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Keywords:

  • malignant pleural mesothelioma;
  • SOCS-1;
  • gene therapy;
  • NF-κB;
  • STAT3

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO-4, H28 and H226) with adenovirus-expressing SOCS-1 vector to examine the effect of SOCS-1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS-1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF-κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS-1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS-1 regulated NF-κB and STAT3 signaling to induce apoptosis in MESO-4 and H226 cells. Furthermore, SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF-κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS-1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed.

Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells of serosal cavities. MPM may be asymptomatic at the early stage and is sometimes observed incidentally during routine chest radiography. Common symptoms include chest pain and dyspnea, which are caused by tumor invasion of the chest wall or pleural effusion and occur late during disease progression. Therefore, complete surgical resection is not applicable in the majority of patients at the time of diagnosis of this disease. Although chemotherapy with cisplatin plus pemetrexed improves survival time for patients with unresectable MPM, the overall median survival time is only 12 months.1 Among molecular-targeted therapies, epidermal growth factor receptor (EGFR) inhibitors and angiogenesis inhibitors have been tested for MPM but without therapeutic benefit.2 MPM is often associated with past exposure to asbestos, in which case there is a long latency period, often exceeding 20 years, between first exposure to asbestos and diagnosis of MPM.3 The number of deaths from MPM is expected to increase in the next 20 years worldwide where heavy use of asbestos has occurred.3–6 Thus, there is a growing need for the development of new therapies to treat this disease.

Inflammation is considered to play a critical role in the development and progression of various cancers, including MPM.7 Asbestos-induced chronic inflammation is implicated in the pathogenesis of MPM. Tumor development and progression induced by an inflammatory response are thought to be mediated by an interaction between proinflammatory cytokines and pathways including NF-κB and STAT3 that promote antiapoptotic signaling.8 It was reported that asbestos caused an increase in the expression of NF-κB and proinflammatory cytokines such as TNF-α and interleukin (IL)-6.9–11 The suppressor of cytokine signaling (SOCS) family proteins participate in the negative regulation of cytokine responses by terminating the activation of multiple signaling pathways.12–14 Among these, SOCS-1 is known as the most potent negative regulator of proinflammatory signaling including NF-κB and STAT3 pathways. SOCS-1 was reported to ubiquitinate NF-κB p65, inducing subsequent degradation by proteasome.15 On the other hand, SOCS-1 interacts with phosphorylated tyrosine residues on JAK kinases to interfere with the activation of STAT proteins.16, 17 Epigenetic silencing of SOCS-1 is detected in human cancers, such as hepatocellular carcinoma, gastric carcinoma, multiple myeloma and pancreatic ductal neoplasm, and is implicated in cancer development.18–22 However, the role of SOCS-1 in MPM have not yet been investigated.

Because MPM locates within the thoracic cavity and rarely displays widespread metastasis, gene transfer to the thoracic cavity makes this tumor uniquely accessible, thus facilitating the direct administration of novel therapeutic agents and subsequent analysis of treatment effects. Clinical trials involving intrapleural administration of adenoviral vectors to patients with MPM have demonstrated that intrapleural gene therapy using adenoviral vectors is safe and well tolerated by patients with MPM.23, 24 In the current study, we demonstrate the silencing of SOCS-1 in MPM cell lines and the antitumor effect of SOCS-1 gene delivery against MPM in vitro and in vivo. Current first-line chemotherapy of MPM is cisplatin plus pemetrexed. Pemetrexed has shown modest activity as single agent in patients with MPM, and treatment with pemetrexed plus cisplatin resulted in superior survival time compared to treatment with cisplatin alone in patients with MPM.1 Therefore, we evaluated combined cisplatin plus pemetrexed, the first-line chemotherapy of this disease, with SOCS-1 gene delivery in the treatment of MPM.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell lines

Mesothelioma cell lines H28 and H226 were purchased from American Type Culture Collection (Manassas, VA). Mesothelioma cell line ACC-MESO-4 (MESO-4) cell lines were purchased from RIKEN BRC cell bank (Tsukuba, Japan). The identity of each cell line was confirmed by DNA fingerprinting via short tandem repeat (STR) profiling on June 30, 2011. The method used for testing was multiplexed PCR amplification of eight STR loci (TH01, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA and TPOX), and amelogenin was performed using the PowerPlexTM16 System (Promega, Madison, WI). PCR-amplified fragments were analyzed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Then the fragments were typed based on allelic ladders. All the cells were cultured in RPMI 1640 (Wako, Osaka, Japan) with 10% fetal calf serum (FCS; HyClone Laboratories, Logan, UT), 100 IU/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan) at 37°C under a humidified atmosphere of 5% CO2.

