Liver cancer is one of the most common cancers worldwide. It is one of the leading causes of cancer death in Taiwanese men and women and that number is increasing. Liver cancer metastasis is a major cause of mortality and concerted efforts are focused on inhibiting tumor metastasis.
Metastasis occurs as a complex multistep process involving cancer cell adhesion, invasion, and migration (Price and Thompson, 2002) requiring proteolytic enzymes to degrade extracellular matrix (ECM) components (Simpson-Haidaris and Rybarczyk, 2001). Matrix metalloproteinases (MMPs) such as MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are the principal ECM-degrading enzymes (Klein et al., 2004). Both MMP enzymes are strongly associated with tumor-cell invasion (Ura et al., 1989). Inhibition of MMP-2 and -9 activities could be targets for preventing cancer cell invasion.
Bufalin, a class of toxic steroids is one of the active ingredients of Chinese traditional medicine called “Chan su.” It was purified from the skin glands of Bufogargarizans or Bufo melanostictus, and it has been officially approved as a treatment for cancer in China (Meng et al., 2009). Numerous studies have shown that bufalin has antitumor effects (Chen et al., 2009a; Qi et al., 2011). Bufalin-induced apoptosis in human cancer cells such as leukemia (Watabe et al., 1998), prostate cancer (Yeh et al., 2003), gastric cancer (Li et al., 2009), osteosarcoma (Yin et al., 2007), endometrial and ovarian cancer (Takai et al., 2008), and colon cancer (Xie et al., 2011). Moreover, it was demonstrated that bufalin inhibited the proliferation of transplantable human hepatocellular carcinoma in nude mice (Han et al., 2007). Bufalin-inhibited migration and invasion in human osteosarcoma U-2 OS cells (Chueh et al., 2011). The purpose of the present study was to determine if bufalin would have antimigration and antiinvasion in a human cancer cell line.
MATERIALS AND METHODS
Materials and Reagents
Bufalin and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). Dulbecco's modified (DMEM) with l-glutamine, fetal bovine serum, penicillin-streptomycin, and trypsin-EDTA were obtained from Invitrogen (Carlsbad, CA). Primary antibodies antiMMP-1 (Cat. MAB13439), antiMMP-9 (Cat. AB19016), antifocal adhesion kinase (FAK) (Cat. 05-537), antiphosphoserine/threonine kinase AKT (Ser473) (Cat. 05-669), and antip-c-jun (Cat.06-659) were purchased from Merck Millipore Corp. (Billerica, MA). Antip-ERK1/2, antip-JNK1/2, antip-p38, antiiNOS, antiCOX-2, antiNF-κB p65, antiVEGF, antiRho A, antiROCK-1, antiRas, antiSOS1, antiGRB2, antiPI3K, antiTIMP1, antiTIMP2, antiAKT, antiphosphoserine/threonine kinase AKT (308), antiMEKK3, antiMKK7, antip-PERK, antiPKC, antiMMP-2, antiMMP-7, and antiuPA and second antibody were obtained from Santa Cruz Biotechnology, (Santa Cruz, CA).
Cell lines and Culture Conditions
The human hepatocellular carcinoma cell line SK-Hep1 was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). SK-Hep1 cells were cultured in DMEM, supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (100 Units/mL penicillin and 100 μg/mL streptomycin) and grown at 37°C under humidified 95% air and 5% CO2 (Lee et al., 2008; Ji et al., 2009).
Determination of Percentage of Viable SK-Hep1 Cells
SK-Hep1 cells (5 × 105 cells/well) were placed in a 12-well plate for 24 h and then incubated with 0, 5, 10, 25, 50, 75, and 100 nM bufalin for 24 h. Cells were harvested and stained with PI (5 μg/mL) then analyzed by flow cytometry (Becton-Dickinson, San Jose, CA) as previously described (Lu et al., 2010; Chiang et al., 2011), and the percentages of viable cells were calculated.
