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CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF-α-induced apoptosis by tumor-associated macrophages.

Kee JY, Ito A, Hojo S, Hashimoto I, Igarashi Y, Tsuneyama K, Tsukada K, Irimura T, Shibahara N, Takasaki I, Inujima A, Nakayama T, Yoshie O, Sakurai H, Saiki I, Koizumi K - BMC Cancer (2014)

Bottom Line: Silencing of IRF8 significantly decreased TNF-α-induced apoptosis.Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis.Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

View Article: PubMed Central - PubMed

Affiliation: Division of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. kkoizumi@inm.u-toyama.ac.jp.

ABSTRACT

Background: Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients.

Methods: We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages.

Results: CXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis.

Conclusions: Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

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Related in: MedlinePlus

Silencing of IRF8 expression by siRNA inhibited TNF-α-induced apoptosis in SL4-CXCL16. (A) Knockdown of IRF8 expression by qRT-PCR and Western blot analysis. (B) Viability of IRF8 knockdown cells stimulated with TNF-α. Cells were seeded in 96-well plates (2 × 103 cells) and stimulated with TNF-α (10 ng/ml) for 0–72 h and then viability was measured by the WST-8 assay. *P <0.05. (C) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml). (D) Effects of IRF8 knockdown on TNF-α-induced apoptotic responses. C, si-Control; I, si-IRF8. All experiments were repeated at least three times.
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Fig4: Silencing of IRF8 expression by siRNA inhibited TNF-α-induced apoptosis in SL4-CXCL16. (A) Knockdown of IRF8 expression by qRT-PCR and Western blot analysis. (B) Viability of IRF8 knockdown cells stimulated with TNF-α. Cells were seeded in 96-well plates (2 × 103 cells) and stimulated with TNF-α (10 ng/ml) for 0–72 h and then viability was measured by the WST-8 assay. *P <0.05. (C) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml). (D) Effects of IRF8 knockdown on TNF-α-induced apoptotic responses. C, si-Control; I, si-IRF8. All experiments were repeated at least three times.

Mentions: The increased sensitivity to TNF-α-induced apoptosis and IRF8 expression apparent in SL4-CXCL16 cells led us to hypothesize that IRF8 expression was related to sensitivity to TNF-α-induced apoptosis. Regulation of TNF-α-induced apoptosis by IRF8 has not been previously reported, even though it regulates Fas-mediated apoptosis [34–38]. When SL4-CXCL16 cells were transfected with IRF8 siRNA (Figure 4A) and stimulated with TNF-α, TNF-α-induced apoptosis was significantly inhibited in IRF8 knockdown cells compared with control siRNA-transfected cells (si-Cont) (Figure 4B and C). The increased activation of caspase-3 and PARP observed in SL4-CXCL16 cells was also decreased in IRF8 knockdown cells (Figure 4D). These results suggested that CXCL16-mediated upregulation of TNF-α-induced apoptosis in IRF8-sensitized SL4-CXCL16 cells occurred via downstream caspase-3 and PARP signaling.Figure 4


CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF-α-induced apoptosis by tumor-associated macrophages.

Kee JY, Ito A, Hojo S, Hashimoto I, Igarashi Y, Tsuneyama K, Tsukada K, Irimura T, Shibahara N, Takasaki I, Inujima A, Nakayama T, Yoshie O, Sakurai H, Saiki I, Koizumi K - BMC Cancer (2014)

Silencing of IRF8 expression by siRNA inhibited TNF-α-induced apoptosis in SL4-CXCL16. (A) Knockdown of IRF8 expression by qRT-PCR and Western blot analysis. (B) Viability of IRF8 knockdown cells stimulated with TNF-α. Cells were seeded in 96-well plates (2 × 103 cells) and stimulated with TNF-α (10 ng/ml) for 0–72 h and then viability was measured by the WST-8 assay. *P <0.05. (C) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml). (D) Effects of IRF8 knockdown on TNF-α-induced apoptotic responses. C, si-Control; I, si-IRF8. All experiments were repeated at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4300614&req=5

Fig4: Silencing of IRF8 expression by siRNA inhibited TNF-α-induced apoptosis in SL4-CXCL16. (A) Knockdown of IRF8 expression by qRT-PCR and Western blot analysis. (B) Viability of IRF8 knockdown cells stimulated with TNF-α. Cells were seeded in 96-well plates (2 × 103 cells) and stimulated with TNF-α (10 ng/ml) for 0–72 h and then viability was measured by the WST-8 assay. *P <0.05. (C) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml). (D) Effects of IRF8 knockdown on TNF-α-induced apoptotic responses. C, si-Control; I, si-IRF8. All experiments were repeated at least three times.
Mentions: The increased sensitivity to TNF-α-induced apoptosis and IRF8 expression apparent in SL4-CXCL16 cells led us to hypothesize that IRF8 expression was related to sensitivity to TNF-α-induced apoptosis. Regulation of TNF-α-induced apoptosis by IRF8 has not been previously reported, even though it regulates Fas-mediated apoptosis [34–38]. When SL4-CXCL16 cells were transfected with IRF8 siRNA (Figure 4A) and stimulated with TNF-α, TNF-α-induced apoptosis was significantly inhibited in IRF8 knockdown cells compared with control siRNA-transfected cells (si-Cont) (Figure 4B and C). The increased activation of caspase-3 and PARP observed in SL4-CXCL16 cells was also decreased in IRF8 knockdown cells (Figure 4D). These results suggested that CXCL16-mediated upregulation of TNF-α-induced apoptosis in IRF8-sensitized SL4-CXCL16 cells occurred via downstream caspase-3 and PARP signaling.Figure 4

Bottom Line: Silencing of IRF8 significantly decreased TNF-α-induced apoptosis.Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis.Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

View Article: PubMed Central - PubMed

Affiliation: Division of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. kkoizumi@inm.u-toyama.ac.jp.

ABSTRACT

Background: Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients.

Methods: We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages.

Results: CXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis.

Conclusions: Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.

Show MeSH
Related in: MedlinePlus