Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin-EDTA were purchased from Corning Life Sciences (Corning, NY, USA). Antibodies against JAK2, p-JAK2 (Y1007/1008), STAT3, p-STAT3 (Y705), ERK, p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, N-cadherin, β-catenin, TCF8/ZEB1, claudin-1, Slug, Snail, and MMP9 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-MMP9 antibody was purchased from Abcam (Cambridge, UK). Anti-β-actin antibody was purchased from Abgent, Inc. (San Diego, CA, USA). Anti-mouse and rabbit IgG-HRP antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The electuary ointment of Huaier (Gaitianli Medicine Co., Ltd., Jiangsu, China) was dissolved in complete medium and then sterilized by filtration with a 0.22-mm filter to get the 10 mg/ml stock solution, which was stored at 4°C for short-term storage.

Tsc1^−/− MEFs and Tsc2^−/− MEFs were previously described (7,19). Cells were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin in 5% CO₂ at 37°C.

Tsc1^−/− MEFs and Tsc2^−/− MEFs were seeded in 96-well plate at a density of 2.0×10³ cells/well. Twenty-four hours later, cells were treated with Huaier aqueous extract at the indicated concentrations of ranging from 0 to 8 mg/ml, and incubated for 12, 24 and 48 h, respectively. Ten microliters CCK-8 reagent (Dojindo, Kumamoto, Japan) was added to each well, and then incubated for 2 h at 37°C. The optical density values were measured at 450 nm using a microplate reader (Perkin-Elmer, Waltham, MA, USA).

Tsc1^−/− MEFs and Tsc2^−/− MEFs were seeded in a 6-well plate at a density of 1,000 cells/well. Twenty-four hours later, cells were treated with Huaier aqueous extract at the indicated concentrations. After 10 days of treatment, the cells were fixed with methanol for 15 min, and then colonies were stained with 0.1% crystal violet. The cell colonies were photographed using a digital camera (Leica Microsystems GmbH, Wetzlar, Germany).

Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min, and stained with Hoechst 33258 (Biyuntian Biotechnology Co., Ltd., Shanghai, China) for 30 min in the dark before washed with PBS again. Then cells were observed under an inverted fluorescence microscope (Leica Microsystems GmbH).

Tsc1^−/− MEFs and Tsc2^−/− MEFs were seeded in 12-well plates at 2.5×10⁵ cells/well. The next day, the cells were treated with Huaier aqueous extract at 4 or 8 mg/ml for 48 h. Apoptosis was analyzed with an Annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. In brief, cells were collected and washed twice with PBS, then resuspended at a concentration of 1×10⁶ cells/ml in binding buffer. Five microliters Annexin V-FITC and 5 µl propidium iodide (PI) were added and incubated for 15 min at room temperature in the dark. Finally, 400 µl the binding buffer was added and then cells were analyzed using a flow cytometer (BD Biosciences).

Tsc1^−/− MEFs and Tsc2^−/− MEFs were seeded in 6-well plates at 5×10⁵ cells/well. After 12-h starvation in serum-free medium, cells were treated with Huaier aqueous extract at the concentrations of 0, 4 and 8 mg/ml for 48 h, and then cells were collected, washed twice with PBS and fixed overnight with 70% ethanol at 4°C. The next day, the fixed cells were centrifuged at 1,000 × g for 5 min and washed twice with PBS, then cells were resuspended with 500 µl binding buffer containing RNaseA and PI (Biyuntian Biotechnology Co., Ltd.). After 30-min incubation at 37°C in the dark, the cells were analyzed by flow cytometry.

Cells were seeded in 12-well plates. The complete medium was replaced with serum-free medium when the cells grew to a subconfluent state. After 12-h starvation, a straight cell-free wound was created with a 10-µl pipette tip and washed twice with PBS, then the cells were maintained in serum-free medium containing Huaier aqueous extract at the concentrations of 0, 2 and 4 mg/ml. The cells were observed and the scratch width was measured at 0, 12 and 24 h. The migration distances were analyzed quantitatively.

