Abstract
Background/Aim: Drug resistance has been a recalcitrant problem for sarcoma patients for many decades. Trabectedin is a second-line chemotherapy for soft-tissue sarcoma that often leads to resistance and death of the patients. The objective of the present study was to address the issue of trabectedin-chemoresistance in HT1080 fibrosarcoma cells by combining recombinant methioninase (rMETase) with trabectedin and examining their efficacy on trabectedin-resistant fibrosarcoma cells in vitro. Materials and Methods: Trabectedin-resistant HT1080 (TR-HT1080) cells were generated by subjecting HT1080 human fibrosarcoma cells to increasing trabectedin concentrations (3.3-8 nM). IC50 values for trabectedin and rMETase were compared for HT1080 and TR-HT1080 cells. TR-HT 1080 cells were placed into four groups to determine synergy of rMETase and trabectedin on TR-HT1080 cells: a control group with no treatment; a group treated with trabectedin (3.3 nM); a group treated with rMETase (0.75 U/ml); and a group treated with both trabectedin (3.3 nM) and rMETase (0.75 U/ml). Results: The IC50 value of trabectedin- on TR-HT1080 cells was 42.9 nM, whereas the IC50 value of trabectedin on the parental HT1080 cells was 3.3 nM, indicating a 13-fold increase. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) was synergistic on TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%) (p<0.05). rMETase increased the efficacy of trabectedin 11-fold on trabectedin-resistant fibrosarcoma cells. Conclusion: The combined administration of trabectedin and rMETase was synergistic on the viability of TR-HT1080 cells in vitro. The combination of rMETase and trabectedin has promising clinical potential for overcoming chemo-resistance of soft-tissue sarcoma.
- Methioninase
- methionine restriction
- DNA binding
- synergistic efficacy
- trabectedin-resistance
- fibrosarcoma
- methionine addiction
- Hoffman effect
Trabectedin attaches to the minor groove of DNA, resulting in the curvature of the minor groove towards the major groove and inhibition of DNA synthesis (1). Trabectedin has been effective in clinical studies for treating soft-tissue sarcoma, and it has been approved as a second-line therapy for soft-tissue sarcoma (2-8). In a phase III clinical trial, 378 patients with advanced liposarcoma or leiomyosarcoma were given trabectedin. Of these patients, 270 patients had disease progression and 258 patients died (9).
Recombinant methioninase (rMETase) targets the fundamental and general hallmark of cancer, methionine addiction, termed the Hoffman Effect (10, 11). Numerous studies have shown that chemotherapy is more effective when combined with rMETase, methionine-free media, or a methionine-depleted diet, due to their synergy (12-53). We have previously shown synergy of rMETase and trabectedin on the HT1080 fibrosarcoma cells in vitro (49).
The present study aimed to investigate whether the combination of rMETase and trabectedin in vitro can overcome trabectedin-resistance.
Materials and Methods
Cell culture. The American Type Culture Collection (Manassas, VA, USA) was the source of the HT1080 cell line. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin.
Regents. Trabectedin was obtained from PharmaMar (Horsham, PA, USA). AntiCancer Inc. (San Diego, CA, USA) produced recombinant methioninase (rMETase). The rMETase production process was as previously described (54).
Establishment of trabectedin-resistant HT1080 (TR-HT1080). TR-HT1080 cells were established by culturing HT1080 in stepwise increasing concentrations (3.3-8 nM) of trabectedin for three months.
Drug sensitivity assay 1 – IC50. The WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan) was used to evaluate cell viability (49). Cells (HT1080 or ER-HT1080) were cultured in 96-well plates (3,000 cells/well) in DMEM (100 μl/well) and incubated at 37°C overnight. HT1080 or TR-HT1080 cells were incubated for 72 h with trabectedin at concentrations ranging from 2 nM to 128 nM or rMETase at concentrations ranging from 0.5 U/ml to 8 U/ml. At the end of the culture period, 10 μl of the WST-8 solution was added to each well, and the plate was further incubated at 37°C for 1 h. Absorption was measured at 450 nm using a microplate reader (Sunrise: Tecan, Mannedorf, Switzerland). The half-maximal inhibitory concentration (IC50) values were determined using ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA). Drug-sensitivity curves were generated using Microsoft Excel for Mac 2016 ver. 15.52 (Microsoft, Redmond, WA, USA). Each experiment was conducted twice in triplicate.
Drug sensitivity assay 2 – Synergy. TR-HT1080 cells were seeded at a density of 3,000 cells per well in 96-well plates. Twenty-four hours later, four treatment groups were established: Control (DMEM); trabectedin (3.3 nM); rMETase (0.75 U/ml); and trabectedin (3.3 nM) plus rMETase (0.75 U/ml). Seventy-two hours later, cell viability was assessed in triplicate as described above.
EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) was used to conduct all statistical analyses (55). Tukey-Kramer analysis was employed to investigate the association between variables. Results with p values ≤ 0.05 were considered significant.
Results
Drug sensitivity assay 1 – IC50 of trabectedin and rMETase on HT1080 and TR-HT1080 cells. The IC50 value of trabectedin on HT1080 was 3.3 nM [data from (49)]. The IC50 of trabectedin on TR-HT1080 was 42.9 nM. The IC50 for rMETase on HT1080 was 0.75 U/ml [data from (44)]. The IC50 for rMETase on TR-HT1080 was 0.93 U/ml (Figure 1).
IC50 of trabectedin and rMETase on HT1080 and TR-HT1080 cells (mean±SD, n=3). A) Sensitivity of HT1080 cells to trabectedin [data from (49)]. B) Sensitivity of TR-HT1080 cells to trabectedin. C) Sensitivity of HT1080 cells to rMETase [data from (44)]. D) Sensitivity of TR-HT1080 cells to rMETase.
Drug sensitivity assay 2 – Synergy. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) had synergistic efficacy on the viability of TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%) (p<0.05) (Figure 2).
Synergy of the combination of trabectedin and rMETase on TR-HT1080 cells. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) had synergistic efficacy on the viability of TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%). TR-HT80: trabectedin-resistant HT1080; rMETase: recombinant methioninase.
Discussion
Trabectedin has moderate clinical efficacy for soft tissue sarcoma as 2nd-line treatment, however, drug resistance can still develop after long periods of treatment (5, 6). The development of drug resistance in cancer cells is a result of complex cellular and molecular processes that ultimately result in the recurrence of the disease and progression (56). Alternative treatments are necessary for patients with resistant soft-tissue sarcoma.
In the present study, TR-HT1080 cells were 13-fold-more resistant to trabectedin than HT1080 cells. However, TR-HT1080 cells were sensitive to rMETase similar to parental HT1080 cells. The combination of trabectedin (3.3 nM) and rMETase (0.75 U/ml) (the IC50 values for HT1080 cells) on TR-HT1080 cells resulted in a 11-fold increase in the efficacy of trabectedin.
In conclusion, the combination of trabectedin plus rMETase had synergistic efficacy on the viability of TR-HT1080 cells in vitro. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin overcoming chemo-resistance of soft-tissue sarcoma.
The efficacy of rMETase is because it targets the fundamental and general hallmark of cancer, methionine addiction, which has clinical potential (22, 29, 46, 57-86).
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, and Joseph Leighton, MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.
Footnotes
Authors’ Contributions
SM and RMH designed the study. QH provided rMETase. SM performed experiments. SM was the major contributor to writing the article and RMH revised the article. KM, BMK, MS, MB, NY, KH, HK, SM, KI, TH, HT, and SD critically read the manuscript.
Conflicts of Interest
The Authors have declared that there are no competing interests in relation to this study.
- Received July 11, 2024.
- Revision received July 29, 2024.
- Accepted July 30, 2024.
- Copyright © 2024 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).
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