Abstract
Background/Aim: Drug-resistant osteosarcoma is a highly aggressive malignancy with limited therapeutic options. Recombinant methioninase (rMETase) targets the methionine addiction of cancer and acts synergistically with many cancer-chemotherapy agents. The present study investigated the synergistic efficacy of the combination of rMETase, chloroquine (CQ) which targets autophagy, and rapamycin (RAPA) which targets mTOR, on human 143B osteosarcoma cells in vitro.
Materials and Methods: 143B human osteosarcoma cells. 143B cells were treated under eight conditions at the IC30 of each agent: untreated control; rMETase alone (0.31 U/ml); CQ alone (61.9 μM); RAPA alone (30.9 μM); rMETase (0.31 U/ml) + CQ (61.9 μM); rMETase (0.31 U/ml) + RAPA (30.9 μM); CQ (61.9 μM) + RAPA (30.9 μM); and rMETase (0.31 U/ml) + CQ (61.9 μM) + RAPA (30.9 μM). Cell viability was measured with the WST-8 reagent.
Results: Each of the single-agents, rMETase, CQ, and RAPA demonstrated moderate cytotoxicity when administered alone to 143B cells. The dual combination of CQ plus RAPA had the highest efficacy compared to single agents and compared to rMETase plus CQ and rMETase plus RAPA, which had moderate efficacy. In contrast, the triple combination of rMETase, CQ, plus RAPA exhibited strong synergistic efficacy, eradicating 143B cells.
Conclusion: The triple combination of rMETase, CQ, and RAPA demonstrated strong synergy and effectively eradicated 143B osteosarcoma cells. Therefore, the triple treatment with rMETase, CQ, and RAPA has potential as a novel, effective therapeutic approach for osteosarcoma.
Introduction
Osteosarcoma is the most common primary malignant bone tumor, primarily affecting children and young adults (1). Despite advances in multimodal treatments such as surgery and combination chemotherapy, the prognosis for patients with drug-resistant osteosarcoma, particularly those with metastatic or recurrent disease, remains poor (2). Therefore, novel therapeutic strategies are urgently needed to improve clinical outcomes.
Recombinant methioninase (rMETase), an enzyme that reduces extracellular methionine levels, has shown efficacy against all major cancers by targeting methionine addiction, known as Hoffman effect (3, 4). Methionine addiction is a general hallmark of cancer, which has an increased requirement for exogenous methionine (5-10) compared to normal cells. Previous studies have demonstrated that methionine depletion can effectively inhibit osteosarcoma cell proliferation and sensitize them to chemotherapy (11-13).
Chloroquine (CQ) is a well-known antimalarial drug that inhibits autophagy, a cellular survival mechanism of cancer cells under stress (14). Autophagy inhibition by CQ has been shown to enhance the efficacy of chemotherapeutic agents and overcome drug resistance in various cancer models (15).
Rapamycin (RAPA), which targets the mammalian target of rapamycin (mTOR) pathway, inhibits cell growth, proliferation, and protein synthesis in cancer cells (16). We have previously shown synergy of RAPA and rMETase including on breast osteosarcoma (17, 18). The mTOR pathway is frequently up-regulated in osteosarcoma, and its inhibition has been reported to improve responses to other therapies (19). In the present study, we determined the synergistic efficacy of the combination of rMETase plus CQ plus RAPA against osteosarcoma cells.
Materials and Methods
Cell culture. 143B osteosarcoma cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin (Corning, Corning, NY, USA).
Reagents. Recombinant methioninase (rMETase) was produced by AntiCancer Inc.(San Diego, CA, USA), by fermentation of recombinant E. coli expressing the Pseudo-monas putida methioninase gene following previously- established procedures (20). CQ diphosphate was obtained from Sigma-Aldrich (St. Louis, MO, USA) and RAPA was obtained from MedChemExpress (Monmouth Junction, NJ, USA).
Drug sensitivity assay 1: Determination of the IC30 of rMETase, CQ and RAPA on 143B osteosarcoma cells. To determine the IC30 of rMETase, CQ and RAPA, 143B cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). 143B cells (3×103 cells per well) were seeded in 96-well plates containing 100 μl of DMEM per well. After incubation, rMETase at concentrations ranging from 0.5 U/ml to 8 U/ml; CQ at concentrations from 12.5 μM to 200 μM; and RAPA at 6.25 μM to 100 μM were added to the wells for 72 h. Following the incubation, WST-8 solution (10 μl) was added to each well, and the plates were incubated for an additional hour at 37°C. The resulting absorbance at 450 nm, which correlates with cell viability, was measured using a microplate reader (SUNRISE, TECAN, Mannedorf, Switzerland). Drug-sensitivity curves were generated using Microsoft Excel (Microsoft, Redmond, WA, USA), and IC50 values were calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA). Each experiment was conducted twice in triplicate.
