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Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells Aeyung Kima , Minsik Sonb , Keun Il Kima , Young Yanga , Eun Young Songc , Hee Gu Leec , Jong-Seok Lima,⁎ a
Department of Biological Science and the Research Center for Women's Diseases, Sookmyung Women's University, Chungpa-Dong, Yongsan-Gu, Seoul, 140-742, Republic of Korea b Department of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea c Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-608, Republic of Korea
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
Thymosin β4 (Tβ4) is a major actin-sequestering protein that has been implicated in the growth,
Received 18 April 2009
survival, motility, and metastasis of certain tumors and is considered an indicator for malignant
Revised version received
progression. Therefore, identifying compounds that can downregulate Tβ4 expression is very
22 May 2009
important for the development of anti-cancer chemotherapies. In this study, we investigated the
Accepted 22 May 2009
effects of elevated cAMP on Tβ4 expression and the metastatic potential of murine B16 melanoma
Available online 3 June 2009
cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tβ4 suppression. We found that treatment with the cAMP-inducing compounds α-MSH (α-melanocyte stimulating
Keywords:
hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tβ4 expression and
Cyclic AMP
regulated EMT-associated genes through the suppression of NF-κB activation in B16F10 cells. Along
Thymosin β4
with decreased Tβ4 expression, the in vitro invasiveness and anchorage-independent growth in a
EMT
semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of
E-cadherin
the α-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control
N-cadherin
cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tβ4
MMP
expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover,
Metastasis
suppression of NF-κB activation by α-MSH or IBMX is critical for inhibiting Tβ4 expression. © 2009 Elsevier Inc. All rights reserved.
Introduction The epithelial–mesenchymal transition (EMT) is a process by which cells lose their epithelial features and concomitantly acquire a mesenchymal phenotype. EMT is critical for both normal embryonic development and for the progression of non-invasive adenomas into malignant and metastatic carcinomas [1]. EMT can be defined by the loss of cell–cell adhesion, the induction of extracellular matrix (ECM) degradation, and cytoskeletal reorga-
nization. E-cadherin expression or function is lost during the progression of most carcinomas [2]. The downregulation of Ecadherin leads to a reduction or loss of cell–cell interactions and the detachment of malignant cells from the primary tumor. Conversely, metastatic phenotypes are impaired by the forced expression of E-cadherin in cultured cancer cells and transgenic mouse models [3]. In malignant progression, E-cadherin can be functionally inactivated by mutation and downregulated by promoter hypermethylation or transcriptional repression [4,5]. It
⁎ Corresponding author. Fax: +82 2 2077 7322. E-mail address: [email protected] (J.-S. Lim). Abbreviations: EMT, epithelial–mesenchymal transition; MMP, matrix metalloproteinase; α-MSH, alpha-melanocyte stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine; VEGF, vascular endothelial growth factor; PKA, protein kinase A; AC, adenyl cyclase; PDE, phosphodiesterase 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.05.024
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has been reported that transcription of E-cadherin is suppressed by factors such as Snail, Slug, δEF1/ZEB1, and SIP1/ZEB2 in different type of cancers including melanoma [6–8]. In addition, it has been demonstrated that Akt-mediated NF-κB activation represses Ecadherin by Snail upregulation and induces EMT [9]. Therefore, identifying the mechanisms that block the loss of E-cadherin expression is critical for preventing malignant progression via suppression of the EMT. Thymosin β4 (Tβ4), a 43-amino acid polypeptide originally described as a thymic maturation factor, is known to stimulate tissue remodeling, cell differentiation, and cell and tissue healing after injury [10,11]. In recent studies, the expression of Tβ4 has been found to induce angiogenesis, accelerate wound healing and enhance metastatic potential of various tumor cells through its effects on the cytoskeleton. For example, Clark and colleagues found that the metastatic potential of murine B16 melanoma cells increased when they repeated an in vivo selection scheme and that Tβ4 levels were elevated in highly metastatic B16F10 cells that were cultured from the lung tumors [12]. Of particular interest is the finding by Cha et al. that the overexpression of Tβ4 increases the number of metastatic tumors and the expression of an angiogenic factor, vascular endothelial growth factor (VEGF) [13]. Furthermore, it has been reported that Tβ4 overexpression induces the loss of E-cadherin, a hallmark of the EMT, and contributes to tumor cell invasion and metastasis. Wang et al. have shown that the overexpression of Tβ4 results in a drastic change in cell growth and motility that is accompanied by a downregulation of Ecadherin [14]. They also have demonstrated that Tβ4 overexpression is tightly associated with an increase in the invasiveness of SW480 colon carcinoma cells through an increase of MMP-7 activity, as well as the distant metastasis of human colorectal carcinomas [15]. In addition, they suggested a novel role for Tβ4 in the promotion of colorectal carcinoma progression through the induction of the EMT in tumor cells by upregulating integrinlinked kinase [16]. The growth rate and paclitaxel-resistance of HeLa cells were increased by Tβ4 overexpression [17], while their apoptosis induced by this drug was accelerated following a decrease in Tβ4 [18]. However, little is known about the upstream molecules or signaling mechanisms that modulate Tβ4 expression and therefore affect cancer progression. Cyclic AMP (cAMP) is a ubiquitous signaling molecule that influences cell proliferation, survival, and differentiation partially through the activation of protein kinase A (PKA) [19]. Intracellular cAMP concentrations are regulated by both adenyl cyclase (AC), which uses ATP to produce cAMP, and phosphodiesterases (PDEs), which catalyze the degradation of cAMP to AMP [20,21]. While the role of cAMP in promoting pigmentation is indisputable, its involvement in cancer progression is controversial dependent on cell type and context. For example, the constitutive activation of cAMP by the mutation of genes involved in cAMP signaling has resulted in endocrine neoplasia [22]. In addition, dibutyryl cAMP has been shown to slightly enhance collagen-mediated keratinocyte migration [23], and an increase of cAMP levels has been shown to increase the expression and activity of MMP-2 [24]. In contrast, it has been reported that increased cAMP inhibits growth factor-mediated MMP-9 induction and keratinocyte migration [25], and treatment with α-melanocyte stimulating hormone (αMSH) potently inhibits both in vitro invasion through a reconstituted basement membrane and the in vivo pulmonary metastasis of highly invasive B16–BL6 murine melanoma cells [26].
However, the molecular mechanisms, especially those based on the regulation of EMT, of the cAMP–PKA pathway that suppresses metastasis of melanoma cells are not well understood. In the present study, we evaluated the influence of intracellular cAMP on Tβ4 expression, the EMT regulation, and the subsequent invasive potential in the highly invasive murine melanoma cell line B16F10 using α-MSH or IBMX as an activator of AC or an inhibitor of PDE, respectively. Our results clearly demonstrate that an increase in intracellular cAMP inhibits the expression of Tβ4 via the suppression of NF-κB activation and blocks the loss of E-cadherin, thereby attenuating the metastatic potential of the melanoma cells.
Materials and methods Mice and cell cultures The murine B16F10 melanoma cell line was maintained as a monolayer culture in Dulbecco's Modified Eagle's Medium (DMEM; Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco/Invitrogen), 100 U/ml penicillin/100 μg/ml streptomycin (Gibco/Invitrogen), in a humidified 5% CO2 incubator at 37 °C. Specific pathogen-free female C57BL/6J mice were purchased from Daehan BioLink (Umsong, Republic of Korea) and were maintained in our animal facility for at least 2 weeks before use. The mice were housed under specific pathogen-free conditions in a barrier facility with a 12hour light/dark cycle.
Antibodies and chemicals α-Melanocyte stimulating hormone (α-MSH), 3-isobutyl-1methylxanthine (IBMX), and forskolin were purchased from Sigma (St. Louis, MO). The specific cell-permeable inhibitor of protein kinase A, KT5720, was obtained from Merck (Darmstadt, Germany) and rhodamine phalloidin, a high-affinity probe for Factin, was purchased from Invitrogen. Recombinant murine TNF-α was obtained from ProSpec Tany TechnoGene (Rehovot, Israel). The anti-IκBα and anti-phospho-IκBα (Ser32) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against thymosin β4 (Tβ4) and E-cadherin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-N-cadherin antibody was purchased from Sigma. Synthetic Tβ4 peptide was provided by Immundiagnostik AG (Bensheim, Germany).
Measurement of melanin content B16F10 melanoma cells were seeded at a density of 2.5 × 105 cells/ 100 mm culture plate. After incubation in the presence of cAMPinducing agents, total melanin content in the cell pellet (1 × 107 cells per sample) was measured. Briefly, the pellets were dissolved in 1 N NaOH/10% DMSO for 1 h at 80 °C, and solubilized melanin was measured at OD475. Melanin content was calculated from a standard curve using synthetic melanin.
Determination of intracellular cAMP level The cAMP concentration was measured using a Direct Cyclic AMP Enzyme Immunoassay Kit produced by Assay Designs™ Inc. (Ann Arbor, MI). Briefly, cells were lysed in 0.1 M HCl to inhibit the
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phosphodiesterase activity, and then the supernatants were collected. Following neutralization and dilution, a fixed amount of cAMP direct conjugate was added to compete with the cAMP in the cell lysates for sites on a rabbit polyclonal antibody immobilized on a 96-well plate. After adding cAMP antibody into each well, the plate was incubated at room temperature for 2 h. Thereafter, the plate was washed and a pNPP (p-nitrophenyl phosphate) substrate solution was added into the wells. After the enzyme reaction was stopped, the optical density was read on a microplate reader at 405 nm. The intensity of the bound yellow color was inversely proportional to the concentration of cAMP in the cell lysates.
