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Death receptor 6 (DR6) is required for mouse B16 tumor angiogenesis via the NF-κB, P38 MAPK and STAT3 pathways.

Yang X, Shi B, Li L, Xu Z, Ge Y, Shi J, Liu Y, Zheng D - Oncogenesis (2016)

Bottom Line: Although death receptor 6 (DR6) is aberrantly expressed in certain cancer cell lines, its function, signaling pathway and potential clinical significance in tumor progression are not well characterized.Deficiency of DR6 changes cytokine expression and secretion; in particular, it inhibits the proinflammatory cytokine interleukin-6 (IL-6), which is able to induce the expression of the angiogenesis-related factors: vascular endothelial growth factor-A, platelet-derived growth factor-β, vascular endothelial growth factor-D and platelet-derived growth factor receptor-α.Further experiments demonstrate that DR6-dependent angiogenesis is involved in the IL-6/P38 MAPK and IL-6/STAT3 pathways.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Although death receptor 6 (DR6) is aberrantly expressed in certain cancer cell lines, its function, signaling pathway and potential clinical significance in tumor progression are not well characterized. We report here that knocking down DR6 in the mouse B16 cell line has no effect on B16 cell death in vitro but suppresses xenograft B16 tumor growth by preventing tumor blood vessel formation in vivo. Deficiency of DR6 changes cytokine expression and secretion; in particular, it inhibits the proinflammatory cytokine interleukin-6 (IL-6), which is able to induce the expression of the angiogenesis-related factors: vascular endothelial growth factor-A, platelet-derived growth factor-β, vascular endothelial growth factor-D and platelet-derived growth factor receptor-α. Further experiments demonstrate that DR6-dependent angiogenesis is involved in the IL-6/P38 MAPK and IL-6/STAT3 pathways. Our novel findings demonstrate for the first time that DR6 expression in B16 cells facilitates tumor growth by accelerating tumor angiogenesis. Moreover, these results suggest that DR6 is involved in three important intracellular pathways that lead to homeostatic angiogenesis in tumor growth.

No MeSH data available.


Related in: MedlinePlus

A lack of DR6 prevents the expression of angiogenesis-related mediators in vitro. (a) B16 cells were transfected with the anti-DR6 siRNA for 72 h, and then subjected to the flow cytometry assays with FITC-Annexin V/propidium iodide staining. (b) The expression of caspase 3 and caspase 6 was detected by western blot assays. (c) mRNA expression of DR6 and several cytokines in B16 cells. The mRNAs were isolated from B16 cells in siRNA-transfected for 48 h, and then examined by real-time PCR. The vertical bar in the middle of the graph is the baseline of each cytokine, which represents the siNC group. The gray bars represent the times of each mRNA expression in the siDR6-transfected group, compared with the siNC group. (d) VEGF-A concentration in B16 cultivated media. The cultured media were subjected to ELISA with the VEGF-A specific antibody. (e) Tumor cells were isolated from tumor tissues by Percoll density gradient centrifugation. The expression of VEGF-D and PDGFR-α protein and VEGF-A and PDGF-β mRNA in the B16 tumors. (f) Exogenous mouse VEGF-A enhances the expression of DR6 in B16 cells. Cells were treated with exogenous VEGF-A (10 ng/ml) for 0–12 h and then subjected to western blotting. GAPDH was used as the loading control. The error bars represent the standard deviation of the mean values obtained from triplicate experiments. P<0.05 is considered to be statistically significant.
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fig3: A lack of DR6 prevents the expression of angiogenesis-related mediators in vitro. (a) B16 cells were transfected with the anti-DR6 siRNA for 72 h, and then subjected to the flow cytometry assays with FITC-Annexin V/propidium iodide staining. (b) The expression of caspase 3 and caspase 6 was detected by western blot assays. (c) mRNA expression of DR6 and several cytokines in B16 cells. The mRNAs were isolated from B16 cells in siRNA-transfected for 48 h, and then examined by real-time PCR. The vertical bar in the middle of the graph is the baseline of each cytokine, which represents the siNC group. The gray bars represent the times of each mRNA expression in the siDR6-transfected group, compared with the siNC group. (d) VEGF-A concentration in B16 cultivated media. The cultured media were subjected to ELISA with the VEGF-A specific antibody. (e) Tumor cells were isolated from tumor tissues by Percoll density gradient centrifugation. The expression of VEGF-D and PDGFR-α protein and VEGF-A and PDGF-β mRNA in the B16 tumors. (f) Exogenous mouse VEGF-A enhances the expression of DR6 in B16 cells. Cells were treated with exogenous VEGF-A (10 ng/ml) for 0–12 h and then subjected to western blotting. GAPDH was used as the loading control. The error bars represent the standard deviation of the mean values obtained from triplicate experiments. P<0.05 is considered to be statistically significant.

Mentions: We first tested the B16 cell apoptosis with knocking down DR6. As assessed by FITC-Annexin V and propidium iodide staining, we did not observe apoptotic cell death after knocking down DR6 expression for 72 h in B16 cells (Figure 3a). Alternative splicing of caspase 3 and caspase 6 proteins, which were reported that had been changed in DR6-expressing neuronal cells,11, 12 presented no change in DR6-deficient B16 cells by western blot assays (Figure 3b). These data indicate that DR6 deficiency suppresses mouse B16 tumor growth in vivo but has no effect on B16 cell death in vitro.


