Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines

H. H J Backus, D. Wouters, C. G. Ferreira, V. M M Van Houten, R. H. Brakenhoff, H. M. Pinedo, G. J. Peters

Research output: Contribution to journalArticle

Abstract

Thymidylate synthase (TS) is an important target for chemotherapy and increased levels are associated with resistance to colorectal cancer chemotherapy. TS can be inhibited by 5-fluorouracil (5-FU) and antifolates, ultimately resulting in apoptosis. We aimed to clarify whether activation of caspases and Fas signalling are crucial for the onset of apoptosis after specific inhibition of TS and whether p53 plays a role in activation of these downstream processes. For this purpose, wild-type (wt) and mutant (mt) p53 colon cancer cell lines, Lovo and WiDr, respectively, transfected with mt- and wt-p53, were treated with the specific TS inhibitor, AG337. Treatment with 10×IC50 values of AG337 for 48 h resulted in S phase arrest in all Lovo and WiDr cells (up to 50% of cells being in S phase), irrespective of their p53 status. After 72 h, the induction of apoptosis was most pronounced in the AG337-sensitive cells. Approximately 30% apoptosis was detected in all of the WiDr cells, 20% in Lovo li (non-functional p53), 12-14% in Lovo 92 and B2 (wt p53) and only 7% in Lovo 175×2 cells (mt p53 transfected). The induction of apoptosis in Lovo cells, as determined using the classical sub-G1 peak after propidium iodide (PI) staining, was associated with an increase in the expression of Fas receptor. In addition, synergistic increases in apoptosis from approximately 10 to 35% after 48 h could be detected after simultaneous treatment of AG337 and the Fas activator antibody, CH11. Only additive effects were measurable in WiDr cells, without an increase in Fas receptor expression. Surprisingly, the Fas inhibitor, ZB4, could not decrease the amount of cell death in both cell lines after AG337 treatment. In contrast, simultaneous exposure of Lovo and WiDr cells to AG337 and inhibitors of caspases 8, 9 and 3 caused a decrease in the number of apoptotic cells compared with AG337 exposure alone. Inhibition of apoptosis by approximately 10-80% in Lovo and approximately 70-80% in WiDr cells could be detected. In conclusion, these results indicate that apoptosis induced after specific inhibition of TS is mediated via the caspases, but without clear involvement of Fas signalling. The status of p53 did not affect the onset of apoptosis by these caspases.

Original languageEnglish (US)
Pages (from-to)1310-1317
Number of pages8
JournalEuropean Journal of Cancer
Volume39
Issue number9
DOIs
StatePublished - Jun 2003
Externally publishedYes

Fingerprint

Thymidylate Synthase
Caspase 9
Caspase 8
Colonic Neoplasms
Apoptosis
Cell Line
Caspases
CD95 Antigens
S Phase
Folic Acid Antagonists
Drug Therapy
Propidium
nolatrexed
Fluorouracil
Caspase 3
Colorectal Neoplasms
Cell Death
Cell Count
Staining and Labeling
Antibodies

Keywords

  • Caspases and AG337
  • Fas
  • p53
  • Thymidylate synthase

ASJC Scopus subject areas

  • Cancer Research
  • Hematology
  • Oncology

Cite this

Backus, H. H. J., Wouters, D., Ferreira, C. G., Van Houten, V. M. M., Brakenhoff, R. H., Pinedo, H. M., & Peters, G. J. (2003). Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines. European Journal of Cancer, 39(9), 1310-1317. https://doi.org/10.1016/S0959-8049(03)00204-1

Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines. / Backus, H. H J; Wouters, D.; Ferreira, C. G.; Van Houten, V. M M; Brakenhoff, R. H.; Pinedo, H. M.; Peters, G. J.

In: European Journal of Cancer, Vol. 39, No. 9, 06.2003, p. 1310-1317.