Immunofluorescence analysis Cells were grown to ∼60% confluency on poly-L-Lysine (0.01% solution, Sigma)-coated cell culture coverslips (Thermanox Plastic Coverslips, NUNC, Rochester, NY) for 24 h and incubated in serum-free medium for an additional 6 h. Cells were then treated with α-MSH or IBMX for 24 h. Cells were fixed with 2% formaldehyde in PBS for 10 min and washed with a PBS/serum solution (10% fetal bovine serum, 0.05% azide in PBS) for 5 min. The fixed cells were then incubated with tetramethylrhodamine (TRITC)-conjugated phalloidin (diluted 1:1000 in PBS/serum containing 0.2% saponin) for 45 min at room temperature. Cells were then washed with a PBS/serum solution three times for 5 min and the coverslips were mounted on glass slides with VECTASHIELD (mounting medium with DAPI, Vector Laboratories, Burlingame, CA).
RNA extraction and RT-PCR Total RNA was extracted from the cells using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions and was reverse transcribed to cDNA using M-MLV reverse transcriptase (Promega, Madison, WI) and an oligo(dT) primer. cDNA aliquots corresponding to 5 μg RNA were analyzed by semi-quantitative PCR. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide (EtBr).
Gelatin zymography The enzymatic activity of MMP-2 and MMP-9 was analyzed by gelatin zymography. Cells were incubated in serum-free medium for 24–48 h and equivalent volumes of the culture supernatants were electrophoresed in a 10% SDS-polyacrylamide gel containing 0.1–0.2% gelatin. The gels were washed twice with washing buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2.5% Triton X-100) and then treated with incubation buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.02% NaN3, 1 μM ZnCl2) for 18–36 h at 37 °C. The gels were then stained with a staining solution (0.05% Coomassie blue, 10% isopropanol, 10% acetic acid) and destained (10% isopropanol, 10% acetic acid). MMPs were detected as transparent bands on the blue background of the Coomassie blue-stained slab gels.
Soft agar colony formation assay In order to measure anchorage-independent cell growth,1 × 104 cells were suspended in 3 ml of DMEM containing 0.3% agar and 10% FBS
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and applied onto 3 ml pre-solidified 0.6% agar in 60-mm culture dishes. Following 2 weeks of incubation, the colonies on soft agar were observed with a phase contrast microscope and photographed.
In vitro tumor cell invasion assay The in vitro invasion assay was performed using a 24-well transwell unit with polycarbonate filters that have a diameter of 6.5 mm and a pore size of 8.0 μm (Corning Costar, Cambridge, MA). For the invasion assay, the transwell was coated with 20 μl of a 1:2 mixture of Matrigel:DMEM (Matrigel; BD Biosciences, Bedford, MA) and used as the intervening invasive barrier in a 24-well plate. Cells (5 × 104) were suspended in serum-free DMEM and added to the upper chamber of the transwell. The lower chamber of the transwell was filled with DMEM supplemented with 5% FBS as a chemoattractant. After incubation for 24 h at 37 °C, cells attached to the upper surface of the filter were completely removed by wiping with a cotton swab and the filters were stained with a 0.2% crystal violet/20% methanol (w/v) solution. Alternatively, the cell number was determined using calcein-acetomethylester (calcein-AM, Molecular Probes, Eugene, OR), which can be cleaved by an intracellular esterase to produce calcein, a strongly fluorescent compound. Fluorescence intensity was measured with a fluorescence microplate reader (PerkinElmer Victor, Fremont, CA). The background fluorescence was corrected by subtracting the value of the control well without cells.
In vivo experimental metastasis assay The in vivo metastatic potential of the cells was measured using the lung colonization assay. In brief, cells were injected into the tail vein of mice at a density of 3 × 105 cells/0.2 ml of PBS. Twenty days later, the mice were sacrificed, lungs were fixed with Bouin's solution (Sigma), and the metastatic colonies on the lung surface were observed macroscopically.
Western blot Whole cell lysates were prepared by lysing the cells on ice in MPER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL) for 30 min. The supernatant was recovered following centrifugation (14,000 g, 20 min, 4 °C) and the protein concentration was determined using the BCA assay. Proteins were visualized after immunoblotting using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech) and a LAS 3000 imaging system (FUJIFILM Corporation, Tokyo, Japan). The band intensity was analyzed using TINA 2.0 software. For the detection of the 5 kDa Tβ4 protein, proteins were electrophoresed in NuPAGE Novex 4–12% Bis–Tris Gels (Invitrogen) using the XCell SureLock Mini-Cell according to manufacturer's instructions.
Transient transfection and luciferase reporter assay Semi-confluent cells that were grown in 12-well plates were transiently transfected with a pNF-κB-Luc reporter plasmid (Stratagene, La Jolla, CA) and the pCMV/β-Gal plasmid using the Lipofectamine 2000 reagent (Invitrogen). pCMV/β-Gal was used for normalizing the transfection efficiency. After incubation for 18 h, cells were harvested in passive lysis buffer and the luciferase activity was measured using a VICTOR3 1420 multilabel counter
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with the luminescence microplate reader set (PerkinElmer Victor) and the Luciferase Assay System according to the manufacturer's instructions (Promega). The β-galactosidase activity was measured using o-nitrophenyl-β-galactopyranoside (ONPG) as a substrate and luciferase activity was calculated relative to the β-galactosidase activity. The transient transfection of dominant negative forms of IKKα (dnIKKα) and IKKβ (dnIKKβ) was also performed using the Lipofectamine 2000 reagent. The transfected cells were cultured in complete medium for 24 h and then used as indicated in each experiment. dnIKKα and dnIKKβ were gifts from Dr Jang-Soo Chun (Gwangju Institute of Science and Technology, Gwangju, Republic of Korea).
Statistical analysis Results are presented as means ± standard deviation (SD). All experiments were repeated at least three times and data were analyzed for statistical significance using Student's t test. P values less than 0.05 were considered significant.
Results cAMP elevating agents inhibit MMP-9 activity, anchorage-independent cell growth, in vitro Matrigel invasion, and in vivo pulmonary metastasis of melanoma cells In order to examine the effects of intracellular cAMP levels on the phenotypic changes observed in malignant cancer, we used αMSH or IBMX to increase cAMP levels in murine B16F10 melanoma cells, which are highly metastatic in syngeneic C57BL/6J mice. As shown in Fig. 1A, the treatment of B16F10 cells with the indicated concentration of α-MSH or IBMX did not affect the rate of cell proliferation. However, it dramatically increased intracellular cAMP level, tyrosinase mRNA expression, and melanin synthesis (Figs. 1A–C). As shown in Fig. 1D, treatment with α-MSH or IBMX almost completely suppressed gelatinolytic MMP-9 secretion in melanoma cells, compared to untreated control cells. In addition, the ability of the cells to form colonies in a semi-solid medium, a marker of anchorage independence that is positively correlated with metastatic potential, was significantly decreased by treatment with α-MSH or IBMX (Fig. 1E). While untreated control cells formed sizable colonies in the soft agar, α-MSH- or IBMX-treated cells had a significantly decreased ability to form colonies in soft agar, including both a smaller number of sizable colonies and a reduced colony size. The inhibition of colony formation was clearly not due to a decreased growth rate as shown in Fig. 1A. Increased melanin synthesis mediated by the increase in intracellular cAMP levels after treatment with α-MSH or IBMX was confirmed as black colonies in soft agar. Along with decreased MMP-9 secretion, the invasive potential, which is determined by the ability of the cell to invade a Matrigel barrier, was significantly suppressed by αMSH or IBMX treatment (Fig. 1F). In order to confirm the inhibitory effect of cAMP elevating agents on tumor metastasis in vivo, we examined the ability of B16F10 cells to colonize the lungs after intravenous injection. Cells treated with α-MSH or IBMX induced a decreased number of colonies in the lung compared to untreated control cells, suggesting that increased intracellular cAMP can inhibit the metastatic potential of B16F10 cells in vivo (Fig. 1G).
cAMP elevating agents elicit a morphological transition and F-actin reorganization Interestingly, changes in cell morphology following α-MSH or IBMX treatment were observed using phase contrast microscopy (Fig. 2A). The increase in cAMP level elicited a morphological transition to contracting cells with short processes and induced an increase of cell–cell contact. In order to analyze the changes in actin polymerization, we examined the cellular distribution of Factin in α-MSH- or IBMX-treated cells by staining with TRITCconjugated phalloidin. Upon plating, untreated control cells spread across the L-lysine-coated slides and the actin filaments were homogeneously distributed throughout the cytoplasm and surrounded the external boundary of the cells. In contrast, compared to untreated control cells, treatment with α-MSH or IBMX decreased the spread of cells on the slides with a visible alteration in shape and reduced the staining intensity for F-actin (Fig. 2B). Interestingly, the morphology of B16F1 melanoma (a low metastatic variant) was distinct from that of B16F10 melanoma (a high metastatic variant), but was similar to α-MSH or IBMX-treated B16F10 cells (Fig. 2C). Moreover, the intracellular cAMP level in B16F1 cells was significantly higher than that in B16F10 cells. It has been reported that EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases (MMPs) [27–29]. Additionally, since the EMT facilitates intravasation of tumor cells into blood or lymph vessels and the subsequent formation of distant metastases, we investigated the inhibitory mechanism of cancer metastasis by cAMP based on the regulation of the EMT.
