Introduction
Malignant neoplasm, generally known as cancer, is currently considered one of the most deadly types of diseases. The leading cause of death in patients with cancer is tumour metastasis. Hepatocellular carcinoma (HCC) is amongst the most common types of malignant tumours in the tropics and East Asian countries, including Taiwan. Surgical resection remains the treatment of choice, but because of the high rate of metastasis and poor prognoses, the number of deaths is almost the same as the number of new cases occurring each year worldwide [
1]. Metastasis of HCC to distinct sites or organs, such as the lymph nodes, lungs, spleen, peritoneum, and bones, is a major cause of death [
2].
Tumour metastasis is a complex process during which cancer cells undergo a series of alterations at intracellular and extracellular levels, including changes that (1) damage intercellular interaction, (2) increase cancer cells and extracellular matrix (ECM) interaction, (3) damage ECM components, and (4) increase the invasion and migration of cancer cells. In addition, metastasis involves the over-expression of proteolytic enzymes, such as matrix metalloproteinases (MMPs) [
3]. MMPs are enzymes involved in ECM degradation in physiological processes, such as tissue remodelling and embryonic development. Studies have shown that MMP-2 and MMP-9 are particularly crucial in tumour metastasis, the process of which requires the decomposition of the ECM [
4,
5]. Furthermore, TPA is a diester of phorbol and a potent tumour promoter often employed in biomedical research to activate the signal transduction enzyme PKC [
6]. PKC isoforms comprise a family of serine/threonine kinases that can be activated as major signalling transduction enzymes to respond to extracellular signals, such as those by diacylglycerol and calcium ions, that are generated by ligand-receptor interactions with the nucleus at the cell surface [
7]. In addition, PKC isoforms are involved in signalling transduction pathways for numerous biological processes, such as proliferation, survival, angiogenesis, metastasis, and tumourigenesis [
8-
10]. Previous studies has demonstrated that TPA controls the expressions of MMPs by modulating the activation of transcription factors, such as NF-kB and AP-1, through phosphatidylinositide-3 kinase/Akt (PI3K/Akt), c-Jun N-terminal kinase (JNK), p38, and ERK signalling pathways [
11]. PI3K activation leads to the phosphorylation of phosphatidyl-inositides, which activates the primary downstream target Akt that apparently plays various crucial roles in regulating cellular growth, differentiation, adhesion, inflammatory reactions, and invasion [
12,
13]. Three major mammalian mitogen-activated protein kinases (MAPKs) are ERK, JNK, and p38. Various MAPKs are activated in response to different extracellular stimuli and have distinct downstream targets, thus serving unique roles in cellular responses. The activation of MAPKs has been shown in other systems to be the mechanism crucial for promoting the production of MMPs, which are vital for cell proliferation, invasion, and neovascularization [
14-
16]. In addition, the gene expressions of MMPs are primarily regulated by transcriptional factors, such as NF-κB and AP-1 (a heterodimer consisting of protein molecules from the Fos and Jun protein families).
Galangin is a naturally occurring flavonoid found in
Alpinia officinarum (lesser galangal). Flavonoids are well known antioxidants, which can protect cells from being damaged by free radicals [
17-
19], and are believed to exert inhibitory effects on cancer cells [
20,
21]. Previous studies have demonstrated that galangin exhibits anti-proliferative and apoptotic effects on the growth of cancer cells that originate from human leukemia cells [
22,
23]. Although galangin may inhibit the growth of various cancers by inducing antiproliferation and apoptosis in cancer cells, the precise effect and related molecular mechanism of galangin involved in the TPA-induced metastatuc feature of HepG2 liver cancer cells remains unclear.
Material and methods
Reagents and antibodies
Galangin (purity ≥ 99%) was purchased from Extrasynthese (Genay, France); dimethylsulfoxide (DMSO), Tris–HCl, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), gelatin, leupeptin, Nonidet P-40, deoxycholic acid and sodium orthovanadate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); A protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA, USA). Dulbecco’s phosphate buffer solution (PBS), fetal bovine serum (FBS), trypsin-EDTA, and powdered Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco-BRL (Gaithersburg, MD, USA). Matrigel was obtained from BD Transduction Laboratories (San Diego, CA, USA). Antibodies against Akt, ERK1/2, JNK/SAPK, and p38 MAPK, proteins, and phosphorylated proteins were purchased from Cell Signalling Technology (Beverly, MA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science (Amersham, UK).
