- Open Access
Pinocembrin suppresses TGF-β1-induced epithelial-mesenchymal transition and metastasis of human Y-79 retinoblastoma cells through inactivating αvβ3 integrin/FAK/p38α signaling pathway
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 21 May 2014
Accepted: 21 July 2014
Published: 12 August 2014
Pinocembrin is the most abundant flavonoid in propolis. In this study, we investigated the antimetastatic effect of pinocembrin on TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of human Y-79 retinoblastoma cells.
Firstly, the results showed that pinocembrin significantly suppresses the TGF-β1-induced abilities of the invasion and migration of Y-79 cells under non-cytotoxic concentration. Pinocembrin decreased TGF-β1-induced expression of vimentin, N-cadherin, αv and β3 integrin in Y-79 cells. Molecular data also showed pinocembrin inhibits the activation of focal adhesion kinase (FAK) and p38α signal involved in the downregulation of enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/-9) induced by TGF-β1. Next, pinocembrin also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB was further observed under pinocembrin treatment.
Presented results indicated that pinocembrin inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and metastasis of Y-79 cells by inactivating the αvβ3 integrin/FAK/p38α signaling pathway. Thus, our findings point to the anticancer potential of pinocembrin against retinoblastoma cells.
Retinoblastoma is the most common primary intraocular tumor in children (0–5 years). In the United States, this disease presents most frequently as unilateral sporadic tumors and less frequently as bilateral hereditary tumors. There are two types of this disease, the genetic-heritable type and non-genetic-non-heritable type. About 55 percent of children with retinoblastoma have the non-genetic type. Children with the inherited form have a high risk of developing other cancers later in life. If untreated, the patients die of intracranial extension and disease dissemination within two years . Human retinoblastoma exhibits the patterns of invasion and metastasis. Direct invasive spreads along the optic nerve to the brain and can also seed the orbital tissue and adjacent bone . Current treatments include enucleation, external beam radiotherapy, cryotherapy, photocoagulation photocoagulation, and chemotherapy . Although these methods achieve survival rates of over 95%, there remains a need for better treatment alternatives to improve visual results and to avoid enucleation in hereditary retinoblastoma [4–6]. Thus, effective chemopreventive treatment for metastasis would have an important impact on retinoblastoma mortality rates.
The metastasis of tumor cells is a complex, multistage process. To facilitate the cell motility, invading cells need to change cell-cell adhesion properties, rearrange the extracellular matrix (ECM) environment, suppress anoikis and reorganize their cytoskeletons . Integrins are a family of transmembrane adhesion receptors comprised of 19α and 8β subunits that interact non-covalently to form up to 24 different heterodimeric receptors. Integrin binds to ECM proteins or integrin cross-linking increases the tyrosine phosphorylation of FAK . FAK is a nonreceptor tyrosine kinase primarily localized to cell-matrix adhesions which acts as a central regulator of focal adhesion influencing cell survival, differentiation, proliferation, migration and tissue remodeling [9–11]. Once localized to sites of transmembrane integrin receptor clustering, tyrosine-phosphorylated FAK plays an important role in signal transduction triggered by diverse extracellular signals  and represents a convergent point for synergistic interaction between signal pathways activated by growth factors and integrins [13–15]. Recently a conjunction between cancer stem cells and epithelial-mesenchymal transition (EMT) has been reported, and it has been shown that morphological and molecular changes occur when epithelial cells lose their characteristics, gain mesenchymal properties, and become motile, playing a critical role in the invasion of cancer cells [16, 17]. The expression of Snail (Snail1), Slug (Snail2), ZEB1 or Twist, along with the expression of mesenchymal markers such as N-cadherin and vimentin, and the loss of E-cadherin are key molecular markers of EMT . Transforming growth factor beta-1 (TGF-β1) has been identified as the main inducer of tumor EMT. The TGF-β1 is a pleiotropic cytokine that can mediate a wide spectrum of cellular effects through a variety of signaling pathways. Several studies have reported that TGF-β1 can promote angiogenesis, facilitate invasion, and suppress the host immune system .
