF contributed equally in the work, performed the experimental procedures, and analyzed the data. JUCR participated in the collection of F. crocata leaves, extraction, and the phytochemical profile of compounds. LCS and MAQ participated in Analysis GC-MS. AEZG and NNT participated in cell culture and analyzed the data of cell proliferation and viability. COP, YCC, MALV and JOO participated in data analysis of cell cycle and apoptosis assays by flow cytometry. MOF and COP performed the western blot analysis. MAMC and PAF conceived the project, designed experiments, data analysis, and wrote the manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Miguel A. Mendoza-Catalán.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Fig. S1. Structures of the molecules identified by GC/MS from the Hex-EFc. The numbers correspond to name of compounds shown in Table 1.

Additional file 2: Fig. S2. Structures of the molecules identified by GC/MS from the Dic-EFc. The numbers correspond to name of compounds shown in Table 1.

Additional file 3: Fig. S3. Structures of the molecules identified by GC/MS from the Ace-EFc. The numbers correspond to name of compounds shown in Table 1.

Additional file 4: Fig. S4. Half-maximal inhibitory concentration (IC50) of leaf F. crocata extracts and fractions on cell proliferation (MTT assay).

Additional file 5: Fig. S5. Effect of Dic-EFc on MCF-10A cell viability after 48 h of treatment. MTT assay; V: vehicle, DMSO. (−): negative control, 5% FBS. (+): positive control, 100 μM Ara-C (cytarabine); Dic-EFc: dichloromethane extract of F. crocata. One-way ANOVA, Dunnett’s test: **p < 0.05, **p < 0.01 and ***p < 0.001 versus V.

Additional file 6: Table S1. Phytochemical profile of the dichloromethane extract fractions of F. crocata leaves.

Additional file 7: Fig. S6. Full Images of the blots shown in Fig. 7. V: Vehicle (DMSO), B: Basal, cells without treatment. Arrow: row of bands corresponding to p53, procaspase-8, procaspase 3 and GAPDH shown in Fig. 7. Red box: data not shown in Fig. 7; The Basal condition was omitted.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sánchez-Valdeolívar, C.A., Alvarez-Fitz, P., Zacapala-Gómez, A.E. et al. Phytochemical profile and antiproliferative effect of Ficus crocata extracts on triple-negative breast cancer cells. BMC Complement Med Ther 20, 191 (2020). https://doi.org/10.1186/s12906-020-02993-6

Download citation

Keywords

  • Ficus crocata
  • Moraceae
  • Breast cancer
  • Apoptosis
  • MDA-MB-231 cells