Cell culture and galangin treatment
Human nonmalignant Chang liver cells, human hepatocellular carcinoma HepG2 cells, and human hepatocellular carcinoma Hep3B cells were maintained in DMEM medium. Human gastric adenocarcinoma AGS cells was maintained in RPMI-1640 medium. Above-mentioned cell lines were obtained from BCRC (Bioresource Collection and Research Center in Hsin-Chu, Taiwan). All cells were cultured at 37°C in a humidified atmosphere of 5% CO2-95% air. In a medium supplemented with 10% FBS and antibiotics (100 U/ml of penicillin and 100 mg/ml of streptomycin). Adherent cells were detached through incubation with trypsin. For galangin treatment, the stock solution of galangin was dissolved in DMSO and sterilised through filtration by using 0.2-μm disc filters. Appropriate amounts of the stock solution (10 mg/ml in DMSO) of galangin were added to the cultured medium to achieve the indicated concentrations.
Cell viability (MTT assay)
To measure the effect of galangin on cell viability, the Chang liver, AGS, Hep3B, and HepG2 cells were seeded in 24-well plates (1 × 105 cells/well) for 16–18 h. The cells were treated with or without various concentrations of galangin (0, 1, 2.5, 5, 10, 15, 20, 25, and 30 μM) for 24 h and 48 h. Treatment at each concentration was repeated three times. To further investigate whether galangin and/or TPA influence cell viability, HepG2 cells were treated with the presence or absence of drugs (70 nM TPA and 5 μM galangin) for 24 h. After the exposure period, the medium was removed and the cells were washed with PBS. The medium was changed, incubated with an MTT solution (5 mg/ml/well) for 4 h, and then removed. Formazan was solubilised in isopropanol and measured spectrophotometrically at 563 nm. The percentage of viable cells was estimated by comparing the number of viable treated cells with the number of viable untreated control cells.
Cell-matrix adhesion assay
HepG2 cells were pretreated with 70 nM TPA and incubated in various concentrations of galangin (0, 1, 2.5, and 5 μM) for 24 h. The cells were seeded at a density of 1 × 105 cells/ml in a 24-well plate, coated with 500 μl of type IV collagen (10 μg/ml), and cultured for 30 min. Nonadherent cells were removed by using PBS washes, and adherent cells were fixed in ethanol. After being stained with 0.1% crystal violet, the fixed cells were lysed in 0.2% Triton X-100, and measured spectrophotometrically at 550 nm.
Immunofluorescence assay
To determine the effect of galangin on cell morphology and actin stress fibers, HepG2 cells (4 × 105 cells/well) were plated in 6-well plates, grown for 16–18 h, stimulated with 70 nM TPA for 12 h, and incubated in various concentrations of galangin (0, 1, 2.5, and 5 μM) for 24 h. After the exposure period, the medium was removed, and the cells were washed with Ca2+/Mg2+ free PBS. The cells were then fixed with 4% paraformaldehyde in Ca2+/Mg2+
−free PBS for 15 min and incubated with 0.5% Triton X-100 in Ca2+/Mg2+ free PBS for 5 min. The cells were incubated with 1% BSA and 0.5% Triton X-100 in Ca2+/Mg2+
−free PBS for 1 h (blocking) and then incubated with 200 U/ml Alexa flour 488-phallodin for 1 h to stain the actin filaments. Fluorescent images were captured using a BX51 fluorescence microscope (Olympus, Tokyo, Japan).
Transwell-chamber invasion and migration assay
The invasion assay was performed by using Hanging Cell Culture-inserts (BD Biosciences; pore size, 8 μm) in a 6-well plate. The ability of HepG2 cells to pass through filters coated with Matrigel was measured using a transwell-chamber invasion assay. Matrigel was diluted to 200 μg/ml with distilled cold-filtered water and applied to the upper surface of the filter inserts. Briefly, HepG2 cells were stimulated with 70 nM TPA for 12 h and incubated in various concentrations of galangin (0, 1, 2.5, and 5 μM) for 24 h. After 24 h, the cells were detached using trypsin and resuspended in a serum-free medium. A medium containing 10% FBS medium was applied to the lower chamber as a chemoattractant, and cells were seeded on the upper filter at a density of 1 × 105 cells/ml in the serum-free medium. The 6-well plate was incubated for 24 h at 37°C in 5% CO2, the filter inserts were removed from the wells, and the cells on the upper surface of the filter were wiped with a cotton-tipped swab. Filters were fixed with methanol for 10 min and stained with Giemsa dye for 1 h. The cells that invaded the lower surface of the filter were counted under a light microscope. The data were expressed as the average number of cells attached to the bottom surface from randomly chosen fields. Each experiment was conducted in triplicate.
To measure the migrative ability of HepG2 cells, cells were seeded on the upper surface of the filter inserts with 8-μm pore polycarbonate filters that were not coated with Matrigel. The migrative cells were treated and measured as described in the invasion assay.
Wound healing assay
To determine cell motility, HepG2 cells (1 × 105 cells/ml) were seeded in a 6-well tissue culture plate by using DMEM medium containing 10% FBS and grown to 80-90% confluence. After the medium was aspirated, the center of the cell monolayer was scraped using a sterile micropipette tip to create a denuded zone (gap) featuring an even width. Subsequently, cellular debris was washed with PBS, and the HepG2 cells were pretreated with 70 nM TPA for 12 h and incubated in various concentrations of galangin (0, 1, 2.5, and 5 μM) for 24 h. Wound closure was monitored and photographed using an Olympus CKX-41 inverted microscope and an Olympus E-410 camera. To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation period. Artificial lines that corresponded to the cutting edges were drawn on pictures of the original wounds and overlain on the pictures of the cultures after incubation. Cells that migrated across white lines were counted in 6 random fields from each triplicate treatment; data were presented as the mean ± standard deviation (SD).
Gelatin zymography assay
The activities of MMP-2 and MMP-9 were assayed by gelatin zymography. HepG2 cells (4 × 105 cells/well) were plated in 6-well plates, stimulated with 70 nM TPA for 12 h, and incubated in various concentrations of galangin (0, 1, 2.5, and 5 μM) for 24 h. The conditioned medium was collected and gelatin zymography was performed to examine the activities of MMP-2 and MMP-9. Samples were mixed with a loading buffer and electrophoresed on an 8% SDS-polyacrylamide gel containing 0.1% gelatin. Electrophoresis was performed at 140 and 110 V for 3 h. Gels were washed twice with a zymography washing buffer (2.5% Triton X-100 in double-distilled H2O) at room temperature to remove SDS. The cells were incubated at 37°C for 12–16 h in zymography reaction buffer (40 mM Tris–HCl (pH 8.0), 10 mM CaCl2, 0.02% NaN3), stained with Coomassie blue R-250 (0.125% Coomassie blue R-250, 0.1% amino black, 50% methanol, 10% acetic acid) for 1 h, and destained with a destaining solution (20% methanol, 10% acetic acid, 70% double-distilled H2O). Nonstaining bands representing the levels of the latent forms of MMP-2 and MMP-9 were quantified through densitometer measurement by using a digital imaging analysis system.
Isolation of total RNA, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA electrophoresis
Total RNA was isolated from HepG2 cells by using the total RNA Extraction Midiprep System (Viogene Bio-Tek Corporation, Taiwan). Total RNA (2 μg) was transcribed to 20 μl cDNA with 1 μl dNTPs (2.5 mM), 1 μl oligo dT (10 pmole/μl), and 1 μl RTase (200 U), 1 μl RNase inhibitor and 5× reaction buffer. The appropriate primers (sense of MMP-2, 5′-GGCCCTGTCACTCCTGAGAT-3′, nt 1337–1356; antisense of MMP-2, 5′-GGCATCCAGGTTATCGGGGA-3′, nt 2026–2007; sense of MMP-9, 5′-AGGCCTCTACAGAGTCTTTG-3′, nt 1201–1220; antisense of MMP-9, 5′-CAGTCCAACAAGAAAGGACG-3′, nt 1700–1683; sense of GADPH, 5′-CGGAGTCAACGGATTGGTGTT-3′, nt 94–126; antisense of 5′-AGCCTTCTCCATGGTTGGTGAAGAC-3′, nt 399–375) were used for PCR amplifications. The PCR was performed by using Platinum Taq polymerase (Invitrogen) under the following conditions: 30 cycles at 94°C for 1 min, 59°C (MMP-2) or 60°C (MMP-9 and GAPDH) for 1 min, 72°C for 1 min, and 72°C for 10 min.
Western blotting assay
The membrane as well as cytosolic and nuclear fractions of cells were prepared as described previously [
24]. Western blotting was performed as follows. Denatured samples (50 μg extracted protein) were resolved on 10-12% SDS-PAGE gels. The proteins were transferred onto nitrocellulose membranes. Nonspecific binding of the membranes was blocked with Tris-buffered saline (TBS) containing 1% (w/v) nonfat dry milk and 0.1% (v/v) Tween-20 (TBST) for longer than 2 h. Membranes were washed 3 times with TBST for 10 min and incubated with an appropriate dilution of specific primary antibodies in TBST overnight at 4°C. The membranes were then washed with TBST and incubated with secondary antibody (i.e., horseradish peroxidase-conjugated goat antimouse or antirabbit immunoglobulin G) for 1 h. After the membranes were again washed three times in TBST for 10 min, the bands were detected by performing ECL by using ECL Western blotting detection reagents and exposed ECL hyperfilm in a FUJFILM Las-3000 mini image-analysis system (Tokyo, Japan). Proteins were quantitatively determined through densitometry by using FUJFILM-Multi Gauge V3.0 software.
Electrophoretic mobility shift assay
Cell nuclear proteins were extracted using a nuclear extract buffer and measured using an electrophoretic mobility shift assay (EMSA). Cells (1 × 105/ml) were collected in a PBS buffer (pH 7.4) and centrifuged at 2000 g for 5 min at 4°C. The cells were lysed with buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF〔pH 7.9〕 containing 5% NP-40) for 10 min on ice and were subsequently subjected to vortexing to shear the cytoplasmic membranes. The lysates were centrifuged at 2000 g for 10 min at 4°C. The pellet containing the nuclei was extracted using high salt buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, and 25% glycerol) for 15 min on ice. The lysates were centrifuged at 13000 g for 10 min at 4°C. The supernatant containing the nuclear proteins was collected and frozen at −80°C until use. The protein content of nuclear fractions was determined using the Bio-Rad protein assay. A 5 μg aliquot of nuclear proteins was mixed with biotin-labeled NF-κB or AP-1 oligonucleotide probes for 15 min at room temperature or with oligonucleotides (sense of NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′, antisense of NF-κB, 3′-TCAACTCCCCTGAAAGGGTCCG-5′; sense of AP-1, 5′-CGCTTGATGACTCAGCCGGAA-3′, antisense of AP-1, 3′-GCGAACTACTGAGTCGGCCTT-5′). DNA probes were added to 10 μl binding reactions containing double-distilled H2O, 5 μg nuclear proteins, 1 μl poly (dI-dC), 1 μl biotin-labeled double stranded NF-κB or AP-1 oliginucleotides, and 2 μl of 10-fold binding buffer into a microcentrifuge tube and incubated for 15 min at room temperature. Specific competition binding assays were performed by adding 200-fold excess of unlabeled probe as a specific competitor. After protein-DNA complexes formation, samples were loaded onto a 6% nondenaturing polyacrylamide gel in a 0.5% TBE buffer and transferred to positively charged nitrocellulose membranes (Milipore, Bedford, MA) by usng a transfer blotting apparatus, and crosslinked in a Stratagene crosslinker. Gel shifts were visualised by first using streptavidin-horseradish peroxidase and then chemiluminescent detection.