cAMP elevating agents decrease thymosin β4 expression and regulate the expression of EMT-related genes Several genes that are selectively upregulated in metastatic mouse and human melanoma cells compared with their non-metastatic counterparts have been identified using DNA microarrays. Clark et al. has reported that the expression of fibronectin, thymosin β4 (Tβ4) and RhoC was increased in all human and mouse melanomaderived metastases [12]. Tβ4 binds to monomeric actin, a component of the cytoskeleton, and may act as an “actin buffer”, preventing the spontaneous polymerization of actin monomers into filaments but supplying a pool of actin monomers when the cell needs filaments. Since several studies have suggested that Tβ4 is involved in wound healing, angiogenesis, and tumor cell metastasis [11,13,15,16], we first examined the level of Tβ4 mRNA level in both B16F1 and B16F10 melanoma cells. As shown in Fig. 3A, the mRNA expression level of Tβ4 was considerably elevated in B16F10 cells compared to B16F1 cells. It has been reported that Tβ4 overexpression triggers an EMT by downregulation of E-cadherin, a type I transmembrane protein important in the formation of adherence junctions [16]. As shown in Fig. 3A, B16F10 cells expressed an increased level of mesenchymal markers, such as vimentin, N-cadherin, and fibronectin, as well as E-cadherin suppressors, such as SIP1 and Slug, compared to B16F1 cells. In contrast, the expression level of epithelial marker, E-cadherin, was low in B16F10 cells. Interestingly, the expression of Tβ4, SIP1, Slug, and mesenchymal markers was significantly reduced following αMSH or IBMX treatment, while E-cadherin mRNA expression was significantly increased. As shown in Fig. 3B, decrease of Tβ4 and Ncadherin, and increase of E-cadherin by α-MSH or IBMX were also
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Fig. 1 – α-MSH or IBMX increases intracellular cAMP and suppresses metastatic potential of B16F10 cells. (A) B16F10 cells were incubated with α-MSH (0.5, 1 μM) or IBMX (50, 100 μM) for 24 h and then cell proliferation was determined using the MTT assay. Melanin content was also determined and represented as percentage value. (B) mRNA expression of tyrosinase was measured by RT-PCR using specific primers and appearance of each cell pellet was shown after centrifugation. (C) The cAMP concentration after treatment with α-MSH (1 μM) or IBMX (100 μM) for 30 min was measured as described in ‘Materials and methods’. (D) Cells were incubated in serum-free media for 24–48 h with 1 μM α-MSH or 100 μM IBMX and the conditioned medium was subjected to gelatin zymography for the detection of MMP-2 and MMP-9 activity. (E) In order to examine anchorage-independent cell growth, the soft agar colony formation assay was performed. The colonies in soft agar were observed using a phase contrast microscope. The relative colony size was calculated after measuring the diameter of at least ten colonies. ⁎P < 0.01 versus untreated control cells. (F) A fixed number of cells were plated in the upper chamber of the Matrigel-coated transwell. After incubation for 24 h, the cells invading to the lower surface of the membrane were stained with a 0.2% crystal violet/20% methanol (v/v) solution and observed using a phase contrast microscope. The invading cells were also examined by using calcein-acetomethylester as a substrate for intracellular esterase. ⁎P < 0.01 versus untreated control cells. (G) In order to evaluate the in vivo metastatic potential of the cells, α-MSH or IBMX-pretreated (24 h) cells were injected into the tail vein of mice (3 × 105 cells/0.2 ml of PBS). Twenty days later, the metastatic colonies on the lung surface were counted macroscopically. Data are representative of three independent experiments.
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Fig. 2 – Changes in the cell morphology and F-actin cytoskeleton following treatment with α-MSH or IBMX. (A) B16F10 cells were incubated with 1 μM α-MSH or 100 μM IBMX for 24 h and then cell morphology was observed using a phase contrast microscope (original magnification, upper × 40, lower ×100). (B) B16F10 cells grown on L-lysine-coated coverslips were treated with 1 μM α-MSH or 100 μM IBMX for 24 h. After fixation in 2% formaldehyde, cells were incubated with TRITC-conjugated phalloidin for visualization of F-actin, mounted on the glass slides with a mounting medium containing DAPI, and observed using a fluorescence microscope. Cell images are representative of two independent experiments (original magnification, × 400). (C) Intracellular cAMP level, cell morphology, and appearance of cell pellet were compared between B16F10 and B16F1.
Fig. 3 – α-MSH or IBMX decreases Tβ4 mRNA expression and regulates EMT-related genes. (A) The mRNA expression of Tβ4 and EMT-related gene was measured by RT-PCR using specific primers in B16F1 and B16F10 cells. After treatment with 1 μM α-MSH or 100 μM IBMX for 18 h, the mRNA expression level of Tβ4 and EMT-related genes in B16F10 cells was also measured by RT-PCR. (B) After treatment with 1 μM α-MSH or 100 μM IBMX for 18 h, the protein levels of Tβ4, E-cadherin, and N-cadherin were examined by Western blot. (C) E-cadherin expression was examined by indirect immunofluorescence labeling using a rabbit anti-E-cadherin Ab and an Alexa 594-conjugated anti-rabbit secondary Ab.
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validated by Western blotting. The increase of E-cadherin expression by cAMP elevating agents was also confirmed by immunofluorescence staining (Fig. 3C). In combination with increased Ecadherin expression, cell–cell contact, which is inversely related with the EMT, was obviously increased by the cAMP elevating agents. These results collectively suggest that the increased intracellular cAMP can effectively inhibit the EMT and this may affect the metastatic potential of melanoma cells.
the mRNA level of Tβ4, SIP1, Slug, and mesenchymal markers, such as vimentin, N-cadherin, and fibronectin, but pretreatment with α-MSH or IBMX completely inhibited the TNF-α-mediated increase of EMT-related gene expression (Fig. 4C). Importantly, α-MSH- or IBMX-pretreated cells maintained the expression of epithelial markers, such as γ-catenin and E-cadherin, even after TNF-α stimulation. These results provide clear evidence for the inhibitory effect of cAMP elevating agents on the EMT process.
TNF-α-induced increase of the gelatinolytic MMP-9 activity and EMT-related gene expression is completely blocked by cAMP elevating agents
cAMP elevating agents suppress TNF-α-induced NF-κB activation, which is critical for the Tβ4 expression
TNF-α is known to stimulate malignant tumor cell migration, invasion, and metastasis via activation of MMPs [30,31]. As expected, secretion of the gelatinolytic MMP-9 by B16F10 cells was significantly increased following TNF-α stimulation. However, pretreatment with α-MSH or IBMX completely inhibited TNF-αinduced MMP-9 secretion (Fig. 4A). The enhanced MMP production is one of the critical factors involved in the EMT. Therefore, we examined the level of the EMT-related genes after TNF-α stimulation and determined the effect of treatment with α-MSH or IBMX prior to TNF-α stimulation. As shown in Fig. 4B, TNF-α stimulation significantly enhanced the protein level of Tβ4 and Ncadherin but significantly decreased the protein level of Ecadherin. In the meantime, pretreatment α-MSH or IBMX almost completely inhibited the TNF-α-induced increase in Tβ4 and Ncadherin expression as well as the decrease in E-cadherin expression. Additionally, TNF-α stimulation dramatically increased
Since NF-κB is an important mediator of TNF-α signal transduction, we examined the effect of the cAMP elevating agents on NFκB activity in TNF-α-treated cells. As reported in previous studies [32,33], NF-κB activity was considerably decreased by α-MSH or IBMX treatment in our system (about 20–35% activity compared to untreated control B16F10 cells, Fig. 5A). In addition, while treatment with TNF-α increased NF-κB activity about two-fold, pretreatment with α-MSH or IBMX almost completely prevented TNF-α-mediated increase in NF-κB activity. The dissociation of the inhibitory subunit IκB, which is mediated by its phosphorylation, and its subsequent degradation are critical steps in NF-κB activation. As shown in Fig. 5B, treatment of B16F10 cells with TNF-α immediately increased the level of IκBα phosphorylation and degradation. However, no detectable IκBα phosphorylation or degradation was observed in cells treated with α-MSH or IBMX prior to TNF-α stimulation. To demonstrate the correlation between NF-κB suppression by α-MSH or IBMX and Tβ4 expres-
Fig. 4 – α-MSH or IBMX inhibits the TNF-α-induced increase in MMP-9 secretion, Tβ4 expression, and regulation of EMT-related genes. (A) Cells were pretreated with 1 μM α-MSH or 100 μM IBMX for 18 h and stimulated with 10 ng/ml TNF-α for additional 24 h. The MMP-2 and MMP-9 activities were determined by gelatin zymography using conditioned medium. (B) After pretreatment with 1 μM α-MSH or 100 μM IBMX for 18 h, cells were stimulated with TNF-α for an additional 24 h and the protein levels of Tβ4, E-cadherin, and N-cadherin were examined by Western blot. (C) Cells were pretreated with 1 μM α-MSH or 100 μM IBMX for 18 h and then stimulated with TNF-α for additional 8 h. The mRNA expression level of Tβ4, the EMT-related genes, and the E-cadherin suppressors was measured by RT-PCR.
