Teniposide (VM-26)- and Etoposide (VP-16-213)-Induced Augmentation of Methotrexate Transport and Polyglutamylation in Ehrlich Ascites Tumor Cells in Vitro1

Home , Teniposide

[CANCER RESEARCH 42, 3648-3653, September 1982] 0008-5472/82/0042-0000$02.00 Teniposide (VM-26)- and Etoposide (VP-16-213)-induced Augmentation of Methotrexate Transport and Polyglutamylation in Ehrlich Ascites Tumor Cells in Vitro1

Jack C. Yalowich,2 David W. Fry, and I. David Goldman3

Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298

ABSTRACT MTX observed in L1210 leukemia-bearing mice in vivo (8, 9, 36). One new dimension in the pharmacology of MTX has been Teniposide (VM-26) and etoposide (VP-16-213) were eval the recognition that mammalian cells rapidly form MTX polyglu uated for their effects on methotrexate transport and metabo tamate derivatives, a biochemical transformation which dimin lism in Ehrlich ascites tumor cells in vitro. VM-26 (10 JUM)does ishes the reversibility of the pharmacological effect of this not alter influx of methotrexate but slows efflux of the drug agent (15, 28, 29, 31 ). It is of interest that Vinca alkaloids, by which leads to an increase in the steady-state level of ex augmenting the exchangeable intracellular MTX level, enhance changeable antifolate. VM-26 stimulation of net methotrexate formation of MTX polyglutamates in vitro (13). Similarly, transport occurs within 5 min of exposure of cells to VM-26 probenecid blocks MTX efflux and enhances the exchangeable concentrations of from 10 to 50 fiM\ removal of extracellular intracellular MTX level (32), an effect that has now been shown VM-26 results in a rapid and complete reversal of this effect. to augment synthesis of MTX polyglutamate derivatives as well As extracellular VM-26 is raised to 50 JUM,the progressive rise (13). in net methotrexate uptake observed is due not only to elevated Accordingly, it might be possible to identify other pharma exchangeable antifolate but to an enlarged nonexchangeable cological agents that block MTX efflux and synergize with this pool of intracellular drug as well, a major portion of which is agent by this mechanism in vivo. Of particular interest in this methotrexate polyglutamyl derivatives. VM-26 does not alter regard are the semisynthetic glucopyranoside derivatives of the intracellular water or the chloride distribution ratio. Glucose podophyllotoxin, teniposide (VM-26) and etoposide (VP-16- partially reverses the stimulatory effect of VM-26 but to a lesser 213). These agents are active against a variety of leukemias extent than glucose reversal of the vincristine-induced stimu and solid tumors (26, 30). VP-16-213 is one of the most active lation of net methotrexate transport. agents against small-cell carcinoma of the lung (4, 22, 27). VP-16-213 also stimulates net methotrexate transport in a Although knowledge of the mechanism of action of these manner qualitatively similar to that of VM-26. However, 100 agents is still incomplete, VM-26 and VP-16-213 do not appear UM VP-16-213 is required to yield the same quantitative stim to alter microtubule function, unlike the parent compound ulation as seen for 10 UM VM-26. VP-16-213-induced augmen podophyllotoxin (25), other aglucone congeners of podophyl tation of Â¡ntracellular methotrexate polyglutamate levels is also lotoxin (24), and Vinca alkaloids such as vincristine, which are demonstrated. well known as inhibitors of microtubule assembly and function. These results indicate that VM-26 and VP-16-213 enhance In addition, these epipodophyllotoxins are able to induce a net cell accumulation of methotrexate and methotrexate poly premitotic (20, 21, 23, 34, 35) blockade of the cell cycle rather glutamyl derivatives in the Ehrlich ascites tumor in vitro at than the mitotic block associated with spindle poisons such as concentrations of these agents which are achieved in vivo. vincristine and vinblastine. Of particular interest is evidence Potential therapeutic synergism between methotrexate and suggesting a partial inhibition by VM-26 of mitochondrial epipodophyllotoxins on this basis is therefore possible and is NADH-linked respiration (18-20), which might in turn alter the currently under evaluation in animal tumor systems. energy-dependent exit of MTX. Further, these epipodophyllo toxins are sustained at much higher blood levels and for longer INTRODUCTION intervals clinically than the Vinca alkaloids, a phenomenon that could maximize this potential epipodophyllotoxin-MTX inter Several laboratories have demonstrated that Vinca alkaloids action in vivo (3, 6). stimulate net transport of MTX* in mammalian cells in vitro (8, The present study demonstrates that VM-26 and VP-16-213 14, 36). This has been attributed largely to a marked inhibition at concentrations achieved in vivo do, in fact, enhance the net of energy-dependent MTX efflux resulting in augmentation of cellular accumulation of MTX by inhibition of the exit of the exchangeable intracellular drug (14). Enhanced net MTX trans drug, an effect that is accompanied by increased accumulation port was associated with enhanced MTX inhibition of thymidyl- of MTX polyglutamate derivatives. ate biosynthesis (17) and has been suggested as one possible factor in the therapeutic synergism between vincristine and MATERIALS AND METHODS ' Supported in part by Grant CA-16906 and a grant from Bristol Laboratories. 2 Supported by NIH Training Grant CA-09340. Chemicals. [3',5',9-3H]MTX was synthesized by Moravek Biochem- 3 To whom requests for reprints should be addressed. icals (City of Industry, Calif.) and purified by DEAE-cellulose chroma- 4 The abbreviations used are: MTX, methotrexate (4-amino-10-methyl-pter- tography (17). [cart>oxy-'4C]lnsulin was obtained from New England oylglutamate); DMSO, dimethyl sulfoxide: poly-y-glutamyl derivatives of metho trexate are indicated as 4-NH2-10-CH3-ReGluâ€ž. Nuclear (Boston, Mass.). Vincristine sulfate was obtained from Flow Received December 10, 1981; accepted May 3, 1982. Laboratories, Inc. (Rockville, Md.). VM-26 and VP-16-213 were pro-

