Glabridin Inhibits Migration,Invasion,and Angiogenesis of Human Non–Small Cell Lung Cancer A549 Cells by Inhibiting the FAK/Rho Signaling Pathway

Abstract

This study reports the antimigration, anti-invasive effect of glabridin, a flavonoid obtained from licorice, in human non–small cell lung cancer A549 cells. Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of A549 cells. In addition, glabridin also decreased A549-mediated angiogenesis. Further investigation revealed that glabridin’s inhibition of cancer angiogenesis was also evident in a nude mice model. Blockade of A549 cells migration was associated with an increase of ανβ3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 527), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked Akt activation, resulting in reduced activation of RhoA and myosin light chain phosphorylation. This study demonstrates that glabridin may be a novel anticancer agent for the treatment of lung cancer in 3 different ways: inhibition of migration, invasion, and angiogenesis.

Introduction

Lung cancer is one of the leading causes of death in the world, and non–small cell lung carcinoma (NSCLC) accounts for approximately 75% to 85% of all lung cancers. ¹ About 30% to 40% of patients with advanced lung cancer will develop metastasis, which is defined as lung tumor cells’ migration to and invasion of other organs, such as bones. ² Metastasized lung cancer is particularly challenging because it is highly resistant to radiation and conventional chemotherapeutic agents.^1,2 Consequently, novel therapeutic agents are needed to deal with the increasing incidence of human lung cancer.

Focal adhesion kinase (FAK)/Src signaling has been demonstrated to play a role in various cellular processes, including the immune function and cell differentiation, survival and motility.^3,4 Abnormal activation of FAK/Src signaling has been seen in invasive tumors. ⁵ Clustering of integrin by extracellular matrix (ECM) ligand can initiate intracellular signaling events, leading to changes of the intercellular parts of receptors and subsequent recruitment and autophosphorylation of FAK, followed by Src docking and phosphorylation.^6,7 Src is activated by phosphorylation at Tyr 416 and inhibited by phosphorylation at Tyr 527. Phosphorylated FAK/Src can activate several signaling cascades, such as extracellular signal–regulated kinases (ERKs) or Akt, resulting in the promotion of cell motility. ⁷

The root of Glycyrrhiza glabra (licorice) has been used for many centuries in Asia and Europe as an antioxidant, antidote, demulcent, expectorant, and a remedy for allergic inflammation, as well as a flavoring and sweetening agent. ⁸ Glabridin, an active isoflavan in the hydrophobic fraction of licorice root,^9,10 is easily incorporated into the gut cells and released to the basolateral side by aglycone form in human and mice.^11,12 It has shown multiple biological activities, such as antibacterial, neuroprotective, antiatherosclerotic, antiosteoporosis, and immunomodulatory activities.^9-17 Studies have shown that glabridin exhibits growth inhibition properties against breast cancer cells. ¹⁷ It also enhances the efficacy of cancer chemotherapy by inhibiting P-glycoprotein and multidrug resistance protein 1 synthesis. ¹⁸ However, the precise antitumorigenic mechanisms of glabridin in cancer migration and invasion still remain largely unknown.

Materials and Methods

Test Compound

Glabridin was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. The purity was >97%, as assessed by high-performance liquid chromatography. Control cultures received the carrier solvent (0.1% DMSO).

Cell Culture and Cell Proliferation Assay

A549 human lung adenocarcinoma cells, type II alveolar epithelial cells (American Type Culture Collection [ATCC] CCL185) were cultured in minimum essential medium (MEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, and 100 units/mL penicillin (Life Technologies, Inc., Grand Island, NY). HUVEC (human umbilical vein endothelial cells) culture was grown in EGM-2 medium (Lonza, Switzerland) at 37°C in an atmosphere of 5% CO₂. To obtain the conditioned medium (CM), cells were seeded 2 × 10⁶ cells/100 mm dish. The following day, the medium was replaced and the supernatants were harvested after 24 hours of incubation. Cell proliferation was assessed by Premixed WST-1 Cell Proliferation Reagent (Clontech Laboratories Inc., Mountain View, CA) according to the manufacturer’s instructions.

Cell Migration and Invasion Assay

Cell migration and invasion assay was conducted using QCM 24-well Cell Migration Assay and Invasion System, as previously described.^6,7 Briefly, 3 × 10⁴ cells were seeded into the top chamber and treated with different concentrations of glabridin. Ten percent FBS was added to the bottom wells for 24 hours as chemoattractant. At the end of the treatment, cells were poststained with CyQuant GR dye in cell lysis buffer for 15 minutes at room temperature. Then, fluorescence of the invaded cells was read using a fluorescence plate reader at excitation/emission wavelengths of 485/530 nm.

