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, 135 (1), 63-72

Interleukin-32 Enhances Cytotoxic Effect of Natural Killer Cells to Cancer Cells via Activation of Death Receptor 3

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Interleukin-32 Enhances Cytotoxic Effect of Natural Killer Cells to Cancer Cells via Activation of Death Receptor 3

Mi H Park et al. Immunology.

Abstract

Studies have demonstrated that the anti-tumour effect of natural killer (NK) cells is successful for patients with several cancers. Although interleukin-32 (IL-32) is endogenously expressed in NK cells, cytolytic function of NK cells against cancer cells has not been fully demonstrated. In the present study, we found that the growth of cancer cells was suppressed when colon cancer cells or prostate cancer cells were co-cultured with NK-92 cells, an NK cell line. We also found that the expression of tumour necrosis factor receptor 2 and death receptor 3 (DR3) was increased in PC3 cells, and the expression of FAS and DR3 was increased in SW620 cells by co-culture with NK-92 cells. However, cancer cell growth inhibition and IL-32 expression were abolished when cancer cells were co-cultured with NK cells transfected with small interfering (si) RNA of IL-32. DR3 expression was also diminished by co-culture with IL-32-specific siRNA-transfected NK-92 cells. Expression of APO3L, a ligand of DR3, was elevated in NK cells that were co-cultured with cancer cells. It was also found that expression of apoptosis-related proteins such as cleaved caspase-3 and bax was increased in cancer cells co-cultured with NK-92 cells, but their expression was abolished by co-culture with IL-32 siRNA-transfected NK-92 cells. Moreover, knockdown of DR3 in co-culture of NK-92 cells with cancer cells by siRNA or antibodies of DR3 and APO3L reversed the growth inhibitory effect of NK-92 cells. In conclusion, our study showed that IL-32 enhanced the cytotoxic effect of NK-92 cells on the cancer cells through activation of DR3 and caspase-3.