In Vitro Wound-Healing Migration Assay
SK-Hep1 cells at a density of 5 × 105 cells/well in six-well plates were allowed to form a confluent monolayer then cells were wounded with a 200-μl pipette tip. Cells were treated with bufalin (0, 5, and 10 nM) and then were incubated in new DMEM medium with 1% FCS for 24 h. Cell from each treatment were examined and photographed using a contrast phase microscope. Cell-free area of each treatment was marked and measured as described previously (Chen et al., 2009b). Cell migration was calculated as the percentage of the remaining cell-free area compared with the area of the initial wound (Chen et al., 2009b).
In Vitro Migration and Invasion Assays
Migration and invasion were determined using a 24-well Transwell insert cell culture chambers for migration a 24-well Matrigel-coated transwell cell culture chamber for invasion. For both assays 8-μm pore filters (Millipore, MA) were coated with 30 μg type I collagen (Millipore, MA) for 1 h then cells at a density of 104 cells/0.4 mL in DMEM medium were placed in the upper chamber and treated with 0.5% DMSO (as a control) or with bufalin (0, 5, and 10 nM) and allowed to migrate for 24/48 h. Nonmigrated cells in the upper chamber were removed by using a cotton swab and filters from all treatments were stained with 2% crystal violet. The migrated cells adherent to the underside of the filter were examined, photographed and counted using a light microscope at 200×. Each treatment including control was assayed twice and three independent experiments were performed as previously described (Lee et al., 2008; Ji et al., 2009). For cell invasion, the lower surface of the filter which penetrated through the matrigel was counted and photographed using a light microscope at 200× as previously described (Lee et al., 2008; Ji et al., 2009).
MMP-2 and -9 Activity
Gelatin zymography was used to determine activity of MMP-2 and -9. SK-Hep1 cells at a density of 2 × 106 cells/well were cultured in serum-free DMEM medium in a 12-well plate then were treated with bufalin (0, 1, 5, 10, and 25 nM) for 48 h. At the end of incubation, the conditioned medium was collected and centrifuged to remove cell debris. Gelatin zymography was performed as previously described (Ji et al., 2009). An equal amount of conditioned media from each treatment and control were separated by electrophoresis on 10% SDS–PAGE containing 0.1% gelatin. After electrophoresis, the gels were soaked in 2.5% Triton X-100 in dH2O twice for a total of 60 min at 25°C, then were incubated in substrate buffer (50 mM Tris–HCl, 5 mM CaCl2, 0.02% NaN3, and 1% Triton X-100, pH 8.0) at 37°C for 18 h (Lee et al., 2008; Ji et al., 2009). Gels were washed with PBS and then stained with 0.1% coomassie brilliant blue. After staining, the areas of lysis were observed as white bands against a blue background (Lee et al., 2008; Ji et al., 2009).
Western Blotting Analysis for Protein Expressions
SK-Hep1 cells at a density of 1 × 106 cells/well were maintained in six-well plates and then incubated with 10 μM bufalin for 6, 12, 24, and 48 h. Cells were harvested and resuspended in ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 2 mM EDTA and 0.1% Triton X-100 for sonication (Ji et al., 2009). The lysed homogenates were centrifuged at 13 000×g for 10 min at 4°C to remove cell debris. Total protein concentrations of the supernatants were determined as previously described (Lu et al., 2012). The levels of the following proteins were determined by Western analysis: p-ERK1/2, p-JNK1/2, p-p38, p-c-JUN, iNOS, COX2, NF-κB p65, VEGF, FAK, Rho A, ROCK-1, Ras, SOS1, GRB2, PI3K, TIMP1, TIMP2, AKT, p-AKT(308), p-AKT (473), MEKK3, MKK7, p-PERK, PKC, MMP-2, -9, -1, -7, and uPA were measured by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting as previously described (Ji et al., 2009; Lu et al., 2012).
