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Keywords:

  • NDV;
  • TRAIL;
  • NK cell;
  • liver cancer

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Newcastle disease virus (NDV) is a potential antitumor agent, and its antitumor effect has been evaluated in preclinical tests. However, the mechanisms of NDV-based antitumor therapy are still not completely clear. In the present study we found that NDV-stimulation enhanced the killing ability of mouse spleen natural killer (NK) cells towards mouse hepatoma cell lines, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays an important role in this tumoricidal activity. NDV stimulation induced up-regulation of TRAIL both at the mRNA and protein levels in NK cells. Blocking TRAIL by antibody (Ab) almost completely eliminated the killing effect of NK cells on hepatoma cell lines. Furthermore, neutralizing interferon (IFN)-γ by Ab could inhibit TRAIL expression and tumoricidal activity of NDV-stimulated NK cells. These results indicated a substantial role of TRAIL as an effector molecule in NDV-induced NK cells mediated tumoricidal activity. The NDV stimulation triggered TRAIL expression in mouse spleen NK cells could be mediated by IFN-γ induction. Anat Rec, 296:1552–1560, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Newcastle disease virus (NDV) is an avian pathogen that belongs to the Rubulavirus genus of the paramyxoviridae family. Beside economic significance in zootechnics, NDV has also drawn attention in the field of clinical research against human cancer. NDV has been shown to replicate selectively in human tumor cells, inducing tumor cells death. It has been suggested that the selective antitumor activity of NDV is based on cancer specific defects in the interferon pathway. Animal models and data from several phase II clinical trials treated with NDV have demonstrated the antitumor effects of the virus and safety for the host. NDV is a good inducer of many cytokines that are active against tumor cells. Several reports indicated that NDV could effectively induce the activation of interferons (IFNs) and tumor necrosis factor (TNF)-α in mouse and human lymphocytes (Lorence et al., 1988; Wertz et al., 1994; Zorn et al., 1994). Three members of the TNF family, TNF-α, Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL), have the ability to kill a variety of tumor cell lines in vitro (Krammer, 1999; Wallach et al., 1999). In contrast to other members of the TNF family, TRAIL administration has been proven safe in previous in vivo trials. In recent years, TRAIL has attracted a great deal of attention as a promising anticancer and cytokine producing reagent (Mérino et al., 2007; Humphreys and Halpern, 2008). TRAIL has been shown to be expressed on the surface of the IFNs-stimulated monocytes and dendritic cells (Fanger et al., 1999; Griffith et al., 1999). In addition, IFN-γ induced TRAIL up-regulation has been observed in mouse liver natural killer (NK) cells, indicating that TRAIL up-regulation may contribute to NK cell cytotoxicity and reduction of liver metastases (Smyth et al., 2001; Takeda et al., 2001). So far, the mechanisms of nonlytic NDV strains causing cell death in tumor cells are not fully clear. Given its efficacy for the selective killing of the abnormal cells, NK cells were surmized to be involved in the antitumor activity induced by nonlytic NDV stimulation. In this study, we examined the tumoricidal activity of NDV-stimulated NK cells. Furthermore, we evaluated whether TRAIL is crucial for the tumoricidal activity of NDV-stimulated NK cells, and if so, what is the mechanism of TRAIL regulation in the NDV-stimulated NK cells.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Animals

Female BALB/c mice at the age of 6–10 weeks were obtained from the laboratory animal center of Guangxi Medical University (Nanning, China). The experiments were performed in accordance with the Animal Experimentation Ethics Committee guidelines.

Cell Lines

The mouse hepatoma cell line Hepa 1-6 was provided by Chinese type culture collection and the Novikoff hepatoma cell line was obtained from our laboratory stock. The cell lines were maintained in suspension under tissue-culture conditions (humidified incubator with 5% CO2 at 37°C). The cells were passaged every 3–4 days in DMEM medium (Hyclone, USA) containing 2 mM l-glutamine, 10% fetal calf serum (Hyclone, USA) and antibiotics (100 IU/mL penicillin and 100 IU/mL streptomycin).

