Baicalein, a natural flavonoid obtained from the rhizome of
Melanoma is a cancer consisting of pigment-forming melanocytes of the skin, which are normally found in the basal layer of the epidermis (Fernandez, 2015). Melanoma is less common than other types of skin cancer; however, it causes the majority of skin-cancer-related deaths. Melanoma is an aggressive cancer with a high propensity for metastasis, and metastatic melanoma is characterized by a high mortality rate (Leiter
Metastasis has been widely recognized as a primary cause of cancer-related mortality. Metastasis is a highly coordinated multistep process, of which degradation of the extracellular matrix (ECM) is an important component (Khasigov
Metastasis is also regulated by the cellular signaling pathways, which have been implicated in a number of cellular functions, including cell survival, adhesion, and invasion. The phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway plays an especially important role in the invasion of cancer cells via regulation of the expression of MMP-2 and -9 by transcriptional factors, including nuclear-factor-kappaB (NF-κB) (Park
In addition, tight junction (TJ) represents one mode of cell-to-cell adhesion in endothelial cells. TJ establishes contact between adjacent cells, which serves as a physical barrier to the maintenance of cell polarity (Schneeberger and Lynch, 2004). Because TJ becomes disorganized or lost while normal epithelial cells are transforming into cancerous cells, the alteration and loss of cell polarity are hallmarks of tumorigenesis in epithelial cells (Soler
Baicalein (5,6,7-trihydroxyflavone) is a flavonoid originally isolated from the rhizome of
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics were purchased from Welgene (Daegu, Korea). Baicalein, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT), hematoxylin and eosin (H&E), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Antibodies against claudin-1 (519000), -2 (516100), -3 (341700), and -4 (329400) were obtained from Calbiochem (San Diego, CA, USA). Akt (sc-8312), phosphorylated (p)-Akt (sc-101629), tissue inhibitors of metalloproteinase (TIMP)-1 (sc-5538) and TIMP-2 (sc-5539), MMP-2 (sc-10736) and MMP-9 (sc-10737), and β-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Amersham Corp. (Arlington Heights, IL, USA). All other chemicals were purchased from Sigma-Aldrich Chemical Co.
B16F10 mouse melanoma cells were obtained from the American Type Culture Collection (Manassas, MD, USA) and maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin) at 37°C in a humidified incubator under an atmosphere of 5% CO2 in air. Baicalein was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 100 mM, which was then diluted with RPMI1640 medium to the desired concentration prior to use.
Cell survival was assessed by performing a standard MTT assay. Briefly, B16F10 cells were plated in 6-well culture plates at a density of 2×105 cells per well. After 24 h, the cells were treated with various concentrations of baicalein for 24 h. Next, the cells were washed twice with phosphate buffered saline (PBS) and incubated with 0.5 mg/ml MTT solution for 3 h. The cells were subsequently washed with PBS and solubilized in DMSO, and the optical density of each well was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 540 nm. The effect of baicalein on cell growth was assessed as the percentage of cell viability, in which the vehicle-treated cells were considered 100% viable.
B16F10 cells were harvested after treatment with or without baicalein for 24 h. The cells were reseeded in each new 6-well plate at a density of 2×102 with complete medium for 15 days. The medium was replaced every 3 days to maintain the cells’ growth. The colonies were then washed twice with PBS, fixed with 3.7% paraformaldehyde, and stained with 0.1% toluidine blue for 30 min at room temperature.
B16F10 cells were grown to confluence on 6-well plates coated with 20 μg/ml of rat tail collagen (BD Biosciences, Bedford, MA, USA). Confluent cells were wounded by scraping with a 200 μl pipette tip. After wounding, the cells were washed twice with PBS and incubated with 1% FBS-containing medium supplemented with various concentrations of baicalein for 24 h. Thereafter, the wounding areas were observed and photographed under an inverted microscope (Carl Zeiss, Deisenhofen, Germany) at 40x magnification.