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Fig. 5 – α-MSH or IBMX suppresses the TNF-α-mediated NF-κB activation critical for the Tβ4 expression. (A) The semi-confluent cells pretreated with α-MSH or IBMX for 18 h were transiently transfected with a NF-κB luciferase reporter construct using Lipofectamine 2000 reagent. After incubation for 4 h, 10 ng/ml of TNF-α was treated and incubated for additional 18 h. NF-κB-induced luciferase activity was measured using a luminometer and the intensity was calculated as relative values with respect to β-galactosidase activity. The data are from three independent experiments carried out in duplicate and are expressed as the mean ± SD. ⁎P < 0.01 versus untreated control cells. (B) After treatment with α-MSH or IBMX for 18 h, cells were stimulated with TNF-α for 30 min. Cell lysates were analyzed by Western blot using anti-pIκBα and anti-IκBα antibodies. (C, D) B16F10 cells were transiently transfected with dnIKKα or dnIKKβ and then treated with 10 ng/ml TNF-α for 18 h. The mRNA and protein expression levels of Tβ4, E-cadherin, and N-cadherin were examined by RT-PCR and Western blot, respectively.
sion, we examined the effect of transient expression of dnIKKα or dnIKKβ on TNF-α-induced Tβ4 expression in B16F10 cells. As shown in Figs. 5C and D, the TNF-α-induced increase in Tβ4 expression was significantly decreased by transiently expressing dnIKKα or dnIKKβ, similar to α-MSH or IBMX pretreatment. These results suggest that suppression of NF-κB activation by increased intracellular cAMP may contribute to the inhibition of Tβ4 expression and the prevention of the EMT process.
Pretreatment with a PKA-specific inhibitor, KT5720, recovers NF-κB activity, and blocks cAMP-mediated Tβ4 suppression and F-actin reorganization The second messenger cAMP and its effector, protein kinase A (PKA), mediate the regulation of many processes including glycogenolysis, ion transport, gene transcription, and cell proliferation and differentiation [19]. In order to elucidate whether activation of cAMP-dependent PKA is involved in the suppression of Tβ4 expression and the reorganization of the actin cytoskeleton, we pretreated B16F10 cells with KT5720, a potent and specific cellpermeable inhibitor of PKA, prior to α-MSH or IBMX treatment. As shown in Fig. 6A, KT5720 completely blocked cAMP-mediated
tyrosinase expression as well as cAMP-mediated Tβ4 downregulation. Furthermore, the morphological transition and Factin reorganization by α-MSH or IBMX treatment were considerably inhibited by pretreatment with KT5720 (Fig. 6B). In particular, pretreatment with KT5720 significantly sustained NF-κB activity even after α-MSH or IBMX treatment (Fig. 6C). To assure whether inhibition of Tβ4 expression was directly induced by cAMP, we examined the effect of another adenyl cyclase activator, forskolin. As shown in Fig. 6D, forskolin also enhanced cell–cell contact and tyrosinase mRNA expression while inhibiting Tβ4 expression and NF-κB activity. These results suggest that PKA negatively regulates NF-κB-mediated expression of Tβ4, which is critical for modulating cell motility and invasion via activating an EMT.
Discussion It has been proposed that the second messenger cAMP can function as an anti-cancer agent by acting as an inhibitor of the G1/S transition in the cell cycle or by inducing cell cycle-specific apoptosis [34,35]. Moreover, recent data have demonstrated that the modulation of intracellular cAMP levels can also regulate some
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Fig. 6 – Pretreatment with a PKA inhibitor, KT5720, blocks the cAMP-mediated Tβ4 suppression, F-actin reorganization, and NF-κB suppression. (A) B16F10 cells were pretreated with 250 nM KT5720 for 12 h and then incubated with α-MSH or IBMX for 18 h. The mRNA expression level of Tβ4 was examined by RT-PCR. KT5720 specifically inhibited the cAMP-mediated increase of tyrosinase expression and Tβ4 suppression as well. (B) Cells were fixed and labeled with TRITC-conjugated phalloidin. (C) NF-κB-induced luciferase activity was measured after pretreatment with 250 nM KT5720 for 12 h prior to α-MSH or IBMX treatment. (D) After treatment with forskolin (10 μM) for 18 h, cell morphology, NF-κB activity, and Tβ4 mRNA expression were examined.
cellular processes involved in cancer metastasis [36]. α-MSH treatment inhibited B16–BL6 melanoma invasion through a reconstituted basement membrane, reduced colony formation by 50%, and inhibited the TNF-α-induced increase in uveal invasion [26,37]. Melanoma invasion is also inhibited by forskolin and/or IBMX, further supporting the hypothesis that melanoma invasion is regulated by cAMP post-receptor signaling [38]. However, there is controversy concerning the role of α-MSH in the progression of melanoma. Malignant melanomas tend to exhibit increased melanogenesis and melanoma patients show high levels of αMSH in both the plasma and tumors [39]. Therefore, the serum tyrosinase level is often used as a diagnostic marker for melanoma and inhibition of melanogenesis has been proposed both as an adjuvant treatment modality and as a measure of the reduced probability of melanoma progression. From these reasons, it is very important to clarify the role of α-MSH in melanoma malignancy, as well as underlying mechanism. Tumor metastasis is a multi-step process that includes cell adhesion to the ECM, proteolytic digestion of the ECM, invasion into the lymph and blood vessels, and migration. The EMT is a program of development that is characterized by the loss of cell– cell adhesion, the repression of E-cadherin expression, and an increase in cell motility. Importantly, the initiation of metastasis has many phenotypic similarities to the EMT. Therefore, it is
thought that the suppression of the EMT is important for the prevention of tumor metastasis [40]. In this study, we examined the inhibitory mechanism of cAMP signaling in tumor metastasis, especially in the regulation of the EMT. As reported earlier, cAMP induced by treatment with α-MSH or IBMX inhibited colony formation in soft agar, suppressed MMP9 activity, and decreased the metastasis of B16F10 melanoma cells to the lungs. In addition, α-MSH or IBMX inhibited TNF-αmediated NF-κB activation and the increased cAMP level elicited visible alterations in the cell morphology and F-actin reorganization. In a number of recent studies, Tβ4 has been shown to play a role in tumor progression, including proliferation, migration, angiogenesis, and metastasis, and it has been shown to induce the EMT and alter the actin cytoskeleton [13,41,42]. Interestingly, we observed significant suppression of Tβ4 expression by α-MSH or IBMX treatment in both a resting state and following TNF-α stimulation. For the first time, the data reported here demonstrate that the elevation of the intracellular cAMP level significantly decreases Tβ4 expression in B16F10 melanoma cells. In our study, cAMP strongly inhibited the expression of SIP1 and Slug and thereby resulted in the increase of E-cadherin expression and the decrease of mesenchymal markers expression. The level of Twist and α-catenin was not altered by cAMP in this setting (data not shown). It is generally believed that increased cAMP and activation
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of PKA leads to PKA-mediated phosphorylation of cAMP response element-binding protein (CREB) and the recruitment of CREBbinding protein negatively affects NF-κB activation [43]. Therefore, we examined whether a functional relationship exists between Tβ4 expression and NF-κB activation. Because NF-κB activation requires the activation of IKKs, we examined the levels of Tβ4, Ecadherin, and N-cadherin after transfection with plasmids encoding dominant negative forms of IKKα and IKKβ. The TNF-αmediated upregulation of Tβ4 and N-cadherin was completely abrogated, and the downregulation of E-cadherin by TNF-α was partially blocked by the transfection of these dnIKK constructs. These results clearly indicate that NF-κB inactivation by cAMP is critical for the suppression of Tβ4 expression and prevention of the EMT. These inhibitory effects of cAMP on Tβ4 expression and NFκB activation were significantly abrogated by a PKA-specific inhibitor, suggesting the involvement of a PKA-dependent pathway in this effect. Tβ4 has been thought to regulate tumor progression in many ways. Cha et al. reported that B16F10 cells infected with a Tβ4expressing adenovirus were more metastatic in vivo than B16F10 cells infected with a control adenovirus. Additionally, they demonstrated that Tβ4 induces VEGF expression, thereby promoting angiogenesis [13]. In addition, Tβ4 has also been reported to have anti-inflammatory activity [44], and it may also promote tumor growth by inhibiting immune surveillance [45]. Similarly, the mechanism by which Tβ4 promotes migration, metastasis, and angiogenesis following Tβ4 overexpression or treatment with synthetic Tβ4 peptide is well known. In our study, treatment of B16F1 cells with a synthetic Tβ4 peptide (AcSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES, 1 ng/ml) induced VEGF-α, vimentin, and slug mRNA expression, and suppressed E-cadherin and γ-catenin mRNA expression (data not shown). Therefore, through the use of anti-sense technologies (e.g., interfering RNAs) or antibodies against Tβ4, it should be possible to treat cancers with a high metastatic potential [42]. However, a detailed understanding of the upstream molecules and signaling pathways that regulate Tβ4 expression is lacking. In summary, the present study furthers an earlier observation that cAMP inhibits metastasis by suppressing the activity of MMPs via inactivation of NF-κB. Interestingly, our finding that cAMP suppresses NF-κB-mediated Tβ4 expression, which results in the inhibition of the EMT and cancer metastasis, suggests a novel mechanism for the anti-metastatic action of cAMP. Therefore, further investigations examining the signaling pathways involved in the regulation of Tβ4 in malignant tumors will provide the basis for identifying new anti-cancer strategies.