Reagents

Recombinant human IFN-γ, TNF-α and IL-6 were purchased from PeproTech (Rocky Hill, NJ). Recombinant-soluble IL-6 receptor (sIL-6R) was obtained from Chugai Pharmaceutical (Tokyo, Japan). Cis-platinum(II) diammine dichloride (cisplatin) was purchased from Sigma (St. Louis, MO). Pemetrexed disodium was purchased from Toronto Research Chemicals (Ontario, Canada). Recombinant-active caspase-3 and the caspase-3 inhibitor z-DEVD-fmk were purchased from BD Pharmingen (San Diego, CA).

Real-time PCR analysis

After 12 hr of serum starvation, mesothelioma cell lines (MESO-4, H28 and H226) and human PBMC were treated with 10 ng/ml of recombinant human IFN-γ (PeproTech) for 15 min. Total RNA was prepared from cells using an RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNAs were synthesized from 200 ng of each total RNA preparation using a Quantitect Reverse Transcription Kit (Qiagen), all according to the manufacturer's instructions. The forward and reverse primers were as follows: for human SOCS-1 forward primer, 5′-AGACCCCTTCTCACCTCTTG-3′ and reverse primer, 5′-GCACAGCAGAAAAATAAAGC-3′; for β-actin, 5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′.25 Primers and cDNA were added to SYBR green premix (Invitrogen, Carlsbad, CA), which contained all the reagents required for PCR. The PCR conditions of SOCS-1 consisted of one cycle at 95°C for 10 min followed by 40–50 cycles of 96°C for 10 sec, 68°C for 15 sec and 72°C for 15 sec; β-actin cycling conditions consisted of one cycle at 95°C for 10 min followed by 40–50 cycles of 96°C for 10 sec, 67°C for 30 sec and 72°C for 30 sec. PCR products were measured continuously using the My IQ™ Single-Color Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA).

Preparation of adenoviruses

Replication-defective recombinant adenoviral vector expressing the mouse SOCS-1 gene was provided by Dr. Hiroyuki Mizuguchi (Osaka University, Osaka, Japan), which was constructed by an improved in vitro ligation method, as described previously.26, 27 An adenoviral vector expressing the LacZ gene was constructed using similar methods. Expression of these genes was regulated by Cytomegalovirus promoter/enhancer and intron A. The viruses were amplified in 293 cells. Viruses were purified by CsCl2 step-gradient ultracentrifugation followed by CsCl2 linear gradient ultracentrifugation. The purified viruses were dialyzed against a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2 and 10% glycerol and were stored at −80°C. Viral particle and biological titers were determined by a spectrophotometrical method28 and by using QuickTiter (Adenovirus Titer Immunoassay Kit, Cell Biolabs, San Diego, CA), respectively. After 24-hr incubation of MPM cells in culture medium containing 10% FCS, adenoviral vectors were infected by distributing suspensions onto cells at a multiplicity of infection (MOI) of 10–160.

MTS assay

MPM cell lines were plated in 96-well plates at a density of 1 × 103 cells per well and incubated in RPMI 1640 medium containing 10% FCS. After 72-hr culture, cell proliferation was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 aqueous nonradioactive cell proliferation assay; Promega). MTS color development was measured and analyzed with a microplate reader Model 680 (Bio-Rad Laboratories) at a wavelength of 450 nm with a reference wavelength of 750 nm. This assay was performed in triplicate.

Apoptosis assay

MPM cells were grown to confluence to attain synchronization in G1 growth phase and subcultured at a lower density (1 × 105 cells in a six-well plate) for 24 hr so that most of the cells were in the S phase. Cells were infected with either adenoviral vector carrying SOCS-1 (AdSOCS-1) or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without cisplatin (10 μM) plus pemetrexed (200 μM), followed by incubation at 37°C for an additional 72 hr. The cells were then trypsinized and collected with the supernatants, followed by determination of cell viability by means of annexin V and 7-amino-actinomycin D (7-AAD) staining (BD Biosciences, San Jose, CA) using the FACSCanto flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, Ashland, OR). This assay was performed in triplicate.

Invasion assay

After 24-hr incubation of MPM cells in RPMI 1640 medium containing 10% FCS, adenoviral vectors were infected by distributing suspensions onto cells at an MOI of 40. Transfected cells were treated with cisplatin and pemetrexed in serum-free medium after 6 hr, and invasion assay was performed 24 hr after treatment using a Cultrex 96-well membrane invasion assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Briefly, treated cells were harvested and seeded into the top chamber coated with 0.5 × BME at 5 × 104 cells per well. RPMI 1640 medium (150 μl) containing 10% FCS were added to each well of the bottom invasion chamber. The device was assembled and incubated at 37°C in an incubator containing 5% CO2. After 36-hr incubation, the bottom plate was measured at 485 nm excitation and 520 nm emission. This assay was performed in triplicate.