Reagents and antibodies
Pinocembrin (purity≧95%) was purchased from Extrasynthese (Genay, France). Poly-D-lysine, DMSO, Tris–HCl, EDTA, SDS, phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), gelatin, crystal violet, leupeptin, Nonidet P-40, deoxycholic acid, sodium orthovanadate, mouse monoclonal antibody specific for αvβ3 integrin, and a selective αvβ3 integrin antagonist cyclic RGD (cycloRGDfV) peptide were purchased from Sigma-Aldrich (St. Louis, MO, USA); the protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Dulbecco’s phosphate buffer solution (PBS), trypsin-EDTA, and RPMI 1640 medium were purchased from Life Technologies, Inc. (Gibco/BRL, Gaithersburg, MD). Matrigel was purchased from BD Biosciences (Bedford, MA). Antibodies against E-cadherin, N-cadherin, vimentin, FAK, FAK (Tyr397, Tyr576, Tyr925), ERK1/2, ERK1/2 (Thr202/Tyr204), JNK1/2, JNK1/2 (Thr183/Tyr185), p38α, and p38α (Thr180/Tyr182) were purchased from Cell Signaling Tech. (Beverly, MA, USA). NF-κB (p50 and p65), β-actin, and C23 antibodies were purchased from BD Transduction Laboratories (San Diego, CA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham GE Healthcare UK Ltd (Buckinghamshire, England).
Cell culture and pinocembrin treatment
The human retinoblastoma Y-79 cell line was obtained from BCRC (Bioresource Collection and Research Center in Hsin-Chu, Taiwan). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2-95% air. In medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics (100 U/ml of penicillin and 100 mg/ml of streptomycin). Y-79 cells were grown in suspension at a concentration of 105-106 cells/ml. For all anti-metastatic examinations, cells were cultured in poly-D-lysine-coated culture plates. The stock solution of pinocembrin was dissolved in dimethyl sulfoxide (DMSO) and sterilized by filtration through 0.2 μm disc filters. Appropriate amounts of stock solution (1 mg/ml in DMSO) of pinocembrin were added to the cultured medium to achieve the indicated concentrations (the final DMSO concentration was less than 0.2%).
Cell viability assay
To evaluate the cytotoxicity of pinocembrin, a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] assay was performed to determine the cell viability . Briefly, cells were seeded at a density of 4 × 104 cells/ml in a 24-well plate for 24 h. Then, the cells were treated with or without pinocembrin under various concentrations for 24 h. Each concentration was repeated three times. Also, to further investigate whether pinocembrin and/or TGF-β1 influence cell viability, Y-79 cells were treated with the presence or absence of drugs (10 ng/ml TGF-β1 and 5 μM pinocembrin) for 24 h. After the exposure period, the medium was removed and followed by washing of cells with PBS. Then, the medium was changed and incubated with MTT solution (5 mg/ml)/well for 4 h. The medium was removed, and formazan was solubilized in isopropanol and measured spectrophotometrically at 563 nm. The percentage of viable cells was estimated by comparing with the untreated control cells.
Flow cytometric assay
The effects of pinocembrin on Y-79 cell-cycle progression and TGF-β1-induced avβ3 integrin expression were determined by flow cytometry using FACScan (Becton Dickinson Immunocytometry Systems, UK). Firstly, to analyze the cell-cycle distribution, the cells were first treated with various concentrations of pinocembrin for 24 h, and then were collected by trypsinization, fixed in 75% absolute ethanol, washed in PBS, and resuspended in 1 ml of PBS containing 0.5 mg/ml RNase A and 0.01 mg/ml propidium iodide (PI) in the dark for 30 min at room temperature. The cell-cycle profiles were analyzed by a flow cytometer. The percentage of cells in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle was analyzed by the ModFit LT 3.0 software (Verity Software, Topsham, ME). Furthermore, the cell surface expression of avβ3 integrin was determined using flow cytometry. Y-79 cells were plated in six-well dishes. The cells were then washed with PBS and detached with trypsin at 37°C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsing in PBS, the cells were incubated with a mouse anti-human antibody against avβ3 integrin (1:100) for 1 h at 4°C. Cells were then washed again and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary IgG (1:100; Leinco Tec. Inc., St. Louis, MO, USA) for 45 min and analyzed by flow cytometry.
Cell-matrix adhesion assay
Y-79 cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin for 24 h. Subsequently, cells were seeded at a density of 1 × 105 cells/ml in a 24-well plate and coated with 500 μl type IV collagen (10 μg/ml); then they were cultured for 30 min. Then, non-adherent cells were removed by PBS washes, and adherent cells were fixed in ethanol. After staining with 0.1% crystal violet, fixed cells were lysed in 0.2% Triton X-100, and measured spectrophotometrically at 550 nm.