Acknowledgments This work was financially supported by the Science Research Center for Women's Diseases of the KOSEF (R11-2005-017-03001) and by the Sookmyung Women's University Research Grants 2008.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells Aeyung Kima , Minsik Sonb , Keun Il Kima , Young Yanga , Eun Young Songc , Hee Gu Leec , Jong-Seok Lima,⁎ a
Department of Biological Science and the Research Center for Women's Diseases, Sookmyung Women's University, Chungpa-Dong, Yongsan-Gu, Seoul, 140-742, Republic of Korea b Department of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea c Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-608, Republic of Korea
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
Thymosin β4 (Tβ4) is a major actin-sequestering protein that has been implicated in the growth,
Received 18 April 2009
survival, motility, and metastasis of certain tumors and is considered an indicator for malignant
Revised version received
progression. Therefore, identifying compounds that can downregulate Tβ4 expression is very
22 May 2009
important for the development of anti-cancer chemotherapies. In this study, we investigated the
Accepted 22 May 2009
effects of elevated cAMP on Tβ4 expression and the metastatic potential of murine B16 melanoma
Available online 3 June 2009
cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tβ4 suppression. We found that treatment with the cAMP-inducing compounds α-MSH (α-melanocyte stimulating
Keywords:
hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tβ4 expression and
Cyclic AMP
regulated EMT-associated genes through the suppression of NF-κB activation in B16F10 cells. Along
Thymosin β4
with decreased Tβ4 expression, the in vitro invasiveness and anchorage-independent growth in a
EMT
semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of
E-cadherin
the α-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control
N-cadherin
cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tβ4
MMP
expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover,
Metastasis
suppression of NF-κB activation by α-MSH or IBMX is critical for inhibiting Tβ4 expression. © 2009 Elsevier Inc. All rights reserved.
Introduction The epithelial–mesenchymal transition (EMT) is a process by which cells lose their epithelial features and concomitantly acquire a mesenchymal phenotype. EMT is critical for both normal embryonic development and for the progression of non-invasive adenomas into malignant and metastatic carcinomas [1]. EMT can be defined by the loss of cell–cell adhesion, the induction of extracellular matrix (ECM) degradation, and cytoskeletal reorga-
nization. E-cadherin expression or function is lost during the progression of most carcinomas [2]. The downregulation of Ecadherin leads to a reduction or loss of cell–cell interactions and the detachment of malignant cells from the primary tumor. Conversely, metastatic phenotypes are impaired by the forced expression of E-cadherin in cultured cancer cells and transgenic mouse models [3]. In malignant progression, E-cadherin can be functionally inactivated by mutation and downregulated by promoter hypermethylation or transcriptional repression [4,5]. It
⁎ Corresponding author. Fax: +82 2 2077 7322. E-mail address: [email protected] (J.-S. Lim). Abbreviations: EMT, epithelial–mesenchymal transition; MMP, matrix metalloproteinase; α-MSH, alpha-melanocyte stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine; VEGF, vascular endothelial growth factor; PKA, protein kinase A; AC, adenyl cyclase; PDE, phosphodiesterase 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.05.024
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has been reported that transcription of E-cadherin is suppressed by factors such as Snail, Slug, δEF1/ZEB1, and SIP1/ZEB2 in different type of cancers including melanoma [6–8]. In addition, it has been demonstrated that Akt-mediated NF-κB activation represses Ecadherin by Snail upregulation and induces EMT [9]. Therefore, identifying the mechanisms that block the loss of E-cadherin expression is critical for preventing malignant progression via suppression of the EMT. Thymosin β4 (Tβ4), a 43-amino acid polypeptide originally described as a thymic maturation factor, is known to stimulate tissue remodeling, cell differentiation, and cell and tissue healing after injury [10,11]. In recent studies, the expression of Tβ4 has been found to induce angiogenesis, accelerate wound healing and enhance metastatic potential of various tumor cells through its effects on the cytoskeleton. For example, Clark and colleagues found that the metastatic potential of murine B16 melanoma cells increased when they repeated an in vivo selection scheme and that Tβ4 levels were elevated in highly metastatic B16F10 cells that were cultured from the lung tumors [12]. Of particular interest is the finding by Cha et al. that the overexpression of Tβ4 increases the number of metastatic tumors and the expression of an angiogenic factor, vascular endothelial growth factor (VEGF) [13]. Furthermore, it has been reported that Tβ4 overexpression induces the loss of E-cadherin, a hallmark of the EMT, and contributes to tumor cell invasion and metastasis. Wang et al. have shown that the overexpression of Tβ4 results in a drastic change in cell growth and motility that is accompanied by a downregulation of Ecadherin [14]. They also have demonstrated that Tβ4 overexpression is tightly associated with an increase in the invasiveness of SW480 colon carcinoma cells through an increase of MMP-7 activity, as well as the distant metastasis of human colorectal carcinomas [15]. In addition, they suggested a novel role for Tβ4 in the promotion of colorectal carcinoma progression through the induction of the EMT in tumor cells by upregulating integrinlinked kinase [16]. The growth rate and paclitaxel-resistance of HeLa cells were increased by Tβ4 overexpression [17], while their apoptosis induced by this drug was accelerated following a decrease in Tβ4 [18]. However, little is known about the upstream molecules or signaling mechanisms that modulate Tβ4 expression and therefore affect cancer progression. Cyclic AMP (cAMP) is a ubiquitous signaling molecule that influences cell proliferation, survival, and differentiation partially through the activation of protein kinase A (PKA) [19]. Intracellular cAMP concentrations are regulated by both adenyl cyclase (AC), which uses ATP to produce cAMP, and phosphodiesterases (PDEs), which catalyze the degradation of cAMP to AMP [20,21]. While the role of cAMP in promoting pigmentation is indisputable, its involvement in cancer progression is controversial dependent on cell type and context. For example, the constitutive activation of cAMP by the mutation of genes involved in cAMP signaling has resulted in endocrine neoplasia [22]. In addition, dibutyryl cAMP has been shown to slightly enhance collagen-mediated keratinocyte migration [23], and an increase of cAMP levels has been shown to increase the expression and activity of MMP-2 [24]. In contrast, it has been reported that increased cAMP inhibits growth factor-mediated MMP-9 induction and keratinocyte migration [25], and treatment with α-melanocyte stimulating hormone (αMSH) potently inhibits both in vitro invasion through a reconstituted basement membrane and the in vivo pulmonary metastasis of highly invasive B16–BL6 murine melanoma cells [26].
However, the molecular mechanisms, especially those based on the regulation of EMT, of the cAMP–PKA pathway that suppresses metastasis of melanoma cells are not well understood. In the present study, we evaluated the influence of intracellular cAMP on Tβ4 expression, the EMT regulation, and the subsequent invasive potential in the highly invasive murine melanoma cell line B16F10 using α-MSH or IBMX as an activator of AC or an inhibitor of PDE, respectively. Our results clearly demonstrate that an increase in intracellular cAMP inhibits the expression of Tβ4 via the suppression of NF-κB activation and blocks the loss of E-cadherin, thereby attenuating the metastatic potential of the melanoma cells.
Materials and methods Mice and cell cultures The murine B16F10 melanoma cell line was maintained as a monolayer culture in Dulbecco's Modified Eagle's Medium (DMEM; Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco/Invitrogen), 100 U/ml penicillin/100 μg/ml streptomycin (Gibco/Invitrogen), in a humidified 5% CO2 incubator at 37 °C. Specific pathogen-free female C57BL/6J mice were purchased from Daehan BioLink (Umsong, Republic of Korea) and were maintained in our animal facility for at least 2 weeks before use. The mice were housed under specific pathogen-free conditions in a barrier facility with a 12hour light/dark cycle.
Antibodies and chemicals α-Melanocyte stimulating hormone (α-MSH), 3-isobutyl-1methylxanthine (IBMX), and forskolin were purchased from Sigma (St. Louis, MO). The specific cell-permeable inhibitor of protein kinase A, KT5720, was obtained from Merck (Darmstadt, Germany) and rhodamine phalloidin, a high-affinity probe for Factin, was purchased from Invitrogen. Recombinant murine TNF-α was obtained from ProSpec Tany TechnoGene (Rehovot, Israel). The anti-IκBα and anti-phospho-IκBα (Ser32) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against thymosin β4 (Tβ4) and E-cadherin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-N-cadherin antibody was purchased from Sigma. Synthetic Tβ4 peptide was provided by Immundiagnostik AG (Bensheim, Germany).
Measurement of melanin content B16F10 melanoma cells were seeded at a density of 2.5 × 105 cells/ 100 mm culture plate. After incubation in the presence of cAMPinducing agents, total melanin content in the cell pellet (1 × 107 cells per sample) was measured. Briefly, the pellets were dissolved in 1 N NaOH/10% DMSO for 1 h at 80 °C, and solubilized melanin was measured at OD475. Melanin content was calculated from a standard curve using synthetic melanin.
Determination of intracellular cAMP level The cAMP concentration was measured using a Direct Cyclic AMP Enzyme Immunoassay Kit produced by Assay Designs™ Inc. (Ann Arbor, MI). Briefly, cells were lysed in 0.1 M HCl to inhibit the
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phosphodiesterase activity, and then the supernatants were collected. Following neutralization and dilution, a fixed amount of cAMP direct conjugate was added to compete with the cAMP in the cell lysates for sites on a rabbit polyclonal antibody immobilized on a 96-well plate. After adding cAMP antibody into each well, the plate was incubated at room temperature for 2 h. Thereafter, the plate was washed and a pNPP (p-nitrophenyl phosphate) substrate solution was added into the wells. After the enzyme reaction was stopped, the optical density was read on a microplate reader at 405 nm. The intensity of the bound yellow color was inversely proportional to the concentration of cAMP in the cell lysates.