SDS-PAGE and Western blot analysis

Whole-cell protein extract was prepared from MPM cells in RIPA buffer [10 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1 mmol/l Na3VO4 and 1× protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. Extracted proteins were resolved on SDS-PAGE and transferred to an Immobilon-P Transfer membrane (Millipore, Bedford, MA). The following antibodies were used: anti-phospho-NFκB p65, 1:1,000; anti-NFκB p65, 1:1000; anti-phospho-STAT3, 1:1,000; anti-survivin, 1:1,000; anti-XIAP, 1:1,000; anti-cleaved caspase-3, 1:500; anti-cleaved caspase-8, 1:500; anti-caspase-9, 1:500; anti-phospho-Akt (Ser473), 1:1,000; anti-Akt, 1:1,000 (all from Cell Signaling Technology, Danvers, MA), anti-STAT3, 1:1,000; anti-Lamin B, 1:500; anti-GAPDH, 1:1,000 (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-FAK (Tyr397), 1:1,000 (Biosource, Camarillo, CA), anti-FAK, 1:1,000 (BD Transduction Laboratories, San Jose, CA), anti-FLIP, 1:1,000 (Enzo life sciences, Plymouth Meeting, PA) and anti-SOCS1 antibody (1:500; IBL, Gunma, Japan), followed by a 1:5,000 dilution of donkey anti-rabbit or 1:5,000 dilution of sheep anti-mouse horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-Sciences, Piscataway, NJ) and visualized with Western Lightning ECL reagent (Perkin-Elmer, Boston, MA).

Nuclear and cytoplasmic extraction

Subcellular protein fractionation was performed using the Proteo JET™ cytoplasmic and nuclear protein extraction kit (Fermentas, Ontario, Canada) and by following the procedures as suggested by the manufacturer. Briefly, ten volumes of cell lysis buffer (with protease inhibitors) were added to one volume of packed cells. After vortexing for 10 sec and incubation on ice for 10 min, cytoplasmic proteins were separated from nuclei by centrifugation at 500 g for 7 min. Isolated nuclei were washed once with 500 μl of the nuclei washing buffer and then collected by centrifugation. The collected nuclear pellets were resuspended in ice-cold nuclear storage buffer, and volume of the nuclear lysis reagents was added to the mixtures to lysis the nuclei by shaking for 15 min at 4°C. Then nuclear lysate was collected after rinsing by centrifugation at 20,000 g for 12 min.

Small interfering RNA transfection

Commercial NF-κB p65 and STAT3 small interfering RNA (siRNA) were obtained from Qiagen. Cells were transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Nonspecific siRNA (Qiagen) was used as a negative control, and the selective silencing of p65 and STAT3 was confirmed by Western blot analysis.

DNA binding assay

NF-κB p65 and STAT3 activities were determined using TransAM Assay kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Briefly, nuclear extract was added to 96-well plates precoated with the oligonucleotide containing NF-κB p65 consensus sequence (5′-GGGACTTTCC-3′) or STAT3 consensus sequence (5′-TTCCCGGAA-3′), which is detected by sandwich ELISA. The absorbance was measured and analyzed with a microplate reader Model 680 (Bio-Rad Laboratories) at a wavelength of 450 nm. This assay was performed in triplicate.

Luciferase assay

MPM cell lines were plated in 24-well plates at a density of 3 × 104 cells per well. After 24-hr incubation of MPM cells in RPMI 1640 medium containing 10% FCS, adenoviral vectors were infected by distributing suspensions onto cells at an MOI of 40. The cells were harvested after 24 hr and were transfected with NF-κB p65 reporter (Stratagene, La Jolla, CA) or STAT3 reporter plasmid and pRL-TK plasmid (Promega) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The STAT3 reporter containing the −478/−229 fragment of the Stat3 promoter downstream of the minimal junB promoter luciferase gene (p478/229-Luc) was kindly provided by Dr. Toshio Hirano (Osaka University). Transiently transfected cells were treated with cisplatin and pemetrexed in serum-free medium after 6 hr, and luciferase activity was determined 24 hr after treatment using a dual-luciferase assay system (Promega) according to the manufacturer's instructions. Relative light units of Firefly luciferase activity were normalized with Renilla luciferase activity. This assay was performed in triplicate.

