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.
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 (IC(50)) values for the resistant cell line for 5-FU is over 148 microM compared to 5 microM 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.
Department of Medicine (D8-4), University of Miami Miller School of Medicine, Miami, FL 33136, USA. spochi@med.miami.edu
Pochi R
PR
Subbarayan
Malancha
M
Sarkar
Grodonoff
G
Nelson
Edilberto
E
Benitez
Shebani
S
Singhal
Bach
B
Ardalan
D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004790:Enzyme Induction / Q000187:drug effects; D005472:Fluorouracil / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D011743:Pyrimidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000378:metabolism
D011743:Pyrimidines; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; D005472:Fluorouracil