Ham's F-12K (Kaighn's) medium, fetal bovine serum (FBS), penicillin-streptomycin solution, 0.25% trypsin, Matrigel and Transwell chambers were purchased from Corning Life Sciences (Corning, NY, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). 3-Methyladenine (3-MA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bafilomycin A1 was obtained from Aladdin Shanghai Biochemical Technology Co., Ltd. (Shanghai, China). Bis-Tris Nu-PAGE gels (4–12%) were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Super ECL Plus was from GE Healthcare (Pittsburgh, PA, USA). β-actin antibody was purchased from Abgent (cat. no. AM1829b; San Diego, CA, USA). Atg3 (cat. no. 3415), Beclin-1 (cat. no. 3495) and Atg5 (cat. no. 12994) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). LC3 antibody was from MBL (cat. no. PM036; Tokyo, Japan). Lamin B1 (cat. no. sc-20682), HRP-labeled goat anti-mouse (cat. no. sc-2005) and goat anti-rabbit (cat. no. sc-2004) antibodies were from Santa Cruz Biotechnology.

The electuary ointment of Huaier was obtained from Gaitianli Medicine Co., Ltd. (Jiangsu, China). It was dissolved in F-12K complete medium to obtain a 10 mg/ml stock solution and was stored at 4°C after sterilization by filtration.

Human prostate cancer PC3 cells were obtained from the Cell Culture Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (Beijing, China). PC3 cells were maintained in Ham's F-12K (Kaighn's) medium containing 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO₂.

PC3 cells were seeded into 96-well plates at a density of 3,500 cells/well. On the following day, the cells were treated with Huaier aqueous extract at the indicated concentrations for different times. Afterwards, 10 µl of CCK-8 was added into each well and incubated at 37°C for 2 h. The optical density (OD) was then measured at 450 nm using a microplate reader (Perkin-Elmer, Waltham, MA, USA).

The scratch assay was performed as described previously (10,18). In brief, PC3 cells were seeded into 12-well plates with complete medium. Cells in a subconfluent state were starved with serum-free medium for 12 h, and then a straight cell-free wound was created using a 10-µl pipette tip. Next, the cells were maintained in serum-free medium containing 4 mg/ml of Huaier extract. The scratch width was measured at 0 and 24 h. The cell migration distances were analyzed quantitatively.

The Transwell system was established as previously described (19,20). PC3 cells (1×10⁵) were resuspended in 200 µl serum-free medium containing Huaier extract (4 mg/ml) and then added to the upper chamber of the Transwell system. The lower chamber was filled with 750 µl complete medium containing 10% FBS. After incubation for 36 h, the cells were removed from the upper surface of the membrane with a cotton swab. Next, the cells on the bottom surface of the membrane were fixed and then stained with crystal violet. The Transwell chamber was washed twice with phosphate-buffered saline (PBS) to remove the dye. The invasive cells were observed and counted on 5 random fields under an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Total RNA was extracted from PC3 cells using E.Z.N.A.^® Total RNA Kit I (Omega Bio-tek, Inc., Norcross, GA, USA) and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Inc., Dalian, China) according to the manufacturer's protocols. cDNA was used as a template for the quantitative PCR using the TransStart Top Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd., Beijing, China). The primer sequences were: human Lamin B1 forward, 5′-TTCTCGAAGCTTGATCTGGG-3′ and human Lamin B1 reverse, 5′-GATCGAGCTGGGCAAGTG-3′; human β-actin forward, 5′-GTTGTCGACGACGAGCG-3′ and human β-actin reverse, 5′-GCACAGAGCCTCGCCTT-3′.

Cells were washed twice with PBS and harvested with lysis buffer [10 mM Tris (pH 6.8), 2% SDS, 10% glycerol and 100 mM DTT], and then cell lysates were boiled for 10 min at 98°C. The levels of indicated proteins were detected by immunoblot analysis as previously described (21).

All siRNAs were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). PC3 cells were seeded in 6-well plates and then transfected with siRNAs targeting Lamin B1, Atg5 or Beclin-1 using Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The siRNA target sequences were: Lamin B1 (human), 5′-CGCGCTTGGTAGAGGTGGA-3′; Atg5 (human), 5′-GGACGAATTCCAACTTGTT-3′; Beclin-1 (human), 5′-CAGTTTGGCACAATCAATA-3′; negative control (NC), 5′-TTCTCCGAACGTGTCACGT-3′.

Cells were collected with 2.5% glutaraldehyde, and then centrifuged (1,000 revolutions, 10 min) and washed twice with PBS. The samples were post-fixed with 1% osmium tetroxide at 4°C for 2 h in the dark and then washed three times with 0.1MPB. After dehydration, the permeation, paraffin embedding and section staining were performed as previously described (22). The ultrastructure of cells was observed under a JEM-1230 transmission electron microscope (JEOL, Ltd., Tokyo, Japan).

PC3 cells treated with 8 mg/ml Huaier exact for indicated times were washed twice with PBS and stained with 10 µg/ml acridine orange for 20 min at 37°C in the dark. The cells were observed under an inverted fluorescence microscope.

Cells seeded on confocal dishes were washed with PBS and fixed in 4% paraformaldehyde for 10 min, and then permeabilized with PBS containing 0.5% Triton X-100 for 20 min. Next, the cells were washed three times with PBS and blocked with 1% BSA for 1 h at 37°C, and then incubated with the primary antibody (1:100) overnight at 4°C. Subsequently, the cells were washed with PBS and then incubated with anti-rabbit FITC-conjugated secondary antibody (1:100) for 1 h at 37°C. Nucleus was counterstained with DAPI in the dark after being washed with PBS. The cells were observed and photographed with a laser scanning confocal microscope (Olympus FV1000; Olympus Corp., Tokyo, Japan).