Mouse xenograft model

All animal experiments were conducted according to the institutional ethical guidelines for animal experimentation of the National Institute of Biomedical Innovation (Osaka, Japan). Female ICR nu/nu mice, 6–7 weeks of age, were obtained from Charles River Japan (Yokohama, Japan). The mice were housed for 7–14 days and allowed ad libitum access to food and water.

For pleural xenograft experiments, cells were resuspended in phosphate buffered saline (PBS) at a density of 1 × 106 cells in a total volume of 150 μl of 1/1 (v/v) PBS/Matrigel (Becton Dickinson). The mice were intrathoracically injected with 150 μl of the cell suspension through a 26-gauge needle. The mice were intrathoracically treated with 5 × 107 pfu/150 μl of AdSOCS-1 or AdLacZ on Days 10, 17 and 24 and intraperitoneally treated with pemetrexed (30 μg/g) on Days 10–14 and 17–21 along with cisplatin (2 μg/g) on Days 10 and 17 after the implantation of 1 × 106 MESO-4 or H226 cells into the pleural space. After 31 days of tumor cell inoculation, the mice were killed and their thoracic spaces examined macroscopically for growths, and tumors detected in the thoracic spaces were removed and weighed.

Immunohistochemistry

Tumors in the thoracic spaces were harvested and paraffin embedded for immunohistochemical analysis using anti-SOCS-1 antibody (Abcam, Cambridge, MA) and anti-NFκB p65 antibody (Santa Cruz Biotechnology). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay [with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining] for apoptosis was performed using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions.

Statistical analysis

Data are shown as mean ± SD for the number of experiments indicated. To test for statistically significant differences between two groups, an unpaired Student's t-test was used. For comparisons among three or more groups, the values were analyzed by one-way ANOVA followed by Fisher's least significant difference post hoc comparisons. Differences were considered significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

MPM cells fail to upregulate SOCS-1 expression in response to IFN-γ

Reports of transcriptional silencing of SOCS-1 in various types of cancer cells prompted us to investigate the levels of SOCS-1 expression in MPM cells. By real-time PCR analysis, we found that three MPM cell lines (MESO-4, H28 and H226) did not upregulate SOCS-1 expression in response to IFN-γ, whereas human PBMC upregulated SOCS-1 expression in response to IFN-γ (Fig. 1a). Furthermore, SOCS-1 transcript was underexpressed in three MPM cell lines (Fig. 1a). NF-κB and STAT3, which are known to be regulated by SOCS-1, were endogenously activated in MESO-4 and H226 cells (Figs. 1b and 1c). Under the condition stimulated by inflammatory cytokines TNF-α or IL-6, NF-κB and STAT3 were activated in all three MPM cell lines (Figs. 1b and 1c). Therefore, we subsequently delivered the SOCS-1 gene to MPM cells to investigate the therapeutic efficacy of SOCS-1 overexpression in MPM.

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Figure 1. Expression of SOCS-1, NF-κB and STAT3 in MPM cell lines (MESO-4, H28 and H226). (a) Induction of the expression of SOCS-1 gene with or without stimulation with IFN-γ was analyzed by real-time PCR analysis. Values shown represent means + SD of triplicate measurements. NS means not significant. (b) Expression of endogenous NF-κB. After 12 h of serum starvation, MPM cells were treated with or without 10 ng/ml TNF-α for 30 min. Whole-cell extracts were analyzed by Western blotting. Human PBMC was prepared as a negative control. (c) Expression of endogenous STAT3. After 12 h of serum starvation, MPM cells were treated with or without 20 ng/ml IL-6 and sIL-6R for 15 min. Whole-cell extracts were analyzed by Western blotting. Human PBMC was prepared as a negative control.

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SOCS-1 gene delivery and cisplatin plus pemetrexed cooperate to exhibit antiproliferative effect in MPM cells

We used a replication-defective recombinant AdSOCS-1 to investigate the role of SOCS-1 in the regulation of MPM cell growth. AdSOCS-1 strongly inhibited cell growth of MESO-4, H28 and H226 cells in a dose-dependent manner (Fig. 2a). This indicates that overexpression of SOCS-1 was required for growth inhibition of MPM cells. Because sufficient transduction efficiency of the adenovirus vector and strong expression of SOCS-1 were detected at an MOI of 40 in MPM cells (Supporting Information Fig. S1), we performed subsequent experiments using AdSOCS-1 at an MOI of 40.

