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Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase-phosphoinositide 3-kinase-dependent anti-apoptotic signaling.

Horiguchi H, Endo M, Miyamoto Y, Sakamoto Y, Odagiri H, Masuda T, Kadomatsu T, Tanoue H, Motokawa I, Terada K, Morioka MS, Manabe I, Baba H, Oike Y - Cancer Sci. (2014)

Bottom Line: Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells.Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells.Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase-nuclear factor of activated T cells (Syk-NFAT) pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.

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Induction of anti-apoptotic BCL-2 family members occurs through phosphoinositide 3-kinase–nuclear factor-κB (PI3K–NF-κB) signaling. (a) Nuclear translocation of NF-κB p65 in SW480/Ctrl and SW480/ANGPTL2-1 cells. Nuclei were counterstained with DAPI. Arrowheads indicate overlap of p65 and DAPI staining. Scale bar = 50 μm. (b,c) Quantification of p65-positive nuclei in (a). The proportion of cells showing nuclear staining for the NF-κB subunit p65 was calculated as a percentage of DAPI-positive total cells in four random fields of view per cell line (n = 4). (d) Relative mRNA expression of the anti-apoptotic BCL-2 family members BCL-2 (left) and BCL-XL (right) in SW480/Ctrl or SW480/ANGPTL2-1cells, either non-treated (Ctrl) or treated with NF-κB (BAY) or PI3K (LY) inhibitors. Data from non-treated SW480/Ctrl cells was set at 1 (n = 3). (e) Percentage of viable SW480/ANGPTL2-1 cells treated with or without various combinations of 5-fluorouracil (5-FU), BAY11-7085 (BAY), and LY294002 (LY) (n = 4). (f) Quantitative analysis of the proportion of apoptotic cells seen among SW480/ANGPTL2-1cells treated with or without various combinations of 5-FU, BAY, and LY based on FACS analysis (n = 3). Error bars show SEM. **P < 0.01. n.s., no statistically significant difference.
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fig04: Induction of anti-apoptotic BCL-2 family members occurs through phosphoinositide 3-kinase–nuclear factor-κB (PI3K–NF-κB) signaling. (a) Nuclear translocation of NF-κB p65 in SW480/Ctrl and SW480/ANGPTL2-1 cells. Nuclei were counterstained with DAPI. Arrowheads indicate overlap of p65 and DAPI staining. Scale bar = 50 μm. (b,c) Quantification of p65-positive nuclei in (a). The proportion of cells showing nuclear staining for the NF-κB subunit p65 was calculated as a percentage of DAPI-positive total cells in four random fields of view per cell line (n = 4). (d) Relative mRNA expression of the anti-apoptotic BCL-2 family members BCL-2 (left) and BCL-XL (right) in SW480/Ctrl or SW480/ANGPTL2-1cells, either non-treated (Ctrl) or treated with NF-κB (BAY) or PI3K (LY) inhibitors. Data from non-treated SW480/Ctrl cells was set at 1 (n = 3). (e) Percentage of viable SW480/ANGPTL2-1 cells treated with or without various combinations of 5-fluorouracil (5-FU), BAY11-7085 (BAY), and LY294002 (LY) (n = 4). (f) Quantitative analysis of the proportion of apoptotic cells seen among SW480/ANGPTL2-1cells treated with or without various combinations of 5-FU, BAY, and LY based on FACS analysis (n = 3). Error bars show SEM. **P < 0.01. n.s., no statistically significant difference.

Mentions: Nuclear factor-κB induces BCL-2 family member genes(22) and is known to be activated by Syk signaling.(10) To further investigate molecular mechanisms underlying ANGPTL2 anti-apoptotic activity, we examined NF-κB activation in SW480/ANGPTL2-1 cells and SW480/Ctrl cells by staining with a p65 antibody (Fig. 4a). Analysis of staining distribution indicated that the proportion of NF-κB subunit p65 found in the nucleus increased in SW480/ANGPTL2 cells compared with control cells, which showed largely cytoplasmic staining (Fig. 4a,b). It has been reported that PI3K activates the NF-κB pathway and induces survival signals.(23) To investigate whether NF-κB activation in SW480/ANGPTL2 cells was due to activation of the PI3K pathway, we assessed p65 translocation to the nucleus in the presence of the PI3K inhibitor LY294002. Treatment of SW480/ANGPTL2-1 cells with LY294002 significantly decreased the proportion of nuclear NF-κB relative to untreated cells (Fig. 4a,c). When we asked whether Syk knockdown would alter NF-κB nuclear translocation in SW480/ANGPTL2-1 cells, we found that knockdown decreased NF-κB nuclear translocation relative to that seen in Scr control cells (Fig. S3a). Taken together, these results suggest that ANGPTL2 overexpression promotes nuclear translocation of NF-κB through the Syk–PI3K pathway in CRC cells.


Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase-phosphoinositide 3-kinase-dependent anti-apoptotic signaling.

Horiguchi H, Endo M, Miyamoto Y, Sakamoto Y, Odagiri H, Masuda T, Kadomatsu T, Tanoue H, Motokawa I, Terada K, Morioka MS, Manabe I, Baba H, Oike Y - Cancer Sci. (2014)

Induction of anti-apoptotic BCL-2 family members occurs through phosphoinositide 3-kinase–nuclear factor-κB (PI3K–NF-κB) signaling. (a) Nuclear translocation of NF-κB p65 in SW480/Ctrl and SW480/ANGPTL2-1 cells. Nuclei were counterstained with DAPI. Arrowheads indicate overlap of p65 and DAPI staining. Scale bar = 50 μm. (b,c) Quantification of p65-positive nuclei in (a). The proportion of cells showing nuclear staining for the NF-κB subunit p65 was calculated as a percentage of DAPI-positive total cells in four random fields of view per cell line (n = 4). (d) Relative mRNA expression of the anti-apoptotic BCL-2 family members BCL-2 (left) and BCL-XL (right) in SW480/Ctrl or SW480/ANGPTL2-1cells, either non-treated (Ctrl) or treated with NF-κB (BAY) or PI3K (LY) inhibitors. Data from non-treated SW480/Ctrl cells was set at 1 (n = 3). (e) Percentage of viable SW480/ANGPTL2-1 cells treated with or without various combinations of 5-fluorouracil (5-FU), BAY11-7085 (BAY), and LY294002 (LY) (n = 4). (f) Quantitative analysis of the proportion of apoptotic cells seen among SW480/ANGPTL2-1cells treated with or without various combinations of 5-FU, BAY, and LY based on FACS analysis (n = 3). Error bars show SEM. **P < 0.01. n.s., no statistically significant difference.
© Copyright Policy - open-access
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fig04: Induction of anti-apoptotic BCL-2 family members occurs through phosphoinositide 3-kinase–nuclear factor-κB (PI3K–NF-κB) signaling. (a) Nuclear translocation of NF-κB p65 in SW480/Ctrl and SW480/ANGPTL2-1 cells. Nuclei were counterstained with DAPI. Arrowheads indicate overlap of p65 and DAPI staining. Scale bar = 50 μm. (b,c) Quantification of p65-positive nuclei in (a). The proportion of cells showing nuclear staining for the NF-κB subunit p65 was calculated as a percentage of DAPI-positive total cells in four random fields of view per cell line (n = 4). (d) Relative mRNA expression of the anti-apoptotic BCL-2 family members BCL-2 (left) and BCL-XL (right) in SW480/Ctrl or SW480/ANGPTL2-1cells, either non-treated (Ctrl) or treated with NF-κB (BAY) or PI3K (LY) inhibitors. Data from non-treated SW480/Ctrl cells was set at 1 (n = 3). (e) Percentage of viable SW480/ANGPTL2-1 cells treated with or without various combinations of 5-fluorouracil (5-FU), BAY11-7085 (BAY), and LY294002 (LY) (n = 4). (f) Quantitative analysis of the proportion of apoptotic cells seen among SW480/ANGPTL2-1cells treated with or without various combinations of 5-FU, BAY, and LY based on FACS analysis (n = 3). Error bars show SEM. **P < 0.01. n.s., no statistically significant difference.
Mentions: Nuclear factor-κB induces BCL-2 family member genes(22) and is known to be activated by Syk signaling.(10) To further investigate molecular mechanisms underlying ANGPTL2 anti-apoptotic activity, we examined NF-κB activation in SW480/ANGPTL2-1 cells and SW480/Ctrl cells by staining with a p65 antibody (Fig. 4a). Analysis of staining distribution indicated that the proportion of NF-κB subunit p65 found in the nucleus increased in SW480/ANGPTL2 cells compared with control cells, which showed largely cytoplasmic staining (Fig. 4a,b). It has been reported that PI3K activates the NF-κB pathway and induces survival signals.(23) To investigate whether NF-κB activation in SW480/ANGPTL2 cells was due to activation of the PI3K pathway, we assessed p65 translocation to the nucleus in the presence of the PI3K inhibitor LY294002. Treatment of SW480/ANGPTL2-1 cells with LY294002 significantly decreased the proportion of nuclear NF-κB relative to untreated cells (Fig. 4a,c). When we asked whether Syk knockdown would alter NF-κB nuclear translocation in SW480/ANGPTL2-1 cells, we found that knockdown decreased NF-κB nuclear translocation relative to that seen in Scr control cells (Fig. S3a). Taken together, these results suggest that ANGPTL2 overexpression promotes nuclear translocation of NF-κB through the Syk–PI3K pathway in CRC cells.

Bottom Line: Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells.Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells.Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase-nuclear factor of activated T cells (Syk-NFAT) pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.

Show MeSH
Related in: MedlinePlus