Figures

Figure 1
Figure 1
Effect of natural killer (NK) cells on the cytotoxicity to prostate and colon cancer cells. (a) Colon cancer cells (SW620) and prostate cancer cells (PC3) were cultured in 24-well plates (5 × 104 cells/well), and then co-cultured with NK-92 cells (5 × 104) for 48 hr. Thereafter, cell growth was measured by direct counting after Trypan blue staining. (b) NK-92 cells (5 × 104) were co-cultured with PC3 or SW620 cells for 48 hr, and then harvested. Death receptor [FAS, tumour necrosis factor receptor 2 (TNFR2) and death receptor 3 (DR3)] expression was detected by Western blotting in PC3 and SW620 cells. (c) NK-92 cells (5 × 104) were co-cultured with PC3 or SW620 cells for 48 hr, and then harvested. DR3 receptor expression was investigated by flow cytometry using anti-DR3 in PC3 and SW620 cells as described in Materials and methods. (d) SW620 and PC3 were cultured into 24-well plates (5 × 104 cells/well), and thereafter co-cultured with NK-92 cells (5 × 104) for 48 hr, and then harvested. Apoptotic protein expression such as cleaved caspase-3 and bax was determined by Western blotting. Relative optical density of specific bands is expressed versus actin amount. The figures are representatives of three experiments with replicates. Values are mean ± SD of three experiments with replicates. *P<0·05 indicates significantly different from the control group.
Figure 2
Figure 2
Expression of interleukin-32 (IL-32), and effects of IL-32 knockdown in NK-92 cells on the expression of death receptor 3 and apoptosis-related proteins of cancer cells. (a) IL-32 is endogenously expressed in NK-92 cells, and its expression by IL-32 plasmid transfection. NK-92 cells (1 × 106) were transfected with the IL-32 plasmid (1·5 μg) by electroporation and then incubated for 48 hr. The IL-32 expression was detected by reverse transcription-PCR (upper panel). NK-92 cells transfected with non-targeting control small interfering (si) RNA or IL-32 siRNA (50 nm/well) by electroporation were incubated for 48 hr. IL-32 expression was detected by Western blotting (lower panel). (b) Effect of silencing endogenous IL-32 expression on the cytotoxic effect of NK-92 cells. SW620 and PC3 were cultured into 24-well plates (5 × 104 cells/well), and then co-cultured with IL-32 siRNA-transfected NK-92 cells (5 × 104 cells) for 48 hr. Thereafter, cell growth was measured by direct counting after Trypan blue staining. Sc-siRNA, IL-32 non-targeting control siRNA. The figures are representatives of three experiments with replicates. (c) Effects of IL-32 knockdown in NK-92 cells on the expression of DR3 of cancer cells. NK-92 cells transfected with non-targeting control siRNA or IL-32 siRNA (50 nm/well) by electroporation for 24 hr were co-cultured with colon cancer cells (SW620) or prostate cancer cells (PC3) for 48 hr. The expression of DR3 was determined by Western blotting. (d) Effects of IL-32 knockdown in NK-92 cells on the expression of apoptosis-related proteins of cancer cells. NK-92 cells transfected with non-targeting control siRNA or with IL-32 siRNA (50 nm/well) by electroporation were co-cultured with SW620 or PC3 cells for 48 hr. The expression of cleaved caspase-3 and bax was determined by Western blotting. Quantitative analysis of immunoblot was performed by Western blot density in PC3 and SW620 cells. Relative optical density of specific bands is expressed versus actin amount. The figures are representatives of three experiments with replicates. Values are mean ± SD of three experiments with replicates. *P<0·05 indicates significantly different from the control group. #P<0·05 indicates significantly different from the co-cultured cell group.
Figure 3
Figure 3
Effect of interleukin-32 (IL-32) on the expression of APO3L in natural killer (NK) cells, and effect of APO3L and death regulator 3 (DR3) knockdown on the growth of cancer cells co-cultured with NK cells. (a) Effect of co-culture on the expression of APO3L. NK-92 cells (5 × 104) were co-cultured with PC3 or SW620 cells for 48 hr. APO3L expression was detected by Western blotting in NK-92 cells. (b) Effect of silencing endogenous IL-32 expression on the expression of APO3L in NK-92 cells. NK-92 cells were transfected with non-targeting control small interfering (si) RNA or IL-32 siRNA (50 nm/well) by electroporation and then incubated for 48 hr. APO3L expression was detected by Western blotting. (c) Effects of DR3 knockdown on the cancer cell growth by co-culture with NK cells. SW620 and PC3 (5 × 104 cells/well) were transfected with non-targeting control siRNA or DR3 siRNA (100 nm/well) for 24 hr. Thereafter co-cultured with NK-92 cells for 48 hr, and cancer cell growth was measured by direct counting after Trypan blue staining. (d) Effects of anti-APO3L in NK-32 cells and anti-DR3 in cancer cells on the cancer cell growth by co-culture with NK cells. Before initiation of co-culture, anti-APO3L (2 or 5 μg/ml) was incubated in NK-92 cells for 2 hr. Pre-incubated NK-92 cells (5 × 104) were co-cultured with PC3 or SW620 cells for 48 hr, and then cancer cell growth was measured by direct counting after trypan blue staining. Alternatively, cancer cells were pre-incubated with anti-DR3 (2 or 5 μg/ml) for 2 hr. Pre-incubated cancer cells (5 × 104) were co-cultured with PC3 or SW620 cells for 48 hr, and then cancer cell growth was measured by direct counting after Trypan blue staining. Quantitative analysis of immunoblot was performed by Western blot density in PC3 and SW620 cells. Relative optical density of specific bands is expressed versus actin amount. The figures are representatives of three experiments with replicates. Values are mean ± SD of three experiments with replicates. *P<0·05 indicates significantly different from the control group. #P<0·05 indicates significantly different from the co-cultured cell group.
Figure 4
Figure 4
Effects of interleukin-32 (IL-32) on the expression of perforin/granzyme B and the level of nitric oxide (NO) of cancer cells co-cultured with natural killer (NK) cells. (a) Effect of co-culture on the expression of perforin and granzyme B. SW620 and PC3 cells were cultured into 24-well plates (5 × 104 cells/well) and then co-cultured with NK-92 cells (5 × 104) for 48 hr. Thereafter, expression of perforin and granzyme B was determined by reverse transcription-PCR in NK-92 cells. (b) Effects of IL-32 knockdown in NK-92 cells on the expression of perforin and granzyme B. NK-92 cells were transfected with non-targeting control small interfering (si) RNA or IL-32 siRNA (50 nm/well) by electroporation and then incubated for 48 hr, thereafter co-cultured with colon cancer cells (SW620) or prostate cancer cells (PC3) for 48 hr. The expression of perforin and granzyme B was determined by reverse transcription-PCR in NK-92 cells. The figures are representatives of three experiments with replicates. (c) Effect of co-culture on the level of nitric oxide. SW620 and PC3 were cultured into 24-well plates (5 × 104 cells/well) and then co-cultured with NK-92 cells (5 × 104) for 48 hr. Thereafter, the nitrite release in the supernatant was assessed by Griess reaction as described in Materials and methods. (d) Effects of IL-32 knockdown in NK-92 cells on the level of nitric oxide. NK-92 cells transfected with non-targeting control siRNA or IL-32 siRNA (50 nm/well) by electroporation were co-cultured with SW620 or PC3 for 48 hr. The nitrite release in the supernatant was assessed by Griess reaction as described in Materials and methods. The figures are representatives of three experiments with replicates. Values are mean ± SD of three experiments with replicates.

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