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The over-expression of the ephrin-A1 ligand receptor EphA2 is associated with the growth and metastatic potential of tumors. Although EphA2 is expressed in a variety of tumors, its expression and function in malignant mesothelioma (MM) remain unknown. The authors hypothesized that expression of the receptor EphA2 in MM cells (MMCs) plays a key role in the growth and haptotactic migration of MM. They also hypothesized that silencing EphA2 expression by using small-interfering RNA (siRNA) inhibits the proliferation and haptotaxis of MMCs and induces apoptosis in MMCs.
The expression of EphA2 in MMCs and in normal pleural mesothelial cells (PMCs) was studied by using real-time quantitative polymerase chain reaction analysis and Western blot analysis. The growth of MMCs was determined with the WST-1 cell-proliferation assay. The haptotactic migration of MMCs and PMCs was determined with a Boyden chamber assay. Expression of caspases was determined with calorimetric assays.
The results demonstrated that silencing the receptor EphA2 by siRNA significantly reduced the proliferation and haptotactic migration of MMCs compared with controls. Over-expression of EphA2 with plasmid pcDNA/EphA2 enhanced the proliferation and haptotaxis of MMCs significantly. Knocking down EphA2 expression initiated caspase-9–mediated apoptosis in MMCs.
Malignant mesothelioma (MM) is a highly aggressive tumor that arises from the mesothelial lining of pleural cavities.1 Approximately 3000 patients are diagnosed with MM annually in United States. MM has been associated with asbestos exposure, and it is expected that the incidence of mesothelioma will increase over the next 10 to 20 years.2 More than 50 years after the first description of mesothelioma as a pathologic entity, satisfactory treatment remains elusive. MM responds poorly to conventional treatments, such as chemotherapy, surgery, and radiotherapy. Hence, identifying novel approaches to understanding the molecular events involved in tumor growth and pathogenesis of MM is important.
Eph receptors are the largest family of trans-membrane proteins with an extracellular domain that is capable of recognizing signals from the cell microenvironment and of influencing cell-cell interaction and migration. Receptor EphA2 and its ligands are members of the family of receptor tyrosine kinases (RTK) and play an important role as modulators of a variety of processes during embryonic development.3, 4 EphA2 is expressed at a minimal level in mature cells, especially epithelial cells.5 In malignancy, the receptor EphA2 and its ligands are over-expressed, and the functional alterations of Eph receptor kinases are prevalent in many tumors.6–10 It is known that MM expresses a variety of RTKs, such as epidermal growth factor receptor (EGFR), c-Met, platelet-derived growth factor receptor, and vascular endothelial growth factor receptor.11–13 However, to our knowledge, the possible presence and function of EphA2 and the expression of its ligands in MM are not known. The over-expression of receptor EphA2 has been implicated in tumor growth, angiogenesis, and metastasis.14–17 Therefore, EphA2 appears to be a potential therapeutic target for the containment of tumor growth. In the current study, we demonstrate that EphA2 is expressed in MM cells (MMCs), that the inhibition of EphA2 expression by using small interfering RNA (siRNA) inhibits tumor cell proliferation and migration, and that the posttranscriptional blocking of EphA2 receptor induces apoptosis in MMCs through the initiation of caspase-9 activity.
MATERIALS AND METHODS
Culture of MMCs and Pleural Mesothelial Cells
The MMC lines crl-2081, crl-5915, and crl-5820 (designated MMC1, MMC2, and MMC3, respectively) were obtained from American Type Culture Collection. The MMCs were grown in McCoy medium (GIBCO-BRL, Baltimore, MD) and fetal bovine serum (FBS) from Harlan Bioproducts (Indianapolis, IN). For human pleural mesothelial cells (PMCs) in primary culture (Clonetics, San Diego, CA), between 4 and 8 passages were used. The cells were resuspended in culture Media-199 (Gibco Laboratories, Grand Island, NY) containing 10% FBS (Atlanta Biologicals), penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were plated in 75-cm2 culture flasks (Corning Costar Corporation, MA) and incubated at 37°C in 5% CO2 and 95% air. The medium was changed on alternate days. The mesothelial cells were characterized by the presence of classic cobblestone morphology, the absence of factor VIII antigen, and the presence of cytokeratin, as reported previously.18 When the cells were confluent, they were trypsinized and seeded into culture flasks/transwell chambers as required for different assays. All experiments were performed on cells from 1 lot.
