Abstract
Background/Aim: Methionine addiction is a metabolic hallmark of cancer. Recombinant methioninase (rMETase) targets methionine addiction and effectively depletes methionine. rMETase has shown synergy with chemotherapeutic agents on numerous types of cancer cells. Chloroquine (CQ), an anti-autophagy agent, has demonstrated anti-cancer efficacy in pre-clinical studies. The present study aimed to evaluate the cancer selectivity and synergistic efficacy of rMETase and CQ in a co-culture model of colon-cancer cells and normal fibroblasts.
Materials and Methods: HCT116 human colon-cancer cells and Hs-27 human normal fibroblasts were co-cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and treated with rMETase (0.1-0.5 U/ml) alone, CQ (10-60 μM) alone, or rMETase at various concentrations in combination with CQ (20 μM). Cell morphology and viability were monitored for six days using phase-contrast microscopy (Olympus IX71). The effects of each treatment on the cancer cells and normal fibroblasts were compared.
Results: rMETase treatment selectively reduced HCT116 viability in a dose-dependent manner while sparing normal fibroblasts. In contrast, high-concentrations of CQ decreased the viability of both cell types, with strong cytotoxicity at ≥40 μM. Combination treatment with rMETase and low-dose CQ (20 μM) produced greater selective efficacy against the cancer cells than rMETase alone, eliminating the cancer cells and without significant inhibition of fibroblast viability.
Conclusion: rMETase has selective efficacy against cancer cells in the presence of normal cells, and its efficacy is significantly enhanced selectively on the cancer cells by CQ. The results of the present study suggest the potential for future clinical application of the combination of rMETase and CQ for cancer treatment.
- Methionine addiction
- Hoffman effect
- recombinant methioninase
- chloroquine
- selective synergy
- co-culture model
- colon-cancer cells
- normal fibroblasts
Introduction
Sugimura et al. demonstrated that cancer growth was inhibited in rat models when methionine was removed from the diet (1). Subsequently, Hoffman et al. observed that cancer cells synthesize normal or elevated methionine levels despite their requirement for exogenous methionine (2). These findings led to the concept of methionine addiction of cancer cells, known as the Hoffman effect (2-21).
Methionine addiction is defined as the excessive utilization of methionine for transmethylation reactions within cancer cells, resulting in their dependency on exogenous methionine (7, 9-12, 14, 15, 20, 21). Methionine restriction of cancer cells induces cell-cycle arrest at the late S/G2 phase (18, 19). Cancer cells, unlike normal cells have a requirement for exogenous methionine (2, 5, 6, 10, 18, 19). An effective strategy to restrict methionine in cancer cells is the use of recombinant methioninase (rMETase), an enzyme that degrades methionine (22).
Methionine deprivation also sensitizes cancer cells to chemotherapy. Our previous studies have demonstrated the synergistic efficacy of rMETase in combination with numerous chemotherapeutic agents and targeted therapies in multiple cancer types (22).
Autophagy degrades cytoplasmic proteins and organelles via lysosomes in a recycling process (23). Chloroquine (CQ) is an autophagy inhibitor. Inhibition of autophagy when combined with chemotherapeutic drugs is effective in various pre-clinical cancer models (24-26).
We have recently demonstrated the synergistic efficacy of rMETase combined with CQ against osteosarcoma cells, triple-negative breast-cancer cells, and colon-cancer cells in vitro (27-29).
The present study aimed to determine the cancer-selectivity of rMETase combined with CQ in a co-culture model of HCT116 colon cancer cells and Hs-27 normal fibroblasts, which more closely simulates the tumor microenvironment (8).
Materials and Methods
Cell culture. HCT116 human colon-cancer cells and Hs-27 human fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA).
The cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 with GlutaMAX™ supplement (DMEM/F-12), further supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO2.
Recombinant methioninase production. rMETase was produced by AntiCancer Inc. (San Diego, CA, USA) via fermentation of recombinant Escherichia coli transformed with the methioninase gene from Pseudomonas putida.
The purification process involved heat treatment at 60°C, polyethylene glycol precipitation, and diethyl-aminoethyl (DEAE) Sepharose fast-flow ion-exchange column chromatography, as previously described (30).
Reagents. CQ diphosphate was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS) at a stock concentration of 10 mM.
Co-culture and treatment of HCT116 colon-cancer cells and Hs-27 normal fibroblasts. HCT116 colon-cancer cells and Hs-27 normal fibroblasts were seeded together in 12-well plates at 5×104 cells each (total 1×105 cells/well) and cultured as described above.
Twenty-four hours after seeding, co-cultures were treated with varying concentrations of rMETase (0.1-0.5 U/ml) or CQ (10-60 μM). Control wells received no treatment.
Cell growth and morphology were monitored via phase-contrast microscopy (IX71, Olympus Corporation, Tokyo, Japan) on days-0, -2, -4, and -6 after treatment. Observations were terminated on day-6, when the control group reached full confluence.
For synergy testing, co-cultures, as described above, were treated with rMETase (0.1-0.5 U/ml) plus a low CQ concentration (20 μM) and monitored for six days.
Results
rMETase selectively targets HCT116 colon-cancer cells in co-culture with Hs27 normal human fibroblasts. In untreated co-cultures, HCT116 colon-cancer cells rapidly overtook the culture, obscuring Hs27 normal fibroblasts, and reached complete confluence by day-6. In contrast, rMETase (0.1-0.5 U/ml) caused a progressive eradication of HCT116 cells, while elongated normal fibroblasts remained viable, particularly at higher rMETase concentrations. However, even at 0.5 U/ml rMETase, some HCT116 colon-cancer cells survived although Hs27 fibroblasts dominated the culture (Figure 1).
Selective cytotoxicity of rMETase against HCT116 colon-colon-cancer cells in co-culture with Hs-27 normal fibroblasts. HCT116 colon-cancer cells and Hs-27 fibroblasts were co-cultured and treated with increasing concentrations of recombinant methioninase (rMETase, 0.1, 0.3, and 0.5 U/ml). Untreated controls reached full confluence by day-6, dominated by HCT116 cells. In contrast, rMETase treatment caused a dose-dependent reduction of HCT116 cells, while elongated Hs27 fibroblasts remained viable. Phase-contrast microscopic images were captured on days-0, -2, -4, and -6 using an Olympus IX71 inverted microscope at 100× total magnification. Representative HCT116 colon-cancer cells are indicated by white arrows and representative Hs-27 normal fibroblasts are indicated by black arrows. rMETase: Recombinant methioninase. Please see Materials and Methods for details.
High-dose CQ eradicates both HCT116 colon-cancer cells and Hs-27 normal fibroblasts in co-culture. CQ treatment (10-60 μM) affected both HCT116 colon-cancer cells and Hs-27 normal fibroblasts. At concentrations ≥40 μM, both cell types were nearly eradicated by day-6 (Figure 2).
Cytotoxic effects of CQ on both HCT116 colon-cancer cells and co-cultured Hs-27 normal fibroblasts. Co-cultures of HCT116 colon-cancer cells and Hs-27 normal fibroblasts were treated with CQ (20, 40, and 60 μM). While untreated controls showed rapid HCT116 overgrowth, CQ treatment reduced the viability of both cell types. At CQ concentrations ≥40 μM, both HCT116 cancer cells and Hs-27 normal fibroblasts were greatly diminished. Phase-contrast microscopic images were obtained on days 0, 2, 4, and 6 using an Olympus IX71 inverted microscope at 100× total magnification. Representative HCT116 colon cancer cells are indicated by white arrows and representative Hs-27 normal fibroblasts are indicated by black arrows. CQ: Chloroquine. Please see Materials and Methods for details.
Low-dose CQ enhances the selective cytotoxic efficacy of rMETase against HT116 colon-cancer cells in co-culture with Hs27 normal fibroblasts. Combining 0.3 U/ml rMETase and 20 μM CQ selectively eradicated the HCT116 colon-cancer cells, leaving healthy proliferating Hs-27 normal fibroblasts (Figure 3).
Synergistic selective cytotoxicity of rMETase and CQ combination treatment on HCT-116 colon-cancer cells and not on co-cultured Hs27 normal fibroblasts. Co-cultures were treated with rMETase (0.1, 0.3, and 0.5 U/ml) alone or in combination with CQ (20 μM) for 6 days. rMETase monotherapy reduced but did not completely eliminate HCT116 cells, even at 0.5 U/ml. In contrast, combination therapy at 0.3 U/ml or 0.5 U/ml rMETase plus 20 μM CQ eliminated HCT116 cells and did not greatly inhibit normal-fibroblast growth, demonstrating strong selective synergistic efficacy of the combination of rMETase and low-dose CQ on the cancer cells. Phase-contrast microscopic images were acquired on day-6 using an Olympus IX71 inverted microscope at 100× total magnification. Representative HCT116 colon-cancer cells are indicated by white arrows and representative Hs-27 normal fibroblasts are indicated by black arrows. rMETase: Recombinant methioninase; CQ: chloroquine. Please see Materials and Methods for details.
Co-cultures are a completely controlled system with cancer and normal cells in the same exact environment together (8).
Discussion
The present findings are consistent with previous reports of synergistic efficacy of rMETase and CQ on osteosarcoma cells, triple-negative breast-cancer cells, and colon cancer cells (27-29). The present study extends prior work by showing selective elimination of cancer cells by the combination of rMETase and CQ co-cultured with normal cells. The present results demonstrate that low-dose CQ can potentiate rMETase activity on cancer cells while minimizing effects on co-cultured normal cells. Co-cultures are a completely controlled system, with cancer and normal cells growing together in the same well (8).
Future studies will validate the present findings in mouse models and, ultimately, clinical trials. The present results have immediate translational potential, as CQ is already clinically approved and rMETase is clinically available as a dietary enzyme supplement (31-34).
rMETase is effective because it targets the fundamental hallmark of cancer (1-21, 35-49), methioninase addiction. Comparison of [11C]methionine and [18F]fluorodeoxy-glucose PET imaging has demonstrated the Hoffman effect of methionine addiction is stronger than the Warburg effect of cancer of glucose addiction, respectively (49).
Acknowledgements
This article is dedicated to the memory of A.R. Moossa, MD; Sun Lee, MD; Professor Philip Miles; Richard W. Erbe, MD; Professor Milton Plesur; Professor Gordon H. Sato; Professor Li Jiaxi; Masaki Kitajima, MD; Shigeo Yagi, Ph.D; Jack Geller, MD; Joseph R Bertino, MD; J.A.R. Mead, Ph.D; Eugene P. Frenkel, MD; Professor I. J. Fidler; John Mendelsohn, 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
JK and RMH designed the study. QH and SL produced rMETase. JK conducted all experiments and wrote the article. RMH revised the article. BMK, KM, YA, YM and MB critically read the manuscript.
Conflicts of Interest
All Authors have no conflicts of interest or financial ties to disclose related to this study.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received August 18, 2025.
- Revision received September 26, 2025.
- Accepted October 2, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