Death receptor 6 (DR6) is required for mouse B16 tumor angiogenesis via the NF-κB, P38 MAPK and STAT3 pathways.

Yang X, Shi B, Li L, Xu Z, Ge Y, Shi J, Liu Y, Zheng D - Oncogenesis (2016)

A lack of DR6 prevents the expression of angiogenesis-related mediators in vitro. (a) B16 cells were transfected with the anti-DR6 siRNA for 72 h, and then subjected to the flow cytometry assays with FITC-Annexin V/propidium iodide staining. (b) The expression of caspase 3 and caspase 6 was detected by western blot assays. (c) mRNA expression of DR6 and several cytokines in B16 cells. The mRNAs were isolated from B16 cells in siRNA-transfected for 48 h, and then examined by real-time PCR. The vertical bar in the middle of the graph is the baseline of each cytokine, which represents the siNC group. The gray bars represent the times of each mRNA expression in the siDR6-transfected group, compared with the siNC group. (d) VEGF-A concentration in B16 cultivated media. The cultured media were subjected to ELISA with the VEGF-A specific antibody. (e) Tumor cells were isolated from tumor tissues by Percoll density gradient centrifugation. The expression of VEGF-D and PDGFR-α protein and VEGF-A and PDGF-β mRNA in the B16 tumors. (f) Exogenous mouse VEGF-A enhances the expression of DR6 in B16 cells. Cells were treated with exogenous VEGF-A (10 ng/ml) for 0–12 h and then subjected to western blotting. GAPDH was used as the loading control. The error bars represent the standard deviation of the mean values obtained from triplicate experiments. P<0.05 is considered to be statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: A lack of DR6 prevents the expression of angiogenesis-related mediators in vitro. (a) B16 cells were transfected with the anti-DR6 siRNA for 72 h, and then subjected to the flow cytometry assays with FITC-Annexin V/propidium iodide staining. (b) The expression of caspase 3 and caspase 6 was detected by western blot assays. (c) mRNA expression of DR6 and several cytokines in B16 cells. The mRNAs were isolated from B16 cells in siRNA-transfected for 48 h, and then examined by real-time PCR. The vertical bar in the middle of the graph is the baseline of each cytokine, which represents the siNC group. The gray bars represent the times of each mRNA expression in the siDR6-transfected group, compared with the siNC group. (d) VEGF-A concentration in B16 cultivated media. The cultured media were subjected to ELISA with the VEGF-A specific antibody. (e) Tumor cells were isolated from tumor tissues by Percoll density gradient centrifugation. The expression of VEGF-D and PDGFR-α protein and VEGF-A and PDGF-β mRNA in the B16 tumors. (f) Exogenous mouse VEGF-A enhances the expression of DR6 in B16 cells. Cells were treated with exogenous VEGF-A (10 ng/ml) for 0–12 h and then subjected to western blotting. GAPDH was used as the loading control. The error bars represent the standard deviation of the mean values obtained from triplicate experiments. P<0.05 is considered to be statistically significant.
Mentions: We first tested the B16 cell apoptosis with knocking down DR6. As assessed by FITC-Annexin V and propidium iodide staining, we did not observe apoptotic cell death after knocking down DR6 expression for 72 h in B16 cells (Figure 3a). Alternative splicing of caspase 3 and caspase 6 proteins, which were reported that had been changed in DR6-expressing neuronal cells,11, 12 presented no change in DR6-deficient B16 cells by western blot assays (Figure 3b). These data indicate that DR6 deficiency suppresses mouse B16 tumor growth in vivo but has no effect on B16 cell death in vitro.

Bottom Line: Although death receptor 6 (DR6) is aberrantly expressed in certain cancer cell lines, its function, signaling pathway and potential clinical significance in tumor progression are not well characterized.Deficiency of DR6 changes cytokine expression and secretion; in particular, it inhibits the proinflammatory cytokine interleukin-6 (IL-6), which is able to induce the expression of the angiogenesis-related factors: vascular endothelial growth factor-A, platelet-derived growth factor-β, vascular endothelial growth factor-D and platelet-derived growth factor receptor-α.Further experiments demonstrate that DR6-dependent angiogenesis is involved in the IL-6/P38 MAPK and IL-6/STAT3 pathways.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Although death receptor 6 (DR6) is aberrantly expressed in certain cancer cell lines, its function, signaling pathway and potential clinical significance in tumor progression are not well characterized. We report here that knocking down DR6 in the mouse B16 cell line has no effect on B16 cell death in vitro but suppresses xenograft B16 tumor growth by preventing tumor blood vessel formation in vivo. Deficiency of DR6 changes cytokine expression and secretion; in particular, it inhibits the proinflammatory cytokine interleukin-6 (IL-6), which is able to induce the expression of the angiogenesis-related factors: vascular endothelial growth factor-A, platelet-derived growth factor-β, vascular endothelial growth factor-D and platelet-derived growth factor receptor-α. Further experiments demonstrate that DR6-dependent angiogenesis is involved in the IL-6/P38 MAPK and IL-6/STAT3 pathways. Our novel findings demonstrate for the first time that DR6 expression in B16 cells facilitates tumor growth by accelerating tumor angiogenesis. Moreover, these results suggest that DR6 is involved in three important intracellular pathways that lead to homeostatic angiogenesis in tumor growth.

No MeSH data available.


Related in: MedlinePlus