To evaluate EphA2 receptor expression in MMCs and PMCs, the cells were analyzed by using confocal laser-scanning microscopy (Zeiss LSM 510, Axiovert 100M; Zeiss, Thornwood, NY), as reported previously.18 In brief, the cells were cultured to confluence on gelatinized glass coverslips and fixed in 5% paraformaldehyde (with 50 mM phosphate buffer) in 50% Tris wash buffer (TWB). The glass coverslips were rinsed 3 times and permeabilized with 1.2% Triton X-100 for 5 minutes, rinsed 3 times, incubated with 1% bovine serum albumin (BSA) in 100% TWB for 1 hour, then stained for the expression EphA2 receptor using primary antibody rabbit anti-EphA2 at 1:150 dilution and secondary antibody goat antirabbit immunoglobulin G conjugated with fluorescein isothiocyanate (FITC) (Zymed Laboratories, San Francisco, CA). Rhodamin phalloidin was used for F-actin staining, and 4,6-diamino-2-phenylindole was used as a nuclear stain.
Total RNA from cultured MMCs and PMCs was purified and diluted with RNase-free water to 100 ng/μL; then, 10 μL of each sample were reverse transcribed into combinational DNA (cDNA). After the reverse transcription reaction was finished, 80 μL of RNase-free water were added to each sample. Ten microliters of diluted cDNA product were mixed with 25 μL of SYBR Green JumpStart Taq ReadyMix, 0.5 μL of internal reference dye, and 14.5 μL of specific oligonucleotide primers (80 nM final concentration) to a total volume of 50 μL for quantification of the real-time polymerase chain reaction (PCR) (Table 1). The quantification of real-time PCR was performed by using the SYBR Green method on the Applied Biosystems 7500 Real Time PCR System with the following profile: 1 cycle at 94°C for 2 minutes; 40 cycles at 94°C for 15 seconds, at 60°C for 1 minute, and at 72°C for 1 minute; and the at acquired fluorescent signal from the elongation step. The real-time PCR products were confirmed by electrophoresis on 2% agarose gels.
Table 1. Primers Used in Quantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis
Gene name (Accession no.)
Forward primer (5′-3′)
Reverse primer (5′-3′)
Product size (base pairs)
EphA indicates ephrin-A ligand receptor.
Data analysis was carried out by using the ABI sequence-detection software using relative quantification. The threshold cycle (Ct), which is defined as the cycle at which PCR amplification reaches a significant value, is expressed as the mean value. The relative expression of messenger RNA (mRNA) was calculated by using the ΔCt method (where ΔCt is the value obtained by subtracting the Ct value of the housekeeping gene β-actin mRNA from the Ct value of the target mRNA). The amount of the target relative to β-actin mRNA was expressed as 2−(ΔCt).
Construction of Vector Containing EphA2 Receptor and Transient Transfection of MMC Lines
The gene-transfer vector pcDNA3.2/V5-DEST was used as an expression vector for the expression of receptor EphA2, and pcDNA3.2/V5/CAT was used as a control vector (Invitrogen, Carlsbad, CA). The pENTR TOPO vector, which contains EphA2 insert, was expanded in OneShot Top10 cells and cloned into the destination vector pcDNA3.2/V5-DEST according to the manufacturer's instructions (Invitrogen). The cloned vector was designated as pcDNA/EphA2, and the control vector or pcDNA/CAT was designated as empty vector. The MMCs and PMCs were transfected with pcDNA/EphA2 and empty vector using lipofectamine-2000 reagent (Invitrogen). The transfected cells were used in further experiments.
siRNA and Treatment of MMCs
The siRNA targeting the receptor EphA2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). MMCs and PMCs were plated into 6-well plates or 24-well plates as required for the experiments. The cells were allowed to adhere for 24 hours. The transfection of siRNA was performed using lipofectamine-2000 (Invitrogen) according to the manufacturer's recommendation. After 4 hours of transfection, the culture medium containing 10% serum was added. The assays were carried out 48 hours posttransfection.
