MCF-7 and A549 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin antibiotics. HPL was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The antibody for β-actin was supplied by Santa Cruz Biotechnology, Inc., Snail antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Twist antibody was obtained from Abcam (Cambridge, MA, USA).

All proliferation assays were based on the MTT method. Cells were seeded in a 96-well plate (1×10⁴ cells/well). Following overnight culture, HPL was added to the cells and further cultured for 24 h at 37°C. Cells cultured without HPL were used as a control. The media was removed and dimethyl sulfoxide was added to the MTT (Sigma-Aldrich; Merck KGaG, Darmstadt, Germany) solubilization solution. Absorbance was measured at 550 nm. For the colony formation assay, single-cell suspensions of 5×10³ cells were seeded onto a 6-well plate and allowed to attach for 24 h at 37°C in culture medium. Cells were then treated with 20 or 200 µM HPL at 37°C. Medium containing HPL was refreshed every two days. Following 10 days, colonies were fixed with 100% methanol for 10 min and stained with 0.1% crystal violet at room temperature. Plates were washed with PBS and imaged.

Migration was assessed by a wound-healing assay. MCF-7 and A549 cells were seeded at 2×10⁴ cells/well and were cultured for 24 h. Following scraping the cell monolayer with a sterile micropipette tip, the wells were washed with PBS, and treated with TGF-β (10 ng/ml, R&D Systems, Inc., Minneapolis, MN, USA) or co-treated with TGF-β (10 ng/ml) and HPL (20 µM) for 24 h at 37°C. The first image of each scratch was acquired at time zero. At 24 h, each scratch was examined and captured at the same location and the healed area was determined. All captured images were obtained by using a light microscope (Eclipse, Ti-S, Nikon Instruments Inc., NY, USA).

The invasion of tumor cells was assessed in Transwell chambers (Corning Incorporated, Corning, NY, USA) with 8 µm pore size, 6.5 mm diameter polyvinylpyrrolidone-free polycarbonated membranes that were coated with 1 mg/ml fibronectin (R&D Systems, Inc.). The cells were seeded onto the upper wells at a concentration 1×10⁵ MCF-7 and A549 cells/well and were cultured for 24 h at 37°C following treatment with TGF-β (10 ng/ml) or co-treatment with TGF-β (10 ng/ml) and HPL (20 µM). The bottom chambers of the Transwell were filled with conditioned medium, DMEM. Following incubation for 24 h, cells were fixed with 100% methanol for 10 min, stained with 0.1% crystal violet for 5 min at room temperature and counted under a light microscope.

MCF-7 and A549 cells were treated with TGF-β (10 ng/ml) or co-treated with TGF-β (10 ng/ml) and HPL (20 µM) for 24 h at 37°C. Following lysis in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), proteins quantified with BCA protein assay kit (Thermo Fisher Scientific, Inc.) were resolved by 10% SDS-PAGE gel and immunoblotted with Immobilon-P transfer membrane (EMD Millipore, Billerica, MA, USA) using primary antibodies including anti-E-cadherin (1:1,000; catalog no. ab184633; Abcam, Cambridge, UK), anti-Snail (1:1,000; Cell Signaling Technology, Inc., catalog no. 3895), anti-Twist (1:1,000; catalog no. ab175430; Abcam) and anti-β-actin (1:1,000; catalog no. sc47778; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Following treatment with secondary antibodies, goat anti-mouse IgG (1:2,000; catalog no. sc2005; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, the immunoreactive bands were visualized using the standard enhanced chemiluminescence method (SuperSignal Est Pico; Thermo Scientific, Inc.).

MCF-7 and A549 cells were grown in 4-chamber slides in serum-free media, and were treated with TGF-β (10 ng/ml) or co-treated with TGF-β (10 ng/ml) and HPL (20 µM) at 37°C. Following 24 h, the cells were fixed with 4% paraformaldehyde for 15 min at 4°C. Cells were washed with PBS containing 0.1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) and incubated with anti-E-cadherin antibody (1:100; catalog no. ab184633; Santa Cruz Biotechnology, Inc.) for 1 h followed by 1 h incubation with FITC-tagged goat anti-mouse IgG (1:200; catalog no. sc2010; Santa Cruz Biotechnology, Inc.), then counter-stained with DAPI for 5 min. All staining were procedures performed at room temperature. Cell images were captured at ×400 magnification on a Leica fluorescence microscope.