Virus

The avirulent, nonlytic NDV 7793 strain (NDV 7793) was obtained from our laboratory stock. A stock of infectious virus was propagated in embryonated chicken eggs, harvested from the allantoic fluid, purified by centrifugation (300–400 g, 30 min, 4°C), and then ultracentrifugation (50,000 g, 60 min, 4°C). The sediment was resuspended in phosphate-buffered saline (PBS) and purified twice over sucrose (35%) via ultracentrifugation (97,000 g, 60 min, 4°C). The virus was resuspended in PBS buffer containing 0.1% ethylene diamine tetra-acetic acid (EDTA). NDV was quantified by a hemagglutination test in which one hemagglutination unit (HU) is defined as the smallest virus concentration leading to visible chicken erythrocyte agglutination. The ultraviolet (UV)-NDV was produced by inactivating NDV with UV light for 20 min (254 nm, 2 mW/cm2, 30 cm).

Reagents

Magnetic-activated cell sorting (MACS) separator, mouse NK cell isolation kit, anti-CD49 (DX5)-FITC monoclonal antibody (mAb), and isotype control antibody (Ab) (FITC-conjugated rat IgM) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Lactate dehydrogenase (LDH) assay kit (CytoTox 96® Nonradioactive Cytotoxicity Assay) was purchased from Promega (CA, USA). Recombinant mouse IFN-γ, anti-IFN-γ Ab, and isotype control Ab (rabbit IgG) were purchased from PeproTech (Rocky Hill, USA). RNA purified kit (RNAsimple Total RNA kit, DP419) was purchased from TianGen (Beijing, China). RT-PCR kit (PrimeScript 1st Strand cDNA synthesis Kit) was purchased from TaKaRa (Dalian, China). Anti-GAPDH (HRP) (ab9385) mAb, antimouse TRAIL (ab10516) mAb and isotype control Ab (mouse IgG1) were purchased from Abcam (Hong Kong, China). PE-conjugated anti-TRAIL (N2B2) mAb and isotype control Ab (PE-conjugated rat IgG2a) were purchased from Santa Cruz (CA, USA). Protease inhibitor and soluble TRAIL enzyme-linked immunosorbent assay (ELISA) kit were purchased from BOSTER (Wuhan, China).

NK Cells Preparation and Activation

Spleen leukocytes were obtained from BALB/c mice by density gradient centrifugation. NK cells were separated from leukocytes by MACS separator. In brief, non-NK cells were magnetically labeled by using a mouse NK cell isolation kit according to the manufacturer's instruction. The magnetically labeled non-NK cells were depleted by retaining them on a MACS column in the magnetic field of a MACS separator, whereas the unlabeled NK cells pass through the column. The ratio of NK-enriched cells was >85% as assessed by flow cytometric analysis by using anti-CD49 (DX5) -FITC mAb. NK cells were stimulated for 12 h with 25 HU/mL NDV 7793 (live or UV-inactivated) or with IFN-γ (500 ng/mL) in DMEM medium. Cells were collected by centrifugation (300–400 g, 10 min, 4°C), washed twice in PBS, and used for flow cytometry or cytotoxicity assay, or supernatants were harvested, and IFN-γ or soluble TRAIL (sTRAIL) concentrations were determined by specific ELISA kits according to manufacturer's protocol.

Cytotoxicity Assay

To quantify the cytotoxic activities of NK cells against mouse hepatoma cells, tumor cells (104 cells / well) were incubated with varying number of NK cells for 4 h. After 4 h incubation, centrifuged the plate at 250 g for 4 min and then transferred 50 μL supernatant from each well to measure the concentration of LDH (a stable cytosolic enzyme that is released upon cell lysis). In some experiments antimouse TRAIL neutralizing mAb (500 ng/mL) was added to the NK cells 20 min before adding tumor cell targets. All cytotoxicity assays were performed in 96-well, round-bottom plates in triplicate. The percentage of NK-induced lysis in each sample was calculated as: 100 × (experimental release – effector spontaneous release – target spontaneous release) / (target maximum release – target spontaneous release).