The data are presented as mean ± SD of triplicate samples. The statistical analysis in this study was evaluated by the Student's t-test and ANOVA using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant result.

CCK-8 assay indicated that Huaier aqueous extract significantly inhibited proliferation of the PC3 cells in a time- and dose-dependent manner (Fig. 1A). The IC50 value of PC3 cells exposed to Huaier for 48 h was 8.18 mg/ml. The wound healing assay is widely used to evaluate the migratory ability of cells in vitro. As shown in Fig. 1B, the migration capacity of PC3 cells was decreased after treatment with Huaier, and the migration inhibition rate of PC3 cells treated with Huaier was 44.68±16.39%. Moreover, the invasion capacity of cells in vitro is frequently examined by using Transwell assay. As shown in Fig. 1C, after treatment of PC3 cells with 4 mg/ml Huaier aqueous extract for 36 h, the number of cells that had successfully passed through the Matrigel-coated membrane was markedly reduced compared with that of the control group cells. Taken together, Huaier markedly suppressed the proliferative and metastatic capability of the human prostate cancer cells.

Lamin B1 is a member of the lamin family making up the nuclear matrix and is abnormally overexpressed in multiple types of cancers including prostate, hepatocellular carcinoma and pancreatic cancer (19,23–25). Moreover, Lamin B1 positively regulates the proliferation and invasion of pancreatic cancer cells (25). Our previous study revealed that as a pro-oncogenic gene, Lamin B1 was dramatically decreased in human hepatoma SKHEP-1 cells exposed to Huaier (10). Intriguingly, qRT-PCR and western blot analysis demonstrated that Huaier aqueous extract significantly inhibited the mRNA and protein levels of Lamin B1 in PC3 cells in a dose-dependent manner (Fig. 2A and B).

Next, we investigated whether downregulation of Lamin B1 contributes to Huaier-mediated inhibition of proliferative and metastatic capacity of PC3 cells by using RNA interference. Transfection with siRNAs targeting Lamin B1 markedly reduced Lamin B1 expression (Fig. 3A). Moreover, depletion of Lamin B1 significantly blunted the proliferation of PC3 cells (Fig. 3B). In addition, reduction of Lamin B1 substantially suppressed the migration and invasion of PC3 cells (Fig. 3C and D). Thus, Lamin B1 positively regulates the proliferation and metastatic potential of human prostate cancer PC3 cells. To further investigate the role of Lamin B1 in the inhibition of proliferative and metastatic potential of PC3 cells in the presence of Huaier, we treated PC3 cells transfected with Lamin B1 siRNAs or negative control siRNAs with Huaier aqueous extract. As depicted in Fig. 4A, depletion of Lamin B1 attenuated the inhibitory effect of Huaier on the proliferation of PC3 cells. Additionally, the suppression of migration and invasion of PC3 cells caused by Huaier treatment were significantly impaired by knockdown of Lamin B1 (Fig. 4B and C). Collectively, decreased Lamin B1 is partially responsible for the inhibition of proliferation and the metastatic potential of PC3 cells in the presence of Huaier.

Autophagy is one of the underlying mechanisms for drug-induced cell death (26,27). In the present study we determined the effect of Huaier on autophagy in PC3 cells. Observation of autophagosomes and other related subcellular structures in the cytoplasm with transmission electron microscope is widely used for autophagy assessment (22,28,29). Ultrastructural changes of PC3 cells treated with Huaier were observed by transmission electron microscope. As shown in Fig. 5A, PC3 cells treated with Huaier exhibited typical characteristics of autophagy: autophagosomes and autolysosomes which contained cytoplasmic components. Moreover, acridine orange staining was used to observe the accumulation of autophagy vesicles. The number of acidic vesicles marked by orange fluorescence increased in response to Huaier treatment in a time-dependent manner (Fig. 5B). A series of autophagy-related proteins participate in different stages of autophagosome formation, such as Atg3, Atg5 and Beclin-1 (30–33). LC3 is an autophagosomal marker protein (34). LC3-II, one form of LC3, accumulates on the membranes of autophagosomes and is widely used as a marker for autophagy evaluation (34,35). Western blot analysis revealed that Huaier treatment led to increase in the expression of Atg3, Atg5, Beclin-1 and LC3-II in a dose- and time-dependent manner in PC3 cells (Fig. 5C and D). Moreover, Huaier treatment markedly increased LC3 puncta in number and intensity in PC3 cells under a fluorescence microscope (Fig. 5E), indicating that the number of autophagosomes was increased in the presence of Huaier. Taken together, Huaier evidently promoted autophagy in PC3 cells.

To investigate whether autophagy was involved in the inhibition of proliferation of PC3 cells by Huaier, we suppressed autophagy with two autophagy inhibitors and RNA interference. As depicted in Fig. 6A and B, inhibition of autophagy with 3-MA or bafilomycin A1 significantly impaired the sensitivity of PC3 cells to Huaier treatment. Moreover, Atg5 and Beclin-1, two essential components involved in autophagosome formation (36), were efficiently knocked down in the PC3 cells transfected with siRNAs (Fig. 6C and D). Consistent with the results of autophagy inhibitors, suppression of autophagy with siRNAs against Atg5 or Beclin-1 remarkably blunted the inhibitory effect of Huaier on the proliferation of PC3 cells (Fig. 6C and D). Collectively, the inhibitory proliferation of PC3 cells exposed to Huaier is partially mediated by activation of autophagy.