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Clinical Cancer Research Vol. 6, 643-653, February 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Methionine Depletion Enhances the Antitumoral Efficacy of Cytotoxic Agents in Drug-resistant Human Tumor Xenografts1

F. Poirson-Bichat, R. A. Bras Gonçalves, L. Miccoli, B. Dutrillaux and M. F. Poupon2

Institut Curie, UMR 147 CNRS-Institut Curie, 75231 Paris Cedex 05, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
 RESULTS
 DISCUSSION
 REFERENCES
 
Efficacy of chemotherapy is limited in numerous tumors by specific cellular mechanisms that inactivate cytotoxic antitumoral drugs, such as ATP-dependent drug efflux and/or drug detoxification by glutathione. In reducing ATP pools and/or glutathione synthesis, it might be possible to enhance the efficacy of drugs affected by such resistance mechanisms. Reduction of the ATP pool and glutathione content is achievable in cancer cells by depleting the exogenous methionine (Met) supply and ethionine. Thus, the rationale for the present study was to use Met depletion to decrease the ATP and glutathione pools so as to sensitize tumors refractory to cytotoxic anticancer drugs. Met depletion was achieved by feeding mice a methionine-free diet supplemented with homocysteine. The effects of Met depletion combined with ethionine and/or chemotherapeutic agents were studied using human solid cancers xenografted into nude mice. TC71-MA (a colon cancer) SCLC6 (a small cell lung cancer), and SNB19 (a glioma) were found to be refractory to cisplatin, doxorubicin, and carmustine, respectively. These three drugs are used to treat such tumors and are dependent for their activity on the lack of cellular ATP- or glutathione-dependent mechanisms of resistance. TC71-MA, SCLC6, and SNB19 were Met dependent because their proliferation in vitro and growth in vivo were reduced by Met depletion. Cisplatin was inactive in the treatment of TC71-MA colon cancer, whereas a methionine-free diet, alone or in combination with ethionine, prolonged the survival of mice by 2-fold and 2.8-fold, respectively. When all three approaches were combined, survival was prolonged by 3.3-fold. Doxorubicin did not affect the growth of SCLC6, a MDR1-MRP-expressing tumor. A Met-deprived diet and ethionine slightly decreased SCLC6 growth and, in combination with doxorubicin, an inhibition of 51% was obtained, with survival prolonged by 1.7-fold. Combined treatment produced greater tumor growth inhibition (74%) in SCLC6-Dox, a SCLC6 tumor pretreated with doxorubicin. Growth of SNB19 glioma was not inhibited by carmustine, but when it was combined with Met depletion, survival duration was prolonged by 2-fold, with a growth inhibition of 80%. These results indicate the potential of Met depletion to enhance the antitumoral effects of chemotherapeutic agents on drug-refractory tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
 RESULTS
 DISCUSSION
 REFERENCES
 
Metabolic anomalies are commonly found in solid tumors, and some of them have been known for decades (1) . Among the metabolic abnormalities recurrently found in cancers, Met3 dependency and alterations of Met metabolism have been found in many, if not in all, types of human tumors (2, 3, 4, 5, 6, 7, 8, 9, 10, 11) . Met dependency leads to the inability of cells to proliferate in culture when Met is absent and replaced by Hcy, one of its metabolic precursors, whereas normal cells grow in such a medium (2) . This difference in the growth of normal cells and Met-dependent tumor cells in Met-Hcy+ medium might be due to the different requirement for transmethylation reactions necessary to maintain their proliferation rate. Methods for reducing in vivo Met intake are to decrease the Met supply in food either using a Met-deprived diet (6) , a methioninase infusion (12 , 13) , or Met-free total parenteral nutrition (14 , 15) . Met-depleted diets effectively decreased metastatic potential and tumorogenicity in an experimental rat model, as previously shown by us (16) and by Guo et al. (8 , 9) .

In recent studies, we have obtained a potentiation of the antitumoral effects of a Met-deprived diet by treating tumor-bearing mice simultaneously with ethionine, a Met analogue. Ethionine might act by inhibiting most methyltransferases and hence lead to DNA hypomethylation, which can affect gene activity, as shown by Razin and Riggs (17) . Ethionine combined with a Met-deprived diet can augment the effects of Met depletion alone in reducing the growth of human prostate tumor (11) and glioma (10) xenografted into nude mice and of Yoshida sarcoma grafted into the rat (9 , 18) . In vitro, we also observed that when tumor cells were cultured in a Met-Hcy+ medium containing ethionine, their ATP and glutathione pools were decreased, and the cell cycle was blocked in S phase-G2, inducing an irreversible arrest of DNA replication.