RT-PCR

RNA was prepared by using the RNAsimple Total RNA kit according to the manufacture's instruction. For isolation, 2 × 106 cells were used. For generation of cDNA total RNA was reverse transcribed by using PrimeScript 1st Strand cDNA synthesis Kit with oligo dT primers (in synthesis of cDNA for β-actin control, random 6 mers primers were used) in 20 μL containing 20 U RNase inhibitor and 500 μM dNTPs. Reverse transcription was performed by using a thermal program of 65°C for 5 min, (30°C for 10 min, when random primers were used), 42°C for 60 min, 95°C for 5 min. Five microliters of this cDNA-containing solution was used for DNA amplification in a PCR instrument (ABI 9700, USA) with 1.25 U of Taq DNA polymerase (TaKaRa, China) in a 50 μL reaction according to the manufacturer instructions. PCR cycle conditions for both TRAIL and β-actin were started with initial denaturation (94°C for 5 min) followed by 35 reaction cycles. Each cycle consisted of a denaturation step (94°C for 30 s), an annealing step (54°C for 30 s), and an elongation step (72°C for 30 s). The reaction was completed with a 72°C elongation for 7 min. PCR products were resolved on 1.5% agarose gels and visualized with ethidium bromide.

Primers were purchased from Sangon Biotech (Shanghai, China). Primer sequences for TRAIL were: sense, 5′-cttacatgtacttcaccaacgaga-3′; and antisense 5′-agtcccagaaatccacatcc-3′, yielding a PCR product of 99 bp.

The amount of each reverse-transcribed mRNA was controlled with a β-actin PCR using the primers: sense, 5′-ccgcagctaggaataatgga-3′; and antisense, 5′-caaatgctttcgctctggtc-3′, yielding a PCR product of 222 bp.

Western Blot Analysis

Purified NK cells were collected through centrifugation (300 g, 10 min, 4°C), and then washed with ice-cold PBS twice. The cell pellets were resuspended in 100 μL lysis buffer per 1 × 107 cells (30 mM Tris-Hcl pH 7.5, 120 mM NaCl, 10% glycerol and 1% Triton X-100) with the addition of protease inhibitor according to the manufacturer's instruction. After 30 min ice-water bath incubation, the lysates were collected and heated to 100°C for 5 min to denaturalize the proteins.

For western blot analysis the resulting lysates were supplemented with 5-fold concentrated standard reducing sample buffer. Subsequently, lysates containing 60 μg of protein, as determined by the bicinchoninic acid method (Enhance BCA protein Assay Kit, Bryotime, China), were separated on SDS-PAGE. After protein transfer onto nitrocellulose membrane by electroblotting, the membrane was blocked with 5% skim milk powder in TBST solution for 1 h at room temperature. Then the membrane was incubated in TBST containing 5% skim milk and 1 μg/mL primary Abs against mouse TRAIL (anti-GAPDH-HRP was used for loading control), rocked gently for 12 h at 4°C. Rinsed the membrane with TBST three times for 5 min, and then incubated with HRP-conjugated secondary Abs diluted 1:2000 in TBST for 1 h at room temperature. After washing three times for 5 min each time with TBST, the blots on membrane were developed by enhanced chemiluminescence (ECL) method using super signal west dura substrate (Thermo Scientific 37071, USA) following the manufacturer's protocol.

Flow Cytometry

TRAIL cell surface expression was determined by incubating cells with PE-conjugated anti-TRAIL (N2B2) mAb. As an isotype control, PE-conjugated rat IgG2a was used. Surface staining was determined on a FACS-Calibur cytometer (BD Biosciences, USA).

ELISA for Soluble TRAIL

For the detection of soluble TRAIL in the supernatants of activated NK cells, we used a specific ELISA kit for TRAIL. Experiments were carried out according to the manufacturer's protocol. Results given are representing means of triplicate cultures.

