Butein suppresses breast cancer growth by reducing a production of intracellular reactive oxygen species

Abstract

Background: Butein has various functions in human diseases including cancer. While anti-cancer effects of butein have been revealed, it is urgent to understand a unique role of butein against cancer. In this study, we demonstrate that butein inhibition of reactive oxygen species (ROS) production results in suppression of breast cancer growth.

Methods: Different breast cancer cell lines were treated with butein and then subjected to cell viability and apoptosis assays. Butein-sensitive or -resistant breast cancer cells were injected into mammary fat pads of immunocompromised mice and then butein was injected. Breast cancer cells were categorized on the basis of butein sensitivity.

Results: Butein reduced viabilities of different breast cancer cells, while not affecting those of HER2-positive (HER2+) HCC-1419, SKBR-3 and HCC-2218 breast cancer cells. Butein reduction of ROS levels was correlated with apoptotic cell death. Furthermore, butein reduction of ROS level led to inhibitions of AKT phosphorylation. N-acetyl-L-cysteine (NAC), a free radical scavenger, also reduced ROS production and AKT phosphorylation, resulting in apoptotic cell death. In contrast, inhibitory effects of both butein and NAC on ROS production and AKT phosphorylation were not detected in butein-resistant HER2+ HCC-1419, SKBR-3 and HCC-2218 cells. In the in vivo tumor growth assays, butein inhibited tumor growth of butein-sensitive HER2+ BT-474 cells, while not affecting that of butein-resistant HER2+ HCC-1419 cells. Moreover, butein inhibition of ROS production and AKT phosphorylation was confirmed by in vivo tumor growth assays.

Conclusions: Our study first reveals that butein causes breast cancer cell death by the reduction of ROS production. Therefore, our finding provides better knowledge for butein effect on breast cancer and also suggests its treatment option.

Figure 1

Butein effect on viabilities of different breast cancer cell lines. (A) Butein structure. (B) Butein inhibition of breast cancer cell viabilities. Breast cancer cells were treated with different doses of butein (0-100 μg/ml) for 48 hours and then subjected to MTT assays. Red bars indicate 50% of cell viability. Experiments were done in quadruplicate and repeated three times independently. Bars indicate the mean ± SEM.

Figure 2

Butein induction of apoptosis. (A) Breast cancer cells were treated with butein at 0-100 μg/ml (left triangles) for 24 hours and then cleavages of PARP and Caspases were examined with appropriate antibodies in western blots. Actin was detected as a loading control. (B) Breast cancer cells were treated with butein at 10 μg/ml for 24 hours and then stained with Annexin V and PI. Cells were then counted using flow cytometry. Experiments were performed in triplicate and data present the mean ± SD.

Figure 3

Butein effects on ROS generation. (A) Butein alteration of ROS level. 100% stacked bars indicate ROS levels in the cells treated with butein or not. Cells were treated with butein at 10 μg/ml for 5 minutes and incubated with H₂DCF-DA for another 1 hour. ROS levels were measured using flow cytometry. Experiments were independently repeated three times. Red arrows indicate ROS levels in HER2⁺ breast cancer cells. (B) Representative data for butein-altered ROS levels in HER2⁺ breast cancer cells either sensitive (BT-474) or resistant (HCC-1419) to butein. Negative control (Neg.) indicates no treatment of ROS indicator. Left triangles indicate the increase of ROS levels. (C) Correlations between percentages of reduced ROS levels and percentages of apoptotic cell death. A red circle indicates butein-resistant and luminal HER2⁺ breast cancer cells. (D-E) Butein effect on cell cycle profiles. Cells were treated with 10 μg/ml of butein for 24 hours and then stained with PI. Data present the mean ± SD for altered percentages of subG1 populations in different breast cancer cells treated with 10 μg/ml of butein for 24 hours (D). Cell populations at different stages of cell cycle were presented as 100% rate (E). Experiments were performed in triplicate.

Figure 4

Butein effect on AKT phosphorylation. (A) Cells were treated with 10 μg/ml of butein for 15 minutes. Relative phosphorylation levels of SRC, ERK and AKT in different breast cancer cell lines were presented in heatmap. (B) Representative data for phosphorylation levels of SRC, ERK, and AKT. Actin was detected as a loading control. (C) NMF analysis to categorize breast cancer cells based on butein-altered phosphorylation levels of SRC, ERK and AKT. (D-E) Cells were treated with 40 μg/ml of butein, 40 mM of NAC or 10 μM of LY294002 for 5 minutes. ROS levels were measured by incubating cells with H₂DCF-DA for another 1 hour (D), and AKT phosphorylation was detected by anti-p-AKT antibody (E). (F) Cells were treated with NAC at 40 mM, LY294002 at 10 μM or butein at 10 μg/ml for 24 hours and then stained with Annexin V and PI. Apoptotic cells were measured by flow cytometry.

Figure 5

Butein inhibition of both ROS production and AKT phosphorylation in vivo. (A) Tumor cells were orthotopically injected into nude mice (n = 5/group). Butein (10 μg/ml) or saline was subcutaneously injected six days after orthotopic tumor injection, which was repeated three times a week. Tumor volumes were measured at indicative time points. Asterisks indicate that P-value is less than 0.05. (B) Mouse body weights were measured at indicative time points. (C) Histopathological observations. BrdU, cleaved Caspase-3 and pAKT were stained with appropriate antibodies. In situ ROS was labeled with H₂DCF-DA. A bar indicates a scale of 40X observation. (D) Levels of phosphorylated and total AKT in xenograft tumor tissues.

Figure 6

A cartoon to summarize butein effect on breast cancer cells. Butein reduces intracellular ROS level, which inhibits AKT phosphorylation. Butein inhibition of ROS production and AKT phosphorylation results in the suppression of breast cancer growth. While breast cancer cells tested here are sensitive to butein, luminal HER2⁺ SKBR-3 and HCC-1419 and HCC-2218 cells are resistant to butein. This resistant phenotype may be related with basal ROS level.