Current chemotherapy of cancer is based on the use of cytostatic and/or cytotoxic agents acting on components and cellular functions that control growth and cellular division. The failure of chemotherapy is often due to direct or indirect target alterations that induce resistance. Various parameters contribute to drug resistance, and some of them are due to the increased expression of ATP-dependent mechanisms such as P-glycoprotein (19 , 20) and MRP proteins (21 , 22) . Drug resistance can also be related to a high glutathione content and overexpression of some enzymes, such as glutathione transferases (23 , 24) . Therefore, a decrease of ATP or of the glutathione pools might counteract the ATP- or glutathione-dependent mechanisms of resistance.

Experiments combining Met depletion with doxorubicin alone, doxorubicin and vincristine, or 5-fluorouracil using Met-free parenteral nutrition were conducted using the Yoshida sarcoma grafted into the rat (14 , 25 , 26) . They showed a potentiation of the antitumoral effect of cytotoxic drugs. Similar assays were conducted using a human gastric cancer grafted into nude mice, combining Met-depletion with 5-fluorouracil (27) . In patients with gastrointestinal tract cancers, it was shown that Met depletion might play a role as a modulator of 5-fluorouracil by decreasing the free-thymidylate synthetase activity (27) .

Met is an essential amino acid, and we have shown that Met depletion is not compatible with long-term survival. Consequently, Met is substituted by Hcy in the diet used in our experiments, thereby allowing animals to survive (6) .

In the present study, Met depletion was induced by a Met-deprived diet and ethionine to decrease the cellular ATP and glutathione content and to sensitize drug-resistant tumors to the effect of chemotherapeutic drugs. This was tested using drug-resistant human tumors xenografted into nude mice expressing Pgp and/or MRP, both ATP-dependent molecular determinants of resistance, and/or a high level of glutathione. We have treated xenografted human cancers, a glioblastoma, a small cell lung cancer, and a colon cancer with a combination of a Met-deprived diet and ethionine with carmustine, doxorubicin, and cisplatin, three drugs used for therapy of such tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Glutathione Assay
 In Vivo Studies
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Proliferation Assay
SNB19, TC71-MA, and SCLC6 were established as monolayer cell lines as described previously (10 , 11) . For assays, cells were maintained in RPMI 1640 (Sigma) supplemented with 10% FCS (Dutscher, Brumath, France). Briefly, cells were plated in 10% FCS-RPMI 1640 for 24 h in a 5% CO2 atmosphere at 37°C at 105 cells/well in 24-well plates (ATGC, Noisy-le-Grand, France) in triplicate. The medium was replaced by a Met-free medium supplemented with dialyzed FCS, 100 µM folic acid, and 1.5 µM hydrocobalamin with 100 µM of Hcy (Met-Hcy+) or Met (Met+Hcy+), with or without 0.5 mg/ml ethionine (all products from Sigma). Cell monolayers were fixed with methanol and then stained with methylene blue. Methylene blue incorporated in fixed cells was solubilized in hydrochloric acid (0.1 M), and the absorbance of each well solution was measured (wavelength, 620 nm) with a spectrophotometer (LP500; J Bio, les Ulis, France). The cell proliferation index was calculated as the ratio of absorbances corresponding to the assay medium to that of controls x 100. Means and SDs were calculated, and statistical analyses were performed with the Student’s t test.

Measurement of ATP Content
ATP content was quantified by a bioluminescence assay using a Lumac biocounter M1500 (Lumac Perstop Analytical, Bezons, France), as described previously (10) . Cells were plated as described above for 24 and 48 h, in Met-Hcy+ or Met+Hcy+ medium with or without ethionine (0.5 mg/ml). Cell extracts were obtained using 1% trichloroacetic acid (Sigma) in water. ATP was measured in a 450-µl mixture of luciferin-luciferase (20 µl of a 40 mg/ml stock solution), ATP standard (both from Sigma) (10 µl; 5.04 x 10-8 M), and 10 µl of the sample to analyze in distilled water. Results are expressed as percentages of ATP nmol/10-6 treated cell extracts divided by ATP nmol/10-6 control cell extracts. Experiments were repeated five times. Means of data were calculated.

PCR for MDR-1 and MRP mRNA Expression
Total RNA of xenografted tumor was extracted with TRIZOL reagent (Life Technologies, Inc.), and RNA was kept snap-frozen in liquid nitrogen and stored until use. MDR-1 and MRP gene expression was analyzed by reverse transcription-PCR (28) . Levels of human-specific actin were measured as an endogenous control for cDNA synthesis. The primers listed below were selected for their specificity and selectivity for human gene sequences and purchased from Oligo Express (Paris, France): (a) mdr1-1, antisense, 5'-ATATGTTCAAACTTCTGCTCCTGA-3'; (b) mdr1-2, sense, 5'-TGTACCCATCATTGCAATAGCAGG-3' (29) ; (c) mrp-1, antisense, 5'-GTACACGGAAAGCTTGAC-3'; (d) mrp-2, sense, 5'-GGTCACGCACAGCATG-3' (29) ; (e) ß2 microglobulin-1, antisense, 5'-GACAAGTCTGAATGCTCCAC-3'; and (f) ß2 microglobulin-2, sense, 5'-TATCCAGCGTACTCCAAAGA-3'.

PCR Conditions.
The final PCR reaction volume was 50 µl. The cDNA solution (2.5 µl) was pipetted into a sterile 0.2-ml tube, and the following mixture containing 5 µl of 10x Taq buffer (Appligene-Oncor, Illkirch, France), 1 µl of dNTP (final concentration, 2.5 mM each), 1 µl of each of the 5' and 3' primers (100 ng/µl), 38.5 µl of water, and 0.5 µl (2.5 units) of Taq polymerase (Appligene-Oncor) was added. After preheating at 94°C (hot start), the tubes were placed in a Perkin-Elmer 2400 thermocycler (Yvelines, France) for 5 min at 94°C followed by: (a) for the MDR-1 gene, 2 cycles (1 min at 94°C, 1 min at 62°C, and 1 min at 72°C) and 38 cycles (1 min at 94°C, 1 min at 60°C, and 1 min at 72°C), and a final elongation at 72°C for 10 min; (b) for the MRP gene, 35 cycles (1 min at 95°C, 1 min at 52°C, and 1.5 min at 72°C) and a final elongation at 72°C for 10 min; and (c) for the ß2 microglobulin gene, 3 min at 94°C, 35 cycles (1 min at 94°C, 30 s at 60°C, and 30 s at 72°C) and 10 min at 72°C.


   Glutathione Assay
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
In Vivo Studies
 RESULTS
 DISCUSSION
 REFERENCES
 
The glutathione assay was performed for the SNB19 (glioma) xenograft in nude mice that were fed a Met-Hcy+ diet combined with ethionine treatment for 5 days. The tumor samples (50 mg) were pulverized with 20% salicylic acid and centrifuged. Tumor cells (106) cultured 24 h in Met-Hcy+ medium without ethionine, as detailed above, were similarly treated. For each assay, 500 µl of the supernatant were mixed with 500 µl of mixture buffer (0.3 M Na2HPO4 (pH 7.5), 10 mM EDTA, and 0.2 mM DTNB) before reading absorbance at 412 nm. The level of absorbance is proportional to the glutathione pool and is expressed relative to the cultured cell number (in vitro) or to the weight (in grams) of tissue (in vivo).


   In Vivo Studies
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Eight- to 10-week-old, Swiss nu/nu male or female mice (20–25 g body weight) bred in the animal facilities of the Curie Institute (Paris, France) were used in these assays. The animals were maintained under specified pathogen-free conditions. Their care and housing were in accordance with the institutional guidelines of the French Ethical Committee (Ministère de l’Agriculture et de la Forêt, Direction de la Santé et de la Protection Animale, Paris, France) and supervised by authorized investigators.

Tumors and Xenotransplantation.
Xenografts were established by implantation of tumor samples into the scapular area of the nude mice. These samples were taken either from a tumor obtained previously by a s.c. injection of 10 x 106 SNB19 glioma cells [obtained from Gross et al. (30) and subcultured in our laboratory] into the flank of nude mice or from human tumor fragments obtained from the clinical samples and established in serial transplantation, such as TC71-MA, a colon cancer (31) , and SCLC6, a SCLC (32) . Tumors were maintained by successive passages from mouse to mouse. A SCLC6-Dox tumor derived from SCLC6 was established in nude mice by i.p. injection of 30 mg/kg/body weight of doxorubicin followed by transplantation 2 h later into a new nude mouse. This procedure was repeated three times before use in the in vivo assays (32) .

Tumor Growth Inhibition Studies.
Mice were grafted with tumor fragments of approximately 15 mm3 in volume. Tumors appeared at the graft site 2–5 weeks later. Mice bearing growing tumors with a volume of 60–100 mm3 were individually identified and randomly assigned to the control or treatment group, and the treatment was started. The animals bearing tumors were sacrificed when their tumor volume reached 2500 mm3, the level defined as ethical sacrifice. Volumes of individual tumors were calculated from the measurements of two perpendicular diameters using a caliper, performed every 2 days. Each tumor volume (V) was calculated according to the following formula (33 , 34) : V = a2 x b/2, where a and b are the smallest and largest perpendicular tumor diameters. RTVs were calculated from the formula: RTV = (Vx/V1), where Vx = the volume on day x, and V1 is the tumor volume at the initiation of therapy (day 1). Growth curves were obtained for each individual tumor by plotting values of RTV against time (expressed as days after the start of treatment). The antitumor activity was evaluated according to three criteria: (a) the tumor growth inhibition, which was calculated according to the following formula: 100 - (RTVt/RTVc) x 100 where RTVt is the mean RTV of the treated group and RTVc is the mean RTV of the control group at the time of optimal response; (b) the tumor growth delay, calculated as the time in days required for the tumors to reach a 15-fold increase in RTV, corresponding to the survival time of treated mice; prolongation of survival was calculated as the ratio between the survival time in the treated group and that of controls; (c) the tumor doubling time was calculated as the delay in days required to double an initial tumor volume of 200 mm3 (size in exponential growth phase).

The statistical significance of the differences between the tumor volumes reached in each group was calculated with the ANOVA test (GraphPad InStat, San Diego, CA) and the Student’s t test.

Formulation and Administration of Diets and Drugs.
Mice were fed either a regular diet (UAR, Villemoisson, France) or a Met-deprived Hcy-supplemented diet in which the proteins were replaced by an amino acid mixture without Met (Met-Hcy+; Refs. 10 and 11 ). Hcy (Sigma, Grenoble, France) was added at 0.4 g/100 g of diet. Ethionine (Sigma) was chlorohydrated and solubilized extemporaneously in water at a concentration of 25 mg/ml, and 0.2 ml (200 mg/kg) of this solution was injected daily by the i.p. route. Cisplatin (Rhône-Poulenc-Rorer, Vitry-sur Seine, France) was solubilized in water at a concentration of 0.15 mg/ml, and 0.2 ml (1 mg/kg) of this solution was injected by the i.p. route for 5 consecutive days every 2 weeks.

Carmustine (Bristol Myers Squibb, Paris, France) was solubilized in NaCl 0.9% at a concentration of 0.33 mg/ml, and 0.1 ml of this solution (1 mg/kg) was injected by the i.p. route for 3 consecutive days. Doxorubicin (Pharmacia, Saint-Quentin-en-Yvelines, France) was solubilized extemporaneously in water at a concentration of 0.5 mg/ml, and 0.25 ml (5 mg/kg) of this solution was injected by the i.p. route once a week for 2 or 3 weeks, as specified in the text.

Combinations of Met Depletion with Anticancer Drugs.
For all combinations, mice were fed a Met-deprived diet (Met-Hcy+) throughout the experiment. For doxorubicin combined treatment, ethionine (200 mg/kg/day, diluted in water) was administered i.p. on days 1 and 2; two h after the second injection of ethionine, doxorubicin (5 mg/kg, diluted in water) was injected i.p. This treatment was repeated weekly for 2 or 3 weeks, as reported in Fig. 3Citation . Different control groups were used, namely, mice treated with Met-Hcy+ diet or ethionine alone (i.e., Met depletion), doxorubicin alone, or 0.9% NaCl. For cisplatin combined treatment, mice fed a Met-Hcy+ diet and receiving ethionine daily were treated with cisplatin (1 mg/kg, diluted in water) for 5 consecutive days/week, every 2 weeks. Four treatment cycles were performed. Different control groups were used, namely, mice treated with ethionine, a Met-Hcy+ diet, or cisplatin alone, a combination of a Met-Hcy+ diet with ethionine or cisplatin or with NaCl. For carmustine combined treatment, mice fed either a Met-deprived diet (Met-Hcy+) and ethionine daily or a regular diet were treated with carmustine (6 mg/kg in 0.9% NaCl) for 3 consecutive days/week for 3 weeks. Different control groups were used, namely, mice treated only with carmustine or with Met-Hcy+ diet-ethionine or with NaCl.



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  		Fig. 3. Tumor growth curves of TC71-MA, a human colon cancer xenografted into nude mice. Top, mice (10 mice/group) were treated with ethionine (200 mg/kg/day) daily; cisplatin (1 mg/kg/day) for 5 consecutive days weekly for 3 weeks; cisplatin + ethionine; or with a Met-Hcy+ diet. Bottom, control mice were fed a regular diet; mice were treated with Met-Hcy+ diet + cisplatin; Met-Hcy+ diet + ethionine; or Met-Hcy+ diet + ethionine + cisplatin.

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
 RESULTS
DISCUSSION
 REFERENCES
 
Biological Characteristics of the Xenografted Human Tumors Used.
All tumors originated from patients before any treatment, and their human origin was checked by karyotype analysis (data not shown). MDR-1 and MRP mRNA were expressed in TC71-MA tumor, a colon cancer, and in SCLC6 and SCLC6-Dox, both SCLCs, but not in SNB19, a xenografted glioma, as shown by reverse transcription-PCR (Fig. 1)Citation . MDR-1 and MRP mRNA expression was not significantly increased in SCLC6-Dox, despite pretreatment with a high dosage of doxorubicin.



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  		Fig. 1. MDR-1 and MRP mRNA expression of the xenografted human tumors used.

 
In Vitro and in Vivo Effects of Met Depletion in Human Tumor Xenografts.
The in vitro proliferation rate of SNB19 cells decreased after several hours of culture in Met-free Hcy-supplemented medium (Fig. 2)Citation . Addition of ethionine to the culture medium reduced the cell proliferation rate of SNB19, and inhibition of cell proliferation by ethionine was greater in the absence of Met. Measurement of ATP pools, performed in parallel with the proliferation assay, showed a drop in ATP after 48 h of culture in Met-free medium and ethionine. Glutathione content was decreased in cells after a 24-h culture in Met-free medium, with or without ethionine, as shown in Fig. 2Citation . When grafted into nude mice, growth of SNB19 glioma was reduced by 26% as compared to the control group (P > 0.05); no prolongation of survival was observed (Table 1)Citation . In vivo Met depletion was obtained in nude mice bearing xenografts by feeding them a Met-free diet for 3 weeks. Treatment started as soon as the tumor size reached a volume of 60–100 mm3. Met depletion with a Met-free diet and ethionine inhibited tumor growth by 53% (P < 0.01) and induced a prolongation in survival of 16 days (1.6-fold; P < 0.05; greater than the control group). This was associated with a reduction in the glutathione content of 500 mm3 SNB19 xenografts after treatment with a Met-free diet and ethionine. A 4-fold decrease in glutathione content was observed after 5 days of treatment in comparison with untreated tumors (Fig. 2)Citation .



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  		Fig. 2. Effects of Met deprivation and ethionine on cell proliferation, ATP pools, and glutathione content of SNB19, a human glioma. Top, in vitro cell proliferation assay in two different media with Hcy, with and without Met, with and without ethionine, added at a concentration of 0.5 mg/ml. Middle, relative content in ATP of SNB19 cultured in Met-containing medium (left) or in Met-free medium (right), as a function of time. Bottom, glutathione content in cells (left) and in tumor tissue (right), in controls, Met-free medium, or diet and ethionine.

 

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  		Table 1 In vitro and in vivo antitumoral effects of Met-free medium or Met-free diets with and without ethionine (Eth)

 
The in vitro proliferation rate of TC71-MA cells decreased after several hours of culture in Met-free Hcy-supplemented medium (Table 1)Citation . Addition of ethionine to the culture medium did not affect the proliferation rate of TC71-MA, which was already very low. Growth of the TC71-MA colon cancer was inhibited (46%; P < 0.05), prolonging the survival of mice by 12 days, which represents a 2.1-fold improvement over controls (Table 2)Citation . The combination of a Met-free diet with ethionine, leading to a more complete Met depletion, inhibited tumor growth by 56% (P < 0.01), prolonging the survival of mice by 2.8-fold. The doubling time of the tumor was 13 and 26 days after feeding mice with a Met-free diet, without or with ethionine, respectively.


