Article pubs.acs.org/JAFC
Inhibitory Effects of Butein on Cancer Metastasis and Bioenergetic Modulation Shih-Chia Liu,†,∥ Chi Chen,‡ Ching-Hu Chung,‡,∥ Po-Chuan Wang,§ Nan-Lin Wu,‡,# Jen-Kun Cheng,‡,⊥ Yu-Wei Lai,□ Hui-Lung Sun,⊗ Chieh-Yu Peng,△ Chih-Hsin Tang,▽,○ and Shih-Wei Wang*,‡ †
Departments of Orthopaedics, #Dermatology, and ⊥Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan Department of Medicine, Mackay Medical College, New Taipei City, Taiwan § Gastroenterology, Mackay Memorial Hospital, Hsinchu, Taiwan □ Division of Urology, Taipei City Hospital Renai Branch, Taipei, Taiwan ⊗ Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, United States △ School of Pharmacy, College of Pharmacy, and ▽Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan ○ Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan ‡
ABSTRACT: Tumor metastasis is the major obstacle for cancer treatment. Previous studies have shown that butein exhibits antiangiogenesis property and anticancer effects in different kinds of human cancer cells. However, the effects of butein on metastasis and energy metabolism of cancer cells are mostly unknown. This study showed that butein significantly inhibited invasion of cancer cells without acting in a cytotoxic fashion. It was further demonstrated that butien dramatically suppressed cancer metastasis by an in vivo CAM-intravasation model. Additionally, butein concentration-dependently repressed the expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA). The study indicated that butein may repress MMP-9 and uPA proteolytic activities and subsequently inhibit cancer metastasis via Akt/mTOR/ p70S6K translational machinery. Moreover, butein may partly suppress cancer metastasis by down-regulating ATP synthesis via both oxidative and glycolytic metabolism. The results suggest that butein is a potential antimetastatic agent worthy of further development for cancer treatment. KEYWORDS: butein, metastasis, bioenergetics
■
INTRODUCTION Cancer is the leading and the second leading cause of death in developed and developing countries, respectively. There are more than 12 million new cancer cases and nearly 8 million cancer deaths worldwide annually.1 Among these deaths, up to 90% are caused by the direct or indirect effects of metastasis. The sequential events of cancer cells translocating from the primary tumor site to the site of dissemination are often termed the invasion−metastatic cascade.2 Proteolytic activities of cancer cells play critical roles in many events of the cascade, including local invasion, intravasation, and extravasation.3 Therefore, the discovery and development of effective agents to suppress cancer metastasis by attenuating the proteolysis of cancer cells are important works for cancer researchers. Among the molecules involving proteolysis, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) have been reported to be overexpressed in cancerous tissues.4,5 MMPs, one of the endopeptidases families, play critical roles in composing and decomposing extracellular matrix (ECM). The production and secretion of MMPs are regulated by growth factors and cytokines, inducing physiological or pathological cell migration and invasion. Previous studies point out that many members of the MMP family participate in the formation and progression of some neural degenerative diseases and cancer.3 MMPs can accelerate the progression of cancer by © XXXX American Chemical Society
decomposing ECM around the cancer cells, changing cell−cell and cell−ECM interactions, and inducing angiogenesis. MMP-9 is an enzyme that can be induced by some growth factors, such as TNF-α, IL-1β, and PDGF, and degrade type IV collagen.6 The expression of MMP-9 is regulated by intracellular signaling pathways such as Akt, ERK1/2, or JNK.7,8 In addition to MMPs, the serine proteinase, u-PA, and cathepsins are crucial for tumor metastasis, which degrades basement membranes and activates pro-MMPs. uPA not only promotes cancer progression by activating plasmin, it also induces the proliferation of cancer cells by interacting with its membrane-bounded receptor, uPAR.9 Previous studies have shown that the prognoses of patients with lower expression levels of MMP-9, uPA, and uPAR in serum or cancer-adjacent tissues are significantly better than those who have higher levels, indicating that MMP-9 and uPA are potent targets for cancer therapeutics.10−12 Mitochondria are important organelles participating in bioenergetic metabolism and cellular homeostasis in mammalian cells.13 Mitochondrial respiration provides an efficient route for ATP production through the oxidative phosphorylation Received: May 20, 2014 Revised: July 29, 2014 Accepted: August 19, 2014
A
dx.doi.org/10.1021/jf502370c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
