Limits...
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.

Show MeSH

Related in: MedlinePlus

CXCL16 overexpression sensitizes SL4 cells to TNF-α-induced cell death. (A) Viability of SL4-Cont and SL4-CXCL16 cells following TNF-α stimulation. Cells were seeded in 96-well plates (2 × 103 cells) stabilized for 1 h and stimulated by TNF-α (10 ng/ml) for 24–48 h. *P <0.05, **P <0.01. (B) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml) for 0-8 h. (C) Effects of CXCL16 expression on the TNF-α-induced apoptotic pathway in SL4 cells. (D) Effects of CXCL16 expression on the TNF-α-induced NF-κB and MAPK signaling pathways in SL4 cells. β-actin was used as a normalization control. All experiments were repeated at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: CXCL16 overexpression sensitizes SL4 cells to TNF-α-induced cell death. (A) Viability of SL4-Cont and SL4-CXCL16 cells following TNF-α stimulation. Cells were seeded in 96-well plates (2 × 103 cells) stabilized for 1 h and stimulated by TNF-α (10 ng/ml) for 24–48 h. *P <0.05, **P <0.01. (B) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml) for 0-8 h. (C) Effects of CXCL16 expression on the TNF-α-induced apoptotic pathway in SL4 cells. (D) Effects of CXCL16 expression on the TNF-α-induced NF-κB and MAPK signaling pathways in SL4 cells. β-actin was used as a normalization control. All experiments were repeated at least three times.

Mentions: We established a stable expression of CXCL16 in SL4, a metastatic mouse CRC cell line, and confirmed its expression (Figure 1). This cell line was cultured in a polyclonal population with a different expression level of CXCL16 in the antibiotic G418 to maintain the heterozygous characteristics of cancer cells. Membrane-bound CXCL16 was confirmed in almost SL4-CXCL16 cells by flow cytometry (Additional file 1). We next performed microarray analysis to compare gene expressions between SL4-Cont and SL4-CXCL16 cells. Significant differences were noted in the expression of TNF and apoptosis-related factors, whereas no changes were observed in metastatic factors (Table 1). On the other hand, soluble CXCL16 did not affect SL4-CXCL16 cells via the paracrine system because expression of CXCR6 (CXCL16 receptor) was not observed on SL4-CXCL16 cells (Additional file 2). When SL4-Cont and SL4-CXCL16 cells were treated with TNF-α, this induced a time-dependent increase in the death of SL4-CXCL16 cells (Figure 2A). We carried out the Annexin V assay to determine whether the TNF-α-induced cell death was apoptosis. As shown in Figure 2B, apoptosis was greater in TNF-α-treated SL4-CXCL16 cells than in SL4-Cont cells. The TNF-α-induced apoptotic response involved the stimulation of several intercellular signaling pathways [32]. Western blot analysis revealed an increase in the cleavage of PARP and caspase-3 in SL4-CXCL16 cells (Figure 2C). In addition, TNF-α-induced activation of TAK1 and its downstream NF-κB decreased, whereas phosphorylation of ERK and JNK increased in SL4-CXCL16 cells (Figure 2D). These results suggested that CXCL16 expression sensitized metastatic CRC cells to TNF-α-induced apoptosis.Figure 1


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)

CXCL16 overexpression sensitizes SL4 cells to TNF-α-induced cell death. (A) Viability of SL4-Cont and SL4-CXCL16 cells following TNF-α stimulation. Cells were seeded in 96-well plates (2 × 103 cells) stabilized for 1 h and stimulated by TNF-α (10 ng/ml) for 24–48 h. *P <0.05, **P <0.01. (B) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml) for 0-8 h. (C) Effects of CXCL16 expression on the TNF-α-induced apoptotic pathway in SL4 cells. (D) Effects of CXCL16 expression on the TNF-α-induced NF-κB and MAPK signaling pathways in SL4 cells. β-actin was used as a normalization control. 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

Fig2: CXCL16 overexpression sensitizes SL4 cells to TNF-α-induced cell death. (A) Viability of SL4-Cont and SL4-CXCL16 cells following TNF-α stimulation. Cells were seeded in 96-well plates (2 × 103 cells) stabilized for 1 h and stimulated by TNF-α (10 ng/ml) for 24–48 h. *P <0.05, **P <0.01. (B) Annexin V assay. Cells were seeded in 6-well plates (2 × 105 cells) and treated with TNF-α (10 ng/ml) for 0-8 h. (C) Effects of CXCL16 expression on the TNF-α-induced apoptotic pathway in SL4 cells. (D) Effects of CXCL16 expression on the TNF-α-induced NF-κB and MAPK signaling pathways in SL4 cells. β-actin was used as a normalization control. All experiments were repeated at least three times.
Mentions: We established a stable expression of CXCL16 in SL4, a metastatic mouse CRC cell line, and confirmed its expression (Figure 1). This cell line was cultured in a polyclonal population with a different expression level of CXCL16 in the antibiotic G418 to maintain the heterozygous characteristics of cancer cells. Membrane-bound CXCL16 was confirmed in almost SL4-CXCL16 cells by flow cytometry (Additional file 1). We next performed microarray analysis to compare gene expressions between SL4-Cont and SL4-CXCL16 cells. Significant differences were noted in the expression of TNF and apoptosis-related factors, whereas no changes were observed in metastatic factors (Table 1). On the other hand, soluble CXCL16 did not affect SL4-CXCL16 cells via the paracrine system because expression of CXCR6 (CXCL16 receptor) was not observed on SL4-CXCL16 cells (Additional file 2). When SL4-Cont and SL4-CXCL16 cells were treated with TNF-α, this induced a time-dependent increase in the death of SL4-CXCL16 cells (Figure 2A). We carried out the Annexin V assay to determine whether the TNF-α-induced cell death was apoptosis. As shown in Figure 2B, apoptosis was greater in TNF-α-treated SL4-CXCL16 cells than in SL4-Cont cells. The TNF-α-induced apoptotic response involved the stimulation of several intercellular signaling pathways [32]. Western blot analysis revealed an increase in the cleavage of PARP and caspase-3 in SL4-CXCL16 cells (Figure 2C). In addition, TNF-α-induced activation of TAK1 and its downstream NF-κB decreased, whereas phosphorylation of ERK and JNK increased in SL4-CXCL16 cells (Figure 2D). These results suggested that CXCL16 expression sensitized metastatic CRC cells to TNF-α-induced apoptosis.Figure 1

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