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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

Correlation of CXCL16 and IRF8 expression in SL4 cells. (A) Expression of IRF8 in SL4-Cont and SL4-CXCL16 cells cultured for 24 h and analyzed by qRT-PCR. These data were normalized to GAPDH. (B) Knockdown of CXCL16 expression. SL4-CXCL16 cells were transfected with siRNA for 36 h to assess the expression of CXCL16 at the mRNA (upper panel) and protein (lower panel) levels. (C) Expression levels of IRF8 in SL4-CXCL16 using siRNA for control and CXCL16. β-actin was used as a normalization control. *P <0.05, **P <0.01. All experiments were repeated at least three times.
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Fig3: Correlation of CXCL16 and IRF8 expression in SL4 cells. (A) Expression of IRF8 in SL4-Cont and SL4-CXCL16 cells cultured for 24 h and analyzed by qRT-PCR. These data were normalized to GAPDH. (B) Knockdown of CXCL16 expression. SL4-CXCL16 cells were transfected with siRNA for 36 h to assess the expression of CXCL16 at the mRNA (upper panel) and protein (lower panel) levels. (C) Expression levels of IRF8 in SL4-CXCL16 using siRNA for control and CXCL16. β-actin was used as a normalization control. *P <0.05, **P <0.01. All experiments were repeated at least three times.

Mentions: IRF8 has been shown to be a key regulator of Fas-mediated apoptosis in various cancer cells [34–36] and metastatic phenotypes in CRC cells [37, 38]; however, the correlation between TNF-α-induced apoptosis and IRF8 expression in CRC cells has not been investigated. We confirmed that IRF8 expression was increased in SL4-CXCL16 cells (Figure 3A). In contrast, IRF8 expression was significantly decreased by CXCL16 knockdown (Figure 3C), although CXCL16 expression was not completely silenced by siRNA (Figure 3B). These results indicated that the expression of IRF8 correlates with CXCL16 expression.Figure 3


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)

Correlation of CXCL16 and IRF8 expression in SL4 cells. (A) Expression of IRF8 in SL4-Cont and SL4-CXCL16 cells cultured for 24 h and analyzed by qRT-PCR. These data were normalized to GAPDH. (B) Knockdown of CXCL16 expression. SL4-CXCL16 cells were transfected with siRNA for 36 h to assess the expression of CXCL16 at the mRNA (upper panel) and protein (lower panel) levels. (C) Expression levels of IRF8 in SL4-CXCL16 using siRNA for control and CXCL16. β-actin was used as a normalization control. *P <0.05, **P <0.01. All experiments were repeated at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4300614&req=5

Fig3: Correlation of CXCL16 and IRF8 expression in SL4 cells. (A) Expression of IRF8 in SL4-Cont and SL4-CXCL16 cells cultured for 24 h and analyzed by qRT-PCR. These data were normalized to GAPDH. (B) Knockdown of CXCL16 expression. SL4-CXCL16 cells were transfected with siRNA for 36 h to assess the expression of CXCL16 at the mRNA (upper panel) and protein (lower panel) levels. (C) Expression levels of IRF8 in SL4-CXCL16 using siRNA for control and CXCL16. β-actin was used as a normalization control. *P <0.05, **P <0.01. All experiments were repeated at least three times.
Mentions: IRF8 has been shown to be a key regulator of Fas-mediated apoptosis in various cancer cells [34–36] and metastatic phenotypes in CRC cells [37, 38]; however, the correlation between TNF-α-induced apoptosis and IRF8 expression in CRC cells has not been investigated. We confirmed that IRF8 expression was increased in SL4-CXCL16 cells (Figure 3A). In contrast, IRF8 expression was significantly decreased by CXCL16 knockdown (Figure 3C), although CXCL16 expression was not completely silenced by siRNA (Figure 3B). These results indicated that the expression of IRF8 correlates with CXCL16 expression.Figure 3

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