Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates

Abstract

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26‐A, C26‐10, C26‐G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5‐fluorouracil (5‐FU) alone or in combination with leucovorin (LV) (2–5‐fold), to the thymidylate synthase (TS) inhibitors, AG337 (2–7‐fold), ZD1694 (3–49‐fold), ZD9331 (3–40‐fold), LY231514 (2–21‐fold) or GW1843U89 (4–29‐fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2–50‐fold) compared with their HF variants cultured in standard medium containing up to 8 μM folic acid. LV could only increase sensitivity to 5‐FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2–7‐fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7‐fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3‐fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5‐FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5‐FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models. Int. J. Cancer 87:771–778, 2000. © 2000 Wiley‐Liss, Inc.

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer

“…We showed that a Spearman's correlation coefficient for TS/DHFR was 0.456 (Po0.001). Backus et al (2000) reported that low TS expression in vitro correlated with increased sensitivity to 5-FU. Several clinical studies have found that patients with low TS gene expression in primary GC correlate with a better tumour response and longer survival after 5-FU or S-1 treatment (Lenz et al, 1996;Ichikawa et al, 2004).…”

“…This situation has created a greater need for diagnostic techniques that can predict clinical outcomes such as tumour response and survival in GC (Ichikawa, 2006). Considerable evidence suggests that the intratumour gene expressions of drug-metabolising enzymes, DNA repair enzymes, or angiogenic enzymes are useful predictors of treatment outcomes such as survival and the response to anticancer drugs (Backus et al, 2000;Ulrich et al, 2003;Marsh and McLeod, 2004). However, the clinical significance of these biomarkers remains unclear, especially in GC.…”

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications

“…Pemetrexed has an additional target at higher concentrations, and the paradigm has been that this should provide a therapeutic advantage. Yet, the IC 50 for raltitrexed against in vitro tumors is one sixth that of pemetrexed (96), and the maximum tolerated dose for raltitrexed in humans is 2 orders of magnitude lower than that of pemetrexed (97). What can account for this marked difference in activity?…”

Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity

“…The cells were incubated for 24 hours to allow sufficient cell adhesion, and all the microplates were incubated for a total of 72 hours after MTX administration. The cytotoxic activity was measured by the sulforhodamine B (SRB) assay according to the manufacture's instructions (12). The SRB test was initiated by fixing the cells via addition of 200 μ L of 10% trichloracetic acid (Sigma, St. Louis, MO) and the plate was incubated for 1 hour at 4℃.…”

“…The cells were incubated for 5 minutes at 37℃ and then they were washed with ice-cold PBS (pH 7.4) and next fixed with 5% TCA solution. After washing with ice-cold PBS (pH7.4), the final cell pellet was solubilized; the radioactivity was estimated by counting via liquid scintillation (11,12).…”