Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

Pedro M. Borralho, Isabel B. Moreira da Silva, Márcia M. Aranha, Cristina Albuquerque, Carlos Nobre Leitão, Clifford J. Steer, Cecília M.P. Rodrigues

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38 Scopus citations

Abstract

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 μM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p < 0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p < 0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p < 0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p < 0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.

Original languageEnglish (US)
Pages (from-to)40-47
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1772
Issue number1
DOIs
StatePublished - Jan 2007

Bibliographical note

Funding Information:
The authors thank all the members of the laboratory for skillful technical assistance. This work was supported by grant 68796/2004 from Fundação Calouste Gulbenkian (to C.M.P.R.). P.M.B. was recipient of a Ph.D. fellowship (BD/24165/2005) from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.

Keywords

  • 5-fluorouracil
  • Apoptosis
  • Fas
  • Mitochondria
  • p53
  • siRNA

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