Confocal Laser Scanning Microscopy
SK-Hep1 cells at a density of 5 × 104 cells/well were placed in four-well chamber slides and treated with bufalin (0 and 10 nM) for 24 h. Cells were then fixed in 3% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 1 h with the blocking of nonspecific binding sites using 2% BSA as previously described (Yang et al., 2012). Cells were stained with antiNF-κB (1:200 dilution as a primary antibody) overnight and then were stained with FITC-conjugated goat antimouse IgG at 1:100 dilution (as the secondary antibody) (green fluorescence), followed by nuclei counterstaining with PI (red fluorescence). Cells were then photomicrographed using a Leica TCS SP2 confocal spectral microscope (Yang et al., 2012).
Student's t-test for comparison of two groups was used and considered significant at the p < 0.05 between bufalin- and control-treated groups. Appropriate comparisons were made using the Student–Newman–Keuls test for multiple comparisons. p < 0.05 was considered statistically significant. Data were expressed as means ± SEM of at least three independent experiments.
Cytotoxicity of Bufalin in SK-Hep1 Cells
An effect of bufalin (0–100 nM) on cell viability of SK-Hep1 was determined using PI staining and flow cytometry. After incubation for 24 h, cell viability was not significantly affected by bufalin (5–10 nM) as compared with untreated control cells (Fig. 1). These results indicate that bufalin is not toxic to SK-Hep1 cells at 5 and 10 nM. However at concentrations reach to 25 nM and higher cell viability was significantly decreased. Therefore, in order to avoid inhibition of cell viability in the following experiments, the concentrations of bufalin between 5 and 10 nM were selected and an incubation time was used for 24 h.
Effects of Bufalin on Migration and Invasion of SK-Hep1 Cells
The wound healing assay was used to investigate the migration of SK-Hep1 cells after bufalin treatment. Results as shown in (Fig. 2), bufalin inhibited the migration of SK-Hep1 cells for 12 and 24 h, in a concentration-dependent manner. We also found similar effects of bufalin on migration and invasion using a transwell assay. Data in [Fig. 3(A)] and [Fig. 3(B)] show that bufalin (5 and 10 nM) significantly decreased both the migration and invasion of SK-Hep-1 cells. Those effects were dose and time dependently.
Bufalin Suppressed Secretion of MMP-2 and MMP-9 in SK-Hep1 Cells
An effect of bufalin on secretion of MMP-2 and MMP-9 from SK-Hep1 cells was determined using a gelatin zymographic assay. It can be seen in (Fig. 4) that bufalin significantly reduced the secretion of MMP-2 and MMP-9 from SK-Hep1 cells. This effect of bufalin was observed at both 24 and 48 h incubation.
Bufalin Alters Levels of Proteins Associated with Migration and Invasion
Effects of bufalin (10 nM) on phosphorylation of MAPKs, ERK1/2, and PI3K/Akt signaling in SK-Hep1 cells for various time periods are shown in (Fig. 5). Bufalin significantly decreased p-c-Jun proteins [Fig. 5(A)], but it did not significantly alter levels of p-ERK1/2, p-JNK, and p-p38. Protein levels of iNOS, COX2, NF-κB and VEGF [Fig. 5(B)] were reduced in a time-dependent manner.
PI3Ks are a group of ubiquitously expressed lipid kinases which are important players in cell signaling. Incubation of SK-Hep1 cells with bufalin (10 nM) caused a time-dependent decrease of AKT levels [Fig. 5(D)] but increased levels of PI3K, p-AKT (308) and p-AKT (473).
We also found that bufalin significantly suppressed FAK, Rho A, ROCK1, SOS1, Ras, and GRB2 levels [Fig. 5(C)] in a time-dependent manner in SK-Hep1 cells. It also was observed that Bufalin decreased protein levels of MEKK3, MKK7 and p-PERK but increased levels of PKC [Fig. 5(E)]. Levels of MMP-2, -9, -1, and uPA were reduced by Bufalin treatment [Fig. 5(F)].