Immunofluorescence analysis Cells were grown to ∼60% confluency on poly-L-Lysine (0.01% solution, Sigma)-coated cell culture coverslips (Thermanox Plastic Coverslips, NUNC, Rochester, NY) for 24 h and incubated in serum-free medium for an additional 6 h. Cells were then treated with α-MSH or IBMX for 24 h. Cells were fixed with 2% formaldehyde in PBS for 10 min and washed with a PBS/serum solution (10% fetal bovine serum, 0.05% azide in PBS) for 5 min. The fixed cells were then incubated with tetramethylrhodamine (TRITC)-conjugated phalloidin (diluted 1:1000 in PBS/serum containing 0.2% saponin) for 45 min at room temperature. Cells were then washed with a PBS/serum solution three times for 5 min and the coverslips were mounted on glass slides with VECTASHIELD (mounting medium with DAPI, Vector Laboratories, Burlingame, CA).
RNA extraction and RT-PCR Total RNA was extracted from the cells using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions and was reverse transcribed to cDNA using M-MLV reverse transcriptase (Promega, Madison, WI) and an oligo(dT) primer. cDNA aliquots corresponding to 5 μg RNA were analyzed by semi-quantitative PCR. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide (EtBr).
Gelatin zymography The enzymatic activity of MMP-2 and MMP-9 was analyzed by gelatin zymography. Cells were incubated in serum-free medium for 24–48 h and equivalent volumes of the culture supernatants were electrophoresed in a 10% SDS-polyacrylamide gel containing 0.1–0.2% gelatin. The gels were washed twice with washing buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2.5% Triton X-100) and then treated with incubation buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.02% NaN3, 1 μM ZnCl2) for 18–36 h at 37 °C. The gels were then stained with a staining solution (0.05% Coomassie blue, 10% isopropanol, 10% acetic acid) and destained (10% isopropanol, 10% acetic acid). MMPs were detected as transparent bands on the blue background of the Coomassie blue-stained slab gels.
Soft agar colony formation assay In order to measure anchorage-independent cell growth,1 × 104 cells were suspended in 3 ml of DMEM containing 0.3% agar and 10% FBS
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and applied onto 3 ml pre-solidified 0.6% agar in 60-mm culture dishes. Following 2 weeks of incubation, the colonies on soft agar were observed with a phase contrast microscope and photographed.
In vitro tumor cell invasion assay The in vitro invasion assay was performed using a 24-well transwell unit with polycarbonate filters that have a diameter of 6.5 mm and a pore size of 8.0 μm (Corning Costar, Cambridge, MA). For the invasion assay, the transwell was coated with 20 μl of a 1:2 mixture of Matrigel:DMEM (Matrigel; BD Biosciences, Bedford, MA) and used as the intervening invasive barrier in a 24-well plate. Cells (5 × 104) were suspended in serum-free DMEM and added to the upper chamber of the transwell. The lower chamber of the transwell was filled with DMEM supplemented with 5% FBS as a chemoattractant. After incubation for 24 h at 37 °C, cells attached to the upper surface of the filter were completely removed by wiping with a cotton swab and the filters were stained with a 0.2% crystal violet/20% methanol (w/v) solution. Alternatively, the cell number was determined using calcein-acetomethylester (calcein-AM, Molecular Probes, Eugene, OR), which can be cleaved by an intracellular esterase to produce calcein, a strongly fluorescent compound. Fluorescence intensity was measured with a fluorescence microplate reader (PerkinElmer Victor, Fremont, CA). The background fluorescence was corrected by subtracting the value of the control well without cells.
In vivo experimental metastasis assay The in vivo metastatic potential of the cells was measured using the lung colonization assay. In brief, cells were injected into the tail vein of mice at a density of 3 × 105 cells/0.2 ml of PBS. Twenty days later, the mice were sacrificed, lungs were fixed with Bouin's solution (Sigma), and the metastatic colonies on the lung surface were observed macroscopically.
Western blot Whole cell lysates were prepared by lysing the cells on ice in MPER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL) for 30 min. The supernatant was recovered following centrifugation (14,000 g, 20 min, 4 °C) and the protein concentration was determined using the BCA assay. Proteins were visualized after immunoblotting using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech) and a LAS 3000 imaging system (FUJIFILM Corporation, Tokyo, Japan). The band intensity was analyzed using TINA 2.0 software. For the detection of the 5 kDa Tβ4 protein, proteins were electrophoresed in NuPAGE Novex 4–12% Bis–Tris Gels (Invitrogen) using the XCell SureLock Mini-Cell according to manufacturer's instructions.
Transient transfection and luciferase reporter assay Semi-confluent cells that were grown in 12-well plates were transiently transfected with a pNF-κB-Luc reporter plasmid (Stratagene, La Jolla, CA) and the pCMV/β-Gal plasmid using the Lipofectamine 2000 reagent (Invitrogen). pCMV/β-Gal was used for normalizing the transfection efficiency. After incubation for 18 h, cells were harvested in passive lysis buffer and the luciferase activity was measured using a VICTOR3 1420 multilabel counter
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with the luminescence microplate reader set (PerkinElmer Victor) and the Luciferase Assay System according to the manufacturer's instructions (Promega). The β-galactosidase activity was measured using o-nitrophenyl-β-galactopyranoside (ONPG) as a substrate and luciferase activity was calculated relative to the β-galactosidase activity. The transient transfection of dominant negative forms of IKKα (dnIKKα) and IKKβ (dnIKKβ) was also performed using the Lipofectamine 2000 reagent. The transfected cells were cultured in complete medium for 24 h and then used as indicated in each experiment. dnIKKα and dnIKKβ were gifts from Dr Jang-Soo Chun (Gwangju Institute of Science and Technology, Gwangju, Republic of Korea).
Statistical analysis Results are presented as means ± standard deviation (SD). All experiments were repeated at least three times and data were analyzed for statistical significance using Student's t test. P values less than 0.05 were considered significant.
Results cAMP elevating agents inhibit MMP-9 activity, anchorage-independent cell growth, in vitro Matrigel invasion, and in vivo pulmonary metastasis of melanoma cells In order to examine the effects of intracellular cAMP levels on the phenotypic changes observed in malignant cancer, we used αMSH or IBMX to increase cAMP levels in murine B16F10 melanoma cells, which are highly metastatic in syngeneic C57BL/6J mice. As shown in Fig. 1A, the treatment of B16F10 cells with the indicated concentration of α-MSH or IBMX did not affect the rate of cell proliferation. However, it dramatically increased intracellular cAMP level, tyrosinase mRNA expression, and melanin synthesis (Figs. 1A–C). As shown in Fig. 1D, treatment with α-MSH or IBMX almost completely suppressed gelatinolytic MMP-9 secretion in melanoma cells, compared to untreated control cells. In addition, the ability of the cells to form colonies in a semi-solid medium, a marker of anchorage independence that is positively correlated with metastatic potential, was significantly decreased by treatment with α-MSH or IBMX (Fig. 1E). While untreated control cells formed sizable colonies in the soft agar, α-MSH- or IBMX-treated cells had a significantly decreased ability to form colonies in soft agar, including both a smaller number of sizable colonies and a reduced colony size. The inhibition of colony formation was clearly not due to a decreased growth rate as shown in Fig. 1A. Increased melanin synthesis mediated by the increase in intracellular cAMP levels after treatment with α-MSH or IBMX was confirmed as black colonies in soft agar. Along with decreased MMP-9 secretion, the invasive potential, which is determined by the ability of the cell to invade a Matrigel barrier, was significantly suppressed by αMSH or IBMX treatment (Fig. 1F). In order to confirm the inhibitory effect of cAMP elevating agents on tumor metastasis in vivo, we examined the ability of B16F10 cells to colonize the lungs after intravenous injection. Cells treated with α-MSH or IBMX induced a decreased number of colonies in the lung compared to untreated control cells, suggesting that increased intracellular cAMP can inhibit the metastatic potential of B16F10 cells in vivo (Fig. 1G).
cAMP elevating agents elicit a morphological transition and F-actin reorganization Interestingly, changes in cell morphology following α-MSH or IBMX treatment were observed using phase contrast microscopy (Fig. 2A). The increase in cAMP level elicited a morphological transition to contracting cells with short processes and induced an increase of cell–cell contact. In order to analyze the changes in actin polymerization, we examined the cellular distribution of Factin in α-MSH- or IBMX-treated cells by staining with TRITCconjugated phalloidin. Upon plating, untreated control cells spread across the L-lysine-coated slides and the actin filaments were homogeneously distributed throughout the cytoplasm and surrounded the external boundary of the cells. In contrast, compared to untreated control cells, treatment with α-MSH or IBMX decreased the spread of cells on the slides with a visible alteration in shape and reduced the staining intensity for F-actin (Fig. 2B). Interestingly, the morphology of B16F1 melanoma (a low metastatic variant) was distinct from that of B16F10 melanoma (a high metastatic variant), but was similar to α-MSH or IBMX-treated B16F10 cells (Fig. 2C). Moreover, the intracellular cAMP level in B16F1 cells was significantly higher than that in B16F10 cells. It has been reported that EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases (MMPs) [27–29]. Additionally, since the EMT facilitates intravasation of tumor cells into blood or lymph vessels and the subsequent formation of distant metastases, we investigated the inhibitory mechanism of cancer metastasis by cAMP based on the regulation of the EMT.