Tsc1^−/− MEFs and Tsc2^−/− MEFs were maintained in serum-free medium for 12 h to reduce the interference of serum. Transwell chambers were placed in 24-well plates to constitute the Transwell system. The Matrigel (BD Biosciences, San Jose, CA, USA) was dissolved in serum-free medium at a ratio of 1:12, and then 60 µl mixture was put to the upper chamber gently. Next the Transwell system was incubated at 37°C for 4 h. The cells were collected and resuspended in serum-free medium containing different concentrations of Huaier aqueous extract. In brief, 1×10⁵ cells suspended in 200 µl serum-free medium containing drug were added to the upper chamber, and 750 µl complete medium containing 10% FBS was added to the lower chamber. After incubation for 12 or 24 h, the cells on the upper surface of the membrane were wiped off with cotton swabs, and the cells on the lower surface of the membrane were fixed with ethanol for 15 min, then stained with crystal violet for 10 min and washed twice with PBS. The successfully invaded cells were observed and photographed on 5 random fields with an inverted microscope.

Cells were washed twice with PBS and harvested with cell lysis solution as previously described (20). The cell lysate was boiled at 98°C for 10 min. The proteins were separated by 4–12% Bis-Tris Nu-PAGE (Invitrogen, Carlsbad, CA, USA) and then transferred onto PVDF membrane. The membrane was blocked with 3–5% skim milk in TBST at room temperature for 1 h, and then incubated with the primary antibodies at 4°C overnight, followed by the incubation with the HRP-conjugated secondary antibody at room temperature for 2 h. Finally, the membrane was washed three times in TBST and detected by chemiluminescence.

The data groups were compared with the two-tailed Student's t-test using GraphPad Prism 5.0 software. The data are presented as mean ± SD. P<0.05 was considered to be statistically significant.

To evaluate the effect of Huaier aqueous extract on Tsc1^−/− or Tsc2^−/− MEFs, we examined cell viability with the CCK-8 assay. Tsc1^−/− or Tsc2^−/− MEFs were treated with Huaier at the indicated concentrations (0, 2, 4, 6 and 8 mg/ml) for 12, 24 and 48 h, respectively. As shown in Fig. 1A and B, Huaier significantly suppressed the proliferation of Tsc2^−/− or Tsc1^−/− MEFs in a time- and dose-dependent manner. The IC50 values of Tsc1^−/− or Tsc2^−/− MEFs exposed to Huaier aqueous extract of various concentrations for 48 h were 3.89 and 3.56 mg/ml, respectively. Furthermore, we performed colony formation assay to assess the effect of Huaier aqueous extract on cell proliferation. Result indicated that Tsc1^−/− or Tsc2^−/− MEFs treated with Huaier aqueous extract formed smaller and fewer colonies in a dose-dependent manner (Fig. 1C).

To investigate whether the inhibitory effect of Huaier aqueous extract was attributed to cell cycle arrest, we performed flow cytometry assay to evaluate cell cycle distribution of Tsc1^−/− or Tsc2^−/− MEFs exposed to Huaier. Tsc1^−/− or Tsc2^−/− MEFs were treated with Huaier aqueous extract for 48 h. As shown in Fig. 2A, Tsc2^−/− MEFs exposed to Huaier showed significantly decreased fraction of the G0/G1 phase and increased fraction of the S phase in a dose-dependent manner. In addition, Tsc1^−/− MEFs treated with Huaier exhibited significantly decreased fraction of the G2/M phase and increased fraction of the S phase (Fig. 2B). Taken together, these results revealed that Huaier-induced S-phase arrest was partially responsible for the proliferation inhibition of Tsc1^−/− or Tsc2^−/− MEFs.

Next we performed Hoechst staining assay to determine the effect of Huaier aqueous extract on apoptosis. Apoptotic cells could be stained with Hoechst 33258 dye. As observed in Fig. 3A, the nuclei in the untreated Tsc1^−/− or Tsc2^−/− MEFs were stained with a less bright blue fluorescence and these two cells appeared to be intact oval shape. Whereas, more condensed or fragmented chromatin were observed in Tsc1^−/− or Tsc2^−/− MEFs exposed to Huaier aqueous extract. To further explore the effect of Huaier on apoptosis in Tsc1^−/− or Tsc2^−/− MEFs, we conducted flow cytometry analysis with Annexin V-FITC/PI staining. As shown in Fig. 3B, the apoptosis rate of Tsc1^−/− or Tsc2^−/− MEFs increased in a dose-dependent manner in response to Huaier. Collectively, Huaier aqueous extract promotes apoptosis of Tsc1^−/− or Tsc2^−/− MEFs.

