In Vivo, In Vitro and In Silico Anticancer Activity of Ilama Leaves: An Edible and Medicinal Plant in Mexico

Abstract

Ilama leaves are an important source of secondary metabolites with promising anticancer properties. Cancer is a disease that affects a great number of people worldwide. This work aimed to investigate the in vivo, in vitro and in silico anticancer properties of three acyclic terpenoids (geranylgeraniol, phytol and farnesyl acetate) isolated from petroleum ether extract of ilama leaves. Their cytotoxic activity against U-937 cells was assessed using flow cytometry to determine the type of cell death and production of reactive oxygen species (ROS). Also, a morphological analysis of the lymph nodes and a molecular docking study using three proteins related with cancer as targets, namely, Bcl-2, Mcl-1 and VEGFR-2, were performed. The flow cytometry and histomorphological analysis revealed that geranylgeraniol, phytol and farnesyl acetate induced the death of U-937 cells by late apoptosis and necrosis. Geranylgeraniol and phytol induced a significant increase in ROS production. The molecular docking studies showed that geranylgeraniol had more affinity for Bcl-2 and VEGFR-2. In the case of farnesyl acetate, it showed the best affinity for Mcl-1. This study provides information that supports the anticancer potential of geranylgeraniol, phytol and farnesyl acetate as compounds for the treatment of cancer, particularly with the potential to treat non-Hodgkin's lymphoma.

Keywords: Annona macroprophyllata; acyclic terpenoids; agri-food waste; cancer; docking; flow cytometry; ilama leaves; morphological analysis.

Figure 1

Weights (in g) of lymph nodes of female (A) and male (B) mice compared with healthy control (HC), control without treatment at 65 days (U-937), methotrexate (MTX), geranylgeraniol (Gg), phytol (PT), and farnesyl acetate (FA). Results were obtained by ANOVA one-way analysis followed by Bonferroni’s test for multiple comparisons. Data are expressed as mean ± SEM, (n = 6); * p < 0.05 vs. U-937, + p < 0.05 vs. MTX, and ^& p < 0.05 vs. healthy.

Figure 2

Apoptotic and necrotic effects of Gg, PT and FA. U-937 cells were exposed to vehicle 0.02% dimethyl sulfoxide (DMSO, untreated control) (A), methotrexate (CC₅₀ 0.243 µM, MTX) (B), geranylgeraniol (CC₅₀ 0.395 µM, Gg) (C), phytol (CC₅₀ 0.296 µM, PT) (D) and farnesyl acetate (CC₅₀ 0.275 µM, FA) (E) and were incubated with annexin-V-FITC/PI and analyzed by flow cytometry. R1 = necrosis, annexin-V-FITC-negative/PI-positive (E−+, K−+ and B−+); R2 = late apoptosis, annexin-V-FITC-positive/PI-positive (E++, K++ and B++); R3 = viable cells, annexin-V-FITC-negative/PI-negative (E−−, K−− and B−−); R4 = early apoptosis, annexin-V-FITC-positive/PI-negative (E+−, K+− and B+−).

Figure 3

Geranylgeraniol and phytol produced an increase in the generation of ROS. U-937 cells were cultured in DMEM medium (A) or exposed to vehicle 0.02% dimethyl sulfoxide (DMSO) (B), 150 µM hydrogen peroxide (H₂O₂) (C), methotrexate (CC₅₀ 0.243 µM) (D), geranylgeraniol (CC₅₀ 0.395 µM) (E), phytol (CC₅₀ 0.296 µM) (F) or farnesyl acetate (CC₅₀ 0.275 µM) (G) and were incubated with 2′-7 dichloro-dihydrofluorescein diacetate, and the fluorescence of reactive oxygen species (ROS) was analyzed by flow cytometry. x-axis: fluorescence scatter (FSC); y-axis: cell counts.

Figure 4

Representative photomicrographs with H&E staining of transversal axillary lymph node. Paracortex area (7 μm) (40X). Healthy control (A); U-937 (B), methotrexate (C), phytol (D), geranylgeraniol (E) and farnesyl acetate (F). Photomicrographs (7 μm) (100X) show cells in apoptosis (black arrows) and necrotic cells (blue arrows).

Figure 5

Molecular docking of the acyclic terpenoid geranylgeraniol (A) and navitoclax (B) bound to Bcl-2.

Figure 6

Molecular docking of the acyclic terpenoid farnesyl acetate (A) and 9EA (B) bound to Mcl-1.

Figure 7

Molecular docking of the acyclic geranylgeraniol terpenoid (A) and axitinib (B) bound to VEGFR-2.

Figure 8

Annona macroprophyllata Donn. Sm.

Figure 9

Structures of farnesyl acetate, phytol, geranylgeraniol and methotrexate.