Decreased expression of hMLH1 correlates with reduced 5-fluorouracil- mediated apoptosis in colon cancer cells

Hideyuki Fujita, Jun Kato, Joichiro Horii, Keita Harada, Sakiko Hiraoka, Hidenori Shiraha, Kohsaku Sakaguchi, Yasushi Shiratori

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Patients with sporadic microsatellite instable colorectal cancers, in most of which the function of the hMLH1 mismatch repair gene is impaired, do not gain a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. However, the effect of hMLH1 on the cytotoxicity induced by 5-FU has not yet been sufficiently confirmed. In this study, we assessed the effect of hMLH1 on cytotoxicity and apoptosis induced by 5-FU using newly developed cell lines. We constructed two cell lines: SW480 (originally hMLH1-proficient), in which the expression of hMLH1 was reduced using a small interfering RNA (siRNA) technique, and HCT116 (originally hMLH1-deficient), in which the expression of hMLH1 can be regulated by doxycycline. Using these cell lines, a clonogenic survival assay, 4′,6-diamidino-2-phenylindole (DAPI) staining and an Annexin-V assay were performed. Moreover, the incorporation of 5-FU into DNA was determined using tritium-labeled 5-FU. In both of our two cell lines, hMLH1-deficient cells exhibited approximately 2.4-fold clonal surviving fraction compared to hMLH1-proficient cells for 10 days after the administration of 5-FU. Additionally, hMLH1-deficient cells treated with 5-FU exhibited 34-45% less apoptosis than hMLH1-proficient cells according to the results of DAPI staining and Annexin-V assay. Furthermore, hMLH1-deficient cells treated with 5-FU exhibited an approximately 2-fold greater incorporation of 5-FU into DNA than control cells, suggesting that the recognition of 5-FU-incorporated DNA is impaired in hMLH1-deficient cells, resulting in reduced apoptosis. Our conclusions were that decreased expression of hMLH1 in colon cancer cells reduced the apoptosis induced by 5-FU, suggesting that hMLH1 is a key determinant of 5-FU chemosensitivity.

Original languageEnglish
Pages (from-to)1129-1137
Number of pages9
JournalOncology reports
Volume18
Issue number5
Publication statusPublished - Nov 2007

Keywords

  • 5-fluorouracil
  • Apoptosis
  • Chemosensitivity
  • Colorectal cancer
  • hMLH1

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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