Next we determined the effect of Huaier aqueous extract on cell morphology. Tsc1^−/− or Tsc2^−/− MEFs were treated with Huaier aqueous extract at a concentration of 4 or 8 mg/ml for 24 h. As shown in Fig. 4, the untreated cells showed plump cell body, homogeneous cytoplasm, and good refraction. However, Huaier treatment led to shrinked cell body and weakened refraction. Moreover, more cells became round and died, which was concomitant with an increase in the concentration of Huaier.

Migration and invasion play a critical role in metastasis of cancer cells (21). To evaluate the migration ability of cells in vitro, we performed in vitro scratch assay. We treated Tsc1^−/− or Tsc2^−/− MEFs with Huaier aqueous extract at the concentrations of 0, 2 and 4 mg/ml, and then measured the scratch width at time-points of 0, 12 and 24 h. As shown in Fig. 5A, the migration distance was decreased in response to Huaier in a dose-dependent manner.

The scratch healing inhibition rates of Tsc2^−/− MEFs and Tsc1^−/− MEFs were 58.57±4.52 and 82.01±13.97% after treatment with Huaier aqueous extract at a concentration of 4 mg/ml for 24 h. Thus, Huaier significantly inhibits the migration of Tsc1^−/− or Tsc2^−/− MEFs. Furthermore, we performed Transwell assay to assess the invasive capacity of Tsc1^−/− or Tsc2^−/− MEFs in vitro. Tsc2^−/− MEFs were treated with Huaier aqueous extract at the concentrations of 4 and 8 mg/ml for 12 h. As observed in Fig. 5B, the number of Tsc2^−/− MEFs invading through the Matrigel-coated membrane was drastically decreased. In addition, the invasive capacity of Tsc1^−/− MEFs was also reduced by treatment with Huaier (Fig. 5B).

The epithelial-mesenchymal transition (EMT) is a critical process in cancer development, which enhances the metastatic capacity of cancer cells (22,23). We therefore investigated the effects of Huaier on EMT of Tsc1^−/− or Tsc2^−/− MEFs. We examined the expression levels of EMT markers by western blotting. As shown in Fig. 5C, Huaier treatment downregulated the protein levels of N-cadherin, β-catenin, Slug, TCF8/ZEB1, Snail, and claudin-1 (24–26) in Tsc1^−/− or Tsc2^−/− MEFs, indicating that Huaier inhibited EMT of Tsc1^−/− or Tsc2^−/− MEFs. MMP-9 has been reported to be important in cancer metastasis (27,28). Here we found that MMP-9 expression was reduced by Huaier treatment in a dose-dependent manner (Fig. 5C). Taken together, Huaier suppressed the metastatic ability of Tsc1^−/− or Tsc2^−/− MEFs in vitro.

JAK2/STAT3 pathway plays an important role in tumorigenesis and metastasis (29,30). Inhibition of JAK2/STAT3 signaling pathway constrains cancer cell growth and induces apoptosis (31,32). As shown in Fig. 6A, Huaier treatment downregulated the protein levels of JAK2, p-JAK2, STAT3, and p-STAT3 in Tsc1^−/− or Tsc2^−/− MEFs, suggesting that Huaier markedly inhibits JAK2/STAT3 signaling pathway in Tsc1^−/− or Tsc2^−/− MEFs. The MAPK pathway plays a crucial role in multiple cellular processes, such as cell proliferation, apoptosis, migration and invasion (33,34). The MAPK super-family consists of three mammalian MAP kinases, ERK, JNK, and p38 MAPK (35). As shown in Fig. 6B, we found that the phosphorylation of ERK and JNK was inhibited by Huaier treatment in these two cell lines, indicating that Huaier restrains the MAPK signaling pathway in Tsc1^−/− or Tsc2^−/− MEFs.