Western Blot Analysis
MMCs and PMCs were cultured on 6-well tissue culture plates (Costar) to confluence. The cells were lysed in lysis buffer, as reported previously.18 Total protein was estimated by using the bichloroacetic acid method (Pierce, Rockford, IL). Equal amounts of protein (20 mg per lane) were loaded. Proteins in the samples were separated onto denaturing sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gels (Bio-Rad) and were transferred electrophoretically onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). The blots were blocked overnight at 4°C with BSA and were incubated with mouse antihuman EphA2, EphA1, EphA3, EphA4, and EphA5 at 1:500 dilution for 1 hour at room temperature (Zymed Laboratories). After washing, they were incubated with the second antibody (horseradish peroxidase-conjugated antimouse immunoglobulin G antibody) at a dilution of 1:1000 for 1 hour. Eph receptors were detected by using enhanced chemiluminescence (Amersham Pharmacia Biotech). Prestained protein markers were included for molecular mass determination (Bio-Rad).
Cell Proliferation Assay
MMC proliferation was assessed by using an assay based on cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases (Roche, Indianapolis, IN). With this assay, an increase in the number of viable cells results in an increase in the overall activity of the mitochondrial dehydrogenases in the sample. The augmentation in enzyme activity leads to an increase in the formazan dye formed. The formazan dye formed was quantified by using a plate reader at 440 nm. MMCs were plated in 96-well microplate at a density of 0.5 × 105 cells per well. The cells were transfected with siRNA for EphA2 or with scrambled siRNA (sc-siRNA) by using lipofectamine 2000 reagent, and a few wells were left untreated. The negative controls received serum-free media, and some of the wells received pcDNA/EphA2. Cells were allowed to incubate for 48 hours. Then, the WST-1 reagent was applied for 4 hours to measure cell proliferation. The cell proliferation was assessed in triplicate. The data are presented as a percentage of negative control proliferation with P values <.05 considered significant. Each experiment was repeated 4 times.
Cell Proliferation by Direct Cell Counts
To monitor MMC proliferation, we also evaluated direct total cell counts, as described previously.19 Briefly, MMCs (5 × 104 cells) and PMCs were plated in 24-well culture plates. In some of the wells, the cells were transfected with different concentrations of siRNA EphA2 or sc-siRNA, and some wells were left untransfected. Some of the wells received pcDNA/EphA2 and empty vector. After 48 hours of incubation, the cultures were trypsinized, and the total cells were counted on a hemacytometer. All experimental conditions were repeated 4 times in triplicate.
Haptotaxis was performed by using Boyden chambers as described previously.20 In brief, the lower side of the filters (8-μm pore size) were coated with BSA (1 mg /mL) and were left overnight at 37°C in humidified air in the presence of 5% CO2. The filters were removed, washed with RPMI, and air dried. To observe the effect of siRNA EphA2, the MMC lines and PMCs were transfected with 10 nM, 50 nM, and 100 nM of siRNA EphA2 or sc-siRNA prior to using the haptotactic assay. Filters were placed into a Boyden chamber, and lower chambers were filled with RPMI with 1% FBS. In some wells, cells that were transfected with pcDNA/EphA2 and empty vector were added. Next, 1 × 105 cells were seeded into the upper chamber and incubated for 6 hours at 37°C. At the end of the incubation, the medium from the upper well was discarded. The upper sides of the filters were scraped to remove the adherent cells. The filters were removed, fixed in formalin, and stained with Diff-Quik (Baxter S.P.A., Rome, Italy). The number of cells that migrated was quantitated by counting the number of cells on the distal surface of the filter under an optical microscope. The results were expressed as haptotactic index, i.e., the number of cells visualized per 10 high-power fields.
Detection of Apoptosis by Annexin-V and Propidium Iodide Staining
MMCs at 80% confluence were transfected with siRNA for EphA2 and sc-siRNA. After 48 hours, detached cells in the medium were collected, and the remaining adherent cells were harvested by trypsinization. The cells (1 × 105) were washed with phosphate-buffered saline and resuspended in 250 μL of binding buffer (Annexin-V/FITC kit; Becton Dickinson, Mountain View, CA) that contained from 10 μL of 20 μg/mL propidium iodide (PI) and 5 μL of Annexin-V/FITC. The data were collected on FACSCalibur (Becton Dickinson). FITC and PI emissions were detected in the FL-1 and FL-2 channels, respectively. Subsequent analyses were done with CellQuest software (Becton Dickinson).
