Research ArticleExperimental Studies
Open Access

Super Methotrexate-resistant Osteosarcoma Cells Retain Their Sensitivity to Recombinant Methioninase: Targeting Methionine Addiction to Overcome Extreme Cancer-Chemotherapy Resistance

YUSUKE AOKI, QINGHONG HAN, YUTARO KUBOTA, NORIYUKI MASAKI, YASUNORI TOME, MICHAEL BOUVET, KOTARO NISHIDA and ROBERT M. HOFFMAN
Anticancer Research March 2025, 45 (3) 929-934; DOI: https://doi.org/10.21873/anticanres.17480

Abstract

Background/Aim: Drug-resistance in osteosarcoma results in a very poor clinical prognosis and has been a recalcitrant problem over many decades. We have previously reported the development of super methotrexate (MTX)-resistant osteosarcoma cells (143B-MTXSR), selected from parental 143B osteosarcoma cells (143B-P) 143B-MTXSR cells were previously selected by culturing the cells with increasing concentrations of MTX, resulting in osteosarcoma cells which are 5,500 times more MTX-resistant than the parental cells, due to extreme over-expression of dihydrofolate reductase (DHFR). In the present study, the potential therapeutic efficacy of methionine restriction, using recombinant methioninase (rMETase), was explored to overcome super MTX-resistant osteosarcoma cells.

Materials and Methods: Previously-selected 143B-MTXSR cells were used for the present study. Sensitivity to methionine restriction by rMETase was determined using the WST-8 assay and compared between 143B-MTXSR and parental 143B-P cells.

Results: 143B-MTXSR cells (rMETase IC50: 0.38 U/ml) were very sensitive to methionine restriction by rMETase, very similar to 143B-P (rMETase IC50: 0.36 U/ml).

Conclusion: rMETase overcame a 5,500-fold MTX-resistance of osteosarcoma cells. The present results suggest methionine restriction by rMETase can be a potential clinical strategy to overcome recalcitrant drug-resistance in osteosarcoma.

Keywords:

Introduction

In osteosarcoma, 60-70% of patients are sensitive to first-line chemotherapy, including methotrexate (MTX), doxorubicin (DOX), and cisplatinum (CDDP). However, patients who develop resistance to these drugs have a very poor prognosis, with the 5-year survival rate approximately 20%, which has not been improved for three decades (1-5). The development of a novel therapeutic strategy is required to overcome the drug-resistance of osteosarcoma.

Methionine addiction is a fundamental and general hallmark of cancer cells, was discovered by one of us (RMH) 50 years ago and is termed the Hoffman Effect (6-11). Larger amounts of exogenous methionine are required by cancer cells, compared to normal cells, despite high levels of endogenous methionine synthesis in the cancer cells, due to overuse of methionine for highly increased transmethylation reactions (12-21). We have previously reported that all types of cancers, including osteosarcoma, are addicted to exogenous methionine (16, 19-24).

We have previously selected super MTX-resistant osteosarcoma (143B-MTXSR) cells from parental 143B osteosarcoma (143B-P) cells by culturing them with stepwise increasing concentrations of MTX, resulting in 143B-MTXSR cells which are 5,500 times more MTX-resistant than the parental cells (25, 26).

143B-MTXSR cells have reduced malignancy, along with an increase of histone H3K9me3 and H3K27me3 methylation (25, 26).

In the present study, we explored a potential therapeutic strategy for extreme drug-resistance in osteosarcoma, by targeting methionine addiction with methionine restriction, effected by recombinant methioninase (rMETase), on 143B-MTXSR osteosarcoma cells compared to their parental 143B-P osteosarcoma cells.

Materials and Methods

Cell culture. The 143B human osteosarcoma cell line, purchased from the American Type Culture Collection (#CRL-8303, Manassas, VA, USA), and the super MTX resistant cells 143B-MTXSR cells (25, 26) were used in the present study. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Corning Inc., Corning, NY, USA), supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1 IU/ml).

Selection of super methotrexate-resistant osteosarcoma cells. Super methotrexate-resistant 143B osteosarcoma cells (143B-MTXSR) were previously selected from parental 143B osteosarcoma cells (143B-P) by culturing the cells the cells with increasing concentrations of MTX (#HY-14519, MedChemExpress, Monmouth Junction, NJ, USA) (0.04 μM to 100 μM) for 12 months (25, 26).

Reagents. Recombinant methioninase (rMETase) (AntiCancer Inc., San Diego, CA, USA) is a homotetrameric enzyme, with a 172-kDa molecular mass. rMETase was produced by fermentation of E. coli engineered with the Pseudomonas putida methioninase gene, as previously reported (27).

rMETase sensitivity assay. 143B-MTXSR and 143B-P cells were cultured in 96-well plates (1.0×103 cells/well) with DMEM (100 μl/well) at 37°C overnight. The cells were treated with increasing concentrations of rMETase for 72 h, as follows: 0 U/ml; 0.025 U/ml; 0.05 U/ml; 0.1 U/ml; 0.2 U/ml; 0.4 U/ml; 0.8 U/ml; 1.6 U/ml. Cell viability was measured with the Cell Counting Kit-8 (#CK04, Dojindo laboratory, Kumamoto, Japan) after rMETase treatment. rMETase-sensitivity cell-survival curves and half-maximal inhibitory concentrations (IC50) were generated and analyzed with Microsoft Excel for Mac ver. 16.86 (Microsoft, Redmond, WA, USA) and ImageJ ver. 1.53k (National Institutes of Health, Bethesda, MD, USA), as previously described (16, 19, 20, 24). Experiments were performed twice.

Results and Discussion

Super MTX-resistant 143B-MTXSR cells were 5,500 times more resistant to MTX than parental 143B-P cells as shown by the MTX IC50 of 147.5 μM on 143B-MTXSR cells, due to extreme overexpression of dihydrofolate reductase (DHFR), compared to the IC50 of 0.027 μM MTX on parental 143B-P cells (Table I) [data from (26)].

View this table:
Table I.

Resistance to methotrexate (MTX) of super methotrexate-resistant 143B osteosarcoma (143B-MTXSR) cells compared to parental 143B osteosarcoma (143B-P) cells [data from (26)], in contrast to the sensitivity to recombinant methioninase (rMETase) of both 143B-MTXSR and 143B-P cells.

Super MTX-resistant 143B-MTXSR cells showed sensitivity to methionine restriction by rMETase, very similar to parental 143B-P, with the following IC50: 143B-MTXSR (0.38 U/ml), and 143B-P (0.358 U/ml) (Table I, Figure 1). These results indicate that super MTX-resistant osteosarcoma cells retain sensitivity to rMETase.

Figure 1.
Figure 1.

Sensitivity to recombinant methioninase (rMETase) of super methotrexate-resistant 143B osteosarcoma (143B-MTXSR) cells and parental 143B osteosarcoma cells (143B-P) cells. Please see Materials and Methods for experimental details. Error bars: Standard deviation of the mean.

First-line neoadjuvant/adjuvant chemotherapy for osteosarcoma, including MTX, DOX, and CDDP, targets cancer cells in the S/G2-phase of the cell cycle, where cancer cells are selectively arrested by methionine restriction (28, 29). MTX also inhibits DHFR, which blocks folate metabolism, and eventually decreases methionine synthesis from homocysteine with 5-methyltetrahydrofolate as the methyl doner, catalyzed by methionine synthase, which is very active in cancer cells (30, 31). Cancer cells are addicted to folate, as well as to methionine (7).

In super MTX-resistant 143B-MTXSR osteosarcoma cells, selected from parental 143B-P osteosarcoma cells by culturing with stepwise increasing concentrations of MTX, histone H3K9me3 and H3K27me3 are over expressed along with reduced malignancy (25). Methionine-independent revertant 143B osteosarcoma cells, selected from methionine-addicted parental osteosarcoma cells by culturing in low-methionine medium, are less malignant, also due to the increase of histone H3K9me3 and H3K27me3 expression (20). In osteosarcoma, histone methylation is unstable during methionine restriction and methionine addiction is linked to malignancy. Thus, methionine addiction can be a therapeutic target to overcome drug-resistance (16-21, 24, 32-39).

In the present study, super MTX-resistant 143B-MTXSR osteosarcoma cells retained high sensitivity to methionine restriction, effected by rMETase, very similar to methionine-addicted parental 143B-P osteosarcoma cells. These results suggest that methionine restriction by rMETase can be a potential clinical strategy for overcoming MTX resistance in osteosarcoma.

Targeting methionine addiction is effective because it is the fundamental hallmark of cancer (6-41). rMETase is showing clinical promise as an oral supplement (42-50).

Acknowledgements

This paper is dedicated to the memory of A. R. Moossa, MD, Sun Lee, MD, Gordon H. Sato, PhD, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Jack Geller, MD, Shigeo Yagi, PhD, J.A.R. Mead, PhD, Eugene P. Frenkel, MD, Professor Sheldon Penman, Professor John R. Raper, John Mendelsohn, MD and Joseph Leighton, MD.

Footnotes

  • Authors’ Contributions

    YA and RMH were involved in study conception and design. YA, YK, and NM were involved in acquisition of data. YA, YT, YK, NM, and RMH analyzed and interpreted data. YA and RMH wrote the manuscript. All Authors reviewed and approved the manuscript.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare in relation to this study.

  • Funding

    This study was funded in part by the Robert M. Hoffman Foundation for Cancer Research and by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists (24K19627).

  • Received January 6, 2025.
  • Revision received January 22, 2025.
  • Accepted January 23, 2025.

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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Super Methotrexate-resistant Osteosarcoma Cells Retain Their Sensitivity to Recombinant Methioninase: Targeting Methionine Addiction to Overcome Extreme Cancer-Chemotherapy Resistance
YUSUKE AOKI, QINGHONG HAN, YUTARO KUBOTA, NORIYUKI MASAKI, YASUNORI TOME, MICHAEL BOUVET, KOTARO NISHIDA, ROBERT M. HOFFMAN
Anticancer Research Mar 2025, 45 (3) 929-934; DOI: 10.21873/anticanres.17480
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