Bufalin Alters Distribution of NF-κB in SK-Hep1 Cells
In order to confirm bufalin inhibited the NF-κB expression levels, cells after treated with bufalin at 10 nM for 24 h then were examined and photographed by using a confocal laser microscope and result are shown in (Fig. 6). The figure indicated that bufalin suppressed the protein levels of NF-κB in SK-Hep1 cells.
In this study, we investigated the antimigration and antiinvasion effects and mechanistic actions of bufalin in human hepatoma SK-Hep1 cells. We found that bufalin significantly inhibited the migration and invasion of SK-Hep1 cells. We also found that bufalin significantly inhibited secretion of MMP-2 and MMP-9. Bufalin reduced levels of certain proteins associated with migration and invasion. These results demonstrated that the antimigration and antiinvasion effects of bufalin were associated with inhibition of enzymatically degradative processes of tumor metastasis.
There are data showing that the SK-Hep1 human hepatocellular carcinoma cell line is invasive and expresses gelatinase activity required for invasion (Hwang and Lee, 2006). We showed that bufalin inhibited the expression of urokinase plasminogen activator (uPA) (55 kDa serine protease) in SK-Hep1 cells (Fig. 5C). It was reported that plasminogen activator inhibitors (PAI-1 and PAI-2) inhibit both receptor-bound and free uPA. One possibility is that uPA may activate a series of protein degradation reactions which regulate or activate MMPs (Liao et al., 2012).
Bufalin inhibited the levels of FAK and Rho A in SK-Hep1 cells. FAK (cytoplasmic kinase) may play an essential role in metastasis through the modulation of tumor cell migration and invasion (Hwang and Lee, 2006; Hwangbo et al., 2010). Bufalin inhibited phosphorylation of ERK1/2. It was reported that FAK and ERK1/2 are important for fibronectin stimulated invasiveness and MMP-9 secretion in ovarian carcinoma cells (Kuo et al., 2009).
Rho A is involved in cell migration (Pan et al., 2007) and active-Rho A may stimulate expression of MMP-9 and promote migration of endothelial cells (Abecassis et al., 2003). We showed that bufalin decreased Rho A protein levels. Bufalin may inhibit migration and invasion of SK-Hep1 cells through the PI3K/AKT-Rho A and Rho A-MMP-9 pathways.
NF-κB activation has been reported to be associated with tumor cell proliferation, survival, angiogenesis, and invasion (Brown et al., 2008). In human breast cancer cells, NF-κB plays a regulatory role in PMA-induced MMP-9 expression (Park et al., 2009). Activation of NF-κB and AP-1 downstream of MAPK or PI3K-Akt pathways is involved in inflammation, cancer-cell adhesion, invasion, metastasis, and angiogenesis of cancer cells (Aggarwal, 2004; Takada et al., 2004). Thus, the suppression of NF-κB activation has been reported to be effective in the prevention and treatment of cancer (Aggarwal, 2004). It was reported that NF-κB is one of the prime components of the intracellular signaling pathways responsible for MMP-2 and MMP-9 (Lee and Lee, 2002). In the present study, bufalin treatment-induced inhibition of NF-κB in SK-Hep1 cells (Fig. 5).
In conclusion, we have demonstrated that bufalin exerts inhibitory effects on the migration and invasion of the human hepatocellular cancer cell line SK-Hep1. Bufalin may be a candidate for developing preventive agents against hepatocellular cancer metastasis. Moreover, we found that effects of bufalin on migration/invasion may occur through inactivation of ERK1/2 and NF-κB signaling pathways via the reducing FAK, Rho A, ROCK-1, uPA, and NF-κB protein levels and increasing PI3K, and phospho-Akt levels. We propose that a consequence of those actions causes a reduction in MMP-2, MMP-9 secretion leading to inhibition of metastasis in cancer cells (Fig. 7). Further studies are needed in order to demonstrate the potential of bufalin as an anticancer agent.