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. 2020 Jun 22;20(1):191.
doi: 10.1186/s12906-020-02993-6.

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

Carlos A Sánchez-Valdeolívar  1 , Patricia Alvarez-Fitz  2 , Ana E Zacapala-Gómez  1 , Macdiel Acevedo-Quiroz  3 , Lorena Cayetano-Salazar  1 , Monserrat Olea-Flores  1 , Jhonathan U Castillo-Reyes  1 , Napoleón Navarro-Tito  1 , Carlos Ortuño-Pineda  1 , Marco A Leyva-Vázquez  1 , Julio Ortíz-Ortíz  1 , Yaneth Castro-Coronel  1 , Miguel A Mendoza-Catalán  4
Affiliations

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

Carlos A Sánchez-Valdeolívar et al. BMC Complement Med Ther. .

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.

Keywords: Apoptosis; Breast cancer; Ficus crocata; MDA-MB-231 cells; Moraceae.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
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
Fig. 2
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
Fig. 3
Fig. 3
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
Fig. 4
Fig. 4
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
Fig. 5
Fig. 5
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
Fig. 6
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
Fig. 7
Fig. 7
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
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