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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) expression decreases 5-fluorouracil (5-FU)-induced apoptosis in colorectal cancer cells. (a) Representative images showing Western blot analysis of SW480 control cells (SW480/Ctrl) and ANGPTL2-expressing SW480 cells (SW480/ANGPTL2-1 and -2) with an ANGPTL2 antibody. Hsc70 serves as an internal control. (b) ANGPTL2 protein concentrations in the culture medium of SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells (n = 3). (c) The relative number of proliferating cells (SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2) at indicated time points during in vitro culture (n = 3). (d) Percentage of viable cells among non-treated (left) or 5-FU treated (right) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells (n = 4). (e) FACS analysis of apoptotic cells among non-treated (upper) or 5-FU-treated (lower) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells based on analysis of 7-aminoactinomycin D (7-AAD) and annexin V. Right panels show quantitative analysis of the percentage of apoptotic (annexin V-positive) cells (n = 3). Error bars show SEM. **P < 0.01.
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fig01: Angiopoietin-like protein 2 (ANGPTL2) expression decreases 5-fluorouracil (5-FU)-induced apoptosis in colorectal cancer cells. (a) Representative images showing Western blot analysis of SW480 control cells (SW480/Ctrl) and ANGPTL2-expressing SW480 cells (SW480/ANGPTL2-1 and -2) with an ANGPTL2 antibody. Hsc70 serves as an internal control. (b) ANGPTL2 protein concentrations in the culture medium of SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells (n = 3). (c) The relative number of proliferating cells (SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2) at indicated time points during in vitro culture (n = 3). (d) Percentage of viable cells among non-treated (left) or 5-FU treated (right) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells (n = 4). (e) FACS analysis of apoptotic cells among non-treated (upper) or 5-FU-treated (lower) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells based on analysis of 7-aminoactinomycin D (7-AAD) and annexin V. Right panels show quantitative analysis of the percentage of apoptotic (annexin V-positive) cells (n = 3). Error bars show SEM. **P < 0.01.

Mentions: To assess a potential role for ANGPTL2 in enhancing survival of CRC cells treated with antineoplastic drugs, we established two independent SW480 lines constitutively expressing ANGPTL2 (SW480/ANGPTL2-1 and -2) and a control line expressing vector only (SW480/Ctrl). Western blotting and ELISA analyses confirmed that both SW480/ANGPTL2-1 and -2 cells showed similarly enhanced expression of ANGPTL2 protein in both cell lysates and in culture medium relative to SW480/Ctrl cells (Fig. 1a,b). However, we observed no significant difference in proliferation among all three lines at 24, 48 and 72 h after plating (Fig. 1c), indicating that ANGPTL2 protein secreted by CRC cells does not alter proliferation under normal growth conditions. Next, we carried out a cell viability assay 24 h after treatment of CRC cells with the apoptosis-inducing drug 5-FU.(17) Constitutive ANGPTL2 expression in SW480/ANGPTL2-1 and -2 cells increased cell viability following 5-FU treatment relative to control drug-treated control cells (SW480/Ctrl) (Fig. 1d). Thus, we used flow cytometry to evaluate the proportion of apoptosis following 5-FU treatment using double-staining with annexin V and 7-AAD. That proportion significantly decreased in SW480/ANGPTL2-1 or -2 compared to SW480/Ctrl cells (Fig. 1e). We also examined cell viability after treatment of cells with other antineoplastic drugs used to treat CRC, including irinotecan (CPT11), cisplatin (CDDP), and mitomycin C (MMC),(2,18) and observed similar results (Fig. S1). These findings suggest that constitutive expression of ANGPTL2 in SW480 cells increases their chemoresistance by inhibiting apoptosis.


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) expression decreases 5-fluorouracil (5-FU)-induced apoptosis in colorectal cancer cells. (a) Representative images showing Western blot analysis of SW480 control cells (SW480/Ctrl) and ANGPTL2-expressing SW480 cells (SW480/ANGPTL2-1 and -2) with an ANGPTL2 antibody. Hsc70 serves as an internal control. (b) ANGPTL2 protein concentrations in the culture medium of SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells (n = 3). (c) The relative number of proliferating cells (SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2) at indicated time points during in vitro culture (n = 3). (d) Percentage of viable cells among non-treated (left) or 5-FU treated (right) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells (n = 4). (e) FACS analysis of apoptotic cells among non-treated (upper) or 5-FU-treated (lower) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells based on analysis of 7-aminoactinomycin D (7-AAD) and annexin V. Right panels show quantitative analysis of the percentage of apoptotic (annexin V-positive) cells (n = 3). Error bars show SEM. **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Angiopoietin-like protein 2 (ANGPTL2) expression decreases 5-fluorouracil (5-FU)-induced apoptosis in colorectal cancer cells. (a) Representative images showing Western blot analysis of SW480 control cells (SW480/Ctrl) and ANGPTL2-expressing SW480 cells (SW480/ANGPTL2-1 and -2) with an ANGPTL2 antibody. Hsc70 serves as an internal control. (b) ANGPTL2 protein concentrations in the culture medium of SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2 cells (n = 3). (c) The relative number of proliferating cells (SW480/Ctrl, SW480/ANGPTL2-1, and SW480/ANGPTL2-2) at indicated time points during in vitro culture (n = 3). (d) Percentage of viable cells among non-treated (left) or 5-FU treated (right) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells (n = 4). (e) FACS analysis of apoptotic cells among non-treated (upper) or 5-FU-treated (lower) SW480/Ctrl, SW480/ANGPTL2-1, or SW480/ANGPTL2-2 cells based on analysis of 7-aminoactinomycin D (7-AAD) and annexin V. Right panels show quantitative analysis of the percentage of apoptotic (annexin V-positive) cells (n = 3). Error bars show SEM. **P < 0.01.
Mentions: To assess a potential role for ANGPTL2 in enhancing survival of CRC cells treated with antineoplastic drugs, we established two independent SW480 lines constitutively expressing ANGPTL2 (SW480/ANGPTL2-1 and -2) and a control line expressing vector only (SW480/Ctrl). Western blotting and ELISA analyses confirmed that both SW480/ANGPTL2-1 and -2 cells showed similarly enhanced expression of ANGPTL2 protein in both cell lysates and in culture medium relative to SW480/Ctrl cells (Fig. 1a,b). However, we observed no significant difference in proliferation among all three lines at 24, 48 and 72 h after plating (Fig. 1c), indicating that ANGPTL2 protein secreted by CRC cells does not alter proliferation under normal growth conditions. Next, we carried out a cell viability assay 24 h after treatment of CRC cells with the apoptosis-inducing drug 5-FU.(17) Constitutive ANGPTL2 expression in SW480/ANGPTL2-1 and -2 cells increased cell viability following 5-FU treatment relative to control drug-treated control cells (SW480/Ctrl) (Fig. 1d). Thus, we used flow cytometry to evaluate the proportion of apoptosis following 5-FU treatment using double-staining with annexin V and 7-AAD. That proportion significantly decreased in SW480/ANGPTL2-1 or -2 compared to SW480/Ctrl cells (Fig. 1e). We also examined cell viability after treatment of cells with other antineoplastic drugs used to treat CRC, including irinotecan (CPT11), cisplatin (CDDP), and mitomycin C (MMC),(2,18) and observed similar results (Fig. S1). These findings suggest that constitutive expression of ANGPTL2 in SW480 cells increases their chemoresistance by inhibiting apoptosis.

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