Statistical Analysis

Each experiment was carried out at least two or three times. LSD (ANOVA), χ2, and t-test were used to analyze the results. The difference in results was regarded as statistically significant if P value was less than 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

NDV-Stimulated NK Cells Kill Mouse Hepatoma Cells

To test whether NDV-stimulated NK cells have the ability to kill mouse hepatoma cells, NK cells were stimulated by NDV for 12 h and then incubated with tumor cells for 4 h. Mortality rates of tumor cells were evaluated using the LDH release assay. Both kinds of tumor cells could be killed by NDV-stimulated NK cells. The specific lysis rate of Hepa 1-6 and Novikoff hepatoma cells was up to 8.2% and 25.1%, respectively (P < 0.01), at the effector to target (E:T) cell ratio of 20:1. However, the sensitivity of these tumor cells for the killing activity of NDV-stimulated NK cells was different. Novikoff cells were much more sensitive to NK-mediated cytotoxicity than the Hepa 1-6 cells (Fig. 1). The Novikoff cell line showed significantly higher lysis rate, compared to the Hepa 1-6 cell line at each E:T cell ratio (P < 0.01). Similar findings were observed in the groups in which UV-NDV- or IFN-γ-stimulated NK cells were used as the effector cells. On the other hand, unstimulated NK cells showed extremely low killing effect toward the tumor cells. In order to investigate the mechanism of NDV triggered NK cell mediated tumoricidal effect in more detail, Novikoff cells were chosen for further studies because cells of the Novikoff line were more sensitive than those of Hepa 1-6.

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Figure 1. Tumoricidal activity of NK cells toward Hepa 1-6 and Novikoff hepatoma cell lines. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h in DMEM medium. NK cells were harvested by centrifugation, and cocultured with Hepa 1-6 or Novikoff hepatoma cells for 4 h at the indicated E:T ratios. As a negative control, unstimulated NK cells were cocultured with target tumor cells for 4 h. As a positive control, IFN-γ (500 ng/mL)-stimulated NK cells were cocultured with target tumor cells for 4 h. LDH method was used to assess the specific lysis rate of Hepa 1-6 and Novikoff hepatoma cells. Data points represent the mean of triplicate wells, and similar results were obtained in three independent experiments. For clarity, SD bars were omitted from the graphs, but were < 10% the value of all points.

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NDV-Stimulated NK Cells Kill Tumor Cell via TRAIL In Vitro

To evaluate the contribution of TRAIL to the tumoricidal activity of NK cells after the stimulation of NDV, UV-NDV or IFN-γ, the influence of anti-TRAIL neutralizing mAb was examined by a cytotoxicity assay using NK cells as the effector and Novikoff cells as the target. As shown in Fig. 2, NK cells stimulated by NDV and IFN-γ exhibited intensive cytotoxicity toward Novikoff cells at the E:T ratio of 5:1, and the specific lysis rate of Novikoff cells were 21.6% and 19.8%, respectively (P < 0.01). After treating with anti-TRAIL mAb, significant decline in the cytotoxicity of NK cells was observed (Fig. 2). The rate of Novikoff cells lysis caused by NDV- and IFN-γ-stimulated NK cells was 3.2% and 2.7%, respectively (P < 0.01). However, compared with the unstimulated control, UV-NDV stimulation had no obvious effect on the tumoricidal activity of NK cells (P > 0.05).

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Figure 2. Neutralizing antibody for TRAIL blocked the tumoricidal activity of NK cells toward Novikoff hepatoma cell line. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h in DMEM medium. NK cells were harvested by centrifugation, and cocultured with Novikoff hepatoma cells at an E:T cell ratio of 5:1 for 4 h with or without supplement of anti-TRAIL neutralizing antibody. LDH method was used to assess the specific lysis rate of Novikoff hepatoma cells. As a negative control unstimulated NK cells were used. As a positive control IFN-γ (500 ng/mL)-stimulated NK cells were used. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. **P < 0.01 versus Unstimulated, ΔΔP < 0.01 versus NK in the same group.