The activities of MMP-2 and -9 were determined by gelatin zymography. Briefly, after treatment with baicalein for 24 h, the media were collected and clarified by centrifugation at 400 g for 5 min at 4°C to remove cells and debris. The cell-free supernatant was mixed with a 2X nonreducing sample buffer (Invitrogen Co., Carlsbad, CA, USA), and electrophoresis was performed using precast gel (10% polyacrylamide and 0.1% gelatin as a protease substrate) (Invitrogen Co.). Following electrophoresis, the gel was washed twice in 2.5% Triton X-100 for 1 h to remove sodium dodecyl sulfate (SDS), subsequently washed in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 1 μM ZnCl2, and 0.02% NaN3 at pH7.5, and incubated in this buffer at 37°C for 24 h. Thereafter, the gel was stained for 1 h with 0.5% (w/v) Coomassie brilliant blue G-250 (Bio-Rad Laboratories, Hercules, CA, USA) and then lightly destained in methanol:acetic acid:water (3:1:6). The areas of gelatinolytic activity were then manifested as horizontal white bands on a blue background (Lee
Transepithelial electrical resistance (TER), a measure of TJ formation, was measured with an Epithelial Tissue Volt-Ohm meter (EVOM; World Precision Instruments, Sarasota, FL, USA) equipped with a pair of STX-2 chopstick electrodes. Briefly, B16F10 cells were seeded in the 8.0 μm pore size insert (upper chamber) of a Transwell® (Corning Costar Corp.) and allowed to reach full confluence, after which fresh medium was substituted for further experiments. Inserts without cells, inserts with cells in medium, and inserts with cells with baicalein were treated with baicalein for 24 h. Electrodes were placed at the upper and lower chambers, and resistance was measured with the EVOM.
Total RNA was isolated from cells using an RNeasy kit (Qiagen, La Jolla, CA, USA) and primed with random hexamers to synthesize complementary DNA using AMV Reverse Transcriptase (Amersham Corp.). The PCR was carried out in a Mastercycler (Eppendorf, Hamburg, Germany) using primers (Table 1). Conditions for the PCR reactions were 1× (94°C for 3 min), 35× (94°C for 45 s; 58°C for 45 s; and 72°C for 1 min), and 1× (72°C for 10 min). Amplification products obtained by PCR were electrophoretically separated on a 1% agarose gel and visualized by ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) staining.
Following treatment with various concentrations of baicalein, the cells were washed with ice-cold PBS and harvested and lysed with a lysis buffer (20 mM sucrose, 1 mM ethylendiaminetetraacetic acid, 20 μM Tris-Cl, pH 7.2, 1 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl2, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, and 2 μg/ml aprotinin) for 30 min at 4°C. The supernatants were collected and protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. For Western blotting, equal amounts of protein extracts were denatured by boiling at 95°C for 5 min in a sample buffer (0.5 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, and 10% β-mercaptoethanol) at a ratio of 1:1. Samples were stored at −80°C or immediately used for immunoblotting. The equal cellular proteins were separated by denaturing SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes (Amersham Corp.). The membranes were then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies, probed with enzyme-linked secondary antibodies for 1 h at room temperature, and detected using an enhanced chemiluminescence (ECL) detection system.
All data are presented as mean ± standard deviation (SD). Significant differences among the groups were determined using the unpaired Student’s
In order to investigate the effect of baicalein on the viability of B16F10 cells, cells were treated with various concentrations of baicalein for 24 h and subjected to an MTT assay. When compared with the untreated control, treatment with 40 μM baicalein resulted in approximately 87% inhibition of cell growth (Fig. 1). However, cell viabilities were not significantly changed by baicalein at concentrations below 40 μM; therefore, a concentration range of baicalein <30 μM was selected for the subsequent experiments.
