Isolation and Anticancer Progression Evaluation of the Chemical Constituents from Bridelia balansae Tutcher

Abstract

The dichloromethane extract of the roots of Bridelia balansae Tutcher (Phyllanthaceae) was found to show potential anticancer activity against HCT116 colorectal cancer cell. Our bioassay-guided phytochemical investigation of the roots of B. balansae led to the identification of 14 compounds including seven lignans (1-7), three phenylbenzene derivatives (8-10), two flavanone (11-12), and two triterpenoids (13-14). Among them, 4'-demethyl-4-deoxypodophyllotoxin (1) is the first aryltetralin lignan compound identified from this plant species. In addition, the stereochemistry of 1 was validated by X-ray crystallography for the first time, and its distinguished cytotoxic effect on HCT116 cells with an IC₅₀ value at 20 nM was induced via an apoptosis induction mechanism. Compound 1 could also significantly decrease the migration rate of HCT116 cells, indicating its potential application against cancer metastasis. The western blot analysis showed that 1 has the potential to inhibit cell proliferation and metastasis. Treatment of 1 resulted in the downregulation of matrix metalloproteinases 2 (MMP2) and p-Akt, while p21 was upregulated. Collectively, the present study on the phytochemical and biological profile of B. balansae has determined the plant as a useful source to produce promising anticancer lead compounds.

Keywords: Bridelia balansae; apoptosis; colorectal cancer; cytotoxicity; lignan compounds.

Figure 1

The chemical structures of compounds 1–14.

Figure 2

The ORTEP drawing of compound 1.

Figure 3

Effects of compound 1 on induction of apoptosis of HCT116 cancer cells. (a) Flow cytometric analysis of HCT116 cancer cells using Annexin V/PI staining. Cells were treated with 1 at the indicated concentrations for 48 h prior to labelling with annexin V and PI. Q1 shows the percentage of necrotic cells, and Q2 and Q4 represent late and early apoptotic cells, respectively. Q3 shows the population of normal cells. (b) Graphical representation of the percentage of apoptotic cells (sum of Q2 and Q4) from three independent replicates’ data as the mean ± SEM; * p < 0.05.

Figure 4

Compound 1 reduced cellular mobilization in HCT116 cells treated with various concentrations of 1. Images were acquired at 0, 24 h after scratching. The dotted lines define the areas lacking cells. Data were collected from 3 independent experiments and only presentative pictures are shown.

Figure 5

Immunofluorescence study of disruption of the microtubule network in HCT116 cells. Microtubules were visualized with an anti-α-tubulin antibody (red). The nuclei of cells were stained with 4′, 6-diamidino-2-phenylindole and thus showed a blue color.

Figure 6

Western blot analysis shows effects of compound 1 on apoptosis and migration regulatory proteins in HCT116 cancer cells (a). The downregulation of p-AKT and MMP2 and upregulation of P21 are shown in a statistic graph (b), which indicating the inhibition of apoptosis and cell migrate. Data were collected from 3 independent experiments and only representative pictures are shown. (All data were analyzed by GraphPad Prism. Unpaired Student’s t test was used when comparing treatment group with control group within the same cell type. p < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01).