Statistical analysis
Data were expressed as the means ± SD of three independent experiments and analysed using the Student’s t-test (Sigmaplot 2001, SPSS Inc., Chicago, IL, USA). Significant differences were established at P ≤ 0.05.
Discussion
For the past several decades, despite evolving management and novel therapies for various types of cancers, the prognosis for several types of cancers remain poor. Because increasingly more researchers have dedicated themselves to tumour research, the mechanisms underlying the oncogenesis and metastasis of cancer have gradually been revealed, and additional anticarcinogenic compounds have been discovered. Consequently, cancer chemoprevention for inhibiting the progression of cancer by using certain compounds or mixtures, has become a potentially viable method for treating cancer. Galangin is type of flavonoid with the highest concentration levels amongst
Alpinia officinarum. Flavonoids are a vast group of heterogeneous polyphenols that are thought to exert positive effects, including cancer prevention, on human health. Various studies have shown that glangin exerts pleiotropic anticancer effects, including preventive, anticarcinogenic, and antiproliferative effects. In particular, glangin influences several processes and plays a crucial roles in regulating various molecular targets, including NF-κB [
25,
26], Smads [
27], peroxisome proliferator-activated receptor γ (PPARγ) [
28] transcription factors, tumour necrosis factor-alpha (TNF-α), interleukins, intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2) [
29-
32], JNK, p38 [
25], and ERK [
26]. Metastasis is a critical characteristic of malignant tumours that requires a series of signal transductions. When these signal pathways are blocked with drugs or natural compounds, the spread of cancer may be inhibited. The purpose of this study was to determine whether galangin can inhibit HepG2 cancer cell invasion, migration and adhesion, and to elucidate its mechanism at a molecular level.
PKCs were shown to play key roles in various cellular responses, including the regulation of gene expression and the effects exerted on the cytoskeleton, cell growth, and differentiation [
33]. At least PKCs have recognised and are typically distributed into three classes, namely conventional (PKCα, βI, βII, γ), novel (δ, ε, ε’, η, θ, μ), and atypical (ζ, λ/ι) PKCs. Amongst these three classes, the activity of conventional PKCs depends on Ca
+2, diacyglycerol (DAG), or a phorbol ester analog of DNA such as TPA [
34-
37]. Isozymes of the PKC family exert various actions in cellular proliferation through a complex network of signal transduction. Previous studied have indicated that PKCα is a crucial signalling molecule for MMP expression in tumor cells [
38,
39]. In addition, PKCδ was shown to activate MMP expression [
40]. We found that galangin inhibited the mRNA expressions and enzymic activity of MMP-2 and MMP-9 in HepG2 cells. These results suggested that decreases in the protein expression of PKCα and PKCδ are vital to the suppression of MMP-2 and MMP-9 expression by galangin in HepG2cells. The activation of PKC can lead to the activation of MAPKs and the PI3K/Akt pathway. Our results revealed that long-term exposure to 70 nM TPA leads to downregulation of PKC-α and PKC-δ in HepG2 cells, indicating that TPA is involved in the ERK1/2 signal transduction pathway. These findings indicated that galangin inhibited TPA-induced MMP-2 and MMP-9 expression through the suppression of the PKC/ERK pathway in HepG2 cells.
The transcription of MMP-2/-9 genes is regulated by upstream sequences, including motifs corresponding to NF-κB and AP-1 binding sites. NF-κB and AP-1 are activated in numerous pathological processes, including inflammation, cancer-cell adhesion, invasion, metastasis, and angiogenesis. Previous studies have reported that the MMP-2/-9 system plays a crucial role in breast cancer growth, invasion, and metastasis. In this study, we found that treating HepG2 cells with galangin resulted in inhibition of NF-κB and AP-1 DNA binding activity, which was accompanied by inhibition of the nuclear translocation of these factors.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
STC, MDS, and YWS designed the experiment; CCT, YCL, and YWS performed experiments, analyzed the data and wrote the manuscript. All authors critically reviewed the manuscript. All authors approved the final version of this manuscript and agreed to be accountable for all aspects of the work.