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In addition, the medium of NK cells also had tumoricidal activity toward Novikoff cells. Stimulated NK cells were incubated in fresh medium for 4 h, and then collected the supernatant of medium for cytotoxicity assay. As shown in Fig. 3, the medium of NK cells that was stimulated by NDV and IFN-γ induced Novikoff cells lysis. The lysis rate was 9.6% and 10%, respectively, which was significantly higher than the unstimulated control (P < 0.01). Compared with unstimulated control cells, no extra tumoricidal activity was observed in the medium of UV-NDV-stimulated NK cells (P > 0.05). However, after treating with anti-TRAIL mAb, the cytotoxicity of these medium were almost abrogated (P < 0.01), with the highest lysis rate of only 1.6% in Novikoff cells (Fig. 3).

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Figure 3. Tumoricidal activity of NK cells medium toward Novikoff hepatoma cell line. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h in DMEM medium. After stimulation, NK cells were harvested by centrifugation and incubated in fresh medium for 4 h, and then collected the supernatant of medium. Novikoff hepatoma cells were added into the resulting supernatant (104 cells/200 μL) and incubated for 4 h with or without supplement of anti-TRAIL neutralizing antibody. LDH method was used to assess the specific lysis rate of Novikoff hepatoma cells. As a negative control, medium of unstimulated NK cells was used. As a positive control, medium of IFN-γ (500 ng/mL)-stimulated NK cells was used. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. **P < 0.01 versus Unstimulated, ΔΔP < 0.01 versus Medium in the same group.

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Up-Regulation of TRAIL in NDV-Stimulated NK Cells

As TRAIL has been identified as one of the effector molecules that mediate the tumoricidal activity in NDV-stimulated NK cells, we sought to explore how TRAIL expression was up-regulated in NK cells with NDV stimulation. We analyzed TRAIL mRNA expression in NK cells by using RT-PCR assay. NK cells were cocultured with NDV for 1, 2, 4, 8, and 12 h, and then the levels of TRAIL mRNA were measured. The TRAIL mRNA could be detected by 1 h, and after NDV stimulation, the mRNA levels increased up to 4 h followed by a slight decrease at 12 h. The TRAIL mRNA was consistently at a low basal level in the unstimulated NK cells (Fig. 4A).

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Figure 4. Expression of TRAIL mRNA in NK cells. (A) NK cells were stimulated with NDV (25 HU/mL) for 1, 2, 4, 8, and 12 h. (B) NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 4 h. Total RNA was isolated from NK cells, and RT-PCR analysis of TRAIL mRNA level was performed. As a negative control, unstimulated NK cells were used (marked by “C” in figure). As a positive control, IFN-γ (500 ng/mL)-stimulated NK cells were used. β-actin was used as a control over the same time course. Similar results were obtained in three independent experiments from different mice and a representative example out of three was shown.

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In addition, we investigated the differences between TRAIL mRNA expression levels in NK cells stimulated with NDV, UV-NDV, or IFN-γ for 4 h. In all three groups, increased TRAIL mRNA levels could be detected by 4 h after stimulation. However, the mRNA expression levels were significantly higher in the NDV-stimulated NK cells, compared to the IFN-γ and UV-NDV-stimulated cells (Fig. 4B).

The protein expression of TRAIL in NK cells was detected by Western blot analysis (Fig. 5). The results showed clear TRAIL protein expression in NK cells, 12 h after stimulation with NDV, UV-NDV, or IFN-γ. Compared to the unstimulated NK cells, significant increase in the protein expression levels was observed in NDV, UV-NDV, or IFN-γ stimulated NK cells.

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Figure 5. TRAIL expression in NK cells. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h. Cells were lysed, TRAIL-specific western blot analysis was performed. As a negative control, unstimulated NK cells were used (marked by “C” in figure). As a positive control, IFN-γ (500 ng/mL)-stimulated NK cells were used. GAPDH was used as a control over the same time course. A single band of approximately 24 kDa of TRAIL was detected in each group. TRAIL expressions in stimulated NK cells (stimulated by NDV, UV-NDV, or IFN-γ) were obviously higher than unstimulated NK cells. Similar results were obtained in three independent experiments from different mice and a representative example out of three was shown.

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The expression of cell surface TRAIL was determined by flow cytometry, and that of soluble TRAIL was determined by ELISA assay in NK cells.

Membrane-Bound TRAIL Induction on NK Cell Surface by NDV

The freshly isolated NK cells were stimulated with NDV, UV-NDV or IFN-γ for 12 h. The cells were subsequently stained with PE-conjugated mAb against TRAIL (N2B2) for 30 min at 4°C. NDV, UV-NDV and IFN-γ stimulation resulted in a remarkable TRAIL expression on the surface of the NK cells, whereas the cell-surface expression of TRAIL in the unstimulated NK cells was only marginally detectable (Fig. 6). Interestingly, membrane expression of TRAIL in the NK cells stimulated with NDV or UV-NDV were higher, compared to the cells stimulated with IFN-γ (positive control).

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Figure 6. TRAIL expression on the surface of NK cells. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h. After stimulation, NK cells were stained with anti-TRAIL-PE, and TRAIL expression was examined using flow cytometry. As an isotype control, NK cells were stained with PE conjugated control IgG2a. As a negative control, unstimulated NK cells were used. As a positive control, IFN-γ (500 ng/mL)-stimulated NK cells were used. Scatter diagram represent 104 gated NK cells. Similar results were obtained in three independent experiments from different mice and a representative example out of three was shown.

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Soluble TRAIL Induction in NK Cells by NDV

The detection of TRAIL in the supernatants of cultured NK cells stimulated with NDV, UV-NDV, or IFN-γ yielded different profiles of induction for soluble TRAIL. NDV or IFN-γ stimulation induced a significantly higher soluble TRAIL release, compared to the unstimulated NK cells (P < 0.01). However, there was no significant difference between UV-NDV-stimulated and unstimulated soluble TRAIL release in NK cells (Fig. 7).

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Figure 7. The concentration of soluble TRAIL in NK cells medium. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h. Supernatant was harvested, and concentration of soluble TRAIL was determined by ELISA assay. As a negative control, unstimulated NK cells were used. As a positive control, IFN-γ (500 ng/mL)-stimulated NK cells were used. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. **P < 0.01 versus Unstimulated, +P < 0.05 versus NDV.

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Role of IFN-γ in TRAIL Expression of NDV-stimulated NK Cells

To investigate the relationship between IFN-γ and TRAIL expression in NDV-stimulated NK cells, production of IFN-γ by NDV-stimulated NK cells was evaluated using ELISA method. The results demonstrated that NDV stimulation induced a significant up-regulation of IFN-γ concentration in the supernatant of NK cell medium (P < 0.01). UV-NDV stimulation also enhanced production of IFN-γ, compared to the unstimulated control (P < 0.05). Although the level of IFN-γ production in the UV-NDV-stimulated cells was relatively lower than the NDV-stimulated cells, there was no significant difference between the two groups of cells (Fig. 8).

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Figure 8. The concentration of IFN-γ in NK cells medium. NK cells were stimulated with NDV (25 HU/mL) or UV-NDV (25 HU/mL) for 12 h. Supernatant was harvested, and concentration of IFN-γ (ng/L) was determined by ELISA assay. As a negative control, unstimulated NK cells were used. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. *P < 0.05 and **P < 0.01 versus Unstimulated.

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Anti-IFN-γ Neutralizing Ab Down-Regulates TRAIL Expression and Tumoricidal Activity of NK Cells

In order to identify whether IFN-γ was involved in the regulation of TRAIL expression in NK cells after NDV stimulation, the effect of anti-IFN-γ neutralizing Ab on TRAIL expression was examined using flow cytometry and ELISA. The results showed that the surface expression of TRAIL in the NDV-stimulated NK cells was inhibited partially by anti-IFN-γ neutralizing Ab. The extent of inhibition on TRAIL surface expression was increased with the dosage of anti-IFN-γ neutralizing Ab and peaked at 10.48% (derived from 35.84% minus 25.36%) (Fig. 9). Meanwhile, after treating with anti-IFN-γ neutralizing Ab at the concentration of 6 μg/mL, a significant suppression in the production of soluble TRAIL was observed in unstimulated and NDV-stimulated NK cells (Fig. 10). The concentration of soluble TRAIL in the medium of unstimulated and NDV-stimulated NK cells fell by 6.6% and 8.8%, respectively (P < 0.05). Moreover, anti-IFN-γ neutralizing Ab down-regulated the tumoricidal activity of NDV-stimulated NK cells. As shown in Fig. 11, the lysis rate in Novikoff cells caused by NDV-stimulated NK cells was 23.2%, whereas the lysis rate was decreased to 15.4% after anti-IFN-γ neutralizing Ab supplementation (P < 0.01).