treatment, cells on the upper side of the filters were mechanically removed by cotton-tipped swabs, then cells on the lower side were fixed in methanol for 15 min and stained with 0.05% crystal violet for 15 min. The invaded or migrated cells were photographed and quantified by counting the number of stained cells under a microscope. In Vivo Metastasis Analysis. The chorioallantoic membrane (CAM) intravasation model was performed to evaluate tumor cell metastasis as described previously.26 After treatment with butein for 12 h, surviving cells (106 cells) were collected and resuspended in 50 μL of serum-free DMEM and inoculated onto the CAM of a 9-day-old chick embryo, in which an artificial air sac was created (designated “upper CAM”). After 48 h of incubation, the lower half of the CAM (designated “lower CAM”) was removed and stored frozen at −80 °C. Genomic DNA of the frozen tissue was purified from lower CAM by the DNA Isolation Kit. To determine human cells in the chick tissues, the primers specific for human alu sequence (5′-ACGCCTGTAATCCCAGCACTT-3′/5′-TCGCCCAGGCTGGAGTGCA-3′) were used to amplify the human alu repeats present in genomic DNA that was isolated from chick tissues. A quantitative measure of amplifiable chick DNA was examined by amplification of chick GAPDH genomic DNA sequence with chGAPDH primers (5′-GAGGAAAGGTCGCCTGGTGGATCG-3′/5′-GGTGAGGACAAGCAGTGAGGAACG-3′). The cycling conditions were 10 min of polymerase activation at 95 °C followed by 30 cycles at 95 °C for 30 s, 58 °C for 45 s, and 72 °C for 45 s and a final incubation at 72 °C for 10 min. Finally, PCR products were conducted on the agarose gel and visualized using ethidium bromide. Cell Viability Analysis. Cells (104 cells/well) were incubated in 96well plates. After 24 h of incubation, the culture medium was removed and treated with butein for 12 or 24 h. Then, cell survival was determined using the MTT colorimetric assay. The absorbance at 550 nm was measured using a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA). MMP-9 and uPA Activity Assay. After treatment, the medium was collected and stored at −80 °C. Then, MMP-9 and uPA activities in the medium were determined and quantified using MMP-9 and uPA ELISA kits (R&D Systems, Minneapolis, MN, USA). Western Blot Analysis. The cellular lysates were prepared according our previous instruction.27 Proteins were resolved by 8− 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis and immunoblotted with specific primary antibodies. Then, the blots were subsequently incubated with the secondary antibody, and the signals were determined using the chemiluminescent assay kit (Amersham, Buckinghamshire, UK). RT-PCR Analysis. Total RNA was extracted from cells by using a TRIzol kit (MDBio Inc., Taipei, Taiwan). First-strand cDNA was synthesized with random primer and moloney murine leukemia virus reverse transcriptase (M-MLV RT). The primer sequences used for amplification were listed as follows: MMP-9, 5′-TGGGCTACGTGACCTATGAC-3′/5′- CAAAGGTGAGAAGAGAGGGC-3′; uPA, 5′AGAATTCTACCGACTATCTC-3′/5′- ATTCTCTTCCTTGGTGTGAC-3′; PAI, 5′-TGGTTCTGCCCAAGTTCTCC-3′/5′-GACAGCTGTGGATGAGGAGG-3′; uPAR, 5′- CCGAGGTTGTGTGTGGGTTA-3′/5′-ATGCATTCGAGGTAACGGCT-3′; TIMP-1, 5′-TTGGCTTCTGCACTGATGGT-3′/5′-TAAATGTCCACGCTAGGGGC-3′; GAPDH, 5′-TGATGACATCAAGAAGGTGGTGAAG3′/5′-TCCTTGGAGGCCATGTGGGCCAT-3′. The cycling conditions were 5 min of polymerase activation at 94 °C followed by 30 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min and a final incubation at 72 °C for 10 min. The PCR products were separated on the agarose gel and visualized using ethidium bromide. Bioenergetic Function Analysis. The XF24 Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to determine the bioenergetic metabolism of cancer cells. Cells (2 × 104 cells/well) were seeded in an XF24 24-well plate and were allowed to grow for 1 day. Culture medium was next replaced 1 h prior to measurement by the assay medium that contained unbuffered DMEM with or without butein and incubated in a 37 °C incubator for 30 min to stabilize the pH and temperature. Then, the plate was transferred into the incubation chamber of the XF24 Analyzer for real-time determination of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
