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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

Effect of 2-chloroadenosine on CXCL16-mediated inhibition of liver metastasis by SL4-CXCL16 cells. (A) M1 macrophage markers and TNF-α were detected by RT-PCR. GAPDH was used as the normalization control. N, normal; T, tumor. (B) Cytotoxicity of macrophage-derived TNF-α and recovery by TNF-α neutralizing antibody in SL4 cells. Cells (5 × 104 cells) were seeded in 24-well plates and a TNF-α neutralizing antibody added (2.5 μg/ml). RAW 264.7 cells (5 × 104 cells) were seeded in migration chambers and co-cultured. Cells were removed from the chambers and their viability was measured by WST-8 assay. *P <0.05, compared with control. #P <0.05, compared with RAW co-culture. (C and D) Restoration of liver metastasis by CXCL16 expression in a macrophage depletion model. 2-Chloroadenosine was dissolved in saline and injected intraperitoneally (50 μg/100 μl) 24 h before tumor inoculation. *P <0.05. All experiments were repeated at least three times.
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Fig6: Effect of 2-chloroadenosine on CXCL16-mediated inhibition of liver metastasis by SL4-CXCL16 cells. (A) M1 macrophage markers and TNF-α were detected by RT-PCR. GAPDH was used as the normalization control. N, normal; T, tumor. (B) Cytotoxicity of macrophage-derived TNF-α and recovery by TNF-α neutralizing antibody in SL4 cells. Cells (5 × 104 cells) were seeded in 24-well plates and a TNF-α neutralizing antibody added (2.5 μg/ml). RAW 264.7 cells (5 × 104 cells) were seeded in migration chambers and co-cultured. Cells were removed from the chambers and their viability was measured by WST-8 assay. *P <0.05, compared with control. #P <0.05, compared with RAW co-culture. (C and D) Restoration of liver metastasis by CXCL16 expression in a macrophage depletion model. 2-Chloroadenosine was dissolved in saline and injected intraperitoneally (50 μg/100 μl) 24 h before tumor inoculation. *P <0.05. All experiments were repeated at least three times.

Mentions: CXCL16 has been reported to play an important role in immunosurveillance as a chemoattractant [12, 13]. We determined the mechanism of the inhibition of liver metastasis by CXCL16 by carrying out RT-PCR to identify the types of cells that infiltrated the liver. As shown in Figure 6A, the levels of M1 macrophage markers such as CD11b, CD11c and F4/80 were increased at the tumor sites. We hypothesized that M1 macrophages were recruited and inhibited liver metastasis through the secretion of TNF-α. The M1 phenotype is typically IL-12, TNF-α, IL-6, CXCL9, CXCL10, CXCL11, CXCL16, IL-8high, whereas the M2 phenotype is IL-10, CXCR1, CXCR2, CCL17, CCL22, CCL24, CCL16, CCL18, CCL1, CCR2high[26]. Among the M1 and M2 macrophage markers, we selected 10 markers and confirmed in RAW264.7 cells using the RT-PCR method. As a result, we found that phenotype of RAW264.7 cells seem to be M1 type macrophages. Therefore, we conducted a co-culture experiment using RAW264.7 cells as a M1 type macrophage (Figure 6B and Additional files 3 and 4). We confirmed this hypothesis by co-culturing SL4-Cont and SL4-CXCL16 with RAW 264.7 cells and then a TNF-α neutralizing antibody was added to detect differences in cell death. Apoptosis of SL4-CXCL16 was significantly increased by co-culture with RAW 264.7 cells (Figure 6B, middle bar), and neutralization of TNF-α significantly inhibited this response (Figure 6B, right bar). This observation confirmed that CXCL16 expression sensitized the metastatic CRC cell line to apoptosis induced by TNF-α secreted by macrophages. Treatment of mice with 2-chloroadenosine, which is used for macrophage depletion [39], restored liver metastasis (Figure 6C) and also significantly increased tumor weight (Figure 6D). Collectively, these results suggest that CXCL16 expression by SL4 cells induced the accumulation of M1 macrophages, which then induced apoptosis in SL4 cells by secreting TNF-α, thereby leading to inhibition of metastasis.Figure 6


