Paradoxical response of malignant melanoma to methotrexate in vivo and in vitro

Abstract

Summary Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations 10 -5M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H-MTX (10-7-10 -6M) was two orders of magnitude less cytotoxic for melanoma than MTX (0-9-10-8 M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX.The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.Reports of clinical results in patients with advanced malignant melanoma treated with high dose methotrexate (MTX) therapy suggest a poor rate of response (Fisher et al., 1979;Karakousis & Carlson, 1979). These reports do not, however, give any details of plasma concentrations of MTX achieved or the period of time over which a high concentration was maintained. An apparent paradox to this picture of lack of response to MTX in vivo is the observation that murine melanoma cells in vitro are rapidly killed by relatively low (10-8 M to 10-9M) concentrations of the agent (Bostock et al., 1979).Possible explanations for these paradoxical findings in vivo and in vitro may include the differences between the MTX concentrations to which cells are exposed and the possibility that folinic acid rescue used routinely in vivo may salvage tumour cells as well as essential marrow and mucosal elements. Differences in MTX transport, and metabolism between melanoma cells in vivo and in vitro may also operate and the activity of intracellular dihydrofolate reductase, and the proportion of cells undertaking DNA synthesis at the time of exposure to methotrexate may also be important variables. The observation that dihydrofolate reductase is identical in certain MTX-resistant and -sensitive wild-type lines of PGl9 murine melanoma (Bostock et al., 1979) indicates that qualitative enzyme differences are not responsible for the acquired resistance in some melanoma mutants.We have investigated these variables by a study in which three patients with advanced malignant melanoma resistant to other cytotoxic drugs were treated with high dose MTX. The plasma concentrations of MTX and MTX metabolites were measured throughout a 24 h period. The relationship of these in vivo metabolites to those found in the culture medium surrounding human and murine m...

Protection of cells from methotrexate toxicity by 7-hydroxymethotrexate

“…The effect of 1 iM 7-OH MTX on uptake of [3H]-MTX by the KM3 cell line was minimal whereas the effect of 100 MM 7-OH MTX was greater and essentially the same for both melanoma and ALL cell lines (Gaukroger et al, 1983). If the data for the uptake of [3H]-MTX is plotted as a first order reaction then a straight line relationship becomes apparent for the initial uptake period (Figure 6).…”

“…The cell lines used and cell culture techniques employed were given previously (Gaukroger et al, 1983). Radiolabelled (Gaukroger et al, 1983) and only details of the efflux measurements will be given here.…”

“…Radiolabelled (Gaukroger et al, 1983) and only details of the efflux measurements will be given here. For unidirectional measurement of fluxes, simple integrated, rate-equation plots were performed (Eilam & Stein, 1973 Figure 1 for the melanoma cell lines B8 and ADLER, and for the ALL cell line KM3.…”

“…In the presence of this metabolite the decrease in the rate of uptake is greater for the melanoma (70 dpm min 1) than for the ALL (128 dpm min -1). (Gaukroger et al, 1983) (Goldman, 1975) Gaukroger et al, 1983) which will tend to maintain intracellular methotrexate levels. (Fabre et al, 1983; and which in combination with a short cell cycle may lead to exhaustion of reduced folate and rapid cell death (Jacobs et al, 1975 (Gaukroger et al, 1983) but have been identified in ALL cells (Fabre et al, 1983).…”

“…A companson was made of the melanoma cell lines ADLER, B8 and BlO with the ALL cell line KM3, which was reported to be methotrexate sensitive in vivo (Schnieder et al, 1977 (Gaukroger et al, 1983). …”