Involvement of PI3K/Akt Signaling Pathway in Hepatocyte Growth Factor–Induced Migration of Uveal Melanoma Cells

Abstract

purpose. Uveal melanoma is the most common primary intraocular malignancy in adult humans. Unlike cutaneous melanoma, uveal melanoma disseminates preferentially to the liver through the hematogenous system. To date, the mechanism underlying this metastatic homing is largely unknown. This study investigated the effect of hepatocyte growth factor (HGF)-triggered signaling pathways to identify the role of HGF and its downstream effectors in inducing the migration of uveal melanoma cells.

methods. Migration of uveal melanoma cells was measured by in vitro wound healing and transwell migration assays. The expression and translocation of c-Met were detected using indirect immunofluorescence. The activation of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways was analyzed using specific antibodies against phospho-ERK1/2 and phospho-Akt. The impact of HGF treatment on the expression of cell adhesion molecules was measured using Western blotting.

results. HGF was found to enhance cell migration, and that HGF-induced migration depends on PI3K/Akt pathway. The activation of PI3K/Akt pathway induced by the HGF/c-Met axis is involved in the downregulation of cell adhesion molecules E-cadherin and β-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration of uveal melanoma cells. On HGF stimulation, receptor c-Met is translocated to the nucleus in a ligand-dependent manner, suggesting that c-Met may modulate the expression of genes involved in melanoma cell migration.

conclusions. Data from this study directly linked the central PI3K/Akt pathway to uveal melanoma migration and pointed to new avenues for therapeutic intervention in hepatic metastasis.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a mesenchymal-derived or stromal-derived multifunctional growth factor with mitogenic, motogenic, and morphogenic activities. ¹ HGF plays an important role in the development and progression of cancer. Particularly, HGF promotes tumor metastasis by stimulating motility and invasion, likely through a number of mechanisms, including decreased cell-cell adhesion, enhanced cell motility, and upregulated proteolytic activity in matrix metalloproteases (MMPs). ^{2 3 4}  

The pleiotropic cellular effects of HGF are transduced through activation of its transmembrane receptor tyrosine kinase c-Met, which is a product of the c-met proto-oncogene. ⁵ On HGF binding, c-Met undergoes dimerization and autophosphorylation on tyrosine residues, generating multidocking sites, which activate diverse intracellular signaling pathways. Extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathways are two important kinase cascades that mediate HGF-induced invasion and metastasis. Specifically, activated c-Met can recruit growth factor receptor-bound protein 2 (Grb2); Grb2 interacts with son of sevenless (Sos) through its Src homology 3 (SH3) domain. ⁶ Sos promotes the activation of rat sarcoma (Ras), which triggers the ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathway by way of Ras-recombinant activated factor (Raf)-MAPK kinase (Mek)1/2. MAPK pathways play an important role in HGF-induced cell proliferation, migration, ⁷ invasion, ⁸ and branching morphogenesis. ⁹ PI3K is coupled to c-Met through interaction of its p85 subunit with the multidocking sites for Met. Activation of PI3K results in the production of inositol 3,4,5-triphosphate, which activates downstream target Akt. Activation of the PI3K/Akt pathway is responsible for cell motility. ⁷  

Uveal melanoma is the most common primary intraocular malignancy in adult humans, and the uvea is the second most common site for melanoma. ¹⁰ Unlike cutaneous melanoma, uveal melanoma disseminates preferentially to the liver through the hematogenous system; such liver homing is the leading cause of death in uveal melanoma patients. ¹¹ To date, the mechanism underlying this liver homing is largely unknown, but growth factors synthesized in the liver may be implicated. Recent evidence has shown that HGF may play an important role in uveal melanoma metastasis. Several studies have reported HGF as stimulating significant invasive responses in uveal melanoma cells, and functional blocking of HGF receptor c-Met completely abolishes this invasive response to HGF. ¹² High levels of HGF expression have been observed in primary uveal melanoma and in melanoma metastatic to the liver. ¹³ The expression of c-Met in uveal melanoma has been shown to correlate with metastatic phenotype. ¹⁴  

In contrast to the extensive studies on cutaneous melanoma, little is known about the molecular pathogenesis of uveal melanoma. This study investigated the effect of HGF on uveal melanoma cell migration and clarified the signal pathways triggered by HGF. Data showed that HGF can enhance migration and that HGF-induced migration depends on PI3K/Akt pathways. Activation of PI3K/Akt pathways induced by the HGF/c-Met axis is involved in the downregulation of cell adhesion molecules E-cadherin and β-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration of uveal melanoma cells. This study demonstrated for the first time that HGF receptor c-Met localizes to the nucleus in a ligand-dependent manner in uveal melanoma cells, which may play an important role in uveal melanoma migration. Furthermore, the central PI3K/Akt pathway in uveal melanoma migration may be important for the design of specific therapeutic strategies and future treatments for the control of metastasis in uveal melanoma. 

