Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression
Introduction
The enzyme thymidylate synthase (TS) catalyses the methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) to 2′-deoxythymidine-5′-monophosphate (dTMP), an essential precursor during DNA synthesis [1]. 5,10-Methylene tetrahydrofolate (CH2-THF) is the limiting methyl donor during this reaction. TS is usually elevated in tumours [2] and is therefore an interesting target for anticancer agents such as the antifolate Pemetrexed (multitargeted antifolate, MTA, ALIMTA, LY231514) (Fig. 1) [3], which inhibits activity of TS by competition with the binding site of CH2-THF of TS. Other targets of Pemetrexed include glycinamide ribonucleotide formyltransferase (GARFT) which is a component of the de novo purine synthesis pathway, and dihydrofolate reductase (DHFR) which is responsible for recycling of tetrahydrofolates. Pemetrexed is currently being evaluated as an anticancer agent for treatment of colorectal, non-small-cell lung cancer and mesothelioma [4], [5]. Resistance to this agent might develop similarly to other antifolates, and might be due to elevated activity or mutations of the target enzyme TS, impaired polyglutamation of antifolates, decreased transport into the cells [3], [4], but also apoptosis regulating proteins may be involved [6], [7]. Pemetrexed and other clinically active antifolates such as ZD9331 [8], ZD1694 (Tomudex, Raltitrexed) (Fig. 1) [9], GW1843U89 [10] and methotrexate (MTX) [11] require transport into the cell by the reduced folate carrier (RFC). The antifolate AG337 (Thymitaq; Fig. 1) [12], [13] enters the cell by passive diffusion, circumventing a possible defect in the function of RFC in cancer cells. Mutated forms of RFC have been detected in cancer, leading to resistance to antifolates [14]. When the antifolates ZD1694, GW1843U89, MTX and Pemetrexed enter the cell, they are polyglutamated by the enzyme folylpolyglutamate synthetase (FPGS) for which most are excellent substrates. This enzyme catalyses the addition of glutamic acid residues to the glutamate residue of antifolates [15]. Polyglutamated antifolates are retained longer in cells, which may enhance antitumour activity. The enzyme folylpolyglutamate hydrolase (FPGH) is responsible for metabolic breakdown of polyglutamates of antifolates leading to decreased retention in cells [16]. The dynamic equilibrium between FPGS and FPGH will determine the polyglutamated form of antifolates [17]. The antifolates AG337 and ZD9331 can not be polyglutamated and may thus be exploited to circumvent resistance to antifolates due to impaired polyglutamation [8], [12], [18].
Exposure of cancer cells in vitro to antifolates has been demonstrated to induce DNA damage, cell cycle arrest and programmed cell death [19]. Furthermore, it is known that antifolates affect the metabolism of methionine. Tetrahydrofolate serves as a methyl donor for synthesis of purines and thymidine as well as in the remethylation cycle of homocysteine to methionine. It has been shown in vivo that MTX treatment reduces intracellular pools of 5-methyltetrahydrofolate leading to a reduced conversion of homocysteine to methionine by methionine synthetase [20]. For Pemetrexed, a strong correlation was observed between plasma levels of homocysteine and the subsequent development of serious drug-related toxicities [21]. Decrease of homocysteine levels via nutritional supplementation (folic acid) led to a better safety profile of Pemetrexed.
In several studies on leukemic and solid tumour cell lines, resistance to antifolates including Pemetrexed was frequently associated with either RFC or FPGS but less frequently with TS [22], [23], [24], [25]. In H630, HCT-8 and CEM cells, resistance to ZD1694 was associated with a decrease in FPGS activity [22], [23], [25]. Defective polyglutamation was also shown in Pemetrexed-resistant L1210 and CEM cells [22], [24]. Impaired RFC transport as a mechanism of resistance was observed in ZD1694-resistant H630 cells, Pemetrexed-resistant L1210 cells and CEM cells resistant to GW1843U89 or ZD9331 [22], [24], [25]. Increased expression of TS was only shown in MCF-7 cells resistant to ZD1694 due to a 20-fold gene amplification [25]. Since Pemetrexed is also developed for treatment of colorectal cancer, we characterised the mechanisms underlying resistance in human colon cancer WiDr cells. For this purpose, we induced resistance to the antifolate Pemetrexed in the colon cancer cell line WiDr either by a short exposure to Pemetrexed concentrations every week, or by a continuous exposure to lower concentrations. The resistant WiDr variants were studied on the level of TS, FPGS and RFC.
Section snippets
Drugs and biochemicals
Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Flow Laboratories (Irvine, Scotland). Both (nondialysed) fetal calf serum (FCS) and Hank’s balanced salt solution (HBSS) were purchased from Gibco Europe. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) was obtained from Biowhitaker. 5-FU was provided by Sigma Chemical Co. ZD1694 was obtained from Zeneca Pharmaceuticals (now Astra Zeneca). MTX was from Pharmachemie. Pemetrexed (LY231514, ALIMTA, MTA) was provided by
Growth inhibition studies
Resistance to Pemetrexed was induced by gradually increasing Pemetrexed concentrations from 2.0 μM for the 4-hr exposure and from 200 nM for the continuous exposure over a period of 19 months resulting in WiDr-4PEM and WiDr-cPEM, respectively. For the WiDr-4PEM cell line, increasing concentrations using steps of 2 μM required 15 months to induce resistance to 10 μM. Thereafter, steps of 20 μM were tolerated to result in resistance to 50 μM which only required 4 months. For WiDr-cPEM, increasing
Discussion
The results in this study indicate that induction of resistance to Pemetrexed in colon carcinoma WiDr cells led to upregulation of TS. All TS related parameters were increased: mRNA and protein expression and TS activity. However, resistance was not related to alterations in the activity of the FPGS or RFC enzymes.
Consistent with the elevated TS activity, Pemetrexed-resistant WiDr variants displayed cross-resistance to other antifolate TS inhibitors such as ZD1694 and AG337. Schultz et al.[33]
Acknowledgements
This study was supported by a grant from the Dutch Cancer Society (VU 96-1240).
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Present Address: Crucell, Leiden, The Netherlands.