Next, we investigated whether baicalein, at noncytotoxic concentrations, influences anchorage-dependent colony formation of B16F10 cells after seeding at a low density. As shown in Fig. 2A, 2B, untreated control B16F10 cells showed rapid proliferation and formed sizable colonies from a single cell. However, baicalein treatment during incubation suppressed the colony-forming activity and reduced the number of sizable colonies and colony sizes in a dose-dependent manner. These data indicate that baicalein blocks the establishment of anchorage-dependent colonies from a single cell at the same level as growth-independent anchorage.
To investigate the inhibitory effect of baicalein on the migration of B16F10 cells, the confluent monolayer was scraped to create a scratch wound, and the cells migrated to the denuded zone and the levels of wound closure area were analyzed after 24 h incubation with baicalein. As shown in Fig. 2C, 2D, the results indicate that treatment with baicalein significantly reduces migration of B16F10 cells to the denuded zone by comparison with control cells in a concentration-dependent fashion, indicating that baicalein inhibits the motility of B16F10 cells.
In addition, we attempted to determine whether the inhibitory effects of baicalein on cell migration are connected to the decreases in B16F10 cell invasion using a Matrigel invasion assay. The results, which are consistent with those of the wound-healing assay, demonstrate that baicalein significantly inhibits the invasion of B16F10 cells and that these effects are dose dependent (Fig. 3).
Because the activation of MMP-2 and -9 is crucial for ECM degradation, which is required for tumor invasion and metastasis (Vu and Werb, 2000; Egeblad and Werb, 2002), gelatin zymography, RT-PCR and Western blot analyses were conducted to assess whether baicalein regulates activation and expression of MMP-2 and -9 in B16F10 cells. As indicated in Fig. 4A, baicalein inhibited the activities of MMP-2 and -9, and that the effects occur in a dose-dependent manner (Fig. 4B, 4C). In contrast, baicalein treatment concentration-dependently increased the levels of TIMP-1 and -2 mRNA and protein compared with the control group (Fig. 4B, 4C). These results indicate that the anti-invasive effect of baicalein is associated with increased TIMPs levels as well as inhibition of both enzyme activities and expressions of MMP-2 and -9 in B16F10 cells.
Because altered TJ leads to a decrease in resistance to electrical current (as measured by TER) and an increase in paracellular permeability (Soler
Because several lines of evidence have implicated the PI3K/Akt signaling pathway in the expression of MMPs and induction of cancer cell metastasis (Park
The endotoxin LPS, a major component of the outer membrane in gram negative bacteria, is one of the most potent activators of the PI3K/Akt signaling pathway for the stimulation of cancer cell metastasis, and its activation mechanism is relatively well established (Hsu
Baicalein is known to exhibit anticancer effects in various types of cancer cells (Lee
Metastasis, the main cause of death in patients with cancer, is a complex multistep process involving cell adhesion, invasion, and migration. MMPs that are highly expressed in various malignant tumors have been recognized as playing an important role in cell motility and invasion via the degradation of ECM components of blood or lymph vessels (Khasigov
On the other hand, there is an association between the loss of cell-cell adhesion structures and metastasis in many cancers. In precancerous lesions, the tissue remodeling performed by the disassembled and disorganized TJs as determined by decreased resistance to TER causes a loss of cell polarity and in turn promotes cancer cell motility and invasiveness (Soler
By regulating the transcriptional activity of MMPs, numerous cell-signaling pathways play a critical role in the regulation of cancer cell migration and invasion (Park
In summary, the study found that baicalein significantly inhibits migration and invasion in B16F10 melanoma cells by suppressing MMP-2 and -9 expression and activity. The study’s data also show that baicalein increases TJ tightening associated with the downregulation of claudin expression, which is associated with inactivation of the PI3K/Akt signaling pathway. Thus, the findings of the present study indicate that baicalein is a potential candidate for the development of chemotherapeutic treatments for melanoma.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (2015R1A2A2A01004633) and the Functional Districts of the Science Belt support program, Ministry of Science, ICT and Future Planning.