Materials and Methods

Reagents

Anti–ERK1/2, anti–phopho-ERK1/2, anti–Akt, anti–c-Met, anti–α-catenin, and anti–β-catenin reagents to be used for Western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–E-cadherin, anti–phospho-c-Met, and anti–phospho-AKT reagents were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human HGF and blocking antibody against c-Met were purchased from R&D Systems (Minneapolis, MN). PI3K inhibitor Ly294002 was purchased from Promega (Madison, WI). 

Cell Culture

The human uveal melanoma cell lines M17 and SP6.5 were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified incubator containing 5% CO₂, as described. ¹⁵  

Immunofluorescence Confocal Microscopy

Expression and location of c-Met in M17 and SP6.5 cells were detected by indirect immunofluorescence. M17 and SP6.5 cells were grown on coated glass coverslips for 48 hours and prestarved overnight in serum-free media. After treatment with or without HGF (40 ng/mL, 30 minutes), cells were fixed in 4% paraformaldehyde solution for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 in Tris-buffered saline (TBS) for 3 minutes After cells were blocked in TBS containing 5% bovine serum albumin (BSA) for 1 hour, primary antibody against c-Met was incubated in the blocking solution for 1 hour. FITC-conjugated secondary antibody was incubated for an additional hour in the blocking solution. After three washes with TBS, cells were stained for 15 minutes with propidium iodide (PI) to display the nuclei. Cells were mounted in a fluorescent mounting medium, and images were captured using a spinning disc confocal microscope (DSU; Olympus, Tokyo, Japan) and saved as digital images. 

Protein Extraction and Western Blotting

M17 and SP6.5 cells (1×10⁵) were seeded and grown in DMEM with 10% FBS in 12-well plates for 48 hours, then starved overnight in DMEM without FBS. After starvation, the cells were treated with HGF at 37°C for different time periods as indicated in the figure legends, then washed with cold phosphate-buffered saline (PBS) and subjected to lysis in a lysis buffer (50 mM/L Tris·Cl, 1 mM/L EDTA, 20 g/L sodium dodecyl sulfate [SDS], 5 mM/L dithiothreitol, 10 mM/L phenylmethylsulfonyl fluoride). Equal amounts of lysate (containing 50 μg protein) and of rainbow molecular weight markers (Amersham Pharmacia Biotech, Amersham, UK) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), then electrotransferred to nitrocellulose membranes. The membranes were blocked with a buffer containing 5% fat-free milk in PBS with 0.05% Tween 20 for 2 hours and incubated overnight with antibody at 4°C. After a second wash with PBS with 0.05% Tween 20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and developed with an electrogenerated chemiluminescence (ECL) detection kit (Pierce, Rockford, IL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. 

Wound-Healing Assay

In vitro wound-healing assay studies were conducted using previously described methods. ¹⁶ Briefly, M17 and SP6.5 cells were grown to confluence on 12-well tissue culture plates for 48 hours, then starved in serum-free DMEM overnight. A “wound” was made by scraping the middle of the cell monolayer with a sterile micropipette tip. Floating cells were removed by extensive washing with PBS. Fresh DMEM was then added to each plate, with or without HGF (40 ng/mL). Cells were photographed using a phase-contrast microscope at 0, 24, 48, and 72 hours after wounding. 