Scratch Wound–Healing Assay

A549 cells were allowed to grow into full confluence in 24-well plates. The following day, a uniform scratch was made down the center of the well using a micropipette tip, followed by washing once with phosphate-buffered saline. Vehicle control and various concentrations of glabridin were added to the respective wells for the indicated times. Photographic imaging was performed using an Olympus 1 × 50 inverted microscope.

Tube Formation Assay

In vitro angiogenesis assay was performed using BD Bio-Coat Angiogenesis System according to the manufacturer’s instructions. Briefly, HUVEC (2 × 10⁴ cells/mL) was seeded onto the Matrigel-precoated well present with or without glabridin in A549 conditioned medium. Tube formation was assessed after 18 hours and photographic imaging was performed using a fluorescent microscope (Nikon Eclipse TE 300, Germany).

Immunoblot/Immunoprecipitation

Cells (8 × 10⁶/dish) were seeded in a 10-cm dish. After 24 hours of incubation, the cells were treated with various concentrations of glabridin for the indicated times. Total cell extracts were prepared in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na₃VO₄, 0.5% NP-40, 1% Triton X-100, 1 mM PMSF, 5 µg/mL aprotinin, 5 µg/mL leupetin). Equivalent amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes. After the membrane was blocked in Tris-buffer saline containing 0.05% Tween 20 (TBST) and 5% nonfat powdered milk, the membranes were incubated with primary antibodies at 4° for 1 to 16 hours. After washing 3 times with TBST for 10 minutes each, the membranes were incubated with horseradish peroxidase–labeled secondary antibody for 1 hour. The membranes were washed again, and detection performed using an enhanced chemiluminescence blotting detection system (Amersham, Piscataway, NJ).

For immunoprecipitation, cell lysates (200 µg of total protein) were incubated with 2 µg of anti-FAK overnight, then 20 µL of protein A-agarose beads (Millipore, Bedford, MA) for 2 hours at 4°C. Association of FAK with Src was detected by incubating the blots with anti-Src antibodies (cell signaling).

Rho Activity Assay

Rho activity was analyzed by a Rho Activation Assay Kit (Upstate Biotechnology, Lake Placid, NY) according to the protocol supplied by the manufacturer. Cells were lysed in 500 µL of 1× Mg²+ lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA, and 2% glycerol) and centrifuged at 12000 rpm for 15 minutes at 4°C. Active (GTP/GTPγS-bound) Rho was isolated by pull-down assay: 50 µL (30 µg) of rhotekin Rho binding domain (RBD) agarose bead slurry (50 mM Tris, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM PMSF, and 1 µg/mL each of aprotinin and leupeptin) were added to each sample containing 500 µg total protein, and incubated for 45 minutes at 4°C with gentle agitation. The rhotekin–RBD agarose bead–Rho complex was then pulled down by centrifugation (10 seconds, 14 000g, 4°C). The supernatant was removed, and the beads were washed 3 times with 0.5 mL lysis buffers. Samples were subjected to immunoblot assay performed with anti-Rho antibody.

Matrigel Plug Angiogenesis Assay

Male nude mice (6 weeks old; BALB/cA-nu (nu/nu)) were purchased from the National Science Council Animal Center (Taipei, Taiwan) and maintained in pathogen-free conditions. Ten mice were randomly divided into 2 groups. A549 cells were trypsinized and resuspended at 3 × 10⁷ cells/mL in serum-free medium. Aliquots of cells (3 × 10⁶ cells) were mixed with 0.4 mL of phenol red-free Matrigel (BD Biosciences, San Jose, CA) and injected into both flanks of each nude mouse. For the glabridin-treated group, 10 µM glabridin was added to the cell suspensions, whereas Matrigel mixed with the medium alone was used as a negative control. Matrigel plugs were removed 15 days after implantation, weighed, and was determined content hemoglobin using a Drabkin’s reagent kit containing sodium bicarbonate, potassium ferricyanide, and potassium cyanide. Hemoglobin level measurements indicated blood vessel formation.

Statistical Analyses

Data were expressed as means ± SD of 3 determinations. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (P < .05) between the means of the 2 test groups were analyzed by Dunnett’s test.

Results

Glabridin Inhibits Migration, Invasion, and Epithelial–Mesenchymal Transition, but Does Not Affect the Proliferation of A549 Cells

We first assessed the effect of glabridin on the viability of A549 cells. As shown in Figure 1A, glabridin did not affect the cell viability of A549 cells at concentrations ranging from 1 to 10 µM.

Figure 1.

Glabridin inhibits lung cancer cell migration and invasion

To examine the effect of glabridin on human lung cancer cell migration, we employed transwell migration and wound-healing assay to characterize the cells’ migration response to glabridin. As shown in Figure 1B, culture medium increased the migration of A549 cells after 24 hours incubation, whereas glabridin treatment decreased the migration of A549 cells in a dose-dependent manner. Furthermore, scratch wound–healing assay also showed that glabridin decreased A549 cells’ migration ability after treatment for the indicated times (Figure 1C).