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Figure 2. Effect of AdSOCS-1, cisplatin and pemetrexed on viability of MPM cell lines (MESO-4, H28 and H226). (a) Effect of AdSOCS-1 on viability of three MPM cell lines. Cells were infected with either AdSOCS-1 or AdLacZ. After a 72-hr culture, viable cell numbers were counted by MTS assay. Values shown represent means + SD of triplicate wells. (b) Effect of cisplatin and pemetrexed on viability of three MPM cell lines. Cells were cultivated in the presence of cisplatin (1–100 μM) or pemetrexed (10–5,000 μM). After 72 hr culture, cell viability was estimated using the MTS assay. Values shown represent means ± SD of triplicate wells. (c) Effect of combined AdSOCS-1 and cisplatin plus pemetrexed on viability of three MPM cell lines. Cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 120-hr culture, cell viability was estimated using the MTS assay. Values shown represent means + SD of triplicate wells.

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We next evaluated the therapeutic efficacy of cisplatin plus pemetrexed in the regulation of MPM cell growth. In three MPM cell lines, these compounds induced a dose-dependent decrease in cell viability with a cytotoxicity of >10 μM for cisplatin and 1 mM for pemetrexed (Fig. 2b). In view of evidence that AdSOCS-1 reduces cell viability, we sought to determine whether combination of pemetrexed, cisplatin and AdSOCS-1 would significantly reduce cell viability in MPM cells. To reveal a synergism between two chemotherapeutic molecules, each compound should be minimally toxic. Therefore, MOI 40 of AdSOCS-1 was combined to slightly cytotoxic doses of cisplatin (10 μM) and pemetrexed (200 μM). These concentrations are also within a dose range that can be achieved for chemotherapy of patients with MPM.

We therefore investigated the therapeutic efficacy of combined AdSOCS-1 and cisplatin plus pemetrexed under the concentrations described above. We found that AdSOCS-1 decreased cell growth inhibited by cisplatin plus pemetrexed (p = 0.0008, p = 0.0016 and p = 0.0121 for MESO-4, H28 and H226, respectively; Fig. 2c). Taken together, these findings suggest that AdSOCS-1 and cisplatin plus pemetrexed cooperated to inhibit cell growth in MPM cells.

SOCS-1 gene delivery combined with cisplatin plus pemetrexed induced apoptosis and inhibited invasion in MPM cells

Next, we investigated the mechanism by which combined AdSOCS-1 and cisplatin plus pemetrexed inhibited cell growth in MPM cells. As light microscopy findings suggested poor cell viability, apoptosis in these cells was tested by means of annexin V and 7-AAD staining using flow cytometry 3 days after the addition of AdSOCS-1 and cisplatin plus pemetrexed to the culture. The results of flow cytometry analysis led to the identification of two types of cells: early apoptotic (AnnexinV+7-AAD) and late apoptotic (Annexin V+7-AAD+). Compared to treatment with AdLacZ, treatment with AdSOCS-1 and cisplatin plus pemetrexed both resulted in elevated apoptosis in three MPM cell lines. Furthermore, AdSOCS-1 increased apoptosis induced by cisplatin plus pemetrexed (p = 0.0002, p = 0.0169 and p = 0.0022 for MESO-4, H28 and H226, respectively; Fig. 3a). These results suggest that AdSOCS-1 and cisplatin plus pemetrexed cooperated to induce apoptosis in MPM cells.

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Figure 3. Apoptosis and cell invasion regulated by AdSOCS-1 combined with cisplatin and pemetrexed in MPM cell lines (MESO-4, H28 and H226). (a) Apoptosis assay. MPM cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 72-hr culture, apoptosis was determined by means of annexin V and 7-AAD staining using flow cytometry. Values shown represent means + SD of triplicate wells. NS means not significant. (b) Cell invasion assay. MPM cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, transfected cells were treated with or without 10 μM cisplatin (C) and 200 μM pemetrexed (P) in serum-free medium after 6 hr and invasion assay was performed 24 hr after treatment. Values shown represent means + SD of triplicate wells. NS means not significant.

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In addition to apoptosis, cell invasion is an important mechanism for inhibition of cell growth. AdSOCS-1 significantly inhibited invasiveness in three MPM cell lines (p < 0.0001, p < 0.0001 and p = 0.0003 for MESO-4, H28 and H226, respectively; Fig. 3b). By contrast, cisplatin plus pemetrexed failed to regulate invasion of H28 and H226 cells (Fig. 3b). Thus, regarding cell invasion, we estimate that AdSOCS-1 played a major role in MPM cells.