Transwell invasion and migration assay
Invasion and migration assay were performed by using Hanging Cell Culture-inserts (BD Biosciences, San Jose, CA; pore size, 8-μm) in 6-well dishes. The ability of Y-79 cells to pass through filters coated with Matrigel (BD Biosciences) was measured by invasion assay. Matrigel was diluted to 200 μg/ml with filtered distill water and applied to the upper surface of the filter inserts. Briefly, Y-79 cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin. After 24 h, cells were detached by trypsin and resuspended in serum-free medium. Medium containing 10% fetal bovine serum was applied to the lower chamber as a chemoattractant, and then cells were seeded on the upper filter at a density of 1 × 105 cells/ml in the serum-free medium. The plates were incubated for 24 h at 37°C in 5% CO2, 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, and then the cells that had invaded the lower surface of the filter were counted under a light microscope. The data are presented as the average number of cells attached to the bottom surface from randomly chosen fields. Each experiment was carried out in triplicate. To measure the migration ability of Y-79 cells, cells were seeded into a transwell with 8 μm pore polycarbonate filters which were not coated with matrigel. Migrating cells were treated with the presence or absence of drugs (TGF-β1 and pinocembrin). Migration assay was measured as described in the invasion assay.
Gelatin zymography assay
Cells (4 × 105 cells/ml) were seeded into the culture and stimulated with 10 ng/ml TGF-β1 for 2 h and then incubated in different concentrations of pinocembrin (0, 1, 2.5, and 5 μM) for 24 h. Subsequently, the conditioned medium was collected and gelatin zymography was performed to examine the activities of MMP-2 and MMP-9. Samples were mixed with loading buffer and electrophoresed on 8% SDS-polyacrylamide gel containing 0.1% gelatin. Electrophoresis was performed at 140 and 110 V for 3 h. Gels were then washed twice in zymography washing buffer (2.5% Triton X-100 in double-distilled H2O) at room temperature to remove SDS, followed by incubation at 37°C for 12–16 h in zymography reaction buffer (40 mM Tris–HCl, 10 mM CaCl2, 0.02% NaN3), stained with Coomassie blue R-250 (0.125% Coomassie brilliant blue R-250, 0.1% amino black, 50% methanol, 10% acetic acid) for 1 h and destained with 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 by densitometer measurement 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 human Y-79 cells using the total RNA Extraction Midiprep System (Viogene BioTek 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), 1 μl RTase (200 U), 1 μl RNase inhibitor and 5 X reaction buffer. The appropriate primers (sense of MMP-2, 5′-GGCCCTGTCACTCCTGAGAT-3′ nt 1337–1356; antisense of MMP-2, 5′-GGCATCCGGTTATCGGGGA-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. PCR was performed with Platinum Taq polymerase (Invitrogen) under the following conditions: 30 cycles of 94°C for 1 min, 59°C (MMP-2) or 60°C (MMP-9 and GAPDH) for 1 min, 72°C for 1 min followed by 10 min at 72°C.
Western blotting assay
The preparation of cytosolic and nuclear fractions of the cells was performed as described previously . Western blotting was performed as follows. The denatured samples (50 μg purified protein) were resolved on 10-12% SDS-PAGE gels. The proteins were then transferred onto nitrocellulose membranes. Non-specific 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 more than 2 h. Membranes were washed with TBST three times for 10 min and incubated with an appropriate dilution of specific primary antibodies in TBST overnight at 4°C. Subsequently, membranes were washed with TBST and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat antimouse or antirabbit IgG) for 1 h. After washing the membrane three times for 10 min in TBST, the bands detection was carried out by enhanced chemiluminescence using ECL Western blotting detection reagents and exposed ECL hyperfilm in FUJFILM Las-3000 mini (Tokyo, Japan). Then proteins were quantitatively determined by densitometry using FUJFILM-Multi Gauge V3.0 software.