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Figure 9. Neutralizing antibody for IFN-γ inhibited TRAIL expression on NDV-stimulated NK cells membrane. NK cells were stimulated with NDV (25 HU/mL) for 12 h supplemented with anti-IFN-γ neutralizing antibody (0.3 or 6 μg/mL). TRAIL expression on NK cells membrane was examined using flow cytometry. Scatter diagrams represent 104 gated NK cells. Similar results were obtained in three independent experiments from different mice and a representative example out of three was shown.

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Figure 10. Neutralizing antibody for IFN-γ inhibited soluble TRAIL expression in NDV-stimulated NK cells. NK cells were stimulated with NDV (25 HU/mL) for 12 h supplemented with anti-IFN-γ neutralizing antibody (0.3 or 6 μg/mL). Supernatant was harvested, and concentration of soluble TRAIL was determined by ELISA assay. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. ΔP < 0.05 versus NK in the same group.

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Figure 11. Neutralizing antibody for IFN-γ down-regulated the tumoricidal activity of NK cells toward Novikoff hepatoma cell line. NK cells were stimulated with NDV (25 HU/mL) for 12 h supplemented with anti-IFN-γ neutralizing antibody (6 μg/mL). NK cells were harvested by centrifugation, and cocultured with Novikoff hepatoma cells at an E:T cell ratio of 5:1 for 4 h. LDH method was used to assess the specific lysis rate of Novikoff hepatoma cells. Bars represent the mean ± SD of triplicate wells, and similar results were obtained in three independent experiments. ΔΔP < 0.01 versus NK in the same group.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

NDV has garnered much attention as a promising tumor biotherapeutic agent. Studies have shown that NDV-infected tumor cells are better at activating NK cells than uninfected tumor cells (Jarahian et al., 2009). However, so far, the mechanisms of the direct activation of NK cells by NDV stimulation are not clearly understood. We show here that NDV-stimulated mouse spleen NK cells exert a potent tumoricidal activity in vitro. We investigated the involvement of TRAIL in regulation of the tumoricidal activity of NK cells. By using neutralizing Ab testing we demonstrated that the TRAIL pathway, mediated by IFN-γ, could act as a crucial effector system utilized by the NDV-stimulated NK cells to kill target tumor cells.

Stimulation of mouse spleen NK cell with NDV 7793 enhanced its killing activity toward mouse hepatoma cell lines in vitro. Although both tumor cell lines could be killed by NDV-stimulated NK cells, the Novikoff cell line was more sensitive to activated NK cells than the Hepa 1-6 cell line (Fig. 1). The sensitivity of tumor cells to activated NK cells might correlate with the surface expression of TRAIL's receptors: DR4 (TRAIL-R1), DR5 (TRAIL-R2), DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4). However, potential contributions of other TNF family members, such as Fas ligand and TNF-α to the tumoricidal activity of NDV-stimulated NK cells cannot be excluded.