Combined SOCS-1 gene delivery and cisplatin plus pemetrexed activate caspase signaling pathways

One of the important pathways in apoptosis is the caspase signaling pathway. Therefore, we investigated the regulation of caspases by AdSOCS-1 and cisplatin plus pemetrexed. We focused on antiapoptotic proteins survivin, FLIP and XIAP, which inhibit caspases.29–31 Combined AdSOCS-1 and cisplatin plus pemetrexed inhibited survivin, FLIP and XIAP in MPM cells and activated caspases-3 and -9 in MESO-4 and H226 cells and caspase-8 in MESO-4 cells (Figs. 4a and 4b). Consistent with the cleavage of FAK and Akt by recombinant caspase-3, AdSOCS-1 and cisplatin plus pemetrexed inhibited FAK in MPM cells and Akt in MESO-4 cells, but AdSOCS-1 alone did not affect phospho-Akt in MESO-4 cells (Figs. 4a4c). Taken together, these findings suggest that combined AdSOCS-1 and cisplatin plus pemetrexed activates caspase signaling pathways.

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Figure 4. Caspase pathway activated by combined AdSOCS-1 and cisplatin plus pemetrexed. (a) Inhibition of antiapoptotic proteins. Three MPM cell lines (MESO-4, H28 and H226) were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 24-hr (upper panel) or 48-hr (lower panel) culture, protein extracts were blotted with indicated antibodies. (b) Activation of caspases. MESO-4 and H226 cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 72-hr culture, protein extracts were blotted with indicated antibodies. (c) Cleavage of FAK and Akt by caspase-3. Protein extracts of MESO-4 cells were incubated with recombinant active caspase-3 with or without z-DEVD-fmk. After 3 hr of incubation, protein extracts were blotted with indicated antibodies.

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SOCS-1 gene delivery regulates NF-κB and STAT3 in MESO-4 and H226 cells

Because it has been reported that antiapoptotic signaling is regulated by the transcription factors NF-κB and STAT3, we investigated the role of AdSOCS-1 and cisplatin plus pemetrexed on NF-κB and STAT3 signaling in MESO-4 and H226 cells. NF-κB was stimulated by the inflammatory cytokine TNF-α (Fig. 5a). AdSOCS-1, but not cisplatin plus pemetrexed, inhibited phosphorylation and nuclear translocation of NF-κB stimulated by TNF-α in MESO-4 and H226 cells (Fig. 5a). In contrast, by NF-κB-dependent luciferase reporter assay and TransAM DNA-binding assay, we found that cisplatin plus pemetrexed cooperated with AdSOCS-1 to inhibit transcriptional and DNA-binding activity of NF-κB in MESO-4 and H226 cells (Fig. 5d). Regarding STAT3 regulation, AdSOCS-1, but not cisplatin plus pemetrexed, inhibited phosphorylation and transcriptional activity of STAT3 in MESO-4 and H226 cells (Figs. 5b and 5d). On the other hand, cisplatin plus pemetrexed cooperated with AdSOCS-1 to inhibit DNA-binding activity of STAT3 in MESO-4 and H226 cells (Fig. 5d). Furthermore, silencing NF-κB expression by siRNA abrogated XIAP in MESO-4 and H226 cells, whereas silencing STAT3 expression abrogated survivin in MESO-4 and H226 cells and FLIP in MESO-4 cells (Fig. 5c). Collectively, these data indicate that combined SOCS-1 gene delivery and cisplatin plus pemetrexed exhibit antitumor effect in MESO-4 and H226 cells partially by inhibiting NF-κB and STAT3 signaling.

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Figure 5. NF-κB and STAT3 signaling is regulated by AdSOCS-1. (a, upper panel) Activation of NF-κB p65 by TNF-α. After 12 hr of serum starvation, MESO-4 cells were incubated with 10 ng/ml TNF-α for 0–90 min. Cytoplasmic and nuclear fraction of protein extracts were blotted with indicated antibodies. (a, lower panel) Inhibition of NF-κB by SOCS-1. MESO-4 and H226 cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 24-hr culture in serum-starved medium, MESO-4 and H226 cells were stimulated with 10 ng/ml TNF-α for 30 min. Cytoplasmic and nuclear fraction of protein extracts were blotted with indicated antibodies. (b) Inhibition of STAT3 by SOCS-1. MESO-4 and H226 cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 24-hr culture, protein extracts were blotted with indicated antibodies. (c) Inhibition of antiapoptotic proteins by NF-κB p65 and STAT3. STAT3, NF-κB p65 or nonspecific siRNA as control was added to MESO-4 and H226 cells. After 24-hr culture, protein extracts were blotted with indicated antibodies. (d) DNA binding and luciferase assay of NF-κB p65 (upper panel) and STAT3 (lower panel). MESO-4 and H226 cells were infected with either AdSOCS-1 or AdLacZ as control at an MOI of 40. After 6 hr from infection, cells were cultivated with or without 10 μM cisplatin (C) plus 200 μM pemetrexed (P). After an additional 24-hr culture, cells were analyzed by DNA binding and luciferase assay. Values shown represent means + SD of triplicate wells. NS means not significant.