Drug sensitivity assay 2: Determination of synergy of rMETase plus CQ plus RAPA in all combinations on 143B osteosarcoma cells. To assess the combined effect of rMETase plus CQ plus RAPA, 143B cells were seeded in 96-well plates at a density of 3×103 cells per well. After 24 h, the cells were treated as follows: control (DMEM only); rMETase (0.31 U/ml, IC30 [data from (13)]); CQ (61.9 μM, IC30); RAPA (30.9 μM, IC30); rMETase (0.31 U/ml, IC30 [data from (13)]) plus CQ (61.9 μM, IC30); rMETase (0.31 U/ml, IC30 [data from (13)]) plus RAPA (30.9 μM, IC30); CQ (61.9 μM, IC30) plus RAPA (30.9 μM, IC30); and rMETase (0.31 U/ml, IC30 [data from (13)]) plus CQ (61.9 μM, IC30) plus RAPA (30.9 μM, IC30). After 72 h of treatment, cell viability was measured as described above.
Statistical analysis. All statistical analyses were performed using EZR software (Jichi Medical University, Saitama, Japan). The Tukey–Kramer test was applied to evaluate relationships between groups, with a significance level set at p<0.05.
Results
IC30 of rMETase alone; CQ alone; and RAPA alone on 143B osteosarcoma cells. The IC30 value of rMETase alone on 143B cells was 0.31 U/ml [data from (13)]. The IC30 of CQ alone on 143B cells was 61.9 μM. The IC30 of RAPA alone on 143B cells was 30.9 μM. (Figure 1).
Determination of IC30 of recombinant methioninase (rMETase); chloroquine; and rapamycin on 143-B osteosarcoma cells. A) IC30 of rMETase on 143-B cells [data from (13)]. B) IC30 of chloroquine on 143-B cells. C) IC30 of rapamycin on 143B cells. Please see Materials and Methods for details.
Synergistic efficacy of all combinations of rMETase, CQ and RAPA on 143B osteosarcoma cells (Figure 2). Compared to the control cells, treatment with rMETase alone (0.31 U/ml, IC30 [data from (13)]) reduced the viability of 143B cells by approximately 19.3% (p<0.05); CQ alone (61.9 μM, IC30) reduced the viability of 143B cells by 27.3% (p<0.05); RAPA alone (30.9 μM, IC30) reduced the viability of 143B cells by 41% (p<0.05). The combination of rMETase (0.31 U/ml, IC30 [data from (13)]) plus CQ (61.9 μM, IC30) reduced the viability of 143B cells by 23.1% (p<0.05); rMETase (0.31 U/ml, IC30 [data from (13)]) plus RAPA (30.9 μM, IC30) reduced the viability of 143B cells by 46% (p<0.05); CQ (61.9 μM, IC30) plus RAPA (30.9 μM, IC30) reduced the viability of 143B cells by 77.9% (p<0.05). The combination of rMETase (0.31 U/ml, IC30 [data from (13)]), CQ (61.9 μM, IC30) and RAPA (30.9 μM, IC30) caused a significantly greater reduction in the viability of 143B cells (approximately 93.6%; p<0.05 compared to all other groups), essentially eradicating the 143B osteosarcoma cells.
Determination of the synergistic efficacy of all combinations of recombinant methioninase (rMETase), chloroquine (CQ), and rapamycin (RAPA) at their IC50 on 143B osteosarcoma cells. Please see Materials and Methods for details.
Discussion
The survival of osteosarcoma patients who develop drug resistance and metastases is very low, below 30% at 5 years, which has not improved in 3 decades (21-23). In the present study, we determined the synergistic efficacy of the combination of rMETase, CQ and RAPA on 143B osteosarcoma cells.
The triple combination of rMETase, CQ, and RAPA showed high synergy and higher efficacy than single agents and dual combinations. The synergy may be due to simultaneous targeting of multiple pathways: methionine addiction (3, 4); mTOR signaling (19); and autophagy (15), which may be interacting.
Clinical implications and future directions. The present findings provide a strong rationale for further preclinical and clinical investigation into the combination of rMETase, CQ, and RAPA on osteosarcoma cells. Future studies should focus on determining the molecular mechanisms of the observed synergy and validating the efficacy of this combination in vivo using patient-derived orthotopic xenograft (PDOX) osteosarcoma mouse models (24). The toxicity of the triple combination needs to be determined in the clinic (25).
Conclusion
The present study demonstrated that the triple combination of rMETase, CQ, and RAPA produces high synergistic efficacy on 143B osteosarcoma cells. The results of metabolic and autophagy-targeting may overcome the limitations of current treatments of drug-resistant metastatic osteosarcoma.
rMETase is effective because it targets the fundamental hallmark of cancer (3-10).
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.
Footnotes
Authors’ Contributions
SM, RMH, and QH 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, MB, NY, KH, HK, SM, KI, TH, HT, and SD critically read the manuscript.
Conflicts of Interest
The Authors declared that there are no competing interests in relation to this study.
- Received January 3, 2025.
- Revision received January 30, 2025.
- Accepted January 31, 2025.
- Copyright © 2025 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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