Cytotoxicity of Triterpene Seco-Acids from Betula pubescens Buds

Abstract

The present study investigated the magnitude and mechanism of the cytotoxic effect on selected cancer cell lines of 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) isolated from downy birch (Betula pubescens) buds by carbon dioxide supercritical fluid extraction and gradient column chromatography. Cell viability in six human cancer lines exposed to these compounds was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by annexin V/propidium iodide staining of gastric cancer AGS and colorectal cancer DLD-1 cells. To evaluate the mechanism of apoptosis, the expression of apoptosis-related proteins was analyzed by Western blot. Compound 1 exhibited non-specific toxicity, while compounds 2 and 3 were specifically toxic to colon and stomach cancer cells. The toxicity of compounds 2 and 3 against these two cell lines was greater than for compound 1. Cleavage of caspase-8, -9, and -3 was found in AGS and DLD-1 cells treated with all three seco-acids, indicating the induction of apoptosis via extrinsic and intrinsic pathways. Therefore, triterpene seco-acids (1-3) decreased cell viability by apoptosis induction. AGS and DLD-1 cells were more susceptible to seco-acids with an oxidized C19 than normal fibroblasts. Hence, it made them a new group of triterpenes with potential anticancer activity.

Keywords: apoptosis; birch buds; cytotoxicity; triterpene seco-acids.

Figure 1

Chemical structure of triterpene seco-acids: 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3).

Figure 2

Triterpene seco-acids decrease cell viability. Effect of 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) on the viability of cancer cells A375, AGS, DLD-1, HeLa, LN229, MD-MB-231 and normal dermal fibroblasts after 24, 48, and 72 h treatment, as assessed by MTT assay. Data are presented as mean ± standard error of the mean (SEM) from three independent experiments. * p < 0.05 compared to control group. The half-maximal inhibitory concentration (IC₅₀) values of seco-acids for the cell lines are shown.

Figure 3

Triterpene seco-acids promote apoptosis in AGS and DLD-1 cells. AGS and DLD-1 cells treated with 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) for 24 h were triple stained with annexin V-FITC conjugate (green fluorescence), propidium iodide (red fluorescence), and Hoechst 33342 (blue fluorescence), and were visualized by fluorescence microscopy. Representative merged photographs are shown. Scale bar = 100 μm. Data are presented as mean ± standard deviation (SD) from three assays. * p < 0.05 compared to control group.

Figure 4

Triterpene seco-acids trigger extrinsic and intrinsic pathways of apoptosis in AGS and DLD-1 cells. AGS and DLD-1 cells were treated with 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) for 24 h, and the expression levels of apoptosis-related proteins were determined by Western blotting. Antibodies against caspase-8, BID, p53, caspase-9, caspase-3, cleaved caspase-3, caspase-7, PARP and cleaved PARP were used. The expression of β-actin served to normalize protein loading.