5-fluorouracil causes endothelial cell senescence: potential protective role of glucagon-like peptide 1

Abstract

Background and purpose: 5-fluorouracil (5FU) and its prodrug, capecitabine, can damage endothelial cells, whilst endothelial integrity is preserved by glucagon-like peptide 1 (GLP-1). Here, we studied the effect of 5FU on endothelial senescence and whether GLP-1 antagonizes it.

Experimental approach: EA.hy926 cells were exposed to 5FU or sera from patients taking capecitabine, with or without pre-incubation with GLP-1. Senescence was identified by expression of senescence-associated β-galactosidase and p16^INK4a and reduced cell proliferation. Soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and CD146 (marker of endothelial injury) were measured by ELISA before and at completion of capecitabine chemotherapy. RT-PCR, western blotting, functional experiments with signalling inhibitors and ERK1/2 silencing were performed to characterize 5FU-induced phenotype and elucidate the pathways underlying 5FU and GLP-1 activity.

Key results: Both 5FU and sera from capecitabine-treated patients stimulated endothelial cell senescence. 5FU-elicited senescence occurred via activation of p38 and JNK, and was associated with decreased eNOS and SIRT-1 levels. Furthermore, 5FU up-regulated VCAM1 and TYMP (encodes enzyme activating capecitabine and 5FU), and sVCAM-1 and CD146 concentrations were higher after than before capecitabine chemotherapy. A non-significant trend for higher ICAM1 levels was also observed. GLP-1 counteracted 5FU-initiated senescence and reduced eNOS and SIRT-1 expression, this protection being mediated by GLP-1 receptor, ERK1/2 and, possibly, PKA and PI3K.

Conclusions and implications: 5FU causes endothelial cell senescence and dysfunction, which may contribute to its cardiovascular side effects. 5FU-triggered senescence was prevented by GLP-1, raising the possibility of using GLP-1 analogues and degradation inhibitors to treat 5FU and capecitabine vascular toxicity.

Linked articles: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Figure 1

5FU induces senescence of endothelial cells. (A, B) Representative pictures and percentage of EA.hy926 cells stained for the senescence markers SA β‐gal (A) and p16^INK4a (B) after no treatment (CTR) or exposure to 5FU. (C) Percentage of BrdU positive EA.hy926 cells in CTR condition and after incubation with 5FU. (D) Representative images of CTR and 5FU‐treated EA.hy926 cells stained for actin to visualize morphology and cytoplasm size. Nuclei are counterstained with DAPI. (E–G) Expression of VCAM1, ICAM1 and TYMP in CTR or 5FU‐treated cells. n = 7 for the experiments depicted in (A, B) and 5 for the other ones. * Statistically significant versus CTR (unpaired t‐test for A–C, Mann–Whitney test for E–G). Magnification of pictures is 400×, bars correspond to 50 μm.

Figure 2

5FU‐elicited senescence is mediated by p38 and JNK and is associated with a down‐regulation of eNOS and SIRT‐1. (A) Representative pictures of the staining for SA β‐gal after no treatment (CTR) or exposure of EA.hy926 cells to 5FU with or without pre‐incubation with the following signalling inhibitors: SB203580 (inhibitor of p38); SP600125 (inhibitor of JNK); PD98059 (inhibitor of ERK1/2); or LY‐294002 (inhibitor of PI3K). Magnification is 200×, and bars are 50 μm. (B) Percentage of SA β‐gal‐positive EA.hy926 cells in the same conditions as in (A). (C–D) Densitometry analysis and representative western blots for eNOS (C) and SIRT‐1 (D) after the treatments described in (A). eNOS and SIRT‐1 protein levels were expressed as optical density (OD) ratio with actin as internal standard. Data are presented as mean ± SEM of five independent replicates. In all graphs, * indicates statistically significant versus CTR and # statistically significant versus 5FU (ANOVA with post hoc Tukey's multiple comparisons test).

