Research ArticleExperimental Studies
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Synergistic Eradication of Fibrosarcoma With Acquired Ifosfamide Resistance Using Methionine Restriction Combined With Ifosfamide in Nude-mouse Models

SEI MORINAGA, QINGHONG HAN, KOHEI MIZUTA, BYUNG MO KANG, MICHAEL BOUVET, NORIO YAMAMOTO, KATSUHIRO HAYASHI, HIROAKI KIMURA, SHINJI MIWA, KENTARO IGARASHI, TAKASHI HIGUCHI, HIROYUKI TSUCHIYA, SATORU DEMURA and ROBERT M. HOFFMAN
In Vivo January 2025, 39 (1) 120-126; DOI: https://doi.org/10.21873/invivo.13809

Abstract

Background/Aim: Ifosfamide is used clinically with doxorubicin as first-line chemotherapy for soft-tissue sarcoma. However, ifosfamide efficacy for soft-tissue sarcoma is limited due to frequent occurence of ifosfamide resistance and thus more effective therapy is needed. The present study aimed to determine the synergy of recombinant methioninase (rMETase) plus ifosfamide against HT1080 human fibrosarcoma cells in vitro. Additionally, the present study also investigated the efficacy of a methionine-restricted diet combined with ifosfamide in nude-mouse models of ifosfamide-resistant HT1080 (IR-HT1080). Materials and Methods: Cell viability for HT1080 human fibrosarcoma cells was determined in four groups in vitro: No treatment control; ifosfamide alone; rMETase alone; and a combination of ifosfamide plus rMETase. HT1080 tumors were established in nude mice subcutaneously. The HT1080 tumor models were treated by administering ifosfamide by intraperitoneal injection twice a week, for a total of 11 doses. Surviving tumors were considered ifosfamide resistant (IR-HT1080). Four groups of IR-HT1080 nude-mouse models were subsequently established: Group 1 was a no-treatment control, Group 2 received ifosfamide, Group 3 was given a methionine-restricted diet (MR), and Group 4 received ifosfamide plus MR. Additionally, two groups of nude mice with parental HT1080 subcutaneous tumors were included: Group 5 was a no-treatment control, and Group 6 received ifosfamide for comparison. Results: The 50% inhibitory concentration (IC50) for ifosfamide against HT1080 cells was 0.38 mM. The IC50 for rMETase was 0.75 U/ml for HT1080 cells (data from [4]). The combination of rMETase (0.75 U/ml) plus ifosfamide (0.38 mM) was synergistic against HT1080 fibrosarcoma cells in vitro. The combination of ifosfamide plus MR eradicated the IR-HT1080 tumors in nude-mouse models, while each treatment alone achieved limited tumor inhibition. Conclusion: The present results suggest the combination of MR and ifosfamide has promising potential for overcoming ifosfamide resistance in future clinical applications.

Key Words:

Ifosfamide, in combination with doxorubicin, has been used clinically as first-line chemotherapy for high-grade soft-tissue sarcoma; however, its effectiveness is often limited by acquired ifosfamide resistance. In a phase III clinical study using ifosfamide plus doxorubicin for advanced or metastatic soft-tissue sarcoma, the median overall survival was 14.3 months and the progression-free survival was 7.4 months (1). Many of the patients in the study developed ifosfamide resistance.

The methioninase gene from Pseudomonas putida was cloned and over-expressed in Escherichia coli, enabling the production of recombinant methioninase (rMETase) (2). Many studies have demonstrated that combining either rMETase, a methionine-free medium, or a methionine-depleted diet with chemotherapy resulted in synergistic efficacy against multiple cancer types in mouse models (3-12).

The aim of the present study was to determine the synergy of rMETase and ifosfamide on fibrosarcoma cells in vitro and on ifosfamide-resistant tumors in nude mice.

Materials and Methods

Cell culture. The HT1080 human fibrosarcoma cell line was acquired from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY, USA) and 1 IU/ml penicillin/streptomycin (GIBCO).

Reagents. Ifosfamide was obtained from Baxter (Deerfield, IL, USA). Recombinant methioninase (rMETase) was produced at AntiCancer Inc. (San Diego, CA, USA). The procedure for producing rMETase has been previously described (2).

Drug sensitivity assay 1: IC50. Cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). HT1080 cells were cultured in 96-well plates (3×103 cells/well) in DMEM (100 μl/well) and incubated at 37°C overnight. The cells were treated with various concentrations of ifosfamide, ranging from 0.125 mM to 2 mM; or rMETase, ranging from 0.5 U/ml to 8 U/ml; for 72 h. After the culture period, 10 μl of the WST-8 solution was introduced into each well. The plates were then incubated for an additional hour at 37°C. Absorption was measured at 450 nm using a microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland). Drug-sensitivity curves were generated using Microsoft Excel for Mac 2016 ver. 15.52 (Microsoft, Redmond, WA, USA). Half-maximal inhibitory-concentration (IC50) values were determined using ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA). Experiments were conducted twice, each in triplicate.

Drug sensitivity assay 2: Synergy. HT1080 human fibrosarcoma cells were seeded at a density of 3×10³ cells per well in 96-well plates. After 24 h, the cells were treated as follows: 1) Control (DMEM), 2) ifosfamide alone (0.38 mM [IC50]), 3) rMETase alone (0.75 U/ml [IC50]), and 4) a combination of ifosfamide (0.38 mM) plus rMETase (0.75 U/ml). After 72 h, the viability of the cells was assessed using the method described in Drug Sensitivity Assay 1, which was performed three times.

Mice. 4-6-week-old athymic nu/nu nude mice from AntiCancer Inc. (San Diego, CA, USA) were used. Every experiment was carried out in accordance with an AntiCancer Institutional Animal Care and Use Committee protocol that was specifically authorized for this study. The procedures and principles outlined in the National Institutes of Health Guide for the Care and Use of Animals were strictly followed. Each experiment was carried out in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0.

Establishment of subcutaneous HT1080 tumors and ifosfamide-resistant HT1080 (IR-HT1080) tumors. Nude mice received a subcutaneous injection of 1×106 HT1080 cells in the right flank. One month post-injection, subcutaneous tumors were detected. The subcutaneously-developed tumors were harvested and finely divided into 3-4 mm3 pieces, which were then inserted into the right flank of additional nude mice. Tumors developed within two weeks. To establish IR-HT1080 tumors, 11 ifosfamide intraperitoneal injections (30 mg/kg, twice a week) were performed on the nude mice with HT1080 tumors. The surviving tumors were regarded as IR-HT1080 tumors.

Treatment study protocol. The IR-HT1080 nude-mouse models were randomized into four groups once the tumor volume exceeded 100 mm³, while the parental HT1080 nude mouse models were divided into two groups: Group 1: untreated control (IR-HT1080); Group 2: ifosfamide [intraperitoneal (IP), 30 mg/kg (twice a week, two weeks)] (IR-HT1080); Group 3: methionine-restricted diet (IR-HT1080); Group 4: ifosfamide [IP, 30 mg/kg (twice a week, two weeks)] plus the methionine restricted diet (IR-HT1080); Group 5: untreated control (HT1080); and Group 6 ifosfamide-treated [IP, 30 mg/kg (twice a week, two weeks)] (HT1080). Each group comprised five mice, respectively. Tumor volume and body weight were measured twice a week. The calculation of tumor volume was performed using the formula: tumor volume (mm3)=length (mm)×width (mm)×width (mm)×1/2.

Methionine restricted diet. A methionine-deficient diet (TD. 210562, Inotiv, Inc., West Lafayette, IN, USA) was used in the present study.

Statistical analysis. EZR software (Jichi Medical University, Saitama, Japan) was used for statistical analyses. Tukey-Kramer analysis was used to analyze the relationship between variables. p≤0.05 was considered to be statistically significant.

Results

Drug sensitivity assay 1: IC50 of ifosfamide alone and rMETase alone on HT1080 cells in vitro. The IC50 of ifosfamide on HT 1080 cells was 0.38 mM. The IC50 of rMETase on HT1080 cells was 0.75 U/ml [data from (4)] (Figure 1).

Figure 1.
Figure 1.

Ifosfamide and rMETase sensitivity of HT1080 fibrosarcoma cells in vitro (mean±standard deviation). A) Sensitivity of HT1080 cells to ifosfamide. B) Sensitivity of HT1080 cells to rMETase.

Drug sensitivity assay 2: Synergy of ifosfamide plus rMETase on HT1080 cells. The combination of ifosfamide [0.38 mM (IC50)] and rMETase [0.75 U/ml (IC50)] in vitro was synergistic on HT1080 cells, significantly more effective than either treatment alone (p<0.05) (Figure 2).

Figure 2.
Figure 2.

Efficacy of the combination of ifosfamide and rMETase, alone and in combination on HT1080 fibrosarcoma cells. Control (DMEM); ifosfamide [0.38 mM (IC50)]; rMETase [0.75 U/ml (IC50)]; ifosfamide [0.38 mM (IC50)] and rMETase [0.75 U/ml (IC50)].

Synergy of ifosfamide and methionine restriction on IR-HT1080 tumors in nude mice. The combination of ifosfamide and a methionine-restricted diet synergistically regressed the IR-HT1080 tumors without causing weight loss, demonstrating significantly greater efficacy than either treatment alone (p<0.05) (Figure 3 and Figure 4). The efficacy of ifosfamide on HT1080 relative to an untreated control tended to be greater than the efficacy of ifosfamide on IR-HT1080, compared to an untreated control (Figure 4).

Figure 3.
Figure 3.