Transwell Migration Assay

M17 and SP6.5 cells were grown in DMEM with 10% FBS to 80% confluence and deprived of serum for 24 hours. The cells were harvested by trypsinization and washed once with D-Hanks solution. After washing, the cells were pretreated for 15 minutes with Ly294002 (10 μM) or c-Met blocking antibody (10 μg/mL) in some experiments. To measure cell migration, culture inserts (Transwell; 8-mm pore size; Costar, High Wycombe, UK) were placed into the wells of 24-well culture plates, separating the upper and the lower chambers. In the lower chamber, 400 μL DMEM containing HGF/SF (40 ng/mL), HGF (40 ng/mL) + Ly294002 (10 μM), or HGF (40 ng/mL) + c-Met blocking antibody (10 μg/mL) was added. Then, 1 ×10⁵ cells were added to the upper chamber. After 24 hours of incubation at 37°C with 5% CO₂, the number of cells that had migrated through the pores was quantified by counting five independent visual fields under the microscope (Olympus) using a 20× objective, and cell morphology was observed by staining with hematoxylin and eosin. Three independent assays were performed. 

Results

Enhancement of Migration of Uveal Melanoma Cells by HGF

The effect of HGF on the motility of uveal melanoma cell lines M17 and SP6.5 was assessed by in vitro wound-healing and transwell migration assays. When a wound was introduced into subconfluent cells, M17 and SP6.5 cells exposed to HGF migrated across the wound in greater numbers than did control cells over 24 to 72 hours. At 48 and 72 hours, wounds were completely closed by migrated M17 cells (Fig. 1A) . In a transwell assay, the ability of cells to migrate to the underside of the inserts was determined by 24-hour response to HGF in the lower chamber. The data showed that HGF induced a significant increase in the number of cells detected on the underside of the inserts. Compared with untreated control cells, the number of migrating cells increased approximately 30-fold in M17 cells and 10-fold in SP6.5 cells (Figs. 1B 1C) . 

HGF-Induced Activation and Translocation of c-Met

HGF exerts its effects through its transmembrane receptor tyrosine kinase c-Met. ¹⁷ To identify the functionality of the c-Met receptor, this study subsequently examined the effects of HGF on the activation of c-Met. As shown in Figure 2A , HGF induced c-Met activation in a time-dependent manner. The phosphorylation of c-Met increased at 5 minutes, reached a peak at 30 minutes, and decreased to baseline at 120 minutes in M17 and SP6.5 cells. In contrast to the change in c-Met phosphorylation, total c-Met expression was unchanged between controls and HGF-treated M17 and SP6.5 cells. Meanwhile, the blocking antibody against c-Met (10 μg/mL) completely abolished the migration of M17 and SP6.5 cells (Fig. 3) , suggesting that activation of c-Met was crucial to the migration induced by HGF in M17 and SP6.5 cells. 

Because subcellular location plays an important role in protein function, we used indirect immunofluorescence to investigate the translocation of c-Met induced by HGF in M17 and SP6.5 cells. For cells not treated with HGF, c-Met was distributed primarily in the cytoplasm of M17 cells and at the membrane periphery of SP6.5 cells (Fig. 2B) . However, treatment of cells with HGF (40 ng/mL, 30 minutes) led to the translocation of c-Met from the cytoplasm to the nucleus in M17 cells and from the cell membrane periphery to the cytoplasm in SP6.5 cells. 

Akt Activation but Not ERK1/2 Activation by HGF/c-Met Interaction

Binding of HGF to c-Met receptors activates multiple intracellular signaling pathways. We next sought to determine what pathways were activated by HGF in M17 and SP6.5 cells. Western blot analysis showed that the addition of HGF to M17 and SP6.5 cells did not change phosphorylation or total protein in ERK1/2 at different points in time (Fig. 4) . Under conditions of serum starvation, we found that ERK1/2 was constitutively activated in M17 and SP6.5 cells. This finding is consistent with previous reports. ^{18 19} However, HGF treatment of M17 and SP6.5 cells resulted in dramatic, rapid activation of Akt in a time-dependent manner. Maximum phosphorylation levels of Akt in M17 and SP6.5 cells treated with HGF were observed at 60 and 30 minutes, respectively, and phospho-Akt returned to near basal levels after HGF treatment of M17 and SP6.5 cells. No significant change in total Akt protein expression was detected over the course of the investigation (Fig. 4) . 

To further confirm that the Akt pathway was activated through the HGF/c-Met axis, a blocking antibody directed specifically against c-Met was used to block HGF/c-Met interaction. As shown in Figure 5A , activation of Akt by HGF was abolished in M17 and SP6.5 cells in a dose-dependent manner. These results suggest that HGF-induced AKT activation occurs specifically through its receptor, c-Met. 