cAMP elevating agents decrease thymosin β4 expression and regulate the expression of EMT-related genes Several genes that are selectively upregulated in metastatic mouse and human melanoma cells compared with their non-metastatic counterparts have been identified using DNA microarrays. Clark et al. has reported that the expression of fibronectin, thymosin β4 (Tβ4) and RhoC was increased in all human and mouse melanomaderived metastases [12]. Tβ4 binds to monomeric actin, a component of the cytoskeleton, and may act as an “actin buffer”, preventing the spontaneous polymerization of actin monomers into filaments but supplying a pool of actin monomers when the cell needs filaments. Since several studies have suggested that Tβ4 is involved in wound healing, angiogenesis, and tumor cell metastasis [11,13,15,16], we first examined the level of Tβ4 mRNA level in both B16F1 and B16F10 melanoma cells. As shown in Fig. 3A, the mRNA expression level of Tβ4 was considerably elevated in B16F10 cells compared to B16F1 cells. It has been reported that Tβ4 overexpression triggers an EMT by downregulation of E-cadherin, a type I transmembrane protein important in the formation of adherence junctions [16]. As shown in Fig. 3A, B16F10 cells expressed an increased level of mesenchymal markers, such as vimentin, N-cadherin, and fibronectin, as well as E-cadherin suppressors, such as SIP1 and Slug, compared to B16F1 cells. In contrast, the expression level of epithelial marker, E-cadherin, was low in B16F10 cells. Interestingly, the expression of Tβ4, SIP1, Slug, and mesenchymal markers was significantly reduced following αMSH or IBMX treatment, while E-cadherin mRNA expression was significantly increased. As shown in Fig. 3B, decrease of Tβ4 and Ncadherin, and increase of E-cadherin by α-MSH or IBMX were also
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Fig. 1 – α-MSH or IBMX increases intracellular cAMP and suppresses metastatic potential of B16F10 cells. (A) B16F10 cells were incubated with α-MSH (0.5, 1 μM) or IBMX (50, 100 μM) for 24 h and then cell proliferation was determined using the MTT assay. Melanin content was also determined and represented as percentage value. (B) mRNA expression of tyrosinase was measured by RT-PCR using specific primers and appearance of each cell pellet was shown after centrifugation. (C) The cAMP concentration after treatment with α-MSH (1 μM) or IBMX (100 μM) for 30 min was measured as described in ‘Materials and methods’. (D) Cells were incubated in serum-free media for 24–48 h with 1 μM α-MSH or 100 μM IBMX and the conditioned medium was subjected to gelatin zymography for the detection of MMP-2 and MMP-9 activity. (E) In order to examine anchorage-independent cell growth, the soft agar colony formation assay was performed. The colonies in soft agar were observed using a phase contrast microscope. The relative colony size was calculated after measuring the diameter of at least ten colonies. ⁎P < 0.01 versus untreated control cells. (F) A fixed number of cells were plated in the upper chamber of the Matrigel-coated transwell. After incubation for 24 h, the cells invading to the lower surface of the membrane were stained with a 0.2% crystal violet/20% methanol (v/v) solution and observed using a phase contrast microscope. The invading cells were also examined by using calcein-acetomethylester as a substrate for intracellular esterase. ⁎P < 0.01 versus untreated control cells. (G) In order to evaluate the in vivo metastatic potential of the cells, α-MSH or IBMX-pretreated (24 h) cells were injected into the tail vein of mice (3 × 105 cells/0.2 ml of PBS). Twenty days later, the metastatic colonies on the lung surface were counted macroscopically. Data are representative of three independent experiments.
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Fig. 2 – Changes in the cell morphology and F-actin cytoskeleton following treatment with α-MSH or IBMX. (A) B16F10 cells were incubated with 1 μM α-MSH or 100 μM IBMX for 24 h and then cell morphology was observed using a phase contrast microscope (original magnification, upper × 40, lower ×100). (B) B16F10 cells grown on L-lysine-coated coverslips were treated with 1 μM α-MSH or 100 μM IBMX for 24 h. After fixation in 2% formaldehyde, cells were incubated with TRITC-conjugated phalloidin for visualization of F-actin, mounted on the glass slides with a mounting medium containing DAPI, and observed using a fluorescence microscope. Cell images are representative of two independent experiments (original magnification, × 400). (C) Intracellular cAMP level, cell morphology, and appearance of cell pellet were compared between B16F10 and B16F1.
Fig. 3 – α-MSH or IBMX decreases Tβ4 mRNA expression and regulates EMT-related genes. (A) The mRNA expression of Tβ4 and EMT-related gene was measured by RT-PCR using specific primers in B16F1 and B16F10 cells. After treatment with 1 μM α-MSH or 100 μM IBMX for 18 h, the mRNA expression level of Tβ4 and EMT-related genes in B16F10 cells was also measured by RT-PCR. (B) After treatment with 1 μM α-MSH or 100 μM IBMX for 18 h, the protein levels of Tβ4, E-cadherin, and N-cadherin were examined by Western blot. (C) E-cadherin expression was examined by indirect immunofluorescence labeling using a rabbit anti-E-cadherin Ab and an Alexa 594-conjugated anti-rabbit secondary Ab.
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validated by Western blotting. The increase of E-cadherin expression by cAMP elevating agents was also confirmed by immunofluorescence staining (Fig. 3C). In combination with increased Ecadherin expression, cell–cell contact, which is inversely related with the EMT, was obviously increased by the cAMP elevating agents. These results collectively suggest that the increased intracellular cAMP can effectively inhibit the EMT and this may affect the metastatic potential of melanoma cells.
the mRNA level of Tβ4, SIP1, Slug, and mesenchymal markers, such as vimentin, N-cadherin, and fibronectin, but pretreatment with α-MSH or IBMX completely inhibited the TNF-α-mediated increase of EMT-related gene expression (Fig. 4C). Importantly, α-MSH- or IBMX-pretreated cells maintained the expression of epithelial markers, such as γ-catenin and E-cadherin, even after TNF-α stimulation. These results provide clear evidence for the inhibitory effect of cAMP elevating agents on the EMT process.
TNF-α-induced increase of the gelatinolytic MMP-9 activity and EMT-related gene expression is completely blocked by cAMP elevating agents
cAMP elevating agents suppress TNF-α-induced NF-κB activation, which is critical for the Tβ4 expression
TNF-α is known to stimulate malignant tumor cell migration, invasion, and metastasis via activation of MMPs [30,31]. As expected, secretion of the gelatinolytic MMP-9 by B16F10 cells was significantly increased following TNF-α stimulation. However, pretreatment with α-MSH or IBMX completely inhibited TNF-αinduced MMP-9 secretion (Fig. 4A). The enhanced MMP production is one of the critical factors involved in the EMT. Therefore, we examined the level of the EMT-related genes after TNF-α stimulation and determined the effect of treatment with α-MSH or IBMX prior to TNF-α stimulation. As shown in Fig. 4B, TNF-α stimulation significantly enhanced the protein level of Tβ4 and Ncadherin but significantly decreased the protein level of Ecadherin. In the meantime, pretreatment α-MSH or IBMX almost completely inhibited the TNF-α-induced increase in Tβ4 and Ncadherin expression as well as the decrease in E-cadherin expression. Additionally, TNF-α stimulation dramatically increased
Since NF-κB is an important mediator of TNF-α signal transduction, we examined the effect of the cAMP elevating agents on NFκB activity in TNF-α-treated cells. As reported in previous studies [32,33], NF-κB activity was considerably decreased by α-MSH or IBMX treatment in our system (about 20–35% activity compared to untreated control B16F10 cells, Fig. 5A). In addition, while treatment with TNF-α increased NF-κB activity about two-fold, pretreatment with α-MSH or IBMX almost completely prevented TNF-α-mediated increase in NF-κB activity. The dissociation of the inhibitory subunit IκB, which is mediated by its phosphorylation, and its subsequent degradation are critical steps in NF-κB activation. As shown in Fig. 5B, treatment of B16F10 cells with TNF-α immediately increased the level of IκBα phosphorylation and degradation. However, no detectable IκBα phosphorylation or degradation was observed in cells treated with α-MSH or IBMX prior to TNF-α stimulation. To demonstrate the correlation between NF-κB suppression by α-MSH or IBMX and Tβ4 expres-
Fig. 4 – α-MSH or IBMX inhibits the TNF-α-induced increase in MMP-9 secretion, Tβ4 expression, and regulation of EMT-related genes. (A) Cells were pretreated with 1 μM α-MSH or 100 μM IBMX for 18 h and stimulated with 10 ng/ml TNF-α for additional 24 h. The MMP-2 and MMP-9 activities were determined by gelatin zymography using conditioned medium. (B) After pretreatment with 1 μM α-MSH or 100 μM IBMX for 18 h, cells were stimulated with TNF-α for an additional 24 h and the protein levels of Tβ4, E-cadherin, and N-cadherin were examined by Western blot. (C) Cells were pretreated with 1 μM α-MSH or 100 μM IBMX for 18 h and then stimulated with TNF-α for additional 8 h. The mRNA expression level of Tβ4, the EMT-related genes, and the E-cadherin suppressors was measured by RT-PCR.