The results are presented as the mean ± standard error, and statistical comparisons between groups were performed using the Student's t-test using SigmaPlot (version 10.0; Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study initially examined the effect of HPL on the proliferation of the MCF-7 and A549 human cancer cell lines. To determine the drug concentration that induced 50% growth inhibition (IC₅₀), cells were treated with various concentrations of HPL (1, 2, 5, 10, 20, 50, 100, 200 and 500 mM) for 24 h and cell viability was evaluated by MTT assay. As presented in Fig. 1A, IC₅₀ values for both cell types were similar (~65 µM). The long-term effects of HPL were determined by culturing MCF-7 and A549 cells with or without HPL for 10 days and then performing colony formation assays. At a concentration of 20 µM, HPL demonstrated a slight inhibitory effect, whereas 200 µM HPL almost completely inhibited colony formation (Fig. 1B). Therefore, 20 µM HPL was considered to be a suitable dose for subsequent experiments.

TGF-β (10 ng/ml) may function as a pro-oncogenic factor through the induction of the EMT process, as previously reported (21). The present study investigated the effects of HPL on cell migration to demonstrate that HPL inhibited TGF-β-induced EMT as EMT is associated with enhanced tumor progression. Cancer cell lines were treated with TGF-β alone, TGF-β plus HPL (20 µM) or HPL alone (20 µM), and wound-healing assays were performed. The TGF-β-treated cancer cells exhibited a ≥1.2-fold increase in migration, whereas treatment with 20 µM HPL inhibited this TGF-β-induced migration by 45% for MCF-7 and 50% for A549 cells (Fig. 2A and B). The inhibition of migration was also observed in the HPL alone treatment group, HPL decreased the migration by 60% for MCF-7 and 65% for A549 cells compared with the untreated control group. These results revealed that HPL inhibited the migration of cancer cells during EMT induced by TGF-β.

The present study next investigated whether HPL inhibited the TGF-β-induced invasiveness of cancer cells. Following treatment with TGF-β alone, the number of invasive cells significantly increased compared with the untreated cells. However, the number of invasive cells was significantly reduced in the cells treated with the combination of TGF-β plus HPL (Fig. 3). The quantitative analysis is presented in Fig. 3. HPL significantly inhibited TGF-β-induced invasion of cancer cells by 50% for MCF-7 and 40% for A549 cells, compared with the untreated control group. These results suggested that HPL inhibits the effect of TGF-β, increasing the invasiveness of human cancer cells, as occurs during the EMT.

To further investigate the effect of HPL on TGF-β-induced EMT, the present study evaluated the expression levels of the EMT-associated protein, E-cadherin, by western blotting (Fig. 4A). The expression of E-cadherin was downregulated in the TGF-β-treated group compared with the controls. However, HPL reversed the TGF-β-induced EMT by reducing E-cadherin expression levels. The present study also determined the E-cadherin expression level in cancer cells by immunofluorescence (Fig. 4B). Consistent with the western blotting results, in the two cell types, E-cadherin was seldom expressed following TGF-β treatment, but was significantly recovered by co-treatment with HPL. Taken together, the western blotting and fluorescence imaging results suggested that HPL has an inhibitory effect on EMT.

Numerous previous studies have reported that drugs may inhibit the invasion and migration of cancer cells by suppressing TGF-β activation and Snail/Twist induction, which results in recovering E-cadherin expression (22,23). It was suggested that the TGF-β signaling pathway is critically involved in the acquisition of EMT by its downstream target, the transcription factor Snail and Twist (24). The present study investigated the expression levels of the Snail and Twist proteins by western blotting to determine whether the effect of HPL described above is associated with the inhibition of the TGF-β-Snail/Twist axis. As presented in Fig. 4A, TGF-β significantly upregulated the expression levels of Snail and Twist proteins, which reduced the expression level of E-cadherin. These effects were reduced by HPL, suggesting that HPL suppressed TGF-β-induced EMT by co-regulating Snail and Twist.