Electrophoretic mobility shift assay (EMSA)
Cell nuclear proteins were extracted with a nuclear extract buffer and measured by electrophoretic mobility shift assay (EMSA) . Cells (1 × 105/ml) were collected in PBS buffer (pH 7.4) and centrifuged at 2000 × g for 5 min at 4°C. 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 this was followed by 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 with 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 centrifugated 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 with Bio-Rad protein assay. Synthetic double-strand oligonucleotides of the consensus NF-κB binding sequence, 5′-AGTTGAGG GGACTTTCCCAGGC-3′ and 3′-TCAACTCCCCTGAAAGGGTCCG-5′, were 5′end- labeled with biotin. Binding reactions containing 5 μg of nuclear proteins, double-distilled H2O, 2 μl 10-fold binding buffer, 2 μg poly (dI · dC) and 2 pmol oligonucleotide probe were incubated for 15 min at room temperature. Specific competition binding assays were performed by adding a 200-fold excess of an unlabeled probe as a specific competitor. Following formation of protein-DNA complexes, samples were loaded on a 6% nondenaturing polyacrylamide gel in 0.5 × TBE buffer and then transferred to positively charged nitrocellulose membranes (Millipore, Bedford, MA, USA) by a transfer blotting apparatus and cross-linked in a Stratagene crosslinker. Gel shifts were visualized with streptavidin-horseradish peroxidase followed by chemiluminescent detection.
Transient transfection and luciferase report gene assays
NF-κB transcriptional activity was measured by NF-κB-luciferase report gene expression. Y-79 cells (4 × 104 cells/well) were plated in six-well plates. The cells were transiently co-transfected with the plasmids, pGL3-NF-κB, pCMV-β-gal and pcDNA3.1 using Lipofectamine Plus according to the manufacturer’s protocol. Briefly, a transfection mixture containing 0.5 μg pGL3-NF-κB and 0.2 μg pCMV-β-gal was mixed with the Lipofectamine Plus reagent and added to the cells. After 8 h, the cells were stimulated with 10 ng/ml TGF-β1 for 2 h and then incubated in 5 μM pinocembrin for 0, 1, 3, 6, and 9 h. Then the NF-κB-luciferase activity was measured according to the manufacturer’s instructions (Promega). Briefly, cells were washed twice with cold PBS and lysed by adding 100 μl 1X reporter lysis buffer (24 mM Tris–HCl (pH 7.8), 2 mM dithiotreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) (Promega, Madison, WI, USA). After centrifugation (13000 × g, 2 min), samples were measured for luciferase activity by using a Sirus Luminometer (Berthold Detection System; OAK Ridge, TN) using 10 μl of cell lysate and 100 μl of luciferase assay reagent (Promega). Luciferase activity was measured with a 10 s delay and 30 s integration time and was normalized to β-galactosidase or Renilla luciferase activity to determine transfection efficiency. The values shown represent an average of three independent transfections and each transfection was carried out in triplicate.
Data were expressed as means ± standard deviation of three independent experiments. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences were established at p ≤ 0.05.
Cytotoxicity of pinocembrin to Y-79 cells
In this study, we first examined the effect of pinocembrin on cell cytotoxicity in Y-79 cells. As shown in Figure 1B, pinocembrin did not affect the cell cytotoxicity of Y-79 cells at concentrations ranging from 0–5 μM. In order to verify this, the dose range was applied in all subsequent experiments to avoid the influence of cell growth and cytotoxicity on the observed papameters, and a series of studies were performed to measure the cell growth and cytotoxicity upon pinocembrin stimulation. Firstly, the effects of pinocembrin on Y-79 cell-cycle progression were measured by flow cytometry (Figure 1C) which revealed that a treatment with 5 μM pinocembrin had no effect on the cell-cycle distribution for 24 h. At higher concentrations (10, 20, 40, 60, 80 and 100 μM), pinocembrin increased the G1 fraction (from 60.26% to 73.57%), and caused an apparent accumulation of the cells in the sub-G1 phase, while decreasing the S fraction in a dose-dependent manner. Therefore, the results demonstrated that a 24 h treatment of pinocembrin at a concentration ranging from 0 to 5 μM had no cytotoxicity to Y-79 cells. Also, as illustrated in Figure 1D, in the presence of TGF-β1, pinocembrin at a concentration of 5 μM did not have any toxic effects on cell viability. Thus, non-cytotoxic concentrations of pinocembrin (0–5 μM) were used in subsequent experiments.