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Fig. 5 – α-MSH or IBMX suppresses the TNF-α-mediated NF-κB activation critical for the Tβ4 expression. (A) The semi-confluent cells pretreated with α-MSH or IBMX for 18 h were transiently transfected with a NF-κB luciferase reporter construct using Lipofectamine 2000 reagent. After incubation for 4 h, 10 ng/ml of TNF-α was treated and incubated for additional 18 h. NF-κB-induced luciferase activity was measured using a luminometer and the intensity was calculated as relative values with respect to β-galactosidase activity. The data are from three independent experiments carried out in duplicate and are expressed as the mean ± SD. ⁎P < 0.01 versus untreated control cells. (B) After treatment with α-MSH or IBMX for 18 h, cells were stimulated with TNF-α for 30 min. Cell lysates were analyzed by Western blot using anti-pIκBα and anti-IκBα antibodies. (C, D) B16F10 cells were transiently transfected with dnIKKα or dnIKKβ and then treated with 10 ng/ml TNF-α for 18 h. The mRNA and protein expression levels of Tβ4, E-cadherin, and N-cadherin were examined by RT-PCR and Western blot, respectively.
sion, we examined the effect of transient expression of dnIKKα or dnIKKβ on TNF-α-induced Tβ4 expression in B16F10 cells. As shown in Figs. 5C and D, the TNF-α-induced increase in Tβ4 expression was significantly decreased by transiently expressing dnIKKα or dnIKKβ, similar to α-MSH or IBMX pretreatment. These results suggest that suppression of NF-κB activation by increased intracellular cAMP may contribute to the inhibition of Tβ4 expression and the prevention of the EMT process.
Pretreatment with a PKA-specific inhibitor, KT5720, recovers NF-κB activity, and blocks cAMP-mediated Tβ4 suppression and F-actin reorganization The second messenger cAMP and its effector, protein kinase A (PKA), mediate the regulation of many processes including glycogenolysis, ion transport, gene transcription, and cell proliferation and differentiation [19]. In order to elucidate whether activation of cAMP-dependent PKA is involved in the suppression of Tβ4 expression and the reorganization of the actin cytoskeleton, we pretreated B16F10 cells with KT5720, a potent and specific cellpermeable inhibitor of PKA, prior to α-MSH or IBMX treatment. As shown in Fig. 6A, KT5720 completely blocked cAMP-mediated
tyrosinase expression as well as cAMP-mediated Tβ4 downregulation. Furthermore, the morphological transition and Factin reorganization by α-MSH or IBMX treatment were considerably inhibited by pretreatment with KT5720 (Fig. 6B). In particular, pretreatment with KT5720 significantly sustained NF-κB activity even after α-MSH or IBMX treatment (Fig. 6C). To assure whether inhibition of Tβ4 expression was directly induced by cAMP, we examined the effect of another adenyl cyclase activator, forskolin. As shown in Fig. 6D, forskolin also enhanced cell–cell contact and tyrosinase mRNA expression while inhibiting Tβ4 expression and NF-κB activity. These results suggest that PKA negatively regulates NF-κB-mediated expression of Tβ4, which is critical for modulating cell motility and invasion via activating an EMT.
Discussion It has been proposed that the second messenger cAMP can function as an anti-cancer agent by acting as an inhibitor of the G1/S transition in the cell cycle or by inducing cell cycle-specific apoptosis [34,35]. Moreover, recent data have demonstrated that the modulation of intracellular cAMP levels can also regulate some
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Fig. 6 – Pretreatment with a PKA inhibitor, KT5720, blocks the cAMP-mediated Tβ4 suppression, F-actin reorganization, and NF-κB suppression. (A) B16F10 cells were pretreated with 250 nM KT5720 for 12 h and then incubated with α-MSH or IBMX for 18 h. The mRNA expression level of Tβ4 was examined by RT-PCR. KT5720 specifically inhibited the cAMP-mediated increase of tyrosinase expression and Tβ4 suppression as well. (B) Cells were fixed and labeled with TRITC-conjugated phalloidin. (C) NF-κB-induced luciferase activity was measured after pretreatment with 250 nM KT5720 for 12 h prior to α-MSH or IBMX treatment. (D) After treatment with forskolin (10 μM) for 18 h, cell morphology, NF-κB activity, and Tβ4 mRNA expression were examined.
cellular processes involved in cancer metastasis [36]. α-MSH treatment inhibited B16–BL6 melanoma invasion through a reconstituted basement membrane, reduced colony formation by 50%, and inhibited the TNF-α-induced increase in uveal invasion [26,37]. Melanoma invasion is also inhibited by forskolin and/or IBMX, further supporting the hypothesis that melanoma invasion is regulated by cAMP post-receptor signaling [38]. However, there is controversy concerning the role of α-MSH in the progression of melanoma. Malignant melanomas tend to exhibit increased melanogenesis and melanoma patients show high levels of αMSH in both the plasma and tumors [39]. Therefore, the serum tyrosinase level is often used as a diagnostic marker for melanoma and inhibition of melanogenesis has been proposed both as an adjuvant treatment modality and as a measure of the reduced probability of melanoma progression. From these reasons, it is very important to clarify the role of α-MSH in melanoma malignancy, as well as underlying mechanism. Tumor metastasis is a multi-step process that includes cell adhesion to the ECM, proteolytic digestion of the ECM, invasion into the lymph and blood vessels, and migration. The EMT is a program of development that is characterized by the loss of cell– cell adhesion, the repression of E-cadherin expression, and an increase in cell motility. Importantly, the initiation of metastasis has many phenotypic similarities to the EMT. Therefore, it is
thought that the suppression of the EMT is important for the prevention of tumor metastasis [40]. In this study, we examined the inhibitory mechanism of cAMP signaling in tumor metastasis, especially in the regulation of the EMT. As reported earlier, cAMP induced by treatment with α-MSH or IBMX inhibited colony formation in soft agar, suppressed MMP9 activity, and decreased the metastasis of B16F10 melanoma cells to the lungs. In addition, α-MSH or IBMX inhibited TNF-αmediated NF-κB activation and the increased cAMP level elicited visible alterations in the cell morphology and F-actin reorganization. In a number of recent studies, Tβ4 has been shown to play a role in tumor progression, including proliferation, migration, angiogenesis, and metastasis, and it has been shown to induce the EMT and alter the actin cytoskeleton [13,41,42]. Interestingly, we observed significant suppression of Tβ4 expression by α-MSH or IBMX treatment in both a resting state and following TNF-α stimulation. For the first time, the data reported here demonstrate that the elevation of the intracellular cAMP level significantly decreases Tβ4 expression in B16F10 melanoma cells. In our study, cAMP strongly inhibited the expression of SIP1 and Slug and thereby resulted in the increase of E-cadherin expression and the decrease of mesenchymal markers expression. The level of Twist and α-catenin was not altered by cAMP in this setting (data not shown). It is generally believed that increased cAMP and activation
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of PKA leads to PKA-mediated phosphorylation of cAMP response element-binding protein (CREB) and the recruitment of CREBbinding protein negatively affects NF-κB activation [43]. Therefore, we examined whether a functional relationship exists between Tβ4 expression and NF-κB activation. Because NF-κB activation requires the activation of IKKs, we examined the levels of Tβ4, Ecadherin, and N-cadherin after transfection with plasmids encoding dominant negative forms of IKKα and IKKβ. The TNF-αmediated upregulation of Tβ4 and N-cadherin was completely abrogated, and the downregulation of E-cadherin by TNF-α was partially blocked by the transfection of these dnIKK constructs. These results clearly indicate that NF-κB inactivation by cAMP is critical for the suppression of Tβ4 expression and prevention of the EMT. These inhibitory effects of cAMP on Tβ4 expression and NFκB activation were significantly abrogated by a PKA-specific inhibitor, suggesting the involvement of a PKA-dependent pathway in this effect. Tβ4 has been thought to regulate tumor progression in many ways. Cha et al. reported that B16F10 cells infected with a Tβ4expressing adenovirus were more metastatic in vivo than B16F10 cells infected with a control adenovirus. Additionally, they demonstrated that Tβ4 induces VEGF expression, thereby promoting angiogenesis [13]. In addition, Tβ4 has also been reported to have anti-inflammatory activity [44], and it may also promote tumor growth by inhibiting immune surveillance [45]. Similarly, the mechanism by which Tβ4 promotes migration, metastasis, and angiogenesis following Tβ4 overexpression or treatment with synthetic Tβ4 peptide is well known. In our study, treatment of B16F1 cells with a synthetic Tβ4 peptide (AcSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES, 1 ng/ml) induced VEGF-α, vimentin, and slug mRNA expression, and suppressed E-cadherin and γ-catenin mRNA expression (data not shown). Therefore, through the use of anti-sense technologies (e.g., interfering RNAs) or antibodies against Tβ4, it should be possible to treat cancers with a high metastatic potential [42]. However, a detailed understanding of the upstream molecules and signaling pathways that regulate Tβ4 expression is lacking. In summary, the present study furthers an earlier observation that cAMP inhibits metastasis by suppressing the activity of MMPs via inactivation of NF-κB. Interestingly, our finding that cAMP suppresses NF-κB-mediated Tβ4 expression, which results in the inhibition of the EMT and cancer metastasis, suggests a novel mechanism for the anti-metastatic action of cAMP. Therefore, further investigations examining the signaling pathways involved in the regulation of Tβ4 in malignant tumors will provide the basis for identifying new anti-cancer strategies.
Acknowledgments This work was financially supported by the Science Research Center for Women's Diseases of the KOSEF (R11-2005-017-03001) and by the Sookmyung Women's University Research Grants 2008.
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