Abstract
Background/Aim: The aim of the present study was to determine the synergy of recombinant methioninase (rMETase) and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison to normal fibroblasts, in vitro. Materials and Methods: HT1080 human fibrosarcoma cells and HS27 human fibroblasts were used for in vitro experiments. Four groups were analyzed in vitro: No-treatment control; eribulin; rMETase; eribulin plus rMETase. Dual-color HT1080 cells which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize cytoplasmic and nuclear dynamics during treatment. Results: Eribulin combined with rMETase greatly decreased the viability of HT 1080 cells. In contrast, eribulin combined with rMETase did not show synergy on Hs27 normal fibroblasts. Eribulin combined with rMETase also caused more fragmentation of the nucleus than all other treatments. Conclusion: The combination treatment of eribulin plus rMETase demonstrated efficacy on fibrosarcoma cells in vitro. In contrast, normal fibroblasts were resistant to this combination, indicating the potential clinical applicability of the treatment.
- Methioninase
- eribulin
- combination
- synergy
- fibrosarcoma
- normal fibroblasts
- in vitro
- methionine addiction
- Hoffman effect
- methionine restriction
Soft tissue sarcomas (STSs) are uncommon tumors that account for 1% of all cancer cases (1). The World Health Organization (WHO) has currently divided this heterogeneous collection of tumors into more than 100 histological subgroups (2).
Eribulin is an anti-tubulin agent that can block mitosis. Eribulin was approved for patients with unresectable or metastatic STSs who had previously received anthracycline-containing therapy (3).
In our laboratory, the Pseudomonas putida methioninase gene was cloned and over-expressed in Escherichia coli, yielding high amounts of recombinant methioninase (rMETase) (4). Numerous studies of rMETase or methionine-free medium or a methionine-depleted diet combined with chemotherapy have demonstrated synergistic efficacy (5-37). The aim of the present study was to determine the synergy of rMETase and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison with normal fibroblasts in vitro.
Materials and Methods
Cell culture. HT1080 and Hs27 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (10-013-CV; Corning, Corning, NY, USA) with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin.
Regents. Eribulin was obtained from Eisai Inc. (Nutley, NJ, USA). Recombinant methioninase (rMETase) was produced in AntiCancer Inc. (San Diego, CA, USA). The method for producing rMETase has been previously reported (4).
Drug sensitivity assay 1: IC50. Cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). Cells (HT1080 or Hs27) were cultured in 96-well plates (3,000 cells/well) in DMEM (100 μl/well) and incubated at 37°C overnight. Cells were treated with increasing concentrations of eribulin, between 0.5 nM and 8 nM; or rMETase, between 0.5 U/ml and 8 U/ml for 72 h. At the end of the culture period, 10 μl of the WST-8 solution were added to each well and the plate was additionally incubated for 1 h at 37°C. Absorption was measured with a microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland) at 450 nm. Drug sensitivity curves were obtained with Microsoft Excel for Mac 2016 ver. 15.52 (Microsoft, Redmond, WA, USA) and half-maximal inhibitory concentration (IC50) values were calculated using ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA). Experiments were performed twice, each in triplicate.
Drug sensitivity assay 2: Synergy. Cells (HT1080 or Hs27) were seeded at 3,000 cells/well in 96-well plates. Twenty-four hours later, four treatment groups were established [1: Control (DMEM); 2: eribulin (IC50); 3: rMETase (IC50); 4: eribulin (IC50) plus rMETase (IC50)]. Seventy-two hours later, cell viability was measured in triplicate as described above.
Nuclear fragmentation assay. To visualize nuclear and cytoplasm dynamics, dual-color HT1080 cells, which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, were generated as previously described (38-43). These cells were observed with an IX71 microscope (Olympus, Tokyo, Japan) at ×200 magnification. The cells were seeded at 300,000 cells/well in 6-well plates. Treatment Groups were as follows: 1: control (DMEM); 2: eribulin (0.15 nM); 3: rMETase (0.75 U/ml); 4: eribulin (0.15 nM) plus rMETase (0.75 U/ml). Forty-eight hours later, the number of fragmented nuclei was counted per field of view in six fields.
All statistical analyses were conducted using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan). Associations between variables were tested using a Tukey-Kramer analysis. p-Values ≤0.05 were considered statistically significant.
Results
Drug sensitivity assay 1: IC50 of eribulin and rMETase on HT1080 and Hs27 cells. The IC50 value of eribulin for HT1080 cells was 0.15 nM. The IC50 of rMETase for HT1080 cells was 0.75 U/ml. The IC50 of eribulin for Hs27 was 0.43 nM. The IC50 of rMETase for Hs27 cells was 0.93 U/ml (Figure 1).
Eribulin and recombinant methioninase (rMETase) sensitivity of HT1080 and Hs27 cells (mean±SD, n=3). A) Sensitivity to eribulin of HT1080 cells. B) Sensitivity to rMETase of HT1080 cells. C) Sensitivity to eribulin of Hs27 cells. D) Sensitivity to rMETase of Hs27 cells.
Drug sensitivity assay 2: Synergy of eribulin and rMETase on HTT1080 and Hs27 cells. The combination of eribulin (0.15 nM) plus rMETase (0.75 U/ml) was the most effective on HT1080 cells (p<0.05). In contrast, eribulin (0.43 nM) plus rMETase (0.93 U/ml) did not show synergy in Hs27 cells (Figure 2A and B).
Efficacy of eribulin and recombinant methioninase (rMETase) and their combination on cancer and normal cells. A) HT1080 cells. 1: Control (DMEM); 2: eribulin (0.15 nM); 3: rMETase (0.75 U/ml); 4: eribulin (0.15 nM) plus rMETase (0.75 U/ml). B) Hs27 fibroblasts. 1: Control (DMEM); 2: Eribulin (0.43 nM); 3: rMETase (0.93 U/ml); 4: Eribulin (0.43 nM) plus rMETase (0.93 U/ml).
Nuclear fragmentation assay. The combination of eribulin (0.15 nM) plus rMETase (0.75 U/ml) caused more fragmentation of the nucleus in HT1080 cells than all other treatment groups (p<0.05) (Figure 3A-C).
Efficacy of eribulin, recombinant methioninase (rMETase) and their combination on nuclear fragmentation in HT 1080 cells. A) Images of nuclei expressing GFP 1: Control (DMEM); 2: eribulin (0.15 nM); 3: rMETase (0.75 U/ml); 4: eribulin (0.15 nM) plus rMETase (0.75 U/ml). Scale bars: 100 μm. B) Nuclear fragmentation quantitation. 1: Control (DMEM); 2: eribulin (0.15 nM); 3: rMETase (0.75 U/ml); 4: eribulin (0.15 nM) plus rMETase (0.75 U/ml).
Discussion
Patients with advanced soft tissue sarcoma have a poor prognosis, with a median overall survival of little under 19 months (44). Patients with metastatic and irresectable soft tissue sarcoma who were treated with eribulin had a median overall survival of 13.2-13.5 months and progression-free survival of 2.6-4.1 months according to the Phase 2 and 3 clinical studies (3, 45). Eribulin is a second-line chemotherapy for soft tissue sarcoma, however, its efficacy is limited.
In 1976, our laboratory first identified methionine addiction as a fundamental hallmark of cancer (46-51). To limit methionine and target methionine addiction, our group developed rMETase. We later discovered that rMETase may be administrated orally (o-rMETase), making it a very practical and safe therapeutic (25, 51).
In 1986 our laboratory discovered synergy of methionine restriction and chemotherapy (5, 50). Many different combinations of rMETase and chemotherapy have shown synergy when used together (6-36). In the present study, we showed the extensive synergy between rMETase and eribulin in HT1080 cells. The mechanism of synergy of tubulin-targeting agents with rMETase is that they kill cancer cells as they enter the G2/M phase after they escape the S/G2 cell-cycle block caused by methionine restriction (50, 52, 53). In contrast, eribulin plus rMETase did not show synergy in Hs27 cells because normal cells are not arrested by rMETase at the IC50 for cancer cells. The methionine addiction of cancer cells causes them to arrest for S/G2 when methionine is restricted (50, 52, 53).
Eribulin targets tubulin and reduces the dynamic stability of microtubules (55). Sampson et al. (56) showed the instability and fragmentation of the nucleus by eribulin. Our group showed that staurosporine caused numerous nuclear fragments on dual-color HT1080 cells (43). Eribulin combined with rMETase caused higher nuclear fragmentation than either agent alone (Figure 3). rMETase may enhance eribulin-induced destabilization of the nucleus by affecting its binding to tubulin.
Oral rMETase is showing promise in the clinic, especially in combination with chemotherapy (15, 22, 52, 69-74)
In conclusion, the synergy of rMETase with eribulin on fibrosarcoma cells has therapeutic potential for soft tissue sarcoma because it targets the fundamental hallmark of cancer in methionine addiction (46-49, 51, 54, 57-68).
Acknowledgements
This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Gordon H. Sato, Ph.D, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, and J.A.R Mead, Ph.D. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.
Footnotes
Authors’ Contributions
SM, HQ, YK, KM, BMK, MS, MB, NY, KH, HK, SM, KI, TH, HT and RMH designed the study. SM performed 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 October 26, 2023.
- Revision received November 20, 2023.
- Accepted November 21, 2023.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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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