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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.To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells.

View Article: PubMed Central - PubMed

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

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Angiopoietin-like protein 2 (ANGPTL2) blocks SW480 cell apoptosis by increasing spleen tyrosine kinase (Syk) expression. (a) Genes increased in SW480/ANGPTL2-1 cells based on RNA sequencing analysis. Data are log2 fold-change relative to SW480/Ctrl cells. (b) Relative Syk mRNA expression in SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells. Data from SW480/Ctrl cells was set at 1 (n = 3). (c) Western blotting analysis of Syk, phosphorylated Syk (p-Syk), and Hsc70 in SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells. Hsc70 serves as an internal control. (d) Western blot analysis of Syk, p-Syk, and Hsc70 in SW480/ANGPTL2-1 cells transduced with three Syk siRNAs (A, B, or C) or with control siRNA (Scr). Hsc70 serves as an internal control. (e) The relative number of proliferating cells (SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells) at indicated time points of in vitro culture (n = 4). (f) Percentage of viable cells remaining after 5-fluorouracil (5-FU) treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells (n = 4). (g) Quantitative analysis of the proportion of apoptotic cells following 5-FU treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells based on annexin V staining (n = 3). Error bars show SEM. *P < 0.05; **P < 0.01.
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fig03: Angiopoietin-like protein 2 (ANGPTL2) blocks SW480 cell apoptosis by increasing spleen tyrosine kinase (Syk) expression. (a) Genes increased in SW480/ANGPTL2-1 cells based on RNA sequencing analysis. Data are log2 fold-change relative to SW480/Ctrl cells. (b) Relative Syk mRNA expression in SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells. Data from SW480/Ctrl cells was set at 1 (n = 3). (c) Western blotting analysis of Syk, phosphorylated Syk (p-Syk), and Hsc70 in SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells. Hsc70 serves as an internal control. (d) Western blot analysis of Syk, p-Syk, and Hsc70 in SW480/ANGPTL2-1 cells transduced with three Syk siRNAs (A, B, or C) or with control siRNA (Scr). Hsc70 serves as an internal control. (e) The relative number of proliferating cells (SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells) at indicated time points of in vitro culture (n = 4). (f) Percentage of viable cells remaining after 5-fluorouracil (5-FU) treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells (n = 4). (g) Quantitative analysis of the proportion of apoptotic cells following 5-FU treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells based on annexin V staining (n = 3). Error bars show SEM. *P < 0.05; **P < 0.01.

Mentions: To examine signaling downstream of ANGPTL2 that might induce anti-apoptotic genes, we carried out RNA sequencing analysis of SW480/ANGPTL2-1 and SW480/Ctrl cells (Fig. 3a). We found that levels of 10 transcripts, including ANGPTL2 itself, were markedly increased in SW480/ANGPTL2-1 compared with SW480/Ctrl cells. As BCL-2 and BCL-XL were induced more robustly in SW480/ANGPTL2 compared to SW480/Ctr cells (Fig. 2), we checked their expression by RNA sequencing but did not observe significant induction of either gene using this method. However, we observed upregulation of Syk, which plays a role in cell survival,(10,12,13) in SW480/ANGPTL2 cells. Real-time PCR analysis validated that Syk expression levels in SW480/ANGPTL2-1 and -2 cells increased compared with levels seen in control SW480/Ctrl cells (Fig. 3b). Tyrosine phosphorylation is required to activate Syk kinase activity and function.(21) Thus, we examined Syk activity in SW480/ANGPTL2-1 and -2 versus control cells by Western blotting with a phospho-Syk antibody. Syk expression and phosphorylation was markedly increased in SW480/ANGPTL2-1 and -2 cells compared with SW480/Ctrl cells (Fig. 3c), suggesting that the enhanced cell survival activity seen in both lines is due to Syk upregulation and activation. Next, we asked whether Syk knockdown by siRNA in ANGPTL2-expressing SW480 cells would reduce cell viability or induce cell apoptosis following 5-FU treatment. We confirmed Syk knockdown levels in SW480/ANGPTL2-1 cells by Western blot analysis. Total Syk and phospho-Syk protein were concomitantly reduced by knockdown (Fig. 3d). We observed no significant difference in proliferation between knockdown versus scrambled control cells at 24, 48 or 72 h after plating in normal growth conditions (Fig. 3e). However, Syk knockdown cells (siRNA/A, B, and C) showed decreased viability following 5-FU treatment compared with scrambled control (siRNA Scr) cells (Fig. 3f). In addition, the proportion of apoptosis significantly increased in siRNA/A, B, and C cells compared to siRNA Scr cells (Fig. 3g). Thus, we conclude that Syk upregulation and activation enhances cell survival in ANGPTL2-overexpressing 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)