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SOCS-1 gene delivery combined with cisplatin plus pemetrexed cooperates to exhibit antitumor activity in a mesothelioma xenograft model

We also evaluated the therapeutic effect of AdSOCS-1 combined with cisplatin plus pemetrexed on the growth of intrathoracically implanted MPM cells in ICR nu/nu mice. Of the MPM cell lines used in this study, we were able to establish MESO-4 and H226 xenograft models. Preliminary experiments revealed that when 1 × 106 MPM cells (MESO-4 or H226) were inoculated into the thoracic space, dissemination of tumors was observed in all mice 31 days after cell implantation. Injection of the AdSOCS-1 combined with cisplatin plus pemetrexed significantly reduced the weight of tumor nodules compared to the weight of those in control AdLacZ-injected animals (Fig. 6b). Furthermore, immunohistochemical analysis indicated that AdSOCS-1 inhibited nuclear translocation of NF-κB and AdSOCS-1 combined with cisplatin plus pemetrexed significantly induced apoptosis in the H226 tissue (Fig. 6c). However, apoptosis in MESO-4 tissue could not be clearly determined. From these results, we conclude that AdSOCS-1 combined with cisplatin plus pemetrexed exhibits antitumor activity not only in vitro but also in vivo in the MPM model. We hope that these findings may lead to the successful clinical application of SOCS-1 for MPM treatment.

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Figure 6. Antitumor effect of AdSOCS-1 combined with cisplatin and pemetrexed in vivo. (a) Gross appearance of MESO-4 and H226 tumors grown orthotopically in the thoracic spaces. Female ICR nu/nu mice were intrathoracically treated with AdSOCS-1 or AdLacZ and intraperitoneally treated with pemetrexed and cisplatin after the implantation of MESO-4 or H226 cells into the pleural space. (b) Each tumor nodule found in the thoracic spaces was also weighed. Values shown represent means + SD of five (MESO-4) or six (H226) mice. NS means not significant. (c) Immunohistochemical analysis of SOCS-1, NFκB p65 and TUNEL (blue fluorescence = DAPI staining for nuclei; cyan fluorescence = TUNEL positivity) in H226 tissue. The mice were treated in the same way as described above.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Malignant mesothelioma represents a great challenge to both clinicians and researchers because of its poor prognosis and remarkable resistance to current therapies. Although there have been some improvements in treatment over the past few years, a better understanding of the molecular basis of the disease and of how to improve treatment is required. In our study, we show that SOCS-1 is silenced in MPM cell lines, and SOCS-1 gene delivery inhibits the proliferation of MPM cells through NF-κB and STAT3 pathways that promote antiapoptotic signaling. Specifically, we demonstrate that SOCS-1 gene delivery cooperates with cisplatin plus pemetrexed to regulate NF-κB signaling and significantly to inhibit tumor growth of MPM in vitro and in vivo. These data provide new insights into the clinical application of SOCS-1 gene delivery for the treatment of MPM.

In accordance with previous reports on several types of cells, we showed that AdSOCS1 regulated NF-κB signaling in MESO-4 and H226 cells. SOCS-1 was reported to ubiquitinate NF-κB p65.15 We demonstrated that AdSOCS-1 suppressed the expression of nuclear NF-κB p65 in MESO-4 and H226 cells. Furthermore, we found that AdSOCS-1 inhibited phosphorylation of NF-κB p65 at Ser-536. Previous studies have demonstrated that phosphorylation of serine 536 in NF-κB p65 is necessary for transcriptional activity of NF-κB.32, 33 In addition to ubiquitination of NF-κB p65, SOCS-1 is known to interact with IL-1R–associated kinase (IRAK), a key molecule in the NF-κB pathway.34 IRAK was reported to activate IκB kinase, which induced the phosphorylation of NF-κB p65 on Ser-536.35 We also demonstrated that cisplatin plus pemetrexed inhibited the transcriptional activity of NF-κB p65 but did not affect its nuclear translocation. A previous study found that cisplatin did not affect NF-κB p65 nuclear translocation but modulated its transcriptional activity.36 Taken together, our findings suggest that AdSOCS-1 cooperates with cisplatin plus pemetrexed to regulate NF-κB signaling by different mechanisms.