Pinocembrin suppresses TGF-β1-induced cell-matrix adhesion, invasion, migration, and epithelial- mesenchymal transition in Y-79 cells via αvβ3 integrin
Pinocembrin suppresses TGF-β1-induced integrin levels in Y-79 cells
Pinocembrin suppresses TGF-β1-induced expressions of MMP-2 and MMP-9 in Y-79 cells
Pinocembrin inhibits TGF-β1-induced phosphorylation of FAK/p38α in Y-79 cells
Pinocembrin inhibits TGF-β1-induced DNA binding activity of NF-κB and the expressions of NF-κB and IκBα in Y-79 cells
Retinoblastoma is the most common primary intraocular tumor in children (0–5 years). It is associated with generally poor prognosis due to its tendency toward local invasion and subsequent metastasis. Recently, antimetastatic agents have been defined as a new class of cancer chemopreventive agent. Pinocembrin is one of the flavonoids with the highest concentration levels in propolis. Flavonoids are a vast group of heterogeneous polyphenols that are thought to have positive effects on human health, including cancer prevention. Various studies have shown that pinocembrin exerts pleiotropic anticancer effects, including preventive, anticarcinogenic, and antiproliferative effects in various in vivo and in vitro models. In particular, pinocembrin could influence several processes and play important roles in the regulation of various molecular targets, including NF-κB , Smad2 and PPARγ transcription factors , Vascular endothelial growth factors , TGF-β1 , necrosis factor-alpha, interleukin-1beta, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 inflammatory cytokines , p38 MAPK , ERK1/2 and PI3K/Akt protein kinases  and other enzymes (COX-2 and MMPs) [39, 36].
Tumor promoting activity of TGF-β1 associated with the induction of EMT has been documented for different tumor types . Several reports have shown that TGF-β1 has been identified as the main inducer of tumor EMT. The EMT induced by TGF-β1 results in the disruption of the polarized morphology of epithelial cells, formation of actin stress fibers, and enhancement of cell migration . The TGF-β1 family of secreted factors is involved in controlling different biological processes, including cell proliferation, differentiation, and apoptosis. Furthermore, conjunction between TGF-β1 signaling and carcinoma cells in cell invasiveness and tumor metastasis has recently been reported . These effects of TGF-β1 are related to its ability to induce EMT and stimulate cell migration. Furthermore, several non-Smad pathways have also been shown to mediate the cellular effects of TGF-β1. These include ERK, JNK, p38 MAPK, and PI3K/Akt. In this study, we have demonstrated that pinocembrin suppresses TGF-β1-induced EMT via the p38α signaling pathway.
We further examined the effect of the expression levels of E-cadherin, N-cadherin, and vimentin. E-cadherin, an adherent junction protein and type I transmembrane glycoprotein, plays a pivotal role in the maintenance of normal tissue architecture and the suppression of cancer invasion . A previous study demonstrated that the loss of E-cadherin-mediated adhesion plays an important role in the transition of epithelial tumors from a benign to an invasive state . More recent evidence indicates that the gain of expression of another adhesion molecule, N-cadherin, is associated with heightened invasive potential in tumor cells. N-cadherin has also been reported to induce a mesenchymal-scattered phenotype associated with reduced E level in a squamous cell carcinoma cell line . Vimentin, a mesenchymal marker, has been was observed in a few cancer cells. It has previously been demonstrated that vimentin expression is substantially increased in cancer cells of hepatic metastasis . Taken together, our results are consistent with the notion that the changes were associated with an increase in E-cadherin expression and a reduction in N-cadherin and vimentin in TGF-β1-treated Y-79 cells. Loss of cell-cell adhesion followed by the dissociation of epithelial structures is a prerequisite for increased cell motility and tumor invasion. These findings raise the possibility that N-cadherin and vimentin contribute directly to the invasive phenotype. In addition to promoting motile and invasive activities of Y-79 cells in vitro, the expressions of N-cadherin and vimentin greatly sensitized Y-79 cells to TGF-β1, stimulating marked increases in cell migration and invasion as well as in MMP-2/-9 production. When Y-79 cells were treated with pinocembrin, there dramatically reduced the activities and expressions of MMP-2/-9. It is of importance to note that MMP-2/-9 plays roles in tumor progression, as over-expression of the MMP-2/-9 gene in transgenic mice led to enhanced tumorigenesis in a breast cancer model . Also, Sasaki et al.  reported that the survival rates of lung cancer patients were poor for those patients with an elevated MMP-2/-9 mRNA expression on the tumor tissue. We have demonstrated that reduction of proteolytically active MMP-2/-9 is involved in pinocembrin mediated-cell invasion and migration. In addition, the transcription of the MMP-2/-9 gene is regulated by upstream sequences, including motifs corresponding to the NF-κB binding element [49, 50]. Here, we also found that pinocembrin inhibits MMP-2/-9 expression through preventing IκBα being phosphorylated and enhancing IκBα protein expression, both leading to inactivation of NF-kB DNA binding activity. A comprehensive analysis of MMP-2/-9 expression in Y-79 cells will be necessary to fully understand the biological properties of Y-79 cells and to promote the development of new therapeutic strategies.
