Ethanolic extract of Brucea javanica inhibit proliferation of HCT-116 colon cancer cells via caspase activation

Abstract

Brucea javanica (L.) Merr. is a well-known plant in Chinese System of Medicine. Its fruits and seeds have been reported to possess curative properties against various ailments. The chemical constituents and biological activity of this plant have been an interesting area in plant and chemistry medicine. The aim of this study is to evaluate the antiproliferative effects of the B. javanica extract against a colon cancer cell line and identification of the chemical components derived from the extract. An ethanolic extract from B. javanica fruits was prepared by cold maceration method, subjected to LC-MS profiling to elucidate the composition abbreviated as BJEE. The extract was screened for the cytotoxicity effects on HCT-116 colon cancer cells via MTT and LDH methods. Additionally, AO/PI staining verified apoptosis features in HCT-116 cells through microscopic analysis. ROS, caspase activity, and gene expression has been performed to identify its possible mechanism of actions which contribute to apoptosis. Output data from this study showed BJEE inhibited the cell proliferation of HCT-116 colon cancer cells at IC₅₀ value of 8.9 ± 1.32 (μg mL^-1) and significantly increased the levels of caspase-8, 9, and 3/7 in treated cells in comparison to untreated. The changes in expression of caspase genes and some apoptosis genes like Bax and Bcl-2 were confirmed using RT-PCR. Phytochemical analysis by LC-MS identified six major active compounds (bruceine D, isobrucein A, quassimarin, C16 sphinganine, phytosphingosine, and enigmol) in BJEE that may play a key role in cell apoptosis. The current study showed BJEE could be a promising agent for colorectal cancer therapy by significant increase in caspase activity level, and up-regulation of the specific apoptotic genes.

Fig. 1. LDH assay showed the cytotoxicity percentage of BJEE towards HCT-116 cells. The data represent the means ± SD (*p ≤ 0.05) of three independent experiments.

Fig. 2. Chromatographic fingerprint analysis of the BJEE. Thirteen major compounds in BJEE were identified by LC/MS Q-TOF. Abbreviations: BJEE, Brucea javanica ethanol extract; LC/MS, liquid chromatography/mass spectrometry.

Fig. 3. Fluorescent micrograph of acridine orange and propidium iodide double-stained human HCT-116 cells. Cells were treated with different concentrations (10, 20, 40 μg mL⁻¹) of BJEE for 24 hours. (A) Untreated HCT-116 cells after 24 hours demonstrated normal structure without prominent apoptosis. (B) Treated cells at concentration of 10 μg mL⁻¹ BJEE showed early apoptosis features, including chromatin condensation. (C) Early and late apoptosis were observed at concentration of 20 μg mL⁻¹ and (D) late apoptosis and bright red colored secondary necrosis was obvious after 24 hours of BJEE treatment at 40 μg mL⁻¹ (magnification: 20×). Abbreviation: VI: viable cells; CC: chromatin condensation; LA: late apoptosis; SN: secondary necrosis; BJEE, Brucea javanica ethanol extract.

Fig. 4. Effect of BJEE on ROS generation in HCT-116 cells. (A) HCT-116 cells were treated with BJEE at IC₅₀ concentration (9 μg mL⁻¹) and DMSO as negative control for 6 hours then stained with DHE dye (red) (magnification: 20×). (B) A bar chart represents significant effect of BJEE elevated ROS production at concentration of 10 to 20 μg mL⁻¹ after 6 hours. The data represents the means ± SD of three independent experiments. *p ≤ 0.05 compared with the untreated group. Abbreviations: BJEE, Brucea javanica ethanol extract; DMSO, Dimethyl sulfoxide.

Fig. 5. Relative expression of caspase-3/7 (A), caspase-8 (B) and caspase-9 (C) in the HCT-116 cells treated with various concentrations of BJEE. Triplicates of each treatment group were used in each independent experiment. The data represent the means ± SD of three independent experiments; statistical significance is expressed as, *p ≤ 0.05. (A) Caspase-3/7, (B) caspase-8 & (C) caspase-9. Abbreviation: BJEE, Brucea javanica ethanol extract.

Fig. 6. Quantitative analysis of gene expression in HCT-116 treated cells with BJEE after 24 hours showed a significant fold differences in Bax, Casp-3, 9 & 8 genes relative to untreated cells. Meanwhile, gene expression of Bcl-2 did not elicit significant changes. Statistical significance is expressed as, *p ≤ 0.05. Abbreviation: BJEE, Brucea javanica ethanol extract.