Folate-restricted Conditions against Mono- and MultilayeredColon Cancer Cell Lines under Synthase Inhibitors Tomudex (ZD1694) and GW1843U89 Determinants of Activity of the Antifolate Thymidylate

The cytotoxicity and metabolic effects of two thymidylate synthase (TS) inhibitors, Tomudex (Raltitrexed, ZD1694) and GW1843U89, were studied in WiDr colon cancer cells under four different growth conditions: as standard monolayers and as postconfluent multilayers grown under either high (WiDr, 8.8 mM folic acid) or low (WiDr/F, 1 nM leucovorin) folate conditions. Both GW1843U89 and ZD1694 were 13–15-fold more active against WiDr/F than WiDr cells when cultured as monolayers (IC50s in WiDr/F cells were 0.22 and 0.39 nM, respectively). WiDr cells were markedly less sensitive to the drugs when grown as multilayers (4–15-fold), in contrast to the WiDr/F cells, which were equally sensitive. However, total growth inhibition could not be achieved in WiDr multilayers (concentration causing total growth inhibition > 10,000 nM), whereas in WiDr/F multilayers, it could be achieved at 0.42 nM ZD1694 and 150 nM GW1843U89. Growth conditions markedly affected the TS levels when using different enzyme assays. At nonsaturating substrate concentrations, the catalytic activity of TS was similar in monoand multilayers grown under high folate conditions but lower in multilayers at saturating concentrations. In cells grown under low folate conditions, TS catalytic activity was 3–6-fold lower in multilayers than in monolayers. This was consistent with a decrease in the number of S-phase cells in multilayers. Western blotting revealed less pronounced (2–3-fold) differences in the TS protein content. Exposure of the cells for 24 h to the drugs increased the TS levels by 4-fold. Because this increase in TS levels might explain the decrease in sensitivity to the TS inhibitors, we measured TS inhibition (TSI) by the drugs in intact cells using the TS in situ assay. GW1843U89 was more active than ZD1694. However, after 4 h of exposure in WiDr/F monoand multilayers, TSI was in the same range for both drugs [50% TSI (TSI50), 0.5–1.7 nM]. In WiDr cells, the TSI50 for ZD1694, but not GW1843U89, was 10 times higher in the multilayers as compared to the monolayers. Despite the increase in TS protein levels, the extent of TSI was similar or even more pronounced in both cell lines grown as either multior monolayers. Because the cells were grown under depleted and folate-rich conditions that may affect folate uptake, we measured folate transport using methotrexate (MTX) as the reference drug for the activity of the reduced folate carrier. MTX uptake was 4-fold lower in multilayers compared to monolayers in both WiDr and WiDr/F cells. Uptake of MTX was 5-fold more effective in WiDr/F cells than in WiDr cells in both monoand multilayers. In conclusion, the resistance of WiDr multilayers to the novel antifolates ZD1694 and GW1843U89 may be due to the high folate medium concentrations, which may be responsible for impaired drug uptake along with less effective TSI. In contrast, WiDr/F monolayers and multilayers were very sensitive to these antifolates. These effects of folate homeostasis may explain some of the variable results seen in treatment of solid tumors with new antifolate TS inhibitors. INTRODUCTION Three-dimensionally cultured tumor cells offer better models for the study of human solid malignancies than monolayers (1, 2). Models such as multicellular spheroids (3), collagen gels (4), histocultures (5, 6), and cells grown as postconfluent multilayers in V-bottomed microtiter plates (7–10) show a much greater mimicry of the complexity of human neoplastic cells than monolayered cell cultures. These factors include drug penetration barriers, cell proliferation gradients, and microenvironmental conditions such as hypoxia and acidic pH. Our in vitro model in V-bottomed microtiter plates enabled us to perform large-scale drug screening with automatic data handling (7–10). In this system, all tested cell lines reached confluence after 5 days of growth, each with a specific pattern of cell stacking from 2–10 layers. Growth and cytotoxicity, as evaluated with cell counting and the SRB and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, demonstrated for most drugs that multilayered plateau phase cultures rather than monolayers showed a resistant phenotype (7, 8, 10, 11) that is more representative to the in vivo situation in animals and patients. Inhibition of TS has been associated with the antitumor activity of 5FU (12). 5FU acts through its metabolite, FdUMP, which inhibits TS by the formation of a ternary complex of TS, FdUMP, and CH2THF (13, 14). TS expression has been reported to be cell cycle dependent (15, 16). TS protein and TS activity levels are higher in proliferating cells than in nonproliferating cells and vary 14–24-fold between exponential and confluent cell populations (17). Several antifolates are potent TS inhibitors and have been evaluated in the clinic (18, 19). In general, these folate analogues act by forming a tightly bound nonactive complex with TS and the natural substrate dUMP (19, 20). ZD1694 (Tomudex, Raltitrexed) and GW1843U89 are soluble, very potent, third-generation TS inhibitors (Ki 5 60 or 0.09 nM, respectively; Refs. 20–24). Cytotoxic activity is dependent on active cellular uptake via the RFC and subsequent metabolism to polyglutamylated forms by FPGS (25). The predominant polyglutamate for ZD1694 is ZD1694-Glu4, which is '60-fold more potent as a TS inhibitor than the parent drug (21–22). Intracellular metabolism of GW1843U89 usually proceeds only to the diglutamate derivative (GW1843U89Glu2; Ref. 24). In addition, polyglutamated derivatives are not readily effluxed from the cells; for GW1843U89, polyglutamylation resulted in a better retention, rather than a higher TSI. Both compounds have shown activity against xenografts of human ovarian and colonic origin (19, 26); Tomudex showed a response rate comparable to that of 5FU 1 LV in Phase III clinical studies (colon cancer), but with a different toxicity profile (27, 28). For GW1843U89, only a Phase I study has been performed (29). GW1843U89 has been tested against multicellular spheroids of the colon cancer cell line WiDr (30), which can be destroyed after Received 4/26/99; accepted 9/3/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the Dutch Platform for Alternatives for Animal Experiments (PAD 92-47) and the Dutch Cancer Society, Grant VU-96-1260. 2 To whom requests for reprints should be addressed, at Department of Oncology, University Hospital Vrije Universiteit, P. O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-444-2633; Fax: 31-20-444-3844; E-mail: [email protected]. 3 The abbreviations used are: SRB, sulforodamine B; LV, leucovorin; TS, thymidylate synthase; 5FU, 5-fluorouracil; RFC, reduced folate carrier; FPGS, folyl polyglutamate synthetase; MTX, methotrexate; BrdUrd, bromodeoxyuridine; LC50, concentration causing 50% cell kill; TGI, concentration causing total growth inhibition; TSI, thymidylate synthase inhibition; TSI50, concentration causing 50% TSI; FdUMP, 5-fluoro-dUMP; CH2THF, 5, 10-methylene-tetrahydrofolate; dUrd, 29-deoxyuridine.