This work was supported by the grant from the Subsidized Project of the Chung Hwa University, Tainan, Taiwan (98-HT-08009).
- Albert DM: Historic review of retinoblastoma. Ophthalmology. 1987, 94 (6): 654-662. 10.1016/S0161-6420(87)33407-4View ArticlePubMedGoogle Scholar
- Advani SH, Rao SR, Iyer RS, Pai SK, Kurkure PA, Nair CN: Pilot study of sequential combination chemotherapy in advanced and recurrent retinoblastoma. Med Pediatr Oncol. 1994, 22 (2): 125-128. 10.1002/mpo.2950220212View ArticlePubMedGoogle Scholar
- Ferris FL, Chew EY: A new era for the treatment of retinoblastoma. ArchOphthalmol. 1996, 114 (11): 1412.Google Scholar
- O’Brien JM: Alternative treatment in retinoblastoma. Ophthalmology. 1998, 105 (4): 571-572. 10.1016/S0161-6420(98)94005-2View ArticlePubMedGoogle Scholar
- Chantada G, Fandiño A, Dávila MT, Manzitti J, Raslawski E, Casak S, Schvartzman E: Results of a prospective study for the treatment of retinoblastoma. Cancer. 2004, 100 (4): 834-842. 10.1002/cncr.11952View ArticlePubMedGoogle Scholar
- Shields CL, Meadows AT, Leahey AM, Shields JA: Continuing challenges in the management of retinoblastoma with chemotherapy. Retina. 2004, 24 (6): 849-862. 10.1097/00006982-200412000-00003View ArticlePubMedGoogle Scholar
- Woodhouse EC, Chuaqui RF, Liotta LA: General mechanisms of metastasis. Cancer. 1997, 180 (8): 1529-1537.View ArticleGoogle Scholar
- Humphries MJ: Integrin structure. Biochem Soc Trans. 2000, 28 (4): 311-339. 10.1042/0300-5127:0280311View ArticlePubMedGoogle Scholar
- Stupack DG: The biology of integrins. Oncology. 2007, 21 (9): 6-12.PubMedGoogle Scholar
- van Nimwegen MJ, van de Water B: Focal adhesion kinase: a potential target in cancer therapy. Biochem Pharmacol. 2007, 73 (5): 597-609. 10.1016/j.bcp.2006.08.011View ArticlePubMedGoogle Scholar
- Mitra SK, Schlaepfer DD: Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr Opin Cell Biol. 2006, 18 (5): 516-523. 10.1016/j.ceb.2006.08.011View ArticlePubMedGoogle Scholar
- Zachary I: Focal adhesion kinase. Int J Biochem Cell Biol. 1997, 29 (7): 929-934.View ArticlePubMedGoogle Scholar
- Reif S, Lang A, Lindquist JN, Yata Y, Gabele E, Scanga A, Brenner DA, Rippe RA: The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem. 2003, 278 (10): 8083-8090. 10.1074/jbc.M212927200View ArticlePubMedGoogle Scholar
- Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA: Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol. 1997, 137 (2): 481-492. 10.1083/jcb.137.2.481PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng DQ, Woodard AS, Tallini G, Languino LR: Substrate specificity of alpha vbeta 3 integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation. J Biol Chem. 2000, 275 (32): 24565-24574. 10.1074/jbc.M002646200View ArticlePubMedGoogle Scholar
- Thiery JP, Acloque H, Huang RY, Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 2009, 139 (5): 871-890. 10.1016/j.cell.2009.11.007View ArticlePubMedGoogle Scholar
- Yang J, Weinberg RA: Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell. 2008, 14 (6): 818-829. 10.1016/j.devcel.2008.05.009View ArticlePubMedGoogle Scholar
- Gavert N, Ben-Ze'ev A: Epithelial-mesenchymal transition and the invasive 407 potential of tumors. Trends Mol Med. 2008, 14 (5): 199-209. 10.1016/j.molmed.2008.03.004View ArticlePubMedGoogle Scholar
- Derynck R, Zhang YE: Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003, 425 (6958): 577-584. 10.1038/nature02006View ArticlePubMedGoogle Scholar