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1982 American Association for Cancer Research. Enhanced MTX Transport by VM-26 and VP-16-213 vided by Bristol Laboratories (Syracuse, N. Y.). Authentic standards of 4-NH2-10-CH3-PteGlu2 and 4-NH2-10-CH3-PteGlu3 were kindly sup 0,0, A, CONTROL I6 plied by Dr. C. M. Baugh (University of South Alabama). All other â€¢,Â»,Â»-HO>JMVM-26 chemicals were of reagent grade. Cells, Media, and Incubation Techniques. Ehrlich ascites tumor cells were grown in male CF-1 mice (Sprague-Dawley, Madison, Wis.) and passed weekly by i.p. inoculation of 0.2 ml of undiluted ascitic fluid. Cells were harvested 7 to 10 days postinoculation, washed, and centrifuged (500 x g for 2 min) twice in 0.85% NaCI solution to separate erythrocytes. The cells were finally suspended in a buffer composed of 136 mM NaCI, 4.4 mM KCI, 16 mM NaHCO3, 1.1 rnw KH2PO4, 1 mM MgCI,, and 1.9 mM CaCI?. The pH was maintained at 7.4 by passing warm and humidified 95% O2-5% CO2 over the cell suspension. Specially designed flasks containing Teflon paddles were utilized to stir the cell suspensions in a 37Â°water bath. Unidirectional fluxes, net uptake, and intracellular binding of [ H]MTX were measured as described previously (14). Transport fluxes were stopped by injec tion of the cell suspension into 10 volumes of 0Â°0.85% NaCI solution