TRAIL is constitutively expressed in many immune cells as a type II membrane protein. The extracellular domain of TRAIL can be proteolytically cleaved from the cell surface producing a soluble form of TRAIL (Liabakk et al., 2002; LeBlanc et al., 2003). Like TNF and Fas ligand, TRAIL also exerts its physiological activity in a biologically active soluble form (Lub-de Hooge et al., 2005). In the present study, TRAIL was up-regulated at both the mRNA and protein levels in NK cells after NDV or UV-NDV stimulation. Both NDV and UV-NDV stimulation could enhance membrane-bound TRAIL expression on NK cells (Fig. 6). However, it seemed that viral replication have some connections with soluble TRAIL production. The expression level of soluble TRAIL induced by UV-NDV stimulation was significantly lower than that of the NDV stimulation (P < 0.05), and showed no difference compared to the unstimulated control (Fig. 7). In addition, high levels of soluble TRAIL secretion of NDV- or IFN-γ-stimulated NK cells was accompanied by significant high levels of cytotoxicity toward tumor cells, whereas UV-NDV stimulation could not improve the tumoricidal activity of NK cells significantly (Figs. 1, 2). To confirm the tumoricidal activity of soluble TRAIL, the medium of NK cells containing soluble TRAIL were collected for the cytotoxicity assay. The medium from NDV- or IFN-γ-stimulated NK cells showed higher tumoricidal activity, compared to the unstimulated NK cells (P < 0.01). No significant difference was observed between the medium from UV-NDV-stimulated and unstimulated NK cells (Fig. 3). These results suggested that soluble TRAIL is an important antitumor factor employed by NK cells, and the difference in boosting up tumoricidal activity of NK cells between NDV and UV-NDV stimulation might be from the different induction of soluble TRAIL in NK cells. Furthermore, the presence of anti-TRAIL neutralizing mAb almost completely blocked the tumoricidal activity of NK cells or the medium containing NK cells, which could indicate the antitumor mechanism of stimulated NK cells. Taken together, TRAIL is critical for the tumoricidal activity of stimulated NK cells and the soluble rather than membrane-bound forms of TRAIL mediate the tumoricidal activity of NDV-stimulated NK cells to a greater extent in vitro.

In addition to NDV and UV-NDV, IFN-γ stimulation also enhanced TRAIL expression in NK cells by up-regulating the TRAIL mRNA level (Figs. 4B, 5). These results are consistent with data obtained in previous studies (Tu et al., 2011). The significant enhancement of IFN-γ secretion in NK cells after NDV stimulation (Fig. 8) indicated that high levels of TRAIL expression in NDV-stimulated NK cells are associated with increased production of IFN-γ. On the other hand, neutralizing IFN-γ by specific Ab inhibited the up-regulation of TRAIL expression in NDV-stimulated NK cells to some degree (Figs. 9, 10), and the ratio of inhibition was increased with the dosage of anti-IFN-γ Ab. Although the complete inhibition was not observed in the present study, the data showed that IFN-γ could play an important role in the regulation of TRAIL expression in NDV-stimulated NK cells.

The tumor microenvironment consists of tumor-infiltrating NK cells, which provide an early source of IFN-γ. It has been found that IFN-γ plays a critical role in NK cells infiltration into the local tumor site and the tumor-infiltrating NK cells mainly suppresses tumor progression through the IFN-γ pathway (Hayakawa et al., 2011). In this study, blocking IFN-γ by anti-IFN-γ neutralizing Ab not only inhibited TRAIL expression, but also down-regulated the tumoricidal activity of NDV-stimulated NK cells (Fig. 11), indicating that IFN-γ is a positive regulatory factor for NDV-stimulated NK cells to elicit an effective antitumor immune response. The constitutive expression of TRAIL upon IFN stimulation has also been found in other types of immunocytes. In human keratinocytes and peripheral blood mononuclear cells, IFN-α was shown to induce TRAIL expression (Zahn et al., 2011), whereas in human monocytes, IFN-β was shown to enhance the levels of both soluble and membrane-bound forms of TRAIL (Ehrlich et al., 2003). In human bone marrow mesenchymal stem cells, IFN-γ could selectively induce apoptosis via the TRAIL-mediated pathway (Du et al., 2012).

Our results demonstrated that one of the antitumor properties of NDV 7793 strain is to trigger the tumoricidal activity of NK cells by TRAIL induction through the IFN-γ pathway. Although TRAIL expression was critical for the antitumor effect of the NDV-stimulated NK cells, the sensitivity of tumor cells to TRAIL-induced death is also an essential component of this phenomenon. This is demonstrated by the fact that the tumor cell line Hepa 1-6 was resistant to TRAIL-expressing NK cells.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

We thank Qiguang Huang, Jinlian Luo and Danni Zhou for excellent technical assistance.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED