Abstract
Background/Aim: The aim of the present study was to determine the extent of methioninase treatment beyond which methionine-addicted cancer cells could no longer be rescued by methionine repletion.
Materials and Methods: Methionine-addicted HCT-116 human colon-cancer cells were treated in vitro with 1, 3, and 6 unit/ml of recombinant methioninase (rMETase) in methionine-containing growth media in 96-well culture dishes for eight days. rMETase was removed from six culture wells at various time points, and the cells were subsequently given normal growth medium containing 100 μM methionine. Cell number was determined every other day using the WST-8 cell-counting kit.
Results: When HCT-116 human colon-cancer cells were treated with 1 unit rMETase/ml, the cells could be fully rescued on days 2, 4, and 6 by removing rMETase and adding normal methionine-containing growth medium. When treated with 3 units of rMETase/ml, the cells could be fully rescued by methionine replenishment after two days of treatment. However, after four and six days of treatment, the cells were partially rescued; the cells did not die, but stopped proliferating. In contrast, treatment with 6 units of rMETase/ml led to proliferation arrest when rescue was attempted on days 2 and 4, and no rescue was possible on day 6, as the cells continued to die.
Conclusion: Three distinct responses to methionine replenishment following rMETase treatment were observed in HCT-116 cells. First, complete recovery and resumption of proliferation occurred with low-dose rMETase at all time points and with short-term treatment at intermediate doses of r-METase. Second, prolonged intermediate-dose treatment or short-term high-dose treatment, resulted in continued proliferation arrest without further cell death. Third, high-dose rMETase treatment for six days led to cell death.
- Methionine rescue
- methionine addiction
- Hoffman effect
- methioninase
- methionine restriction
- colon cancer
- HCT-116 cells
Introduction
Methionine addiction is the fundamental and general hallmark of cancer, termed the Hoffman effect (1-17). Sugimura et al. (1) first demonstrated that cancers are methionine dependent by removing one amino acid at a time from the diet of rats with the Walker 256 carcinoma and found that removal of methionine slowed tumor growth more than when other amino acids were removed. There was no further research in this area for another 14 years until Chello and Bertino showed that murine lymphoma cells had a very high methionine requirement for growth (2). In 1974, Halpern et al. (3) showed that substituting the methionine precursor homocysteine for methionine in the culture medium arrested cancer cells, but not normal cells. In 1976, we found that cancer cells were addicted to methionine: Cancer cells synthesized normal or greater than normal amounts of methionine but still required an exogenous source of methionine (4-6).
We subsequently isolated methionine independent revertants from methionine-addicted cancer cells, which were less malignant, thereby demonstrating methionine addiction is linked to malignancy (7-10). The basis of methionine addiction are the elevated transmethylation reactions in cancer cell (8-13).
We also found that methionine restriction enhanced the response of cancer cells to chemotherapy due to the selective S/G2 phase cell-cycle arrest, which is the cell cycle phase targeted by chemotherapeutic drugs (14). Recently, we showed that recombinant methioninase (rMETase) treatment can cause cancer cells to undergo catastrophic death (15).
In the present study, we determined the extent of rMETase treatment of cancer cells that allows the cells to be rescued by methionine replenishment, above which the cancer cells reach a point of no return.
Materials and Methods
Cell culture. The HCT-116 cell line, derived from human colorectal carcinoma, was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (GIBCO) and 1 IU/ml of penicillin-streptomycin antibiotic mixture (GIBCO). All experiments were conducted using 96-well plates. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Recombinant methioninase treatment and methionine rescue. When HCT-116 cells reached confluence, recombinant L-methionine-α-deamino-γ-mercaptomethane- lyase (rMET-ase) was administered at concentrations of 1, 3, or 6 U/ml to four 96-well plates per concentration group. rMETase, a methionine-degrading enzyme, originally derived from Pseudomonas putida, was produced through fermentation of recombinant Escherichia coli, transformed with the Pseudomonas putida methioninase gene at AntiCancer Inc. (16). At 2, 4, and 6 days following rMETase treatment, rMETase-containing medium was replaced with methionine-containing DMEM in six wells per group at each time point, to determine methionine rescue.
Cell viability. Cell viability was assessed at 2, 4, 6, and 8 days after rMETase treatment by adding 10 μl of the WST-8 Cell-Counting-Kit reagent (Dojindo Laboratory, Kumamoto, Japan) to each well. After a 70-min incubation, optical density was measured at 450 nm using a spectrophotometer (Sunrise™, Tecan Inc. Mannedorf, Switzerland). Relative cell survival was calculated by comparing each treated group to the untreated control group. At each time point and rMETase concentration, cell viability was evaluated in six replicate wells.
Results
The response of HCT-116 cells to methionine replenishment after rMETase treatment (methionine rescue) was dependent on the concentration and time of rMETase treatment. Higher concentrations and prolonged exposure to rMETase reduced the HCT-116 cells’ ability to recover.
Treatment of HCT-116 human colon cancer cells with rMETase at a concentration of 1 unit/mL demonstrated that the growth-inhibitory effect of methionine depletion was fully reversible at all time points. Specifically, upon removal of rMETase and replenishment of methionine with standard growth medium, the cells exhibited complete recovery of proliferative capacity when methionine replenishment was performed on days 2, 4, or 6 following initial rMETase treatment (Figure 1A).
Rescuability of cancer cells by methionine replenishment after treatment with rMETase at various doses and time periods. HCT-116 human colon cancer cells were cultured in 96-well plates in DMEM and rMETase was added at 1 U/ml (A), 3 U/ml (B), and 6 U/ml (C). Rescue was performed by refeeding cells with complete DMEM growth medium containing 100 μM L-methionine. Please see Materials and Methods for details. Data represent cell survival percentages (mean±standard deviation).
In contrast, when the rMETase concentration was increased to 3 units/