Figure 1.
IL-27 induces TRAIL expression in human melanomas and inhibits their tumor growth partly in a TRAIL-dependent manner.
(A) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (10 ng/ml) for 0, 20, and 60 min. Cell lysate was then prepared and subjected to Western blotting with anti-pY-STAT1, anti-pY-STAT3, anti-total STAT1, and anti-total STAT3. Similar results were obtained in two independent experiments. (B) These melanoma cell lines were stimulated with increasing doses of IL-27 (0–100 ng/ml) for 72–96 h in triplicate and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means ± SD. *, †, and ‡ indicate p<0.01, p<0.0001, and p<0.00001, respectively, compared with 0 ng/ml IL-27. Similar results were obtained in three independent experiments. (C) These melanoma cell lines were stimulated with increasing doses of IL-27 (0–100 ng/ml) for 24 h. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in more than three independent experiments. (D) These melanoma cell lines were stimulated with IL-27 (100 ng/ml) for 48 h, and then analyzed for cell surface expression of TRAIL by FACS using PE-labeled anti-TRAIL (solid line) and its control Ab (plain line with shading). Similar results were obtained in two independent experiments. (E) These melanoma cell lines were stimulated with IL-27 (10 and 100 ng/ml) in the presence of anti-TRAIL neutralizing Ab or its control Ab (10 µg/ml) for 72–96 h in triplicate and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means ± SD. * indicates p < 0.05, compared with control Ab. Similar results were obtained in three independent experiments.
Figure 2.
IL-27 and TLR3 agonist poly(I:C) cooperatively inhibits tumor growth of human melanomas.
(A) Human melanoma cell lines (SK-MEL-13, -28 and -37) were stimulated with IL-27 (10 ng/ml) and/or poly(I:C) (10 µg/ml) for 72-96 h in triplicate and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means ± SD. *, †, ‡, §, ¶, and || indicate p<0.05, p<0.01, p<0.001, p<0.00001, and p<0.000001, respectively, compared with 0 ng/ml IL-27 and 0 µg/ml poly(I:C). Similar results were obtained in thee independent experiments. (B) Morphology was evaluated using light microscopy. A representative result of SK-MEL-27 from among the three human melanoma cell lines is shown. Similar results were obtained in more than five independent experiments. (C) These melanoma cell lines were stimulated with IL-27 (10 ng/ml) and/or poly(I:C) (10 µg/ml) for 48 h. Then, apoptosis was assessed by FACS analysis of cells stained with Annexin V-FITC and PI. A representative result of SK-MEL-27 from among the three human melanoma cell lines is shown. Similar results were obtained in three independent experiments. (D) Percentage of each population was calculated. Data are shown as means ± SD of three independent experiments. * indicates p < 0.05, compared to no stimulation.
Figure 3.
IL-27 enhances TLR3 expression in human melanomas, which could account for the cooperative effect between IL-27 and poly(I:C).
(A-C) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (1, 3, 10 ng/ml) and/or poly(I:C) (1 µg/ml) for 24 h or the indicated times. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in more than two independent experiments. (D and E) Cell lysate was also prepared after the stimulation for 48 h and subjected to Western blotting with antibodies against TLR3, RIG-I, MDA5 and β-actin. Similar results were obtained in more than two independent experiments. (F) SK-MEL-37 cells were transfected with siRNA specific to TLR3 or control siRNA for 24 h. These cells were then stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 µg/ml) for a further 24 h, and total cell lysate was prepared and subjected to Western blot using anti-TLR3 and anti-β-actin. (G) The siRNA-transfected cells were also stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 µg/ml) for a further 48 h and pulsed with 3H-thymidine for the last 8 h in triplicate. 3H-thymidine incorporation was measured, and relative proliferation (%) to that of respective unstimulated cells was calculated. Data are shown as means ± SD. * indicates p < 0.05 compared with no siRNA and control siRNA. Similar results were obtained in two independent experiments.
Figure 4.
IL-27 and poly(I:C) cooperatively induce TRAIL expression in human melanomas and inhibit their tumor growth partly in a TRAIL-dependent manner.
(A) Human melanoma cell lines (SK-MEL-13, -28, and -37) were stimulated with IL-27 (1, 3, 10 ng/ml) and/or poly(I:C) (0 or 1 µg/ml) for 24 h. Total RNA was then extracted and subjected to RT-PCR analysis. Similar results were obtained in three independent experiments. (B) These melanoma cell lines were stimulated with IL-27 (10 ng/ml) and/or poly(I:C) (10 µg/ml) for 48 h, and then total cell lysates were prepared and subjected to Western blot analysis using anti-TRAIL and anti-β-actin. Relative expression level of TRAIL was determined by the intensity of each band of TRAIL and β-actin. Similar results were obtained in two independent experiments. (C) These cells were also analyzed for cell surface expression of TRAIL by FACS using PE-labeled anti-TRAIL (solid line) and its control Ab (plain line with shading). Similar results were obtained in more than two independent experiments. (D) These melanoma cell lines were stimulated with IL-27 (10 ng/ml) and poly(I:C) (1 µg/ml) in the presence of anti-TRAIL neutralizing Ab or its control Ab (10 µg/ml) for 72–96 h in triplicate and pulsed with 3H-thymidine for the last 24 h, and 3H-thymidine incorporation was measured. Data are shown as means ± SD. * indicates p < 0.05, compared with control Ab. Similar results were obtained in three independent experiments.
Figure 5.
IL-27 and poly(I:C) cooperatively inhibit in vivo tumor growth of human melanoma in immunodeficient mice.
Immunodeficient NOD/SCID mice were s.c. injected with human melanoma cells of the SK-MEL-37 cell line, and treated by weekly i.v. injections with PBS alone, IL-27 (1 µg), poly(I:C) (30 µg), or IL-27 (1 µg) plus poly(I:C) (30 µg) from 1 week postengraftment. Tumor growth was monitored weekly and expressed as volume (mm3). Data are shown as means ± SD. * indicates p < 0.05, compared with PBS. Similar results were obtained in two independent experiments.
Figure 6.
Hypothetical model of the pathway by which IL-27 and the combination of IL-27 and poly(I:C) induce TRAIL up-regulation and inhibits tumor growth in human melanomas.
IL-27 induces IRF-1 expression through WSX-1/STAT1 signaling, resulting in up-regulation of TRAIL expression. IL-27 also augments TLR3 expression in IRF-1-dependent and -independent manner. Thus, due to the augmented TLR3 expression by IL-27, IL-27, and a synthetic TLR3 agonist, poly(I:C), cooperatively enhance TRAIL expression and greatly inhibit tumor growth partly in a TRAIL-dependent manner.