The combination of ifosfamide plus methionine restriction eradicated ifosfamide-resistant HT1080 fibrosarcoma (IR-HT1080) in nude mice. A) Time course efficacy of methionine-restricted diet (MR) and ifosfamied alone and in combination on ifosfamide-resistant (IR)-HT1080 tumors in nude mice. Ifosfamide was also tested on HT1080 tumors compared to a no-treatment control. B) IR-HT1080 and HT1080 tumor volume on day 14 for each treatment group. Data are shown as the mean±standard deviation. Please see Materials and Methods for dose, route and schedule of ifosfamide treatment.

Figure 4.
Figure 4.

Effect of the ifosfamide and a methionine-restricted (MR) diet alone and in combination on the body weight of nude mice with ifosfamide-resistant HT1080 (IR-HT1080) and parental HT1080 tumors. Data are shown as the mean±standard deviation. Untreated control (IR-HT1080); ifosfamide (IR-HT1080); methionine-restricted diet (MR) (IR-HT1080); ifosfamide plus methionine restriction (IR-HT1080); untreated control (HT1080); ifosfamide (HT1080).

Discussion

Ifosfamide, an alkylating agent classified as an oxaza-phosphorine, is extensively utilized in the treatment of lung cancer, soft tissue sarcoma, osteosarcoma, and non-Hodgkin’s lymphoma (14). Ifosfamide is currently regarded as the second most-effective agent on soft-tissue sarcomas (1, 15, 16). A retrospective study indicated that ifosfamide rechallenge may be a viable therapeutic option for patients who previously responded or achieved stable disease with ifosfamide therapy. Additionally, clinical efficacy was achieved in patients who were rechallenged with ifosfamide in the second, third, and fourth lines of treatment (17). However, once ifosfamide resistance was established, the prognosis for the patient was poor.

In 1976, our laboratory discovered methionine addiction as a fundamental hallmark of cancer (18-21). A wide variety of combinations of methionine restriction and chemotherapy drugs have shown synergistic efficacy (3-12). Oral rMETase (o-rMETase) has shown promise on high-stage recalcitrant cancer in challenging clinical cases (22-30).

In the present study, we showed that the combination of rMETase and ifosfamide had synergistic efficacy on HT1080 human fibrosarcoma cells in vitro. Ifosfamide is believed to be a pro-drug that is activated in the liver (31). However, in the present study, ifosfamide was found to be active in vitro, but at relatively high concentrations, and had significantly enhanced activity in combination with rMETase. We have previously observed synergy between rMETase and eribulin, trabectedin, docetaxel, and doxorubicin on HT1080 cells in vitro (4, 8, 9, 11-13).

In the present study, we established an IR-HT1080 nude-mouse model to mimic clinical cases by selecting HT1080 subcutaneous tumors that continued to grow despite frequent intraperitoneal administrations of ifosfamide.

A previous gene analysis study showed differential expression of EPB41L3, GADD45A, IER3, OXCT1, UBE2L6, UBE2A, ALPL, and EFNB2 in ifosfamide-resistant osteo-sarcoma, correlating with a more aggressive tumor behavior (32). IR-HT1080 tumors may have abnormalities in some of these genes, which will be investigated in future studies.

In the present study the combination of a methionine-restricted diet and ifosfamide showed extensive synergy for IR-HT1080 tumors in a nude-mouse model with apparent eradication of the tumors (Figure 3).

Methionine restriction is effective because it targets the fundamental cancer hallmark of methionine addiction (18-21, 33-59).

The present study suggests promising clinical potential for the combination of methionine restriction and ifosfamide as a synergistic approach to treating drug-resistant soft-tissue sarcoma.

Acknowledgements

This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, Ph.D., J.A.R Mead, Ph.D., Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, Joseph Leighton, MD and John Mendelsohn, MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.

Footnotes

  • Authors’ Contributions

    SM, and RMH designed the study. SM performed experiments. SM was a major contributor to writing the manuscript and RMH revised the paper. HQ, KM, BMK, MB, NY, KH, HK, SM, KI, TH, HT, SD critically read the manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare no competing interests in relation to this study.

  • Received September 30, 2024.
  • Revision received October 15, 2024.
  • Accepted October 16, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Synergistic Eradication of Fibrosarcoma With Acquired Ifosfamide Resistance Using Methionine Restriction Combined With Ifosfamide in Nude-mouse Models
SEI MORINAGA, QINGHONG HAN, KOHEI MIZUTA, BYUNG MO KANG, MICHAEL BOUVET, NORIO YAMAMOTO, KATSUHIRO HAYASHI, HIROAKI KIMURA, SHINJI MIWA, KENTARO IGARASHI, TAKASHI HIGUCHI, HIROYUKI TSUCHIYA, SATORU DEMURA, ROBERT M. HOFFMAN
In Vivo Jan 2025, 39 (1) 120-126; DOI: 10.21873/invivo.13809
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