(pH 7.4). The cell fraction was separated by centrifugation (500 x g 60 90 for 2 min) and washed twice with 0Â°0.85% NaCI solution. The washed Minutes pellet was aspirated into the tip of a Pasteur pipet, extruded onto a Chart 1. Effects of VM-26 on total cell antifolate at 3 different extracellular polyethylene tare, and dried overnight at 70Â°. The dried pellets were MTX concentrations. Cells were incubated with 0.2% DMSO (control) or with 10 UM VM-26 in 0.2% DMSO for 5 min before addition of [3H]MTX at the indicated weighed on a Cahn electrobalance (Cahn Instruments, Paramount, Calif.), placed in a scintillation vial, and dissolved in 0.25 ml of 1 N final concentrations. KOH for 1 hr at 70Â°. The digest was neutralized with 0.25 ml of 1 N HCI, and 4 ml of Ready-Solv (Beckman Instruments, Inc., Irvine, Calif.) were added. Radioactivity was determined in a Beckman LS-230 nonexchangeable fraction of the monoglutamate, and the level scintillation spectrometer, and counting efficiencies were determined of MTX polyglutamate derivatives. using [3H]- or [14C]toluene internal standards. Chart 2 illustrates the dose dependence of the VM-26 effect Determination of Intracellular Water and Chloride Distribution on the various compartments of Â¡ntracellular drug. As the VM- Ratio. Intracellular water was determined from the difference between 26 concentration is increased from 5 to 50 Â¡J.M,there is a the wet and dry weights of a cell pellet, less the ['"C]inulin space as progressive increase in total antifolate accumulation. More described elsewhere (14). specifically, this chart illustrates the increase in the exchange The chloride distribution ratio (the ratio of the concentration of able intracellular drug level, as well as the nonexchangeable chloride in the intracellular to extracellular water) was determined as component which reflects, in part, MTX polyglutamate deriva described elsewhere (14) and utilized as an indication of changes in tives (see below). At lower concentrations of VM-26, a steady membrane potential. state for intracellular antifolate is attained. However, at high Analysis of Intracellular MTX and Its Polyglutamate Derivatives. Portions of washed cell pellets which had been incubated with [3H]MTX concentrations (50 /IM), a steady state is not achieved over the were extracted with 1 ml of 10% trichloroacetic acid. The trichloro- interval of observation. Hence, total levels of the different MTX acetic acid extracts were neutralized by adding 0.175 ml of 1 mM KOH components at high concentrations of VM-26 may be under and 0.35 ml of 1 mM K2HPO4 (pH 7.0) to 0.7 ml of the sample. Analyses estimated. At low VM-26 concentrations the intracellular drug were performed with an Altex Model 332 gradient liquid Chromatograph component most dramatically augmented is exchangeable an equipped with a Model 210 injector and a 5-fim particle size ultrasphere tifolate, while at the highest concentrations there is a marked ODS column (25 x .46 cm inside diameter) (Altex, Berkeley, Calif.). increase in the nonexchangeable drug. One ml of the neutralized extracted was injected onto the column, and Effects of VM-26 on MTX Influx. Studies were undertaken separations were achieved by a modification of the procedure of to evaluate the effects of VM-26 on the bidirectional fluxes of Cashmore et al. (7), as reported in detail elsewhere (12). Authentic MTX. Because of the low solubility of VM-26, DMSO was used standards of MTX, 4-NH2-10-CH3-PteGlu2, and 4-NH2-10-CH3-PteGlu3 were included in each sample and monitored with an UV detector. as a solvent. Influx of MTX is 35% greater in the absence of Intracellular polyglutamates were quantitated by evaluating the per 0.2% DMSO, but there is no further inhibition of influx as the centage of each derivative from the Chromatographie analysis and the DMSO concentration is increased to 0.8%. At VM-26 and VP- total 3H in each pellet, as determined in units of nmol per g dry weight. 16-213 concentrations to 20 Â¡Â¿M,noinhibition of MTX influx is observed. At 50 and 100 JUM,VM-26 and VP-16-213 influx of MTX is inhibited in excess of that produced by the DMSO RESULTS solvent alone. However, inhibition is less than 25% at 100 Â¡Ã•M. Hence, at low VM-26 concentrations that enhance net uptake Effects of VM-26 on MTX Influx and Net Transport. Chart of MTX (Chart 1), influx of MTX is unchanged, suggesting that 1 illustrates that 10 JIMVM-26 augments net uptake of 1, 2, or there must be an inhibitory effect on MTX efflux under these 5 /IM MTX with little apparent effect on the initial uptake conditions to account for the marked changes in net MTX velocity. As the extracellular MTX concentration is increased, transport. the stimulatory effect of VM-26 is increased. The onset of VM- Effects of VM-26 on Net MTX Efflux. Chart 3 illustrates an 26 enhancement of net MTX uptake is rapid. At 10 to 50 fiM experiment in which cells loaded with MTX in the presence or VM-26, net uptake of MTX is induced within 5 min (not shown). absence of 10 /IM VM-26 were resuspended into MTX-free As is demonstrated below, these effects of VM-26 on net MTX buffer with or without 10 Â¡IMVM-26, respectively. DMSO was uptake reflect an increase in the free intracellular drug level, present at the same concentration under both conditions. Efflux

SEPTEMBER 1982 3649

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1200 2400 3600 Seconds Chart 3. Effect of VM-26 on MTX efflux. Cells were incubated with 10 pu VM- 26 in 0.2% DMSO or with DMSO alone, along with 2 /IM [3H]MTX. Fifty min later, cell fractions were washed free of extracellular MTX, cell total antifolate was 5 IO 20 50 5 IO 20 50 5 IO 20 50 measured, and the cells were resuspended in a large volume of MTX-free buffer. VM-26I/JU) VM-26-treated cells were again exposed to VM-26 and the control cells were exposed to the same level of DMSO alone. Over the next 70 min, the intracellular Chart 2. Effects of VM-26 concentration on total, nonexchangeable. and antifolate level was measured. The points at Time zero indicate the mean Â±S.E. exchangeable antifolate levels in Ehrlich ascites tumor cells. Cells were incubated of 5 measurements of the steady-state levels of antifolate in VM-26-treated and with various concentrations of VM-26 at a constant DMSO solvent level for 5 control cells prior to resuspension in MTX-free buffer, inset, log of the fraction of min, following which 2 /IM | H|MTX was added. After 55 min. 5 measurements of exchangeable antifolate remaining in the cell as a function of time after resus- the steady-state intracellular methotrexate level were obtained. The cell fractions pension into MTX-free buffer. MTX, . 0 is the exchangeable drug level prior to were then separated by centrifugation and resuspended in a large volume of removal of extracellular MTX at Time zero and MTX. is the exchangeable level at MTX-free buffer. The VM-26-treated cells were again exposed to VM-26 and the times following resuspension in drug-free buffer. control cells were suspended in buffer containing DMSO alone. After 65 min of incubation in MTX-free buffer. 5 measurements of the nonexchangeable intra cellular antifolate level were obtained. Exchangeable antifolate was determined from the difference between the total cell steady-state level and the nonexchange able level. Column, mean of the average values from 3 separate experiments performed on different days; bars, S.E. ^.A

Â£6 VM-26"* of exchangeable intracellular MTX5 was slowed by VM-26. +GLUCOSEÂ° I A* There was, in addition, a small increase in the nonexchangea oA"AÂ° o ble component. In Chart 3, (inset) the log of the exchangeable 5 A60 intracellular MTX level (total â€”nonexchangeable) is plotted as ^Ai AA a function of time after suspension into MTX-free buffer in control and VM-26-treated cells. In 5 experiments, performed on 5 separate days, there was a 65.0 Â±4.7% (S.E.) decline in the rate constant for efflux of exchangeable MTX in the pres ence of VM-26. Reversibility of the Effect of VM-26 on Net MTX Transport. Cells were exposed to VM-26 for 15 min and either continued

in the presence of this agent or exposed to VM-26-free buffer. i25 i i i i i After an additional 15 min, MTX was added and net uptake was 30 40 50A 70 monitored. When VM-26 is removed prior to the exposure to Minutes MTX, the stimulatory effect is eliminated, indicating that this Chart 4. Effect of 5 HIM glucose on net uptake of MTX in the presence or effect of the VM-26 is completely reversible. absence of 10 /IM VM-26. Cells were exposed to 10 /IM VM-26 and/or glucose for 5 min prior to addition of [3H]MTX to achieve a final MTX extracellular Effects of Glucose on Stimulation of Net MTX Uptake by concentration of 2 Â¡a*. VM-26. As reported previously (10, 14), glucose alone pro duces a small depression of net MTX transport (Chart 4). Other studies demonstrated that glucose reversed the stimulatory glucose was significantly less ( p < 0.01 ) than the 24.3 Â±1.3% effects of inhibitors of aerobic metabolism on MTX transport inhibitory effect of glucose on vincristine augmentation of net MTX transport (not shown). (11, 16). As observed previously for vincristine (14), glucose Effects of VM-26 on Intracellular Water, Transmembrane partially reversed the effects of VM-26. In 5 experiments, the inhibitory effect of glucose on VM-26 augmentation of net MTX Chloride Distribution Ratio, Nonexchangeable Antifolate, and the Transmembrane Electrochemical-Potential Differ transport (17.5 Â± 1.8%) was significantly greater than the ence for Exchangeable Intracellular MTX. While VM-26 en depression of net MTX transport by glucose alone, 11.5 Â± 1.5%, p < 0.05 (Chart 4). This reversal of the VM-26 effect by hances net MTX transport, it has little effect on other physical properties of cells under these experimental conditions. As 5 Under these experimental conditions, the major portion of intracellular drug illustrated in Table 1, VM-26 does not alter the ratio of the dry that leaves the cell is MTX monoglutamate. to wet weight of a cell pellet. Nor is there an alteration in the

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Table 1 Effects of VM-26 (10 Â¡IM)or the 0.2% DMSO solvent on physical properties of Ehrlich ase/fes tumor cells

Table 2 Effect of VM-26 on the steady-state exchangeable intracellular MTX concentration and the transmembrane electrochemical-potential difference for MTX This analysis is derived from 5 experiments, one of which is illustrated in Chart 3. concentrationNonexchangeable

cell (nmol/g cell (fiM)0.684 drywt)5.473 cellwt)2.863 (nmol/g day wt)2.610(nmol/g dry Â± 0.077C Control Â± 0.168 Â± 0.234 Â± 0.061 Â± 0.031 Â± 0.138 VM-26Total 7.322 Â± 0.134MTX 3.057 Â± 0.037[MTX1/IMTX],,Exchangeable4.265 Â± 0.137[MTX],8 1.117 Â± 0.036Measure0.3420.559 Â± 0.018Expected0.2230.223Measured/expected1.5342.570 Â± 0.080 [MTX1, concentration of exchangeable MTX in the intracellular compartment. [MTX1 is calculated by dividing exchangeable cell MTX by H2OÂ¡/DW(Table 1). [MTX],, concentration of MTX in the extracellular compartment. Under these conditions, the major portion of exchangeable intracellular antifolate is MTX monoglutamate. "The "expected" distribution ratio for MTX is derived from the Nernst equation and refers to the distribution ratio of MTX (a bivalent aniÃ³n) that would be expected if the transport system were passive (nonenergy dependent). This calculation is based on the membrane potential computed from the chloride distribution ratio indicated in Table 1 (10). c Mean Â±S.E.

extracellular space (the ratio of the extracellular water to wet Table 3 weight) or the intracellular water to dry weight. Similarly, there Comparative effects of VM-26, VP-16-213, and vincristine on net MTX uptake is no change in the intracellular chloride level or the chloride antifolate of control levelwt)4.53 (nmol/g dry level141.8 distribution ratio, a parameter that varies with changes in the Â± 0.10a membrane potential (10, 33). Likewise, the 0.2% DMSO sol Control + VM-26 (10 /IM) 6.43 Â±0.12 vent does not alter these cellular properties. Table 2 illustrates + VP-1 6-21 3(1 00 UM) 6.34 Â± 0.08 139.8 the effects of VM-26 on transmembrane gradients for MTX. + Vincristine (1 0 MM)Steady-state 6.52 Â± 0.05% 143.9 The increase in total cell antifolate induced by VM-26 in this Mean Â± S.E. of the average value from 3 experiments performed on experiment is largely due to an increase in the exchangeable separate days. Four to 5 determinations were performed in each experiment. intracellular MTX concentration. The Nernst equation was used to predict the expected passive distribution ratio for MTX, a 220 bivalent aniÃ³n, based upon the membrane potential estimated from the chloride distribution ratio (10). It can be seen that the 0MTX-polyglutamotes 200 ratio of the measured to expected distribution ratio for MTX increases from 1.5 in the absence of VM-26 to 2.5 in the o ISO presence of this agent. Hence, VM-26 produces a large in o crease in the transmembrane electrochemical-potential differ " I60 ence for MTX. Comparison of the Effects of VM-26, VP-16-213, and Vin- I40 cristine on Net MTX Uptake. VM-26 stimulation of net MTX uptake is more potent than that of VP-16-213 and is compa 120 rable to that observed with equimolar vincristine. A concentra tion of 100 fiM VP-16-213 produces an augmentation of net IOO MTX uptake comparable to that observed with 10 U.MVM-26 or 10 /IM vincristine (Table 3). Effects of VM-26 and VP-16-213 on the Formation of MTX lO^JM VÃŒUM lOjJM M);IM VM-26 VP-I6-2I3 Polyglutamate Derivatives. Chart 5 illustrates the accumula Chart 5. Effect of VM-26 or VP-16-213 on the intracellular levels of MTX and tion of cell MTX and MTX polyglutamate derivatives in the MTX polyglutamate derivatives. Cells were incubated 3 hr with 5 mM L-glutamine, presence or absence of 10 and 50 U.MVM-26 or VP-16-213. In 5 MM[3H]MTX, and 10 or 50 JIMof the indicated epipodophyllotoxins, after which these experiments, 5 mM L-glutamine was present to enhance the intracellular 3H was extracted and analyzed by high-pressure liquid chromatography as described in "Materials and Methods." The data are expressed as synthesis of polyglutamate derivatives (12). In the presence of a percentage of the control cells exposed to the DMSO solvent alone. Column, VM-26, there was a concentration-dependent increase in both mean of 3 experiments performed on separate days; bars, S.E. the monoglutamate and polyglutamate levels. Only at 50 /ÃŒM VP-16-213 were increased levels of MTX and MTX polygluta- utilized. This suggests that the epipodophyllotoxins do not mates observed. There was no statistically significant differ directly affect polyglutamate formation but rather act by in ence (p > 0.2) between the increase in MTX and MTX polyglu creasing exchangeable MTX monoglutamate substrate which, tamate levels at each concentration of VM-26 or VP-16-213 in turn, increases the rate of polyglutamylation. Table 4 indi-

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Table 4 Effect of 50 Â¡tu VM-26 on intracellular levels of MTX and MTX pofyglutamate derivatives Cells were incubated with 5 .MM| H |MTx and 5 HIM L-glutamine for 60 min, following which samples were taken for analysis of total intracellular [3H]antifolate. Exchangeable and nonexchangeable antifolate levels were determined by measurements of MTX and MTX polyglutamate derivatives after cells were incubated in MTX-free buffer for 70 min. antifolateTotalControl Cell polygluta- mates1 (nmol/g dry 0.08 Â± weight) VM-26 (nmol/g dry 12.40 Â± 0.31 5.61 Â± 0.17 6.78 Â± 0.19 3 54 Â± 0.28 weight) Increase (nmol/g dry 6.52Â±Â± 0.33Nonexchangeable2.882.73 Â±0.080.20Exchangeable3.013.77Â±Â±0.170.32MTX 20252Â±Â±0.050.28 weight)5.88 a Mean Â±S.E. of 4 experiments performed on separate days.