Angiopoietin-like protein 2 (ANGPTL2) blocks SW480 cell apoptosis by increasing spleen tyrosine kinase (Syk) expression. (a) Genes increased in SW480/ANGPTL2-1 cells based on RNA sequencing analysis. Data are log2 fold-change relative to SW480/Ctrl cells. (b) Relative Syk mRNA expression in SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells. Data from SW480/Ctrl cells was set at 1 (n = 3). (c) Western blotting analysis of Syk, phosphorylated Syk (p-Syk), and Hsc70 in SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells. Hsc70 serves as an internal control. (d) Western blot analysis of Syk, p-Syk, and Hsc70 in SW480/ANGPTL2-1 cells transduced with three Syk siRNAs (A, B, or C) or with control siRNA (Scr). Hsc70 serves as an internal control. (e) The relative number of proliferating cells (SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells) at indicated time points of in vitro culture (n = 4). (f) Percentage of viable cells remaining after 5-fluorouracil (5-FU) treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells (n = 4). (g) Quantitative analysis of the proportion of apoptotic cells following 5-FU treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells based on annexin V staining (n = 3). Error bars show SEM. *P < 0.05; **P < 0.01.
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fig03: Angiopoietin-like protein 2 (ANGPTL2) blocks SW480 cell apoptosis by increasing spleen tyrosine kinase (Syk) expression. (a) Genes increased in SW480/ANGPTL2-1 cells based on RNA sequencing analysis. Data are log2 fold-change relative to SW480/Ctrl cells. (b) Relative Syk mRNA expression in SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells. Data from SW480/Ctrl cells was set at 1 (n = 3). (c) Western blotting analysis of Syk, phosphorylated Syk (p-Syk), and Hsc70 in SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells. Hsc70 serves as an internal control. (d) Western blot analysis of Syk, p-Syk, and Hsc70 in SW480/ANGPTL2-1 cells transduced with three Syk siRNAs (A, B, or C) or with control siRNA (Scr). Hsc70 serves as an internal control. (e) The relative number of proliferating cells (SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells) at indicated time points of in vitro culture (n = 4). (f) Percentage of viable cells remaining after 5-fluorouracil (5-FU) treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells (n = 4). (g) Quantitative analysis of the proportion of apoptotic cells following 5-FU treatment of SW480/ANGPTL2-1/Scr, SW480/ANGPTL2-1/A, SW480/ANGPTL2-1/B, or SW480/ANGPTL2-1/C cells based on annexin V staining (n = 3). Error bars show SEM. *P < 0.05; **P < 0.01.
Mentions: To examine signaling downstream of ANGPTL2 that might induce anti-apoptotic genes, we carried out RNA sequencing analysis of SW480/ANGPTL2-1 and SW480/Ctrl cells (Fig. 3a). We found that levels of 10 transcripts, including ANGPTL2 itself, were markedly increased in SW480/ANGPTL2-1 compared with SW480/Ctrl cells. As BCL-2 and BCL-XL were induced more robustly in SW480/ANGPTL2 compared to SW480/Ctr cells (Fig. 2), we checked their expression by RNA sequencing but did not observe significant induction of either gene using this method. However, we observed upregulation of Syk, which plays a role in cell survival,(10,12,13) in SW480/ANGPTL2 cells. Real-time PCR analysis validated that Syk expression levels in SW480/ANGPTL2-1 and -2 cells increased compared with levels seen in control SW480/Ctrl cells (Fig. 3b). Tyrosine phosphorylation is required to activate Syk kinase activity and function.(21) Thus, we examined Syk activity in SW480/ANGPTL2-1 and -2 versus control cells by Western blotting with a phospho-Syk antibody. Syk expression and phosphorylation was markedly increased in SW480/ANGPTL2-1 and -2 cells compared with SW480/Ctrl cells (Fig. 3c), suggesting that the enhanced cell survival activity seen in both lines is due to Syk upregulation and activation. Next, we asked whether Syk knockdown by siRNA in ANGPTL2-expressing SW480 cells would reduce cell viability or induce cell apoptosis following 5-FU treatment. We confirmed Syk knockdown levels in SW480/ANGPTL2-1 cells by Western blot analysis. Total Syk and phospho-Syk protein were concomitantly reduced by knockdown (Fig. 3d). We observed no significant difference in proliferation between knockdown versus scrambled control cells at 24, 48 or 72 h after plating in normal growth conditions (Fig. 3e). However, Syk knockdown cells (siRNA/A, B, and C) showed decreased viability following 5-FU treatment compared with scrambled control (siRNA Scr) cells (Fig. 3f). In addition, the proportion of apoptosis significantly increased in siRNA/A, B, and C cells compared to siRNA Scr cells (Fig. 3g). Thus, we conclude that Syk upregulation and activation enhances cell survival in ANGPTL2-overexpressing 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.To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus