Alpha-mangostin inhibits intracellular fatty acid synthase and induces apoptosis in breast cancer cells

Abstract

Background: Fatty acid synthase (FAS) has been proven over-expressed in human breast cancer cells and consequently, has been recognized as a target for breast cancer treatment. Alpha-mangostin, a natural xanthone found in mangosteen pericarp, has a variety of biological activities, including anti-cancer effect. In our previous study, alpha-mangostin had been found both fast-binding and slow-binding inhibitions to FAS in vitro. This study was designed to investigate the activity of alpha-mangostin on intracellular FAS activity in FAS over-expressed human breast cancer cells, and to testify whether the anti-cancer activity of alpha-mangostin may be related to its inhibitory effect on FAS.

Methods: We evaluated the cytotoxicity of alpha-mangostin in human breast cancer MCF-7 and MDA-MB-231 cells. Intracellular FAS activity was measured by a spectrophotometer at 340 nm of NADPH absorption. Cell Counting Kit assay was used to test the cell viability. Immunoblot analysis was performed to detect FAS expression level, intracellular fatty acid accumulation and cell signaling (FAK, ERK1/2 and AKT). Apoptotic effects were detected by flow cytometry and immunoblot analysis of PARP, Bax and Bcl-2. Small interfering RNA was used to down-regulate FAS expression and/or activity.

Results: Alpha-mangostin could effectively suppress FAS expression and inhibit intracellular FAS activity, and result in decrease of intracellular fatty acid accumulation. It could also reduce cell viability, induce apoptosis in human breast cancer cells, increase in the levels of the PARP cleavage product, and attenuate the balance between anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. Moreover, alpha-mangostin inhibited the phosphorylation of FAK. However, the active forms of AKT, and ERK1/2 proteins were not involved in the changes of FAS expression induced by alpha-mangostin.

Conclusions: Alpha-mangostin induced breast cancer cell apoptosis by inhibiting FAS, which provide a basis for the development of xanthone as an agent for breast cancer therapy.

Figure 1

Effect of α-mangostin on the viability of MCF-7 and MDA-MB-231 cells. A Chemical structure of α-mangostin. B MCF-7 and MDA-MB-231 cells were treated with 0, 1, 2, 3, 4, 6, 8, and 10 μM α-mangostin for 24 h and 48 h. Cell viability was then determined by the CCK-8 assay as described in Materials and Methods. The percentage of cell viability was calculated as the ratio of α-mangostin treated cells to control cells. Data represented the mean ± SD of three independent experiments.

Figure 2

Apoptotic effect of α-mangostin in MCF-7 and MDA-MB-231 cells. A α-Mangostin induced apoptosis in MCF-7 and MDA-MB-231 cells as assessed by PARP cleavage (note intact PARP at 116 kDa and its cleavage product at 89 kDa) B MCF-7 and MDA-MB-231 cells were double-stained with annexin V and PI and analyzed by flow cytometry. The gate setting distinguished between living (bottom left), necrotic (top left), early apoptotic (bottom right), and late apoptotic (top right) cells. C The expression of Bax and Bcl-2 were determined by immunoblotting. MCF-7 and MDA-MB-231 cells were treated with α-mangostin at different concentrations for 24 h and equal amounts of lysates were immunoblotted with the corresponding antibody. Blots were reprobed for GAPDH as a loading control. In all cases, shown gels were representative of those obtained from at least two independent experiments.

Figure 3

α-Mangostin down-regulated FAS expression and inhibited intracellular FAS activity in breast cancer cells. A Representative Western blots showing a dose-dependent inhibition of FAS expression levels in MCF-7 and MDA-MB-231 cells after treating with α-mangostin at 24 h. B Relative FAS activity was measured by spectrophotometrically monitoring oxidation of NADPH at 340 nm. Data were expressed as means ± SD (n = 3). *p < 0.05 compared to control (0 μM); **p < 0.01 compared to control (0 μM).

Figure 4

Silence of FAS enhanced the cytotoxicity of α-mangostin in breast cancer cells. A Breast cancer cells were transfected with siRNA-targeting FAS for 72 h. Equal amounts of total protein from MCF-7 and MDA-MB-231 cells transfected with siRNA-targeting FAS or with the control were subjected to immunoblotting analyses with antibodies against FAS or GAPDH. B Effect of siRNA-induced silencing of FAS expression on cytotoxicity induced by α-mangostin. Cytotoxicity of α-mangostin was enhanced by siRNA-induced reduction of FAS expression levels. Data represented the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared with the same concentration of α-mangostin in control-transfected cells.

Figure 5

Effects of α-mangostin on PI3K/AKT and MAPK/ERK1/2 signaling pathways in breast cancer cells. A α-Mangostin induced FAS blockade led to down-regulate p-AKT in both MCF-7 and MDA-MB-231 cells. B α-Mangostin induced ERK1/2 activation in both MCF-7 and MDA-MB-231 cells. Cells were treated with α-mangostin at different concentrations for 24 h and equal amounts of lysates were immunoblotted with the corresponding antibody. Blots were reprobed for GAPDH as a loading control. In all cases, shown gels were representative of those obtained from at least two independent experiments.

Figure 6

α-Mangostin down-regulated phosphorylation of FAK in breast cancer cells. Tyr³⁹⁷ phosphorylation of FAK in both MCF-7 and MDA-MB-231 cells were decreased after treated with α-mangostin at different concentrations for 24 h. Equal amounts of lysates were immunoblotted with the corresponding antibody. Blots were reprobed for GAPDH as a loading control. In all cases, shown gels were representative of those obtained from at least two independent experiments.

Figure 7

Inhibitory effect of α-mangostin on the amount of intracellular fatty acid. Intracellular free fatty acid was detected by Free Fatty Acid Quantification Kit. Data were expressed as means ± SD (n = 3). The experiments were repeated twice. **p < 0.01 significantly different from control (0 μM); ***p < 0.001 significantly different from control (0 μM).