- Medić-Sarić M, Rastija V, Bojić M: Recent advances in the application of high performance liquid chromatography in the analysis of polyphenols in wine and propolis. J AOAC Int. 2011, 94 (1): 32-42.PubMedGoogle Scholar
- Pepeljnjak S, Jalsenjak I, Maysinger D: Flavonoid content in propolis extracts and growth inhibition of Bacillus subtilis. Pharmazie. 1985, 40 (2): 122-123.PubMedGoogle Scholar
- Del Rayo CM, Sanchez B, Quiroz H, Contreras JL, Mata R: Pinocembrine: a bioactive flavanone from Teloxys graveolens. J Ethnopharmacol. 1991, 31 (3): 383-389. 10.1016/0378-8741(91)90022-6View ArticleGoogle Scholar
- Santos AC, Uyemura SA, Lopes JL, Bazon JN, Mingatto FE, Curti C: Effect of naturally occurring flavonoids on lipid peroxidation and membrane permeability transition in mitochondria. Free Radic Bio Med. 1998, 24 (9): 1455-1461. 10.1016/S0891-5849(98)00003-3View ArticleGoogle Scholar
- Sala A, Recio MC, Schinella GR, Manez S, Giner RM, Cerda-Nicolas M, Rosi JL: Assessment of the anti-inflammatory activity and free radical scavenger activity of tiliroside. Eur J Pharmacol. 2003, 461 (1): 53-61. 10.1016/S0014-2999(02)02953-9View ArticlePubMedGoogle Scholar
- Liu R, Gao M, Yang ZH, Du GH: Pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia-reperfusion both in vivo and in vitro. Braun Res. 2008, 1216: 104-115.View ArticleGoogle Scholar
- Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65 (1–2): 55-63.View ArticlePubMedGoogle Scholar
- Ito H, Duxbury M, Benoit E, Clancy TE, Zinner MJ, Ashley SW, Whang EE: Prostaglandin E2 enhances pancreatic cancer invasiveness throughan Ets-1-dependent induction of matrix metalloproteinase-2. Cancer Res. 2004, 64 (20): 7439-7446. 10.1158/0008-5472.CAN-04-1177View ArticlePubMedGoogle Scholar
- Ma W, Lim W, Gee K, Aucoin S, Nandan D, Kozlowski M, Diaz-Mitoma F, Kumar A: The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide stimulated human macrophages. J Biol Chem. 2001, 276 (17): 13664-13674.PubMedGoogle Scholar
- Pechkovsky DV, Scaffidi AK, Hackett TL, Ballard J, Shaheen F, Thompson PJ, Thannickal VJ, Knight DA: Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. J Biol Chem. 2008, 283 (19): 12898-12908. 10.1074/jbc.M708226200View ArticlePubMedGoogle Scholar
- Pfaff M, Tangemann K, Müller B, Gurrath M, Müller G, Kessler H, Timpl R, Engel J: Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins. J Biol Chem. 1994, 269 (32): 20233-20238.PubMedGoogle Scholar
- Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS: Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NFkappa B activation. Mutat Res. 2001, 480–481: 243-268.View ArticlePubMedGoogle Scholar
- Bharti AC, Aggarwal BB: Nuclear factor-kappa B and cancer: its role in prevention and therapy. Biochem Pharmacol. 2002, 64 (5–6): 883-888.View ArticlePubMedGoogle Scholar
- Wang K, Ping S, Huang S, Hu L, Xuan H, Zhang C, Hu F: Molecular mechanisms underlying the in vitro anti-inflammatory effects of a flavonoid-rich ethanol extract from chinese propolis (poplar type). Evid Based Complement Alternat Med. 2013, 2013: 127672.PubMed CentralPubMedGoogle Scholar
- Kao HF, Chang-Chien PW, Chang WT, Yeh TM, Wang JY: Propolis inhibits TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells via PPARγ activation. Int Immunopharmacol. 2013, 15 (3): 565-574. 10.1016/j.intimp.2012.12.018View ArticlePubMedGoogle Scholar
- Sang H, Yuan N, Yao S, Li F, Wang J, Fang Y, Qin S: Inhibitory effect of the combination therapy of simvastatin and pinocembrin on atherosclerosis in ApoE-deficient mice. Lipids Health Dis. 2012, 11: 166. 10.1186/1476-511X-11-166PubMed CentralView ArticlePubMedGoogle Scholar