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)

Effect of 2-chloroadenosine on CXCL16-mediated inhibition of liver metastasis by SL4-CXCL16 cells. (A) M1 macrophage markers and TNF-α were detected by RT-PCR. GAPDH was used as the normalization control. N, normal; T, tumor. (B) Cytotoxicity of macrophage-derived TNF-α and recovery by TNF-α neutralizing antibody in SL4 cells. Cells (5 × 104 cells) were seeded in 24-well plates and a TNF-α neutralizing antibody added (2.5 μg/ml). RAW 264.7 cells (5 × 104 cells) were seeded in migration chambers and co-cultured. Cells were removed from the chambers and their viability was measured by WST-8 assay. *P <0.05, compared with control. #P <0.05, compared with RAW co-culture. (C and D) Restoration of liver metastasis by CXCL16 expression in a macrophage depletion model. 2-Chloroadenosine was dissolved in saline and injected intraperitoneally (50 μg/100 μl) 24 h before tumor inoculation. *P <0.05. All experiments were repeated at least three times.
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Fig6: Effect of 2-chloroadenosine on CXCL16-mediated inhibition of liver metastasis by SL4-CXCL16 cells. (A) M1 macrophage markers and TNF-α were detected by RT-PCR. GAPDH was used as the normalization control. N, normal; T, tumor. (B) Cytotoxicity of macrophage-derived TNF-α and recovery by TNF-α neutralizing antibody in SL4 cells. Cells (5 × 104 cells) were seeded in 24-well plates and a TNF-α neutralizing antibody added (2.5 μg/ml). RAW 264.7 cells (5 × 104 cells) were seeded in migration chambers and co-cultured. Cells were removed from the chambers and their viability was measured by WST-8 assay. *P <0.05, compared with control. #P <0.05, compared with RAW co-culture. (C and D) Restoration of liver metastasis by CXCL16 expression in a macrophage depletion model. 2-Chloroadenosine was dissolved in saline and injected intraperitoneally (50 μg/100 μl) 24 h before tumor inoculation. *P <0.05. All experiments were repeated at least three times.
Mentions: CXCL16 has been reported to play an important role in immunosurveillance as a chemoattractant [12, 13]. We determined the mechanism of the inhibition of liver metastasis by CXCL16 by carrying out RT-PCR to identify the types of cells that infiltrated the liver. As shown in Figure 6A, the levels of M1 macrophage markers such as CD11b, CD11c and F4/80 were increased at the tumor sites. We hypothesized that M1 macrophages were recruited and inhibited liver metastasis through the secretion of TNF-α. The M1 phenotype is typically IL-12, TNF-α, IL-6, CXCL9, CXCL10, CXCL11, CXCL16, IL-8high, whereas the M2 phenotype is IL-10, CXCR1, CXCR2, CCL17, CCL22, CCL24, CCL16, CCL18, CCL1, CCR2high[26]. Among the M1 and M2 macrophage markers, we selected 10 markers and confirmed in RAW264.7 cells using the RT-PCR method. As a result, we found that phenotype of RAW264.7 cells seem to be M1 type macrophages. Therefore, we conducted a co-culture experiment using RAW264.7 cells as a M1 type macrophage (Figure 6B and Additional files 3 and 4). We confirmed this hypothesis by co-culturing SL4-Cont and SL4-CXCL16 with RAW 264.7 cells and then a TNF-α neutralizing antibody was added to detect differences in cell death. Apoptosis of SL4-CXCL16 was significantly increased by co-culture with RAW 264.7 cells (Figure 6B, middle bar), and neutralization of TNF-α significantly inhibited this response (Figure 6B, right bar). This observation confirmed that CXCL16 expression sensitized the metastatic CRC cell line to apoptosis induced by TNF-α secreted by macrophages. Treatment of mice with 2-chloroadenosine, which is used for macrophage depletion [39], restored liver metastasis (Figure 6C) and also significantly increased tumor weight (Figure 6D). Collectively, these results suggest that CXCL16 expression by SL4 cells induced the accumulation of M1 macrophages, which then induced apoptosis in SL4 cells by secreting TNF-α, thereby leading to inhibition of metastasis.Figure 6

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