(OXPHOS) machinery. Reprogramming energy metabolism has been demonstrated to be a cancer hallmark.14 Most cancer cells exhibit high glycolysis capacity and low mitochondrial respiration even in the presence of oxygen, which is recognized as the “Warburg effect”. Compelling evidence suggests that the Warburg effect might result from mitochondrial dysfunction, activation oncogenes (e.g., Ras and Myc), oncogenic alterations (e.g., c-myc and p53), or environmental stresses (e.g., hypoxia and inflammation).15 Recent studies demonstrate that glycolysis not only generates ATP but also provides carbon-containing intermediates (such as pyruvate and glucose-6-phosphate) for cancer progression. These intermediates can be diverted to amino acid and ribose for nucleotide to supply the nutrients that the fast-growing cancer cells need. In addition, the overexpression of glycolysis in cancer cells will produce a lot of lactate, which can acidify the tumor microenvironment.16 The acidity of the microenvironment has been reported to promote cancer metastasis, which is probably mediated by pH-dependent activation of proteases, such as MMPs and cathepsins, that degrade the tumor extracellular matrix.17 Thus, targeting both mitochondrial OXPHOS and glycolysis to selective inhibition of energy metabolism of cancer cells may be an attractive strategy for the treatment of metastatic cancer. Butein (3,4,2′,4′-tetrahydroxychalcone) is a chalcone derivative extracted from the heartwood of Dalbergia odoriferau. Butein can also be identified in the barks of Rhus verniciflua and Semecarpus anacardiu.18 The extract of natural chalcone butein has been found with reported benefits including antioxidant, antiinflammatory, and antirestenosis activities.19−21 Emerging studies have shown that butein exhibits an antiangiogenesis property and antiproliferation and apoptotic effects against numerous lines of cancer cells.22−25 However, the effects of butein on cancer metastasis and the energy metabolism of cancer cells are not fully understood. The purpose of this study was to determine the antimetastatic effect and mechanism of butein in vitro and in vivo.
■
MATERIALS AND METHODS
Materials. Butein was purchased from Extrasynthese Corp. (Genay, France). Crystal violet and other chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). Rabbit monoclonal antibodies specific for p-ERK1/2, ERK1/2, and p-p70S6K, as well as mouse monoclonal antibody specific for p-IκBα, were obtained from Cell Signaling Technologies (Boston, MA, USA). Rabbit monoclonal antibodies specific for p-Akt, Akt, p-mTOR, p-NF-κB/p65, MMP-9, and GAPDH were obtained from Epitomics (Burlingame, CA, USA). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibody specific for uPA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other cell culture reagents were purchased from Gibco (Grand Island, NY, USA). Cell Culture. The human hepatocellular carcinoma (HCC) cell line SK-Hep-1 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). SK-Hep-1 cells were maintained in humidified air containing 5% CO2 at 37 °C with DMEM containing 10% FBS, penicillin (100 units/mL), streptomycin (100 μg/mL), and Lglutamine (2 mM). In Vitro Metastasis Analysis. Cell invasion and migration assays were performed using Transwell inserts (Corning, Corning, NY, USA) to assess in vitro metastasis of cancer cells. For invasion assay, filters were precoated with 30 μL of Matrigel (BD Biosciences, Bedford, MA, USA) for 30 min. Approximately 5 × 104 cells in 100 μL of serum-free medium were placed in the upper chamber, and 500 μL of the same medium containing 10% FBS in the absence or presence of butein was placed in the lower chamber in both invasion and migration assays. After 12 h of B
dx.doi.org/10.1021/jf502370c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 1. Effect of butein on cancer metastasis in vitro and iv vivo. SK-Hep-1 cells were seeded onto the upper chamber consisting of 8 mm pore size filters and then treated with the indicated concentration of butein for 12 h in medium containing 10% FBS as a chemoattractant in the lower chamber for the analysis of cell invasion (A) and migration (B). (C) SK-Hep-1 cells were treated with the indicated concentrations of butein for 12 and 24 h in medium containing 10% FBS, and the cell viability was determined using the MTT assay. (D) SK-Hep-1 cells were treated with the indicated concentrations of butein for 12 h and then subjected to CAM-intravasation assay coupled with the Alu PCR-based assay. The cancer cells in the chick embryo lower CAM were determined using PCR of the human genome-specific Alu sequence with chick GAPDH specific primers as the control. The quantitative detection for the relative amount of cell metastasis was represented and normalized against chick GAPDH. Data are expressed as the mean ± SEM of four independent experiments. (∗∗) p < 0.01 and (∗∗∗) p < 0.001 compared with the control group. luciferases were measured by DLR assay system (Promega, Madison, WI, USA) using FlexStation 3 (Molecular Devices). Statistics. Data are presented as the mean ± standard error of mean (SEM). Statistical analyses of data were done with one-way ANOVA followed by Student’s t test. The difference is significant if the p value is