Caspase 8 and Caspase 9 Activity Analysis
Caspase activity was measured by calorimetric assay according to the manufacturer's instructions (R&D systems, Inc., Minneapolis, MN). Briefly, cells were transfected with EphA2 siRNA and sc-siRNA; and, after 24 hours, cell lysates were incubated with reaction buffer and the appropriate calorimetric substrate at 37°C for 2 hours. Color development was quantified by measuring absorbance at 405 nm.
The unpaired Student t test was used to determine statistical significance. Differences were considered significant at P values <.05.
MMCs Express Receptor EphA2
Eph RTK expression was evaluated in 3 MMC cell lines and in normal PMCs. The expression of Eph receptors, such as EphA1, EphA2, EphA3, EphA4, and EphA5, was tested. Among the 5 Eph receptors tested, only receptor EphA2 showed high expression in all 3 MMC lines. MMC1 also showed high expression for EphA4 and EphA5 compared with MMC2 and MMC3. The MMC1 and MMC2 cell lines showed strong bands for EphA2 receptor in Western blot analysis compared with the MMC3 cell line and the PMCs. However, significantly lower expression of EphA2 was observed in normal PMCs. β-actin was used as a loading control to demonstrate equal loading of protein (Fig. 1).
Expression of mRNA Levels in MMC Lines
To investigate the transcription-translation of EphA2, we measured the mRNA levels in MMC1, MMC2, MMC3 cell lines and in PMCs by using real-time reverse transcription-PCR, as illustrated in Figure 2. The Eph receptors product was normalized to β-actin. The fold increase in EphA2 mRNA levels was 18-fold in the MMC1 cell line, 12-fold in the MMC2 cell line, 10-fold in the MMC3 cell line, and 0.5-fold in the PMCs. The levels of mRNA were correlated with protein expression in the cell lines MMC1 and MMC3. However, the PMCs showed low levels of EphA2 mRNA. The low levels of protein were correlated with low levels of mRNA in PMCs. These results suggest that a posttranslational regulatory mechanism also contributes to the high levels of EphA2 protein in MMCs. In MMC1, an 18-fold increase in the expression of EphA2 mRNA was observed compared with other cell lines, and increases of 12-fold and 10-fold increases were observed in the MMC2 and MMC3 cell lines. Weak expression levels of EphA1, EphA4, and EphA5 were observed, whereas EphA3 levels were undetectable. A significantly lower expression or no expression of mRNA for Eph receptors was observed in PMCs.
Immunofluorescence Staining for Receptor EphA2 in MMCs and PMCs
We analyzed the expression of receptor EphA2 by using confocal microscopy (Fig. 3). MMCs showed very strong EphA2 receptor expression compared with normal PMCs. Cell-cell contact was reduced greatly in all MMC lines. The actin cytoskeleton was disrupted, as indicated by diffuse staining for F-actin filaments, compared with normal PMCs (Fig. 3B). PMCs showed lower expression of EphA2.
siRNA EphA2 Significantly Down-Regulates the Expression of EphA2
MMCs were transfected with varying concentrations of siRNA EphA2 (10 nM, 50 nM, and 100 nM). A significant down-regulation of EphA2 receptor was observed in MMC1 in a concentration-dependent manner. siRNA EphA2 at concentrations of 50 nM and 100 nM knocked down EphA2 receptor expression 60%, and 75%, respectively, compared with controls and sc-siRNA. However, we did not observe a significant effect of transfection with siRNA for EphA2 on PMCs (Fig. 4).