Next, we also assessed the effect of glabridin on lung cancer cell invasion. Compared with vehicle-treated cells, culture medium increased A549 cells’ invasion capability. However, glabridin treatment attenuated cell invasion in a dose-dependent manner (Figure 1D).

Epithelial–mesenchymal transition (EMT) is a crucial progression in the development of invasive cancer cells. ¹⁹ We assessed the effect of glabridin on EMT markers. Glabridin treatment increased E-cadherin levels, and decreased vimentin (Figure 1E).

Glabridin Inhibits Angiogenesis In Vitro and In Vivo

Because angiogenesis has been proven to be involved in cancer invasion and metastasis, we also tested whether glabridin inhibits angiogenesis in in vitro and in vivo models. As shown in Figure 2A, the conditioned medium of A549 cells caused the formation of the capillary-like HUVEC structures, but this phenomenon was blocked by glabridin treatment.

Figure 2.

Glabridin inhibits angiogenesis in vitro and in vivo

Next, we assessed the effect of glabridin on cancer angiogenesis by an in vivo model. A549 cells were mixed with Matrigel and injected into the flank of nude mice. Tissue sections showed that glabridin decreased the formation of functional blood vessels, characterized as red blood cell–containing capillary structures within the Matrigel plug (Figure 2B). In addition, relative angiogenesis was assayed by the hemoglobin content of the Matrigel plug. Compared with the Matrigel mixed with medium alone, angiogenesis of A549 cells was greatly increased, and hemoglobin levels in the Matrigel plugs containing A549 cells were higher than in Matrigel alone. Glabridin treatment inhibited A549 cell–induced angiogenesis, and hemoglobin levels in the glabridin-treated plugs were significantly lower than those of the solvent-treated plugs (Figure 2C). These results suggest that glabridin treatment decreases angiogenesis of A549 cells in an in vivo model.

Glabridin Inhibits Integrin Levels by Increasing Degradation in A549 Cells

Because integrins play an important role in cell migration,3 we assessed the intergrin expression in A549 cells after glabridin treatment. As shown in Figures 3A and 3B, glabridin decreased the expression of αν and β3 integrin in A549 cells.

Next, we assessed whether glabridin decreased the amount of integrin by increasing protein degradation. Cells were pretreated with proteasomal inhibitor MG-132 (10 µmol/L) and then treated them with glabridin for 3 hours. The results showed that glabridin decreased the levels of integrins (αν and β3). This effect was restored by MG-132 (Figure 3C), suggesting that glabridin increased integrin degradation in human NSCLC A549 cells.

Figure 3.

Glabridin increased the degradation of integrins

Glabridin Inhibits FAK/Src Signaling in A549 Cells

FAK is essential for the regulation of integrin-mediated cell adhesion and migration of cancer cells. ³ We therefore assessed the effect of glabridin on FAK signaling. As shown in Figure 4, glabridin decreased the phosphorylation of FAK at Tyr 397, 576, and 925 sites in A549 cells. However, glabridin did not cause any change in the protein levels of total FAK. Exposure of A549 cells to glabridin decreased active form Src (Tyr 416 phosphorylation) and increased inactive phosphorylation Src at Tyr 527. Similar responses were observed for the phosphorylated forms of FAK downstream targets, AKT (Thr 308 and Ser 473; Figure 4B). In addition, the association of FAK and Src increased in a time-dependent manner in glabridin-treated A549 cell, as detected by immunoprecipitation assay (Figure 4C).

Figure 4.

Glabridin inhibited FAK/Src signaling pathway

Mitogen-activated protein kinase (MAPK) family is also a target of FAK/Src signaling, so we next investigated the phosphorylation of JNK, p38 and ERK1/2 in A549 cells. The data showed that glabridin could not decrease the phosphorylation of JNK, p38 and ERK1/2 in A549 cells (Figure 4D).

Glabridin Decreases the Phosphorylation of Myosin Light Chain and Inhibits RhoA Activity in A549 Cells

We assessed the effect of glabridin on the phosphorylation of myosin light chain (MLC), which is involved in controlling cell migration. Glabridin decreased the phosphorylation of MLC in A549 cells (Figure 5A). In addition, glabridin also decreased the activity of Rho in A549 cells (Figure 5B).

Figure 5.

Glabridin decreases the phosphorylation of myosin light chain (MLC), and inhibits RhoA activity

Discussion

Lung cancer has been one of the leading causes of death in the world, and is associated with very poor prognoses even after tumor resection.^1,2 In our study, we have found that glabridin effectively inhibits lung cancer and the migration and invasion of blood endothelial cells concomitant with inhibition of integrin/FAK/Src pathway, and effectively inhibits in vivo tumor cell angiogenesis.