Requirement of Activation of PI3-Kinase for HGF-Induced Akt Activation and Cell Migration

Akt is a downstream target of PI3K. However, some studies have indicated that Akt is activated by HGF, not only in a PI3K signaling pathway-dependent manner but also through PI3K-independent mechanisms. ²⁰ Therefore, we next examined whether PI3K involved the activation of AKT by HGF in M17 and SP6.5 cells. When M17 and SP6.5 cells were treated with different concentrations of a PI3K inhibitor, Ly294002, HGF-induced Akt activation in M17 and SP6.5 cells was inhibited in a dose-dependent manner (Fig. 5B) . This suggests HGF activates Akt in a PI3K-dependent manner. 

To further analyze whether PI3-kinase activity was necessary for HGF-induced cell migration, we performed transwell migration assays in the presence of the PI3K inhibitor Ly294002. As shown in Figure 3 , 10 μM Ly294002 partially abolished HGF-induced migration of M17 and SP6.5 cells. Taken together, these results demonstrated that activation of the PI3K/Akt pathway is involved in HGF-induced migration of uveal melanoma cells. 

Involvement of the PI3K/Akt Pathway via HGF/c-Met Axis in Regulation of Cell Adhesion Molecules

Accumulating evidence indicates that changes in expression or function of cell adhesion molecules can lead to loss of cell-cell contacts and gain of cell motility, each of which contributes to tumor invasion and metastasis. Therefore, we next examined the impact of HGF treatment on the expression of cell adhesion molecules. As shown in Figure 6 , when M17 and SP6.5 cells were treated with different concentrations of HGF for 24 hours, protein levels of E-cadherin and β-catenin were decreased in a dose-dependent manner, whereas α-catenin expression was unchanged compared with expression in untreated cells (Fig. 6) . To further demonstrate that activation of the PI3K/Akt pathway through the HGF/c-Met axis is involved in the downregulation of E-cadherin and β-catenin, PI3K inhibitor Ly294002 and an antibody blocking against c-Met were added to M17 and SP6.5 cells. The result revealed that both Ly294002 (10 μM) and c-Met blocking antibody (10 μg/mL) inhibited the decrease of E-cadherin and β-catenin induced by HGF (Figs. 7A 7B) . 

Discussion

Unlike cutaneous melanoma, which typically metastasizes to lymph nodes, lungs, and brain, uveal melanoma disseminates preferentially to the liver through the hematogenous system; such liver metastasis is the leading cause of death in patients with uveal melanoma. Previous studies have documented the functional importance of HGF in liver metastasis of uveal melanoma. ^{12 13 14 21} However, to date, the molecular mechanism of HGF-mediated uveal melanoma metastasis is still unknown. This study showed that HGF treatment of uveal melanoma cells results in PI3K-dependent activation of Akt specifically through its receptor, c-Met. Functionally, HGF induces c-Met–dependent cell migration, and Akt activation is involved in HGF-induced migration of uveal melanoma cells. Activation of the PI3K/Akt pathway induced by the HGF/c-Met axis is involved in the downregulation of E-cadherin and β-catenin. 

c-Met is a transmembrane receptor tyrosine kinase of 190 kDa. It is a disulfide-linked heterodimer consisting of a 45-kDa extracellular α-subunit and a 145-kDa β-subunit that spans the plasma membrane and contains the catalytic region with tyrosine kinase activity. Binding of HGF induces c-Met dimerization and autophosphorylation. ²² Several transmembrane receptors have been reported to translocate to the nucleus, including receptors for insulin, nerve growth factor, growth hormone, interleukin-1, c-erbB4, and HER-2/neu. These receptors can function as transcription factors to activate gene expression in the nucleus, and they affect the biological behavior of cells. ²³ The subcellular location of c-Met has been a matter of considerable scientific debate. Some studies have found that treatment of malignant pleural mesothelioma cells with HGF leads to the internalization of c-Met from the plasma membrane, ²⁴ whereas other research groups have shown c-Met expression not only at the cell membrane but also in the cytoplasm and the nuclei of breast cancer cells. ²⁵ This study showed that HGF induced the activation of c-Met in a time-dependent manner; such activation is necessary for the migration of M17 and SP6.5 cells because the blocking antibody against c-Met abolishes such migration. However, c-Met is found in different subcellular locations in M17 and SP6.5 cells. In non-HGF–treated cells, c-Met is distributed primarily in the cytoplasm of M17 cells and in the membrane periphery of SP6.5 cells; in contrast, treatment of cells with HGF led to the translocation of c-Met from the cytoplasm to the nucleus of M17 cells and from the cell membrane periphery to the cytoplasm of SP6.5 cells. In addition, this study showed that after HGF treatment, the number of migrating M17 cells was significantly (threefold) greater than that of SP6.5 cells. It is unknown how c-Met is translocated to the nucleus or whether the nuclear location of c-Met is implicated in the increased migration potential of uveal melanoma cells. Therefore, the subcellular location of c-Met and its function in uveal melanoma deserve further in-depth investigation. 

