<i>In vitro</i> activity of novel antifolates against human squamous carcinoma cell lines of the head and neck with inherent resistance to methotrexate

Laan, Bernard F. A. M. van der; Jansen, Gerrit; Kathmann, G. A. M.; Westerhof, G R; Schornagel, J. H.; Hordijk, G. J.

doi:10.1002/ijc.2910510613

Cited by 21 publications

(10 citation statements)

References 16 publications

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“… FPGS activity in HF and LF colon cancer and HNSCC cell lines. Extracts of colon cancer and HNSCC cell lines (Van der Laan et al ., 1992) were used for the determination of the FPGS activity with 250 μM MTX as a substrate. The FPGS activity was expressed as pmol 3 H‐glutamate incorporated/hr/mg protein.…”

Section: Resultsmentioning

confidence: 99%

“…The HNSCC cell lines were cultured in “standard folate” RPMI 1640 medium (containing 2.2 μM folic acid) and supplemented with 10% FCS. Each of the 7 cell lines described above were adapted in a period of 5–6 months to grow at low folate conditions (WiDr/F, C26‐10/F, C26‐A/F, C26‐G/F and 11B/F, 14C/F and 22B/F) (Van der Wilt et al ., 1995; Van der Laan et al ., 1992) and cultured in folate‐free RPMI 1640 supplemented with 10% dialyzed FCS, 2 mM glutamine and 2.5 nM LV (for the murine colon cancer cells), 1.0 nM LV (for WiDr/F) and 0.5 nM LV (for the HNSCC cells). The cell lines cultured in “standard folate” medium will be further referred to as HF (high folate) cells, whereas cells grown under low folate conditions will be referred to as LF (low folate) cells.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2000

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2000

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“…23,24), and WiDr (colon; ref. 25) were either grown in standard RPMI 1640 (Cambrex BioScience) containing 2.3 Amol/L supraphysiologic folic acid (HF) supplemented with 10% FCS (Greiner Bio-One) and 20 mmol/L HEPES (Cambrex BioScience) or adapted to more physiologic concentrations of folates (1-5 nmol/L folic acid or leucovorin; LF) by gradually decreasing the folate concentrations in the medium over a period of up to 4 months, depending on the cell line, followed by monitoring these cells under these conditions for at least 1 year.…”

Section: Chemicalsmentioning

confidence: 99%

Cellular folate status modulates the expression of BCRP and MRP multidrug transporters in cancer cell lines from different origins

Lemos

Kathmann²,

Giovannetti³

et al. 2009

Molecular Cancer Therapeutics

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As cellular folate levels seem to have a different effect on cancer cells from different origins, we extended our initial study to a broader panel of cancer cells. BCRP and MRP1-5 expression was determined in KB, OVCAR-3, IGROV-1, ZR75-1/R/MTX, SCC-11B, SCC-22B, and WiDr either grown in standard RPMI 1640 containing 2.3 Mmol/L supraphysiologic concentration of folic acid [high folate (HF)] or adapted to more physiologic concentrations [1-5 nmol/L folic acid or leucovorin; low folate (LF)]. Compared with the HF counterparts, KB LF cells displayed 16.1-fold increased MRP3 and OVCAR-3 LF cells showed 4.8-fold increased MRP4 mRNA levels along with increased MRP3 and MRP4 protein expression, respectively. A marked increase on BCRP protein and mRNA expression was observed in WiDr LF cells. These cells acquired f2-fold resistance to mitoxantrone compared with the HF cell line, a phenotype that could be reverted by the BCRP inhibitor Ko143. Of note, WiDr cells expressed BCRP in the intracellular compartment, similarly to what we have described for Caco-2 cells. Our results provide further evidence for an important role of cellular folate status in the modulation of the expression of multidrug resistance transporters in cancer cells. We show that up-regulation of intracellularly localized BCRP in response to adaptation to LF conditions may be a common feature within a panel of colon cancer cell lines. Under these circumstances, folate supplementation might improve the efficacy of chemotherapeutic drugs by decreasing BCRP expression. [Mol Cancer Ther 2009;8(3):655 -64]

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Section: Discussionmentioning

confidence: 99%

“…Recent reports have shown that a number of human cell lines derived from solid tumors display increased sensitivity to antifolates and 5‐FU when grown in low folate medium (43,44). This is associated with reduced TS protein and activity levels as well as FdUMP binding sites (44). These cells also increase their RFC activity from two‐ to seven‐fold, but do not alter their folylpolyglutamate synthetase (FPGS) levels even though in some cases they do show an accumulation of MTX triglutamates.…”

Section: Discussionmentioning

confidence: 99%

Severe folate restriction results in depletion of and alteration in the composition of the intracellular folate pool, moderate sensitization to methotrexate and trimetrexate, upregulation of endogenous DHFR activity, and overexpression of metallothionein II and folate receptor alpha that, upon folate repletion, confer drug resistance to CHL cells

et al. 2002

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DC-3F/FA3 cells (FA3) were derived from antifolate-sensitive CHL cells by selection for growth in folate-free media containing 15 pM [6S]-5CHOFH4. These cells undergo a 30-fold decrease in intracellular folates, overexpress folate receptor alpha (FR alpha) and metallothionein II, and display increased sensitivity to the dihydrofolate reductase (DHFR) targeted anti-folates methotrexate (MTX) and trimetrexate (TMTX), which can be attributed primarily to the folate pool status. Upon folate repletion by growth in 15 nM [6S]-5CHOFH4, they display a 5- and 10-fold increase in resistance to both drugs, respectively, even though folate pools are restored by only 43%. Enforced overexpression of FR alpha in transfectants cultured in nanomolar folate did not confer resistance to MTX but did support a modest 2-fold increase in resistance to TMTX. Enforced overexpression of MTII had a similar effect, but when both were overexpressed together no increase in resistance beyond that conferred by each one separately was noted, suggesting that both confer resistance to TMTX through a common downstream mechanism. Analysis of three independent low folate selected clones, FA3, FA7, and FA14, showed that each had a 5- to 6-fold increase in DHFR activity accompanied by a similar increase in DHFR protein level. However, no differences were detected in the DHFR gene copy number or in the steady-state amount of DHFR mRNA, suggesting that a posttranscriptional mechanism was responsible for the increase in DHFR expression.

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In vitro activity of novel antifolates against human squamous carcinoma cell lines of the head and neck with inherent resistance to methotrexate

Cited by 21 publications

References 16 publications

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

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

Cellular folate status modulates the expression of BCRP and MRP multidrug transporters in cancer cell lines from different origins

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