Research output: Contribution to journalArticle

Backus, HHJ, Wouters, D, Ferreira, CG, Van Houten, VMM, Brakenhoff, RH, Pinedo, HM & Peters, GJ 2003, 'Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines', European Journal of Cancer, vol. 39, no. 9, pp. 1310-1317. https://doi.org/10.1016/S0959-8049(03)00204-1
Backus, H. H J ; Wouters, D. ; Ferreira, C. G. ; Van Houten, V. M M ; Brakenhoff, R. H. ; Pinedo, H. M. ; Peters, G. J. / Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines. In: European Journal of Cancer. 2003 ; Vol. 39, No. 9. pp. 1310-1317.
@article{b9b2dd20548147d59b3258896b0790a2,
title = "Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines",
abstract = "Thymidylate synthase (TS) is an important target for chemotherapy and increased levels are associated with resistance to colorectal cancer chemotherapy. TS can be inhibited by 5-fluorouracil (5-FU) and antifolates, ultimately resulting in apoptosis. We aimed to clarify whether activation of caspases and Fas signalling are crucial for the onset of apoptosis after specific inhibition of TS and whether p53 plays a role in activation of these downstream processes. For this purpose, wild-type (wt) and mutant (mt) p53 colon cancer cell lines, Lovo and WiDr, respectively, transfected with mt- and wt-p53, were treated with the specific TS inhibitor, AG337. Treatment with 10×IC50 values of AG337 for 48 h resulted in S phase arrest in all Lovo and WiDr cells (up to 50{\%} of cells being in S phase), irrespective of their p53 status. After 72 h, the induction of apoptosis was most pronounced in the AG337-sensitive cells. Approximately 30{\%} apoptosis was detected in all of the WiDr cells, 20{\%} in Lovo li (non-functional p53), 12-14{\%} in Lovo 92 and B2 (wt p53) and only 7{\%} in Lovo 175×2 cells (mt p53 transfected). The induction of apoptosis in Lovo cells, as determined using the classical sub-G1 peak after propidium iodide (PI) staining, was associated with an increase in the expression of Fas receptor. In addition, synergistic increases in apoptosis from approximately 10 to 35{\%} after 48 h could be detected after simultaneous treatment of AG337 and the Fas activator antibody, CH11. Only additive effects were measurable in WiDr cells, without an increase in Fas receptor expression. Surprisingly, the Fas inhibitor, ZB4, could not decrease the amount of cell death in both cell lines after AG337 treatment. In contrast, simultaneous exposure of Lovo and WiDr cells to AG337 and inhibitors of caspases 8, 9 and 3 caused a decrease in the number of apoptotic cells compared with AG337 exposure alone. Inhibition of apoptosis by approximately 10-80{\%} in Lovo and approximately 70-80{\%} in WiDr cells could be detected. In conclusion, these results indicate that apoptosis induced after specific inhibition of TS is mediated via the caspases, but without clear involvement of Fas signalling. The status of p53 did not affect the onset of apoptosis by these caspases.",
keywords = "Caspases and AG337, Fas, p53, Thymidylate synthase",
author = "Backus, {H. H J} and D. Wouters and Ferreira, {C. G.} and {Van Houten}, {V. M M} and Brakenhoff, {R. H.} and Pinedo, {H. M.} and Peters, {G. J.}",
year = "2003",
month = "6",
doi = "10.1016/S0959-8049(03)00204-1",
language = "English (US)",
volume = "39",
pages = "1310--1317",
journal = "European Journal of Cancer",
issn = "0959-8049",
publisher = "Elsevier Limited",
number = "9",

}

TY - JOUR

T1 - Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines

AU - Backus, H. H J

AU - Wouters, D.

AU - Ferreira, C. G.

AU - Van Houten, V. M M

AU - Brakenhoff, R. H.

AU - Pinedo, H. M.

AU - Peters, G. J.

