Chronic exposure of colorectal cancer cells in culture to fluoropyrimidine analogs induces thymidylate synthase and suppresses p53. A molecular explanation for the mechanism of 5-FU resistance

Subbayan Pochi, Malancha Sarkar, Grodonoff Nelson, Edilberto Benitez, Shebani Singhal, Bach Ardalan

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Resistance to chemotherapy is a major issue in treating malignant diseases. 5-Fluorouracil (5-FU) is the drug of choice in managing colorectal cancer (CRC) patients. However, 5-FU resistance leads to eventual treatment failure. Therefore, delaying or reversing the onset of 5-FU resistance will benefit these terminally ill patient populations. A metabolite of 5-FU irreversibly binds thymidylate synthase (TS) thus inhibiting its activity. Many studies demonstrated that these resistant patients had an increased intratumoral TS level. We used TS-siRNA to reduce TS and resensitize HT29FU CRC cells back to this uracil analogue. We exposed the CRC cell line HT29 to an increasing concentration of 5-FU or 5-fluorouridine (FUR) and established a derivative cell line (HT29FU and HT29FUR). Using real-time polymerase chain reaction (PCR) and Western immunodetection assays, we analyzed the expression of TS and p53 mRNA and protein in control and experimental groups. Cytotoxicity to 5-FU was determined by reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT assay) or trypan blue dye exclusion assay. The HT29FU and HT29FUR cells have a distinct morphology: they are generally asteroid shaped. The half maximal inhibitory concentration (IC50) values for the resistant cell line for 5-FU is over 148 μM compared to 5 μM for the sensitive parental cell line. The resistant cell lines expressed more of TS and less of p53. TS-siRNA suppressed TS only. Other pathways were not significantly altered. It also marginally (20%) re-sensitized resistant cells to 5-FU. Restoration of partial sensitivity to 5-FU by TS-siRNA reiterates the primacy of the DNA synthesis pathway in 5-FU mode of action. We speculate that the short half-life of the transiently transfected siRNA may contribute to the marginal restoration of sensitivity. By integrating TS-siRNA expression vector into the genome and regulating its expression, we may be able to reverse 5-FU resistance and make the cells as sensitive as the parental cell line.

Original languageEnglish
Pages (from-to)1149-1156
Number of pages8
JournalAnticancer Research
Volume30
Issue number4
StatePublished - Apr 1 2010

Fingerprint

Thymidylate Synthase
Fluorouracil
Colorectal Neoplasms
Cell Culture Techniques
Small Interfering RNA
Cell Line
Minor Planets
Terminally Ill
Trypan Blue
Uracil
Treatment Failure
Inhibitory Concentration 50
Half-Life
Real-Time Polymerase Chain Reaction
Coloring Agents
Genome

Keywords

  • Chemoresistance
  • HT29
  • mRNA quantification
  • Real-time PCR
  • Translational regulation

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Chronic exposure of colorectal cancer cells in culture to fluoropyrimidine analogs induces thymidylate synthase and suppresses p53. A molecular explanation for the mechanism of 5-FU resistance. / Pochi, Subbayan; Sarkar, Malancha; Nelson, Grodonoff; Benitez, Edilberto; Singhal, Shebani; Ardalan, Bach.

In: Anticancer Research, Vol. 30, No. 4, 01.04.2010, p. 1149-1156.

Research output: Contribution to journalArticle

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abstract = "Resistance to chemotherapy is a major issue in treating malignant diseases. 5-Fluorouracil (5-FU) is the drug of choice in managing colorectal cancer (CRC) patients. However, 5-FU resistance leads to eventual treatment failure. Therefore, delaying or reversing the onset of 5-FU resistance will benefit these terminally ill patient populations. A metabolite of 5-FU irreversibly binds thymidylate synthase (TS) thus inhibiting its activity. Many studies demonstrated that these resistant patients had an increased intratumoral TS level. We used TS-siRNA to reduce TS and resensitize HT29FU CRC cells back to this uracil analogue. We exposed the CRC cell line HT29 to an increasing concentration of 5-FU or 5-fluorouridine (FUR) and established a derivative cell line (HT29FU and HT29FUR). Using real-time polymerase chain reaction (PCR) and Western immunodetection assays, we analyzed the expression of TS and p53 mRNA and protein in control and experimental groups. Cytotoxicity to 5-FU was determined by reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT assay) or trypan blue dye exclusion assay. The HT29FU and HT29FUR cells have a distinct morphology: they are generally asteroid shaped. The half maximal inhibitory concentration (IC50) values for the resistant cell line for 5-FU is over 148 μM compared to 5 μM for the sensitive parental cell line. The resistant cell lines expressed more of TS and less of p53. TS-siRNA suppressed TS only. Other pathways were not significantly altered. It also marginally (20{\%}) re-sensitized resistant cells to 5-FU. Restoration of partial sensitivity to 5-FU by TS-siRNA reiterates the primacy of the DNA synthesis pathway in 5-FU mode of action. We speculate that the short half-life of the transiently transfected siRNA may contribute to the marginal restoration of sensitivity. By integrating TS-siRNA expression vector into the genome and regulating its expression, we may be able to reverse 5-FU resistance and make the cells as sensitive as the parental cell line.",
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