- Gao M, Zhu SY, Tan CB, Xu B, Zhang WC, Du GH: Pinocembrin protects the neurovascular unit by reducing inflammation and extracellular proteolysis in MCAO rats. J Asian Nat Prod Res. 2010, 12 (5): 407-418. 10.1080/10286020.2010.485129View ArticlePubMedGoogle Scholar
- Liu R, Wu CX, Zhou D, Yang F, Tian S, Zhang L, Zhang TT, Du GH: Pinocembrin protects against β-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondrion-mediated apoptosis. BMC Med. 2012, 10: 105. 10.1186/1741-7015-10-105PubMed CentralView ArticlePubMedGoogle Scholar
- Chen CS, Wu CH, Lai YC, Lee WS, Chen HM, Chen RJ, Chen LC, Ho YS, Wang YJ: NF-kappaB-activated tissue transglutaminase is involved in ethanol-induced hepatic injury and the possible role of propolis in preventing fibrogenesis. Toxicology. 2008, 246 (2–3): 148-157.View ArticlePubMedGoogle Scholar
- Rossi A, Ligresti A, Longo R, Russo A, Borrelli F, Sautebin L: The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages. Phytomedicine. 2002, 9 (6): 530-535. 10.1078/09447110260573164View ArticlePubMedGoogle Scholar
- Gold LI: The role for transforming growth factor-beta (TGF-beta) in human cancer. Crit Rev Oncog. 1999, 10 (4): 303-360.PubMedGoogle Scholar
- Oft M, Heider KH, Beug H: TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis. Curr Biol. 1998, 8 (23): 1243-1252. 10.1016/S0960-9822(07)00533-7View ArticlePubMedGoogle Scholar
- Bandyopadhyay A, Zhu Y, Cibull ML, Bao L, Chen C, Sun L: A soluble transforming growth factor beta type III receptor suppresses tumorigenicity and metastasis of human breast cancer MDA-MB-231 cells. Cancer Res. 1999, 59 (19): 5041-5046.PubMedGoogle Scholar
- Joo YE, Rew JS, Choi SK, Bom HS, Park CS, Kim SJ: Expression of e-cadherin and catenins in early gastric cancer. J Clin Gastroenterol. 2002, 35 (1): 35-42. 10.1097/00004836-200207000-00009View ArticlePubMedGoogle Scholar
- Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA: Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol. 2000, 148 (4): 779-790. 10.1083/jcb.148.4.779PubMed CentralView ArticlePubMedGoogle Scholar
- Islam S, Carey TE, Wolf GT, Wheelock MJ, Johnson KR: Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell–cell adhesion. J Cell Biol. 1996, 135 (6): 1643-1654. 10.1083/jcb.135.6.1643View ArticlePubMedGoogle Scholar
- Nakajima S, Doi R, Toyoda E, Tsuji S, Wada M, Koizumi M, Tulachan SS, Ito D, Kami K, Mori T, Kawaguchi Y, Fujimoto K, Hosotani R, Imamura M: N-cadherin expression and epithelial- mesenchymal transition in pancreatic carcinoma. Clin Cancer Res. 2004, 10 (12): 4125-4133. 10.1158/1078-0432.CCR-0578-03View ArticlePubMedGoogle Scholar
- Rudolph-Owen LA, Chan R, Muller WJ, Matrisian LM: The matrix metalloproteinase matrilysin influences early stage mammary tumorigenesis. Cancer Res. 1998, 58 (23): 5500-5506.PubMedGoogle Scholar
- Sasaki H, Yukiue H, Moiriyama S, Kobayashi Y, Nakashima Y, Kaji M, Kiriyama M, Fukai I, Yamakawa Y, Fujii Y: Clinical significance of matrix metalloproteinase-7 and Ets-1 gene expression in patients with lung cancer. J Surg Res. 2001, 101 (2): 242-247. 10.1006/jsre.2001.6279View ArticlePubMedGoogle Scholar
- Aguirre Ghiso JA, Alonso DF, Farias EF, Gomez DE, de Kier Joffe EB: Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur J Biochem. 1999, 263 (2): 295-304. 10.1046/j.1432-1327.1999.00507.xView ArticlePubMedGoogle Scholar
- Westermarck J, Kahari VM: Regulation of matrix metalloproteinase expression in tumor invasion. The FASEB J. 1999, 13 (8): 781-792.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.