cates that even after a short period of incubation (1 hr) 50 /IM is distinct from the structural property which determines bind VM-26 markedly enhances the accumulation of intracellular ing to microtubular elements. MTX polyglutamates. In fact, the increase of MTX polygluta- Of particular interest are the much higher blood levels that mates (2.50 nmol/g, dry weight) accounts for the major portion VM-26 and VP-16-213 achieve in clinical regimens in compar of the rise in nonexchangeable cell MTX (2.73 nmol/g, dry ison to the Vinca alkaloids. Potential interactions between weight) under these conditions. Vinca alkaloids and MTX in humans in vivo is limited by the low vincristine blood level achieved and its rapid rate of decline (6). DISCUSSION If the effects of epipodophyllotoxins on MTX transport are of the same order of potency in human tumor cells as in the These studies demonstrate that VM-26 and VP-16-213, sim Ehrlich tumor and the higher drug blood levels are available to ilar to the Vinca alkaloids, enhance MTX transport in the Ehrlich the cell in vivo, then the extent and duration of this pharma ascites tumor cell. Although an order of magnitude less potent cological interaction in vivo might be considerably greater than than VM-26, VP-16-213 acts in a qualitatively similar manner. that obtained with the Vinca alkaloids. The differences between these epipodophyllotoxins may be The potential importance of the epipodophyllotoxin-mediated due to differences in the intracellular levels achieved by these stimulation of MTX transport in vivo is of particular interest in 2 agents. For instance, levels of VM-26 in L1210 leukemia view of the recently recognized new element in MTX pharma cells are 10-fold greater than those of VP-16-213 (1, 2). This cology, the formation of MTX polyglutamate derivatives. This difference has been suggested to be based upon greater influx transformation of MTX involving the addition of a homopolymer and tighter intracellular binding of VM-26 in comparison to VP- chain of y-linked glutamyl residues appears to result in the 16-213 or a greater lipophilicity of the thenylidene side chain retention of these active antifolate derivatives within the cell, of VM-26 (1, 2). which diminishes the reversibility of the pharmacological ef Below 20 /IM, VM-26 was found to have a negligible effect fects of this antifolate as the extracellular MTX level falls (12, on MTX influx; the striking augmentation of the exchangeable 13,15, 31 ). Hence, this interaction between epipodophyllotox intracellular drug level was based upon a reduction of the efflux ins and MTX which results in enhanced accumulation of MTX of this antifolate in a manner similar to that observed previously polyglutamate derivatives may be a basis for enhanced efficacy for vincristine (14). Stimulation of net MTX uptake by VM-26 is when these agents are combined in the proper sequence in also similar to that observed with a variety of metabolic poisons clinical regimens. Studies are now in progress to evaluate (11, 16) and was reduced by glucose, although this reversal potential synergism between 4-aminoantifolates and epipodo by glucose is quantitatively less than that for Vinca alkaloids phyllotoxins in in vivo animal tumor systems. and inhibitors of oxidation. Hence, stimulation of net MTX uptake by VM-26 and VP-16-213 appears to be based, at least REFERENCES in part, upon an inhibition of energy metabolism. This is con sistent with evidence that epipodophyllotoxins partially block 1. Allen, L. The role of drug disposition kinetics on cellular transport of the antineoplastic agent VM-26. Drug Metab. Rev., 8. 119-135, 1978. mitochondrial NADH-linked respiration (18-20), an effect, like 2. Allen. L. M. Comparison of uptake and binding of two epipodophyllotoxin that on net MTX uptake, which is rapidly reversed when extra glucopyranosides, 4'-demethyl-epipodophyllotoxin thenylidene-/Ã®-D-gluco- cellular epipodophyllotoxin is removed (18, 19). side and 4-demethyl-epipodophyllotoxin ethylidene-/}-D-glucoside. in the L1210 leukemia cell. Cancer Res., 38. 2549-2554, 1978. While sharing with vincristine similar effects on transport of 3. Allen, L. M., and Creaven, P. J. Comparison of the human pharmacokinetics MTX, epipodophyllotoxins irreversibly block human lymphoid of VM-26 and VP-16. two antineoplastic epipodophyllotoxin glucopyranoside derivatives. Eur. J. 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SEPTEMBER 1982 3653

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1982 American Association for Cancer Research. Teniposide (VM-26)- and Etoposide (VP-16-213)-induced Augmentation of Methotrexate Transport and Polyglutamylation in Ehrlich Ascites Tumor Cells in Vitro

Jack C. Yalowich, David W. Fry and I. David Goldman

Cancer Res 1982;42:3648-3653.

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