On HGF binding, c-Met undergoes dimerization and autophosphorylation of tyrosine residues, generating multidocking sites; this process activates diverse intracellular signaling pathways, including PI3K/Akt, MAPK ERK1/2, p38, and signal transducer and activation of transcription 3 (STAT3). Among the components analyzed, significant and time-dependent activation of Akt was observed. We showed clearly that HGF-induced PI3K-dependent activation of Akt specifically through its receptor, c-Met, and PI3K/Akt pathways activated by HGF were involved in the migration of uveal melanoma cells. Both c-Met–specific blocking antibody and PI3K inhibitor Ly294002 significantly inhibited not only the activation of AKT but also the migration induced by HGF in uveal melanoma cells. These findings agree with other results obtained from studies of fibrosarcoma, pancreatic cancer and fibroblasts. ^{26 27 28 29} Recently, however, two studies have shown that an Akt isoform, Akt1, suppressed the invasive migration of breast cancer cells. ³⁰ Therefore, it seems that the relationship between the activation of the PI3K/AKT pathway and tumor migration is cell type dependent. In addition, no significant regulation of p38, nuclear factor kappa B (NFκB), STAT3, or focal adhesion kinase (FAK) was observed though Western blot analysis in uveal melanoma cells after HGF treatment (data not shown). However, constitute activation of ERK1/2 was observed in M17 and SP6.5 cells. The finding is consistent with previous reports that activation of the MAPK pathway is a common event in uveal melanomas, which is responsible for the proliferation of uveal melanoma cells. ³¹ Therefore, it suggests that growth and migration may be separable events mediated by different factors, pathways, and mechanisms. HGF is most involved in promoting migration. 

Increasing evidence indicates that the disruption of normal cell-cell adhesion endows primary tumor cells with invasive and metastatic potential by increasing cell motility. E-cadherin is a member of the calcium-dependent transmembrane protein family, which forms a key component of adherent junctions and plays a major role in the establishment of cell-cell adhesion. β-catenin binds directly to E-cadherin and α-catenin, linking this complex directly or indirectly to the actin-based cytoskeleton. The E-cadherin-catenin complex plays important roles in many processes, including regulation of cell polarity, formation of junctional complexes, and migration of cells. Previous reports showed that E-cadherin is expressed in uveal melanoma. ^{32 33} The present study demonstrated that activation of the PI3K/Akt pathway induced by the HGF/c-Met axis was involved in the downregulation of E-cadherin and β-catenin by prolonged HGF stimulation. This finding is consistent with previous reports that active Akt downregulates the expression of E-cadherin and β-catenin in squamous cell carcinoma lines. ³⁴ Because HGF-induced migration and downregulation of E-cadherin and β-catenin were completely inhibited by the same concentration of blocking antibody against c-Met, this study concluded that HGF should be sufficient to attenuate cell-cell adhesion and to promote enhanced motility and migration of uveal melanoma cells. In addition, activated β-catenin translocates to the nucleus and drives gene transcription. It has been reported that the intracellular kinase domain of Met is essential for tyrosine phosphorylation and nuclear translocation of β-catenin in normal rat hepatocytes. ³⁵ However, we failed to detect the significant nuclear translocation of β-catenin after HGF treatment in M17 and SP6.5 cells (data not shown). We conclude that the nuclear translocation of β-catenin does not play an important role in the migration of M17 or SP6.5 after HGF treatment. 