PY - 2003/6

Y1 - 2003/6

N2 - Thymidylate synthase (TS) is an important target for chemotherapy and increased levels are associated with resistance to colorectal cancer chemotherapy. TS can be inhibited by 5-fluorouracil (5-FU) and antifolates, ultimately resulting in apoptosis. We aimed to clarify whether activation of caspases and Fas signalling are crucial for the onset of apoptosis after specific inhibition of TS and whether p53 plays a role in activation of these downstream processes. For this purpose, wild-type (wt) and mutant (mt) p53 colon cancer cell lines, Lovo and WiDr, respectively, transfected with mt- and wt-p53, were treated with the specific TS inhibitor, AG337. Treatment with 10×IC50 values of AG337 for 48 h resulted in S phase arrest in all Lovo and WiDr cells (up to 50% of cells being in S phase), irrespective of their p53 status. After 72 h, the induction of apoptosis was most pronounced in the AG337-sensitive cells. Approximately 30% apoptosis was detected in all of the WiDr cells, 20% in Lovo li (non-functional p53), 12-14% in Lovo 92 and B2 (wt p53) and only 7% in Lovo 175×2 cells (mt p53 transfected). The induction of apoptosis in Lovo cells, as determined using the classical sub-G1 peak after propidium iodide (PI) staining, was associated with an increase in the expression of Fas receptor. In addition, synergistic increases in apoptosis from approximately 10 to 35% after 48 h could be detected after simultaneous treatment of AG337 and the Fas activator antibody, CH11. Only additive effects were measurable in WiDr cells, without an increase in Fas receptor expression. Surprisingly, the Fas inhibitor, ZB4, could not decrease the amount of cell death in both cell lines after AG337 treatment. In contrast, simultaneous exposure of Lovo and WiDr cells to AG337 and inhibitors of caspases 8, 9 and 3 caused a decrease in the number of apoptotic cells compared with AG337 exposure alone. Inhibition of apoptosis by approximately 10-80% in Lovo and approximately 70-80% in WiDr cells could be detected. In conclusion, these results indicate that apoptosis induced after specific inhibition of TS is mediated via the caspases, but without clear involvement of Fas signalling. The status of p53 did not affect the onset of apoptosis by these caspases.

AB - Thymidylate synthase (TS) is an important target for chemotherapy and increased levels are associated with resistance to colorectal cancer chemotherapy. TS can be inhibited by 5-fluorouracil (5-FU) and antifolates, ultimately resulting in apoptosis. We aimed to clarify whether activation of caspases and Fas signalling are crucial for the onset of apoptosis after specific inhibition of TS and whether p53 plays a role in activation of these downstream processes. For this purpose, wild-type (wt) and mutant (mt) p53 colon cancer cell lines, Lovo and WiDr, respectively, transfected with mt- and wt-p53, were treated with the specific TS inhibitor, AG337. Treatment with 10×IC50 values of AG337 for 48 h resulted in S phase arrest in all Lovo and WiDr cells (up to 50% of cells being in S phase), irrespective of their p53 status. After 72 h, the induction of apoptosis was most pronounced in the AG337-sensitive cells. Approximately 30% apoptosis was detected in all of the WiDr cells, 20% in Lovo li (non-functional p53), 12-14% in Lovo 92 and B2 (wt p53) and only 7% in Lovo 175×2 cells (mt p53 transfected). The induction of apoptosis in Lovo cells, as determined using the classical sub-G1 peak after propidium iodide (PI) staining, was associated with an increase in the expression of Fas receptor. In addition, synergistic increases in apoptosis from approximately 10 to 35% after 48 h could be detected after simultaneous treatment of AG337 and the Fas activator antibody, CH11. Only additive effects were measurable in WiDr cells, without an increase in Fas receptor expression. Surprisingly, the Fas inhibitor, ZB4, could not decrease the amount of cell death in both cell lines after AG337 treatment. In contrast, simultaneous exposure of Lovo and WiDr cells to AG337 and inhibitors of caspases 8, 9 and 3 caused a decrease in the number of apoptotic cells compared with AG337 exposure alone. Inhibition of apoptosis by approximately 10-80% in Lovo and approximately 70-80% in WiDr cells could be detected. In conclusion, these results indicate that apoptosis induced after specific inhibition of TS is mediated via the caspases, but without clear involvement of Fas signalling. The status of p53 did not affect the onset of apoptosis by these caspases.

KW - Caspases and AG337

KW - Fas

KW - p53

KW - Thymidylate synthase

UR - http://www.scopus.com/inward/record.url?scp=0037565292&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037565292&partnerID=8YFLogxK

U2 - 10.1016/S0959-8049(03)00204-1

DO - 10.1016/S0959-8049(03)00204-1

M3 - Article

C2 - 12763222

AN - SCOPUS:0037565292

VL - 39

SP - 1310

EP - 1317

JO - European Journal of Cancer

JF - European Journal of Cancer

SN - 0959-8049

IS - 9

ER -