Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells

Abstract

Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR)/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway.

Conflict of interest statement

Competing Interests: Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. (Jiangsu, China). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. Huaier extract reduced cell viability and triggered intracellular vesicular organelles in breast cancer cells.

(A, C and E) The cytotoxic effect of Huaier extract was measured using the MTT assay. Cells were treated with different concentrations of Huaier extract over indicated time periods and subjected to the MTT assay. The viability of untreated cells was considered 100%. The experiments were performed in triplicate and data is presented as the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01. (B, D and F) Micrographs show the appearance of vesicular organelles in breast cancer cells after 48 h of exposure to Huaier extract. Arrows indicate the intracellular vacuoles. Bars, 10 μm. (G) AO/EB staining of T47D cells was performed to detect apoptosis and necrosis induced by Huaier extract. Bars, 50 μm. (H) Representative TUNEL staining (red fluorescence) of MDA-MB-231 cells treated with or without Huaier extract. Bars, 50 μm.

Fig 2. Huaier extract induced autophagy in breast cancer cells.

(A) Representative electron micrographs of breast cancer cells treated with or without 4 mg/ml Huaier extract for 48 h. Bars, 100nm. (B) Acidic vesicular organelles induced by Huaier extract were stained with MDC. Bars, 10 μm. (C) Aggregation of LC3B in Huaier-treated cells. Cells were treated with or without 4 mg/ml Huaier extract and stained with the LC3B antibodies using immunofluorescence staining. Puncta represent the autophagosome formation. Bars, 10 μm. (D) Cell lysates were harvested after incubation with different concentrations of Huaier extract for 48h. β-actin was used as a loading control. (E) Cells in suspension were labeled with acridine orange and quantified using flow cytometry. FL1-H indicates green color intensity (cytoplasm and nucleus), whereas FL3-H shows red color intensity (AVO). Cells in up quadrants were considered AVO-positive. Results shown are representative of three independent experiments.

Fig 3. The effect of autophagy inhibition on the cytotoxicity of Huaier extract.

MDA-MB-231 (A and B), MDA-MB-468 (C and D) and MCF7 (E and F) were pretreated with 3-MA or CQ before being exposed to Huaier extract for the indicated time. The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01.

Fig 4. LC3B siRNA blocks Huaier-mediated autophagic cell death.

(A) LC3B expression was knocking-down by siRNA. MDA-MB-231 and MCF7 cells were transfected with siLC3B. siControl was used as the negative control. (B) Huaier-mediated autophagy was blocked by LC3B siRNA. MDA-MB-231 cells were treated with siControl, siControl+Huaier, siLC3B, or siLC3B+Huaier. Autophagy was detected by flow cytometry. (C and D) The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01.

Fig 5. Huaier extract inhibited the activity of mTOR/S6K pathway.

(A and B) Cells were treated with various concentrations of Huaier extract for the indicated time. Cell lysates were prepared and detected via Western blotting. The effect of Huaier extract on the levels of mTOR, p70S6K, S6, 4E-BP1 and their phosphorylated forms are shown. (C) AMPK was activated after Huaier treatment in a dose-dependent manner. Results are representative of three independent experiments.

Fig 6. Huaier extract inhibited the growth of breast cancer cells in vivo by inducing autophagy.

(A) MDA-MB-231 cells were subcutaneously implanted into the right flank of BALB/c nu/nu mice as described in the materials and methods section. Mice with xenograft tumors were photographed after treatment with PBS (control), Huaier alone or in combination with chloroquine (15 mg/kg once daily) on day 32. (B) The mean volumes of xenograft tumors were measured at 4-day intervals with calipers. Growth curves of xenograft tumors are shown. (C) Representative micrographs of immunohistochemical staining using anti-LC3B antibody. Bars, 30 μm. (D) Western blot analysis of protein expression in tumors taken from mice.