Abstract
Background/Aim: Trabectedin is a DNA-binding agent that has shown moderate efficacy for soft-tissue sarcomas. We have previously shown that methionine restriction enhances trabectedin efficacy on both parental and trabectedin-resistant HT1080 (TR-HT1080) cells in vitro. The aim of the present study was to determine whether fibrosarcoma cells that acquire trabectedin resistance become more malignant but maintain sensitivity to methionine restriction in vivo.
Materials and Methods: TR-HT1080 was established by culturing HT1080 cells in stepwise increasing concentrations of trabectedin. An in vitro wound-healing invasion assay was used to compare malignancy of HT1080 and TR-HT1080. In vivo, six groups were established: G1-G4 (TR-HT1080): G1, untreated control; G2, trabectedin treatment; G3, methionine-restricted diet; G4, methionine-restricted diet combined with trabectedin; G5, untreated control of parental HT1080; and G6, trabectedin treatment of parental HT1080.
Results: The IC50 of trabectedin was previously determined to be 3.3 nM for the parental HT1080 cells and 42.9 nM for trabectedin-resistant HT1080 cells, representing a 13-fold increase. Wound-healing invasion assays in vitro showed a more rapid wound-closure ratio in TR-HT1080 cells than in parental cells, suggesting increased malignancy compared to the parental cells. The volume of untreated TR-HT1080 tumors grew more rapidly than that of HT1080 tumors, indicating a higher malignancy of TR-HT1080 tumors. The IC50 of recombinant methioninase was previously determined as 0.75 U/ml for HT1080 and 0.93 U/ml for TR-HT1080 cells. Methionine restriction was highly effective on TR-HT1080 tumors, decreasing tumor growth by 4-fold.
Conclusion: TR-HT1080 cells acquired high malignancy by in vitro selection for trabectedin resistance. However, methionine restriction overcame trabectedin resistance in vivo, strongly inhibiting tumor growth, which should be further investigated in the clinic.
- Methionine restriction
- trabectedin
- HT1080
- fibrosarcoma
- trabectedin-resistance
- malignancy
- methionine addiction
- Hoffman effect
Introduction
Soft-tissue sarcomas are heterogeneous neoplasms originating from mesenchymal tissues, often have resistance to standard chemotherapy and radiation therapy in the clinic.
Trabectedin was originally derived from a sea squirt and is now produced by a semisynthetic process using Safracin B, which is obtained from fermenting Pseudomonas fluorescensa (1). Trabectedin has demonstrated potential in the treatment of soft-tissue sarcomas by binding to DNA and disrupting the transcription processes (2, 3). Although trabectedin has demonstrated efficacy in certain cases, resistance to trabectedin can develop over time, which significantly limits its clinical utility (4). The commercial name of trabectedin is Yondelis.
We have previously shown that HT1080 and trabectedin-resistant HT1080 (TR-HT1080) are sensitive to recombinant methioninase in vitro, as are all cancer cells tested, compared to normal fibroblasts (5-8).
In the present study, we show that TR-HT1080 cells have acquired greater malignancy but retain sensitivity to methionine restriction in vivo.
Materials and Methods
Cell culture. The HT1080 human fibrosarcoma cell line was from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured 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.
Reagents. Trabectedin was obtained from PharmaMar (Horsham, PA, USA).
Establishment of trabectedin-resistant HT1080 (TR-HT1080). Trabectedin-resistant HT1080 cells (TR-HT1080) were previously established by culturing parental HT1080 cells in progressively increasing concentrations (3.3-8 nM) of trabectedin over three months (6).
Wound healing invasion assay. HT1080 and TR-HT1080 cells were seeded in 6-well plates with standard DMEM culture medium and incubated overnight at 37°C. Wounds were generated by scratching the cell monolayers with a 200 μl pipette tip. After scratching, the plates were rinsed twice with phosphate-buffered saline (PBS; #MB1039-1X, BioPioneer Inc., San Diego, CA, USA) to eliminate detached cells, and then re-incubated with DMEM at 37°C. The wound areas were visualized under a light microscope (Olympus IX71, Olympus Corporation, Tokyo, Japan) at zero, four and eight hours post-wounding. Each experiment was conducted in triplicate and repeated twice.
Mice. Athymic nu/nu nude mice aged 4-6 weeks were from AntiCancer Inc. (San Diego, CA, USA). All animal experiments were conducted following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of AntiCancer Inc. The procedures adhered to the guidelines set by the National Institutes of Health (NIH) for the Care and Use of Laboratory Animals and followed the ARRIVE guidelines 2.0.
Methionine-restricted diet. A methionine-deficient diet (TD. 210562, Inotiv, Inc., West Lafayette, IN, USA) was used in the present study.
Establishment of subcutaneous HT1080 and TR-HT1080 tumors. To establish subcutaneous tumors, nude mice were injected in the right flank with 1×106 HT1080 or TR-HT1080 cells. One month post-injection, resulting subcutaneous tumors were excised, sectioned into 3-4 mm3 fragments, and re-implanted into the right flank of additional nude mice. Two weeks following implantation, tumors were established for experimental use.
Treatment protocol. The TR-HT1080 nude mouse models were randomized into four groups once the tumor volume exceeded 100 mm3: Group 1: untreated control; Group 2: trabectedin [intravenous (iv), 0.15 mg/kg (once a week, two weeks)]; Group 3: methionine-restricted diet; Group 4: trabectedin [iv, 0.15 mg/kg (once a week, two weeks)] and a methionine-restricted diet; The parental HT1080 cells comprised groups 5 and 6: Group 5: untreated control; and Group 6 trabectedin [iv, 0.15 mg/kg (once a week, two weeks)]. Each group contained five mice. Tumor volume and body weight were measured twice a week. The calculation of tumor volume was performed using the formula: tumor volume (mm)=length (mm) × width (mm)× width (mm) ×1/2.
Statistical analysis. Statistical analyses were conducted using EZR software (Jichi Medical University, Saitama, Japan). The relationships between variables were analyzed using the Welch’s t-test, with a p-value of ≤0.05 considered statistically significant.
Results
Increased in vitro wound healing by TR-HT1080 cells. A wound-healing invasion assay in vitro was performed to assess the cell invasion capability of HT1080 and TR-HT1080 cells. TR-HT1080 cells had a 2.2 fold more rapid wound-closure rate than HT1080 cells (p<0.05) (Figure 1). These results suggest that TR-HT1080 cells had a greater level of malignancy in vitro than the parental cells.
Trabectedin-resistant HT1080 (TR-HT1080) cells have more rapid wound healing than parental HT1080. Black lines indicate the boundaries f the wound of the cells grown in Dulbecco’s Modified Eagle’s Medium (DMEM). Wounds were made by scratching the cell monolayers with a 200 μl pipette tip. Scale bar: 100 μm. Magnification: 40×. Please see Materials and Methods for details.
TR-HT1080 tumor growth in nude mice was greater than HT1080. In vivo experiments further confirmed the greater malignancy of TR-HT1080 cells. Over 14 days in nude mice, the volume of TR-HT1080 tumors, without treatment, increased 4.4 fold more than HT1080 tumors, indicating that TR-HT1080 tumors had more malignancy (Figure 2).
Tumor growth of untreated trabectedin-resistant HT1080 (TR-HT1080) tumors increased compared to HT1080 tumors, indicating that TR-HT1080 tumors had more malignancy. Tumors were grown subcutaneously in nu/nu nude mice. Please see Materials and Methods for details.
Trabectedin resistance in vivo. The volume of HT1080 tumors treated with trabectedin was inhibited 4.6 fold compared to TR-HT1080 tumors, indicating that TR-HT1080 tumors were more resistant to trabectedin (Figure 3).
Trabectedin-resistant HT1080 (TR-HT1080) tumors with trabectedin treatment grew more than HT1080 tumors with trabectedin treatment in nude mice, indicating that TR-HT1080 tumors were more resistant to trabectedin. Please see Materials and Methods for details.
Efficacy of methionine restriction on TR-HT1080 and HT1080 tumor growth. Methionine restriction had substantial efficacy in inhibiting TR-HT1080 tumor growth. In mice fed a methionine-restricted diet, TR-HT1080 tumor growth decreased significantly (4-fold reduction, p<0.05) compared to a regular diet (Figure 4) without causing more than 10% weight loss, demonstrating its tolerability (Figure 5). However, the combination of methionine restriction with trabectedin treatment did not result in additional synergistic efficacy against TR-HT1080 tumors (Figure 4).
Efficacy of a methionine-restricted diet (MR) and trabectedin, alone, and in combination on trabectedin-resistant HT1080 (TR-HT1080) tumors in nude mice. Please see Materials and Methods for details.
Effect of the treatment protocol on the body weight of nude mice with trabectedin-resistant HT1080 (TR-HT1080) and parental HT1080 tumors. Data are shown as the mean±standard deviation. Please see Materials and Methods for details.
Discussion
Acquired resistance to trabectedin a is a recalcitrant clinical problem. We have previously established trabectedin-resistant HT1080 fibrosarcoma cells (TR-HT1080) with a 13-fold increase in IC50 – from 3.3 nM [data from (5)] in parental HT1080 cells to 42.9 nM in TR-HT1080 cells [data from (6)]. In the present study, we have shown that trabectedin resistance was associated with enhanced invasiveness in vitro, as shown by an increased wound closure ratio in TR-HT1080 cells compared to the parental HT1080, which is consistent with findings in other models of trabectedin-resistant cancer cells (4). We also demonstrated a 4.4 fold increase in tumor growth volume for TR-HT1080 compared to parental HT1080 over 14 days in nude mice. This result indicates that trabectedin resistance caused TR-HT1080 to be more malignant than parental HT1080. Previous results have suggested that drug resistance can enhance cancer cells to be more malignant (9, 10).
The present results demonstrate that methionine restriction alone inhibited TR-HT1080 tumor growth in nude mice by 4-fold. rMETase inhibited TR-HT1080 cells similar to HT1080 in vitro (5, 6). rMETase sensitized chemoresistant cancer cells to various chemotherapeutic drugs (6, 8, 11-15).
In conclusion, the present study suggests that methionine restriction may serve as an effective therapy for trabectedin-resistant sarcomas. Further investigation is warranted to elucidate the molecular mechanisms of the relationship between cancer drug resistance and increased malignancy. Additionally, clinical studies are necessary to assess the efficacy of methionine restriction as a therapeutic approach for trabectedin-resistance soft-tissue sarcomas.
Methionine restriction is effective because it tragets the fundamental hallmark of cancer (16-36). rMETase is showing promise in clinical caner as an oral supplement (37-48).
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, PhD, 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. HQ, KM, BMK, MB, NY, KH, HK, SM, KI, TH, HT, and SD critically reviewed the manuscript. SM performed the experiments. SM was a major contributor to writing the manuscript and RMH revised the paper. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
- Received December 5, 2024.
- Revision received December 15, 2024.
- Accepted December 16, 2024.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
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).