Consistent with previous studies on several types of cells, we found that AdSOCS-1 inhibited the phosphorylation of STAT3 at Tyr-705 in MESO-4 and H226 cells. On the other hand, we were not able to determine the effect of cisplatin plus pemetrexed on STAT3. Cisplatin plus pemetrexed did not affect the phosphorylation and transcriptional activity of STAT3, but inhibited DNA-binding activity of STAT3. A previous study indicated that cisplatin has minimal effects on STAT3 signaling.37 Thus, we need further studies to clarify cisplatin plus pemetrexed involvement in the regulation of STAT3 signaling.

NF-κB and STAT3 are known to promote antiapoptotic signaling.8 In MESO-4 and H226 cells, we found that NF-κB and STAT3 regulate antiapoptotic proteins survivin, XIAP in MESO-4 and H226 cells and FLIP in MESO-4 cells, consistent with the results of previous studies.38–40 The inhibition of these antiapoptotic proteins activates caspase-3, -8, and -9.29 Taken together, our results suggest that SOCS-1 gene delivery cooperates with cisplatin plus pemetrexed to induce apoptosis via NF-κB and STAT3 pathways.

We demonstrated that recombinant human active caspase-3 cleaved FAK and Akt and that combined SOCS-1 gene delivery and cisplatin plus pemetrexed inhibited FAK and Akt expression in MPM cells. FAK and Akt are known to be the substrates of caspase-3.41, 42 Specifically, SOCS-1 gene delivery and cisplatin plus pemetrexed strongly suppressed FAK expression in MPM cells. Interactions of SOCS-1 with FAK through the Src homology 2 domain have been reported to promote polyubiquitination and subsequent degradation of FAK.43 We found that AdSOCS-1 significantly inhibited cell invasion in MPM cells. Because of an important role of FAK in cell invasion, we estimated that AdSOCS-1 strongly suppressed cell invasion partially through caspase and ubiquitin-mediated regulation of FAK.

In addition to NF-κB and STAT3 signaling, SOCS-1 is reported to regulate multiple signaling pathways including FAK, p38 MAPK and p53 pathways.43–45 In our study, we consider that AdSOCS-1 inhibited cell growth in H28 cells partially by regulating these signaling pathways because NF-κB and STAT3 were not endogenously activated in H28 cells. On the other hand, the evidence for functional activity of SOCS-1 in tumor cell is controversial. SOCS-1 is reported to have different functions depending on types of tumor cells. For example, overexpression of SOCS-1 inhibits apoptosis in human acute T-cell leukemia cell lines (Jurkat cells).46 Furthermore, SOCS-1 silencing suppresses cell proliferation and invasion in murine melanoma cells.47 Therefore, the role of SOCS-1 in tumor progression is thought to be cell type specific.

In contrast, recently developed tyrosine kinase inhibitors (TKIs) tend to target specific abnormalities in cancer cells and exhibit surprising effects on patients having these abnormalities. For example, EGFR-TKIs, such as gefitinib, exhibit up to a 70% response rate against non-small cell lung cancer harboring somatic mutations of the EGFR gene.48, 49 These TKIs have been tested for MPM but without therapeutic benefit.2 This is partially explained by the fact that multiple receptor tyrosine kinases are frequently activated in most MPM cells.50 Therefore, SOCS-1, which regulates multiple signaling in different manners, is expected to be an attractive candidate for the treatment of MPM.

In this study, we examined only the cooperative effect of AdSOCS-1 and cisplatin plus pemetrexed. However, SOCS-1 gene delivery combined with chemotherapeutics other than cisplatin plus pemetrexed might represent increased antitumor effect. It is of interest to investigate the effect of SOCS-1 gene therapy combined with drugs such as gemcitabine, etoposide, doxorubicin and vinorelbine, because they have mechanisms of action distinct from those of cisplatin and pemetrexed. Moreover, these new combinations might be useful as second- or third-line therapies for patients with MPM with poor response to the first-line therapy, cisplatin plus pemetrexed. Further preclinical analyses are required to elucidate the optimal combination therapy with SOCS-1 gene delivery and chemotherapeutics.

In conclusion, we demonstrated the antitumor effect of SOCS-1 gene delivery in MPM. Furthermore, we showed the efficacy of combined SOCS-1 gene delivery and cisplatin plus pemetrexed in vitro and in vivo. The results of the clinical application of SOCS-1 for MPM treatment are eagerly anticipated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (K. Iwahori), a Grant-in-Aid from the Ministry of Health, Labour and Welfare, Japan (T. Naka) and a grant from the Kansai Biomedical Cluster Project in Saito, which is promoted by the Knowledge Cluster Initiative of the Ministry of Education, Culture, Sports, Science and Technology, Japan (T. Naka). The authors thank Y. Ito, N. Kawakami and Y. Kanazawa for their secretarial assistance.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IJC_27611_sm_SuppInfo.doc48KSupporting Information

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