siRNA EphA2 Significantly Inhibits the Proliferation of MMCs
A colorimetric assay (WST-1) was performed to assess the effect of siRNA on cellular proliferation in the 3 MMC lines. MMCs were transfected with various concentrations (10 nM, 50 nM, and 100 nM) of receptor EphA2 siRNA, and the proliferation of cells was evaluated. MMCs showed significant down-regulation of proliferation when they were transfected with siRNA EphA2 in a concentration-dependent manner compared with sc-siRNA. At a 10-nM concentration of siRNA EphA2, an insignificant decrease in cell proliferation was observed. At a 50-nM concentration of siRNA EphA2, a 53.84 ± 3.91% decrease was observed in the proliferation of MMC1 cells, and a 32.60 ± 2.53% decrease in the proliferation of MMC2 cells was observed (Fig. 5A,B). At a 100-nM concentration of siRNA EphA2, a maximum decrease in proliferation was observed in MMC1 cells (67.70 ± 5.98%) and in MMC2 cells (51.25 ± 4.91%) compared with sc-siRNA. The cell line MMC3 showed minimum effect (Fig. 5C). MMC3 is a less aggressive cell line, and the expression of EphA2 in MMC3 was lower compared with expression in the MMC1 and MMC2 cell lines. The transfection of MMCs with plasmid-containing pcDNA/EphA2 significantly up-regulated the proliferation of MMCs. It is noteworthy that, in the MMC3 cell line, transfection with plasmid-containing pcDNA/EphA2 increased proliferation significantly compared with the other MMC lines. However, PMCs did not show any significant effect (Fig. 6). These data suggest that EphA2 receptor expression is involved in the growth of MMCs. To confirm the growth of MMCs, we also used a cell-growth assay, which produced similar effects (Fig. 6B). With that assay, interleukin 8 (50 ng/mL) was used as a positive control, and a remarkable increase was observed in the proliferation of the MMC1 and MMC2 cell lines. The relative percent increase observed was 48.25 ± 4.42% in the MMC1 cell line (P <.001 vs. control) and 23.84 ± 2.92% in the MMC2 cell line (P <.001 vs. control). MMC3 cells and PMCs did not show any significant effect compared with the control.
EphA2 Mediates Haptotactic Activity in MMCs
Haptotactic migration was evaluated in all 3 MMC lines and in PMCs. Silencing interference RNA for EphA2 significantly down-regulated the haptotactic activity of MMCs in all 3 cell lines in a concentration-dependent manner (Fig. 7A). A significant inhibition (P <.05) of haptotaxis of the MMC1, MMC2, and MMC3 cell lines was observed at siRNA EphA2 concentrations of 50 nM and 100 nM compared with sc-siRNA. The over-expression of EphA2 by transfecting the MMC lines with pcDNA/EphA2 significantly increased the haptotactic migration in the MMC lines compared with empty vector. The percent increase observed over control was 40.36 ± 4.08% in the MMC2 cell line and 162.50 ± 23.35% in the MMC3 cell line. It is noteworthy that the stable transfectants of cell line MMC3 showed a 2.6-fold higher response compared with empty vector transfectants. PMCs did not show any significant change in haptotactic activity (Fig. 7B). The results suggest that, when the MMC3 cell line was transfected with plasmid (pcDNA/EphA2), it attained aggressive behavior and increased haptotactic migration compared with other 2 MMC cell lines. This also demonstrated the functional significance of receptor EphA2 in the modulation of migratory behavior of MMCs.
Knock-Down of EphA2 by siRNA Induces Apoptosis through Caspase 9 Activation in MMCs
When MMC1 cells were transfected with siRNA EphA2 (50 nm), a significant increase in apoptotic cells (24.48 ± 2.57%; P <.05) was observed with annexin-V staining compared with sc-siRNA (Fig. 8). Silencing EphA2 resulted in the activation of caspase 9 activity in MMC1 cells. However, caspase 8 activity did not show any significant increase in activity compared with caspase 9 (Fig. 9A). Western blot analysis for caspase 9 showed procaspase 9 (≈ 45 kDa) and an active caspase 9 band (≈ 10 kDa). The active caspase 9 band was stronger in the MMC1 cells that were treated with siRNA EphA2 compared with sc-siRNA. These results suggest that knock down of the EphA2 gene in MMC1 cells initiated apoptotic signaling through the activation of caspase 9, which is a candidate of intrinsic pathway (Fig. 9B).