Overexpression of integrins in malignant cells contributes to cancer progression and metastasis by increasing tumor cell survival proliferation, migration, and invasion.^3,20 The integrin levels of normal and neoplasm tissues differ considerably. Several types of integrins, such as ανβ3, ανβ6, and α5β1, are usually present at low or undetectable levels in most epithelial tissue, whereas they are highly upregulated in cancers. ³ In contrast to quiescent endothelial cells, integrin ανβ3 is highly expressed in tumor angiogenic endothelial cells. ²¹ Moreover, the expression of integrins such as ανβ3, ανβ5, α5β1, α6β4, α4β1, and ανβ6 is correlated with cancer progression, patient survival and metastasis. ²² Our results show that glabridin decreased the amount of integrin αν and β3 in A549 cells. In addition, proteosome inhibitor MG-132 reversed glabridin-mediated downregulation of αν and β3 integrins in A549 cells, suggesting that glabridin can inhibit integrins, at least for ανβ3, by increasing the degradation of these integrins.

FAK/Src signaling has been implicated in extracellular matrix (ECM)/integrin-mediated signaling pathways, and plays an important role in tumor metastasis by increasing cell migration and invasiveness.^6,23 Autophosphorylated FAK (Tyr 397) binds to the SH2 domain of Src, relieving inhibitory interaction and leading to activation of Src. Conversely, activated Src phosphorylates additional sites on FAK, including residues Tyr 576 and Tyr 577, resulting in further increased activity of FAK. ²⁴ Activated FAK (Tyr 397)/Src (Tyr 416) transducts signaling through multiple downstream targets, such as PI3K/AKT and Ras/ERK1/2 cascades in cancer cells. ⁷ FAK also binds SH domain of PI3K, which in turn activates AKT kinase, thereby promoting cell migration by regulating various cell movement proteins.^7,23 The formation of FAK/Src complex allows Src to phosphorylate Tyr 925 on FAK to mediate its interaction with Grb2 (growth factor receptor–bound protein 2), leading to the activation of Ras-ERK signaling pathway. ²⁴ In our study, we found that glabridin decreased the phosphorylation of FAK at 397, 576, and 925 sites, and increased inactive phosphorylation Src at Tyr 527. Glabridin also caused that release of Src from FAK, resulting in the inactivation of FAK/Src complex. In addition, glabridin also decreased the phosphorylation of FAK/Src downstream kinase, Akt, in human NSCLC A549 cells. These data suggest that the cooperation of FAK/Src with Akt plays a crucial role in glabridin-mediated cell migration in human lung cancer.

Activation of Akt promotes cytoskeletal rearrangements and cell movement by the activation of guanine exchange factors P-Rex1 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1), Vav (CG7893 gene product from transcript CG7893-RB), Tiam1(T-cell lymphoma invasion and metastasis 1), and Sos (Son of sevenless), which in turn activate GTPase protein Rho family protein.^25,26 Rho family GTPases are important regulators of the cytoskeleton, and affect cell adhesion and migration. Activation of RhoA promotes actin stress fiber formation, focal adhesions–related protein clusters, cell body movement, and cell rear detachment.^27,28 Upregulation of RhoA in HUVEC significantly enhanced morphogenetic changes and cytoskeletal reorganization, resulting in enhanced cell migration and angiogenic capacity. ²⁹ Aberrant regulation of Rho proteins is associated with metastasis by promoting cancer angiogenesis and tumor cell motility. ²⁹ One of the major downstream effectors of RhoA with regard to the cell migration is Rho kinase (ROCK), which can phosphorylate myosin light chain (MLC) at Thr 18/Ser 19 and subsequently trigger myosin contraction, leading to cell migration. ³⁰ Our results show that glabridin treatment decreases the activation of Akt, followed by a decrease of Rho activity. In addition, exposure to glabridin also decreased the phosphorylation of myosin light chain at Thr 18/Ser 19. These findings indicate that glabridin can be an effective inhibitor of AKT/Rho/MLC signaling in lung cancer cells. FAK has been demonstrated to promote cell migration by decreasing RhoA activity. ³¹ In contrast, FAK has also been reported to stimulate RhoA activation by an increase of p190RhoGEF phosphorylation. It remains unknown why both FAK and loss of FAK can stimulate RhoA activation. This may be because of the fact that FAK may regulate RhoA via multiple mechanisms in cell migration. ³²

Conclusion

To summarize, we have provided evidence demonstrating that glabridin inhibits cancer cell migration and invasion as well as angiogenesis in vivo. Glabridin inhibits the ability of A549 to migrate and invade by decreasing integrin levels and sequentially reducing the activation of FAK/Src/AKT/Rho in these cells. Therefore, glabridin is a potentially useful antiinvasive agent in the treatment of human lung carcinoma.