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  		Table 2 Antitumoral effects of cisplatin, Met-free diet, and ethionine on the growth of TC71-MA, a human colon cancer grafted into nude mice

 
The in vitro proliferation rate of SCLC6 cells decreased after several hours of culture in Met-free Hcy-supplemented medium (Table 1)Citation . Addition of ethionine to the culture medium did not change that of SCLC6 (Table 1)Citation . Growth of both SCLC6 and SCLC6-Dox was not significantly reduced by Met depletion (Table 1)Citation , and administration of ethionine had no effect on SCLC6-Dox, whereas it moderately slowed the growth of SCLC6 (see Fig. 4Citation ).



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  		Fig. 4. Tumor growth curves of SCLC6, a human SCLC xenografted into nude mice and its variant, SCLC6-Dox, obtained after three successive injections of 30 mg/kg i.p. of doxorubicin followed by transplantation 2 h later. Top, control mice with SCLC6 (10 mice/group) were fed a regular diet; mice were treated with doxorubicin (5 mg/kg/day) once a week; Met-Hcy+ diet + ethionine; or Met-Hcy+ diet + ethionine + doxorubicin. Bottom, mice with SCLC6-Dox (10 mice/group) were treated with doxorubicin (5 mg/kg/day) once a week; Met-Hcy+ diet + ethionine; or Met-Hcy+ diet + ethionine + doxorubicin.

 
Combination of Met Depletion with Cisplatin for the Treatment of TC71-MA Xenograft, a Colon Cancer.
Cisplatin did not inhibit the growth of TC71-MA (Table 2Citation and Fig. 3Citation ). Ethionine alone or combined with cisplatin given to mice fed a regular diet had no effect. When cisplatin was administered to mice fed a Met-free diet, the growth inhibition was 56% (46% with the diet alone), and the survival was prolonged by 2.8-fold (2.1-fold with the diet alone). A similar effect was obtained in mice fed a Met-free diet and treated with ethionine (Fig. 3)Citation . A superior effect was obtained by treating the tumor-bearing mice with the combination of a Met-free diet, ethionine, and cisplatin, which resulted in a growth inhibition of 61% and prolonged survival by 3.3-fold, although this was only slightly different from the antitumoral effects of the Met-free diet with ethionine or the Met-free diet with cisplatin. It was clear that the Met-free diet enhanced the antitumoral efficacy of the two other compounds used.

Combination of Met Depletion with Doxorubicin for the Treatment of SCLC6 Xenograft, a Small Cell Lung Cancer.
SCLC6, a SCLC expressing Pgp, MRP, and glutathione, was refractory to the effect of doxorubicin. SCLC6-Dox tumors, pretreated with doxorubicin, expressed the same determinants of resistance and were also resistant to doxorubicin when tumor-bearing mice were fed a regular diet (Table 3Citation and Fig. 4Citation ). Doxorubicin reduced the growth of SCLC6 tumors when mice were fed a Met-free diet and received ethionine. Indeed, a 51% growth inhibition (P < 0.01) was seen, and survival of mice was prolonged by 1.7-fold, whereas SCLC6 growth was slowed slightly by the diet plus ethionine. Similar observations were made with SCLC6-Dox tumors; an antitumoral effect of doxorubicin was obtained by the combination of doxorubicin, a Met-free diet, and ethionine, leading to a growth inhibition of 74% (P < 0.01) and a prolonged survival (1.7-fold; Fig. 4Citation ). These results were all the more striking with SCLC6-Dox because it was not inhibited at all by the Met depletion.


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  		Table 3 Antitumoral effects of doxorubicin, Met-free diet, and ethionine on the growth of SCLC6, a human SCLC established in nude mice, and on the growth of SCLC6-Dox, derived from SCLC6 after treatment with doxorubicin, as detailed in "Materials and Methods"

 
Combination of Depletion with Carmustine for the Treatment of SNB19 Xenograft, a Glioblastoma.
A limited antitumoral effect of carmustine alone was obtained in SNB19 glioma (30% tumor growth inhibition; Table 4Citation and Fig. 5Citation ). The combination of carmustine with the Met-deprived diet and ethionine enhanced the antitumoral effect of the Met deprived diet-ethionine association (80% and 46% tumor growth inhibition, respectively), leading to a prolongation of mice survival of 2-fold in the group receiving the triple therapy.