In conclusion, this study has shown for the first time that activation of the PI3K/Akt pathway induced by the HGF/c-Met axis plays a key role in the migration of uveal melanoma cells. The central role of the PI3K/AKT pathway in uveal melanoma migration may be important in the design of specific therapeutic strategies and future treatments to control the metastasis of uveal melanoma. 

 Supported by Grant Y206770 from the Zhejiang Foundation of Natural Sciences.

 Submitted for publication July 31, 2007; revised September 16 and October 7, 2007; accepted November 26, 2007.

 Disclosure: M. Ye, None; D. Hu, None; L. Tu, None; X. Zhou, None; F. Lu, None; B. Wen, None; W. Wu, None; Y. Lin, None; Z. Zhou, None; J. Qu, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effect of HGF/SF on the migration of uveal melanoma cells. (A) In vitro wound-healing assay of uveal melanoma cell lines. Scratches were made in the monolayer, and the cells were cultured for 0, 24, 48, or 72 hours in DMEM without serum or in DMEM containing 40 ng/mL HGF/SF. The scratched regions were photographed under a phase-contrast microscope (×100). Representative areas from triplicate experiments are presented. (B) Transwell migration assay of uveal melanoma cell lines. M17 and SP6.5 cells were serum starved for 24 hours and plated in DMEM without (Ctrl) or with HGF (40 ng/mL). Cell morphology was observed by staining with hematoxylin and eosin. (C) The number of cells that had migrated through the pores was quantified by counting five independent visual fields using a 20× microscope objective. Three independent assays were performed.

Figure 2.

 

Activation and translocation of c-Met induced by HGF. (A) M17 and SP6.5 cells were treated with 40 ng/mL HGF for 0, 5, 15, 30, 60, or 120 minutes. Cell lysates were prepared and used for Western blot analysis with phospho-c-Met, total c-Met, or GAPDH antibody to investigate c-Met activation. (B) M17 and SP6.5 cells on glass coverslips were serum starved overnight and stimulated with HGF (40 ng/mL) for 30 minutes. Cells were fixed with paraformaldehyde and stained for c-Met (green), with phosphatidylinositol (PI) as nuclear counterstain (red).

Figure 3.

 

c-Met and Akt activation is required for HGF-induced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was carried out. Blocking antibody against c-Met (10 μg/mL) or PI3K inhibitor Ly29002 (10 μM) was added, together with HGF, in the lower chamber. (A) Cell morphology was observed by staining with hematoxylin and eosin. (B) The number of cells that had migrated through the pores was quantified by counting five independent visual fields using a 20× microscope objective. Three independent assays were performed.

Figure 4.

 

Activation of Akt and ERK1/2 induced by HGF. M17 and SP6.5 cells were treated with 40 ng/mL HGF for 0, 5, 15, 30, 60, or 120 minutes. Cell lysates were prepared and used for Western blot analysis with phospho-ERK1/2, total ERK, phospho-Akt, total Akt, or GAPDH antibody to investigate Akt and ERK1/2 activation.

Figure 5.

 

Effects of specific inhibitor and blocking antibody on activation of Akt induced by HGF. (A) M17 and SP6.5 cells were preincubated for 2 hours with 10 μg/mL blocking antibody against c-Met or (B) different concentrations of PI3K inhibitor Ly294002, followed by treatment with or without 40 ng/mL HGF for 30 minutes. Cell lysate preparation and Western blot analysis were performed.

Figure 6.

 

HGF induces the downregulation of E-cadherin and β-catenin. M17 and SP6.5 cells were treated with different concentrations of HGF for 24 hours. Whole cell protein extracts were subjected to Western blotting using anti–E-cadherin, anti–β-catenin, and anti–α-catenin antibodies. GAPDH was used as a loading control.

Figure 7.

 

PI3K inhibitor and blocking antibody against c-Met inhibited the downregulation of adhesion molecules by HGF. (A) M17 and SP6.5 cells were preincubated for 2 hours with different concentrations of PI3K inhibitor Ly294002 or (B) 10 μg/mL blocking antibody against c-Met, followed by treatment with or without 40 ng/mL HGF for 24 hours. Cell lysate preparation and Western blot analysis were performed. GAPDH was used as a loading control.

The authors thank Chenghan Huang (New York Blood Center) for critical reading of this manuscript.