In the current study, we demonstrated that MMCs express the RTK EphA2. The results indicated that immunofluorescence staining for EphA2 expression was higher in all 3 MMC cell lines that were tested compared with PMCs. Silencing expression of the EphA2 receptor gene by using siRNA significantly down-regulated expression of the EphA2 receptor gene in MMCs compared with sc-siRNA. Moreover, the proliferation and haptotactic migration of MMCs decreased significantly. Knock down of EphA2 gene induced apoptosis through the induction of caspase 9 activity. The biologic response was lower in the cell line MMC3 and in normal PMCs compared with the cell lines MMC-1 and MMC-2. These findings from the current study suggest that low receptor EphA2 expression in the cell line MMC3 may be responsible for the poor response observed in these cells, and this may be related to poor growth and invasive capacity. The transfection of MMCs with plasmid pcDNA/EphA2 increased the proliferation and haptotactic migration of MMCs, and particularly of the MMC3 cells. However, we observed a very low level of transfection efficiency in PMCs for EphA2 receptor expression. Consequently, transfected PMCs did not show significant increases in proliferation or haptotactic migration.
EphA2 is altered functionally in tumor cells. Normal cells generally have highly stable cell-cell contacts, and EphA2 binds to its ligands, which are anchored to the cell membrane, and, thus, regulates the level of EphA2 protein.21–23 In malignancy, cell growth is highly deregulated, and the cross-talk between the adjacent cells is reduced markedly. The interaction between the receptor EphA2 and its membrane-bound ligand is decreased and results in the accumulation of EphA2 protein in the cytoplasm, as observed by immunofluorescence staining. The aggressive growth and resistance to apoptosis may be associated with the oncogenic potential of the protein EphA2. Previous reports have indicated that blocking EphA2 signaling with ligand-binding agents inhibits tumor growth.24–26 Antibodies to EphA2 and a short peptide that binds to the EphA2 receptor currently are being investigated as therapeutic agents.27, 28
EphA2 protein is expressed in all the MMCs with little or no expression in normal mesothelial cells. To our knowledge, this is the first report to demonstrate receptor EphA2 expression in MM. Earlier studies reported the expression of EphA2 in several cancers, including prostrate, breast, gastric, and ovarian cancers.7, 10, 14, 15 Our current results indicate an association between the level EphA2 receptor expression and aggressive tumor growth and migration. The high level of EphA2 expression in the MMC1 and MMC2 cell lines was associated with increased proliferation and migration. Knock down of the EphA2 gene by using siRNA markedly inhibited proliferation and haptotaxis in MMCs. We speculate that receptor EphA2 expression on the surface of MMCs may be responsible for its aggressive behavior and local tissue invasion. Moreover, the receptor EphA2 is a novel biomarker and is a potential therapeutic target for containing the growth of MM.
Malignant cells frequently are resistant to apoptotic stimuli, and several lines of evidence suggest that the inhibition of apoptosis function may enhance tumor development.29, 30 MM traditionally is associated with occupational exposure to asbestos fibers.31, 32 However, recent reports suggest that genetic susceptibility and Simian virus 40 infections also cause the modifications in cell signaling events, particularly the induction of cell survival pathways and inhibition of apoptosis that favors the growth of MM.33, 34 Activation of caspases is 1 of the key events in the process of initiation of apoptosis. Caspases 8 and 9 have been associated with the initiation of apoptosis and are called initiator caspases. Caspase 9 is an initiator caspase in the intrinsic pathway for apoptosis, and caspase 8 is an initiator caspase in the extrinsic pathway. In the current study, procaspase 9 levels were decreased in samples that were transfected with siRNA EphA2; and a strong, active caspase 9 band was observed. This suggests that blocking the expression of receptor EphA2 induced apoptosis by activating caspase 9 signaling.
It is noteworthy that the destruction of specific mRNA using siRNA is a powerful tool in the analysis of protein function. To our knowledge, this is the first in vitro study to show that silencing EphA2 receptor inhibits the growth and migration of MMCs. Silencing the EphA2 gene also induced apoptosis in MMCs through the activation of caspase 9. The overexpression of receptor by using vector that contained EphA2 significantly up-regulated the proliferation and migration behavior in the MMC3 line. These results demonstrate the potential for using siRNA to knock down the oncogenic protein EphA2 to treat patients with MM.
We acknowledge the technical help of Xiao hong Wang.