Figure 3

Sera from patients receiving capecitabine cause endothelial cell senescence and contain higher concentrations of sVCAM‐1 and CD146. (A) Representative images (left) and percentage (right) of SA β‐gal positive EA.hy926 cells after incubation with sera obtained from patients with radically resected colorectal cancer either before (Bas) or at the end of six cycles of chemotherapy with capecitabine (S6), in the latter case with or without pretreatment with SB203580 (inhibitor of p38), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK1/2) or LY‐294002 (inhibitor of PI3K). Magnification 200×, bars correspond to 50 μm. (B–D) Concentrations of sVCAM‐1, sICAM‐1 and CD146 in sera of patients used in the experiment depicted in (A); n = 5. * and # indicate statistically significant versus Bas and S6, respectively (ANOVA with post hoc Tukey's multiple comparisons test for A, paired t‐test for B–D).

Figure 4

Glucagon‐like peptide 1 (GLP‐1) protects against 5FU‐initiated senescence. (A) Representative pictures (left) and percentage (right) of SA β‐gal positive EA.hy926 cells after no treatment (CTR) or exposure to 5FU, GLP‐1, E9 (GLP‐1 receptor antagonist) and/or H‐89 (inhibitor of PKA) as indicated. (B) Representative pictures (left) and percentage (right) of EA.hy926 positive for SA β‐gal after CTR or exposure to 5FU, GLP‐1, PD98059 (inhibitor of ERK1/2), LY‐294002 (inhibitor of PI3K) and/or L‐NAME (inhibitor of NOS) as indicated. Magnification of pictures is 200×, and bars are equal to 50 μm. Data are mean ± SEM of five independent experiments; * indicate statistically significant versus CTR, # statistically significant versus 5FU and § statistically significant versus GLP‐1 + FU.

Figure 5

Glucagon‐like peptide 1 (GLP‐1) antagonizes eNOS and SIRT‐1 down‐regulation in response to 5FU. (A, B) Densitometry analysis and representative western blots for eNOS (A) and SIRT‐1 (B) in EA.hy926 cells after no treatment or exposure to 5FU, GLP‐1, E9 (GLP‐1 receptor antagonist) and/or H‐89 (inhibitor of PKA) as indicated. For each condition, optical density (OD) of eNOS and SIRT‐1 bands was normalized to that of actin as internal control. (C, D) Densitometry analysis and representative western blots for eNOS (C) and SIRT‐1 (D) in EA.hy926 cells after no treatment or exposure to 5FU, GLP‐1, SB203580 (inhibitor of p38), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK1/2), LY‐294002 (inhibitor of PI3K) and/or L‐NAME (inhibitor of NOS) as indicated. Data were analysed by ANOVA, and the P for trend values are shown above the graphs. Post hoc comparisons were not drawn because of F > 0.05 and significant variance inhomogeneity.

Figure 6

ERK 1/2 is implicated in 5FU toxicity on endothelial cells. (A) Representative images and percentage of EA.hy926 cells stained for SA β‐gal after the following treatments: no treatment (CTR); exposure to 5FU; pre‐incubation with GLP‐1 followed by exposure to 5FU; transfection with ERK1/2 siRNA (siERK), pre‐incubation with GLP‐1 and then exposure to 5FU; transfection with sham siRNA (sham); transfection with siERK. Magnification of pictures is 200×, and bars correspond to 50 μm. (B, C) Representative western blots for ERK1/2 and eNOS (B) and optical density (OD) of the protein bands normalized to that of actin (C) after the treatments described in (A). Graphs show mean ± SEM of five independent experiments. Comparisons were made by means of ANOVA followed by post hoc Tukey's multiple comparisons test. * Statistically significant versus CTR, # statistically significant versus 5FU and ¤ statistically significant versus siERK + GLP‐1 + FU.

Figure 7

Glucagon‐like peptide 1 (GLP‐1) counteracts senescence triggered by sera from patients taking capecitabine. Representative pictures (upper part) and percentage (lower part) of SA β‐gal positive EA.hy926 cells after no treatment or incubation with sera obtained from patients with radically resected colorectal cancer either before (Bas) or at the end of six cycles of chemotherapy with capecitabine (S6), in the latter case with or without pretreatment with GLP‐1, E9 (GLP‐1 receptor antagonist), H‐89 (inhibitor of PKA), PD98059 (inhibitor of ERK1/2), LY‐294002 (inhibitor of PI3K) and/or L‐NAME (inhibitor of NOS) as indicated. Magnification 200×, bars 50 μm. * Statistically significant versus CTR, # statistically significant versus S6 and § statistically significant versus GLP‐1 + S6.