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  		Table 4 Antitumoral effects of carmustine, Met-free diet, and ethionine on the growth of SNB19, a human glioma established in nude mice

 


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  		Fig. 5. Tumor growth curves of SNB19, a human glioma xenografted into nude mice. Tumor-bearing mice (10 mice/group) were treated with carmustine (6 mg/kg/day) for 3 consecutive days weekly, for 3 weeks; Met-Hcy+ diet + ethionine; or Met-Hcy+ diet + ethionine + carmustine; control mice were fed a regular diet.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
 RESULTS
 DISCUSSION
REFERENCES
 
Met depletion of cells induced in vitro by substitution of Met by Hcy in the culture medium and the addition of ethionine reduced cell proliferation of cancer lines and decreased ATP and glutathione pools. ATP is required for the function of drug efflux pumps, such as Pgp encoded by the MDR-1 gene and MRP, which are involved in the resistance to numerous compounds including doxorubicin. Glutathione and glutathione transferases, which are known to be constantly elevated in tumors as compared to normal tissues, detoxify xenobiotics including the drugs doxorubicin, cisplatin, and carmustine used in this study. Met depletion might allow recovery of the antitumoral efficacy of these cytotoxic agents in drug-refractory tumors by decreasing their resistance potential. In vivo, animals can be starved of Met by replacing the standard diet with a Met-free regimen and simultaneous administration of ethionine. Our results confirm this hypothesis.

We and others have previously described the Met dependency of tumor cells (3 , 11 , 35 , 36) . This led us to design a therapeutic approach using a Met-deprived diet and ethionine, a Met analogue, both contributing to Met depletion in animals. The Met-deprived diet was well tolerated by nude mice when Hcy was added to substitute for Met. Hcy is a precursor in endogenous Met synthesis and is used efficiently by normal cells to maintain their metabolism, but not by tumor cells. Ethionine treatment, combined with a Met-deprived diet, was not toxic up to a daily dose of 1 g/kg body weight. In all tumors tested from various origins, tumor growth inhibition was observed when ethionine treatment was associated with the Met-deprived diet (11) , reaching a value of 80% with significant tumor growth delay, prolonging survival times by 2–3-fold in tumor-bearing animals. Such an antitumoral effect was confirmed in the present study in nude mice bearing TC71-MA, a human colon cancer, and SNB19, a glioblastoma.

Met deprivation acts by reducing proliferation of tumor cells, and this inhibition is not counteracted by Hcy but is reinforced by ethionine. Met depletion decreases the glutathione content, irreversibly blocks cells in S phase and G2 of the cell cycle, and induces apoptosis (10) . Combined with ethionine, it induced a drop in ATP pools. Decreases in glutathione induced in the absence of Met have been described previously (37 , 38) . In the present study, we show that the combination of a Met-deprived diet and ethionine induced a depletion of the glutathione pool in tumors xenografted into nude mice.

The fundamental basis of the antiproliferative effect of Met depletion in tumor cells might be partly due to dNTP imbalance induced by folate deviation toward endogenous Met synthesis, which is responsible for the S-phase blockade. The intracellular Met concentration determines the metabolic priority of folate (39 , 40) . Under normal conditions, folates are used for endogenous Met synthesis and purine and pyrimidine bases. Thymidine, which is essential for DNA reparation and replication, is the methylated analogue of uracil. This methylation reaction, which is catalyzed by thymidylate synthetase, specifically requires 5,10-tetrahydrofolate as a methyl donor. When the regular diet is replaced by a Met-deprived diet, folate is diverted away from DNA synthesis to the resynthesis of Met. As a result, there is an imbalance in the dNTP pool. This, in turn, is known to promote an accumulation of DNA strand breaks (41) , impair DNA repair (42) , and lead to apoptotic death (43 , 44) . DNA strand breaks and replication arrest (45) could also be induced by ethionine treatment. Ethionine, the Met analogue used, is metabolized to S-adenosylethionine and thus might transethylate DNA, RNA, and phospholipids. The DNA ethylation by S-adenosylethionine could prevent polymerase recognition of the ethylated cytosine and induce the S-phase blockade by a replication arrest or DNA strand breaks. DNA hypomethylation induced by Met depletion (46 , 47) can also induce an accumulation of DNA strand breaks (41) .

We decided to combine metabolism-targeted therapy and Met depletion, which induces a decrease in glutathione and ATP content, with chemotherapeutic agents, whose efficacy could inversely depend on the expression of Pgp, MRP, or glutathione. The choice of the cytotoxic agent to be combined with Met depletion was dependent on the type of tumor. For SCLC6, a small cell lung cancer, the association of doxorubicin with Met depletion was based on the drug-refractory phenotype of SCLC6, which expresses Pgp and MRP proteins (48) . The activity of Pgp could be responsible for chemoresistance, and we showed previously that it could be reversed by verapamil (49) . Expression of MRP and of glutathione S-transferase {pi} probably contributes to the resistance of SCLC6. Doxorubicin is used for treating patients (50) , even if it is not the reference treatment of SCLC. SCLC6, a doxorubicin-resistant tumor, was a suitable model to show the capacity of increasing the sensitivity to doxorubicin by Met depletion. Indeed, cell chemosensitivity to doxorubicin is dependent on its intracellular accumulation, with doxorubicin resistance being induced by the alteration of membrane transport such as ATP-dependent mechanisms of efflux or by a high glutathione concentration (51) . The decrease of ATP and glutathione pools induced by Met depletion in the presence of ethionine might lead to the recovery of doxorubicin chemosensitivity. Both SCLCs (SCLC6 and SCLC6-Dox) were very resistant to doxorubicin and expressed MDR1, MRP, and GST{pi}, yet they responded to the combination of doxorubicin with Met depletion by a significant inhibition of tumor growth and prolonged survival, whereas doxorubicin alone had little or no activity. The results observed could also be explained by the DNA intercalation and topoisomerase II inhibition of doxorubicin, which could potentiate the effects of the hypomethylation induced by the Met depletion.

Cisplatin has been used in the treatment of colon cancer (52 , 53) . TC71-MA, a human colon cancer, was poorly sensitive to cisplatin. Several arguments led us to combine cisplatin with Met depletion. Cisplatin interferes with Met transport and acts as an inhibitor of amino acid entry. This was demonstrated in brain tissue (54) , and this could contribute to augment Met depletion in tumor tissue. Alternatively, cisplatin detoxification was found to require glutathione, which forms complexes with this heavy metal (55) , and furthermore, efflux of glutathione-cisplatin complexes is driven by MRP, an ATP-dependent transporter (22) that could be reduced by Met depletion. The TC71-MA tumor displayed a high level of glutathione and expressed MRP, like the majority of colon cancers, and this could explain the lack of efficacy of cisplatin. The association of a Met-deprived diet with cisplatin enhanced the antitumoral efficacy of cisplatin. We hypothesize that the simultaneous administration of a Met-deprived diet decreases the glutathione pool, thereby decreasing the formation of cisplatin-glutathione complexes and hence sensitizing TC71-MA to cisplatin.

Carmustine is the reference drug for the treatment of glioma (56 , 57) . However, its efficacy is limited, like all nitrosoureas, by glutathione detoxification, and a relationship between the response of tumors to carmustine and their glutathione content has been described previously (58) . We hypothesize that the effect of Met depletion and ethionine in decreasing the amount of glutathione could be responsible for the potentiation of the antitumoral efficacy of carmustine observed in SNB19 xenografts.

In conclusion, the potentiation of the antitumoral effect of a Met-deprived diet, ethionine, and chemotherapy could be explained, at least in part, by a decrease in glutathione and available ATP pools induced by Met depletion and ethionine administration, which in turn diminished the resistance potential of cancer cells to the cytotoxic agents. The three tumors are representative of solid tumors refractory to conventional therapy, yet they displayed significant responses to chemotherapy when combined with Met depletion.


   ACKNOWLEDGMENTS
 
We are grateful to V. Bordier and C. Alberti for excellent technical assistance in animal experimentation. We thank Dr. S. Agrawal for helpful critical review and reviewing the English used in this report.


   FOOTNOTES
 
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 Supported by the Association sur les Tumeurs Cérébrales (ARTC), the Association pour la Recherche sur le Cancer (ARC), and Luxembourg government Grant R/D BFR 95/035. Back

2 To whom requests for reprints should be addressed, at Institut Curie, UMR 147 CNRS-Institut Curie, 26 rue d’Ulm, 75231 Paris Cedex 05, France. Phone: 33-1-42-34-66-67; Fax: 33-1-42-34-66-74; E-mail: mfpoupon{at}curie.fr Back

3 The abbreviations used are: Met, methionine; Hcy, homocysteine; RTV, relative tumor volume; Pgp, P-glycoprotein; dNTP, deoxynucleoside triphosphate; SCLC, small cell lung cancer; MRP, multidrug related protein. Back

Received 5/24/99; revised 11/ 1/99; accepted 11/ 1/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Glutathione Assay
 In Vivo Studies
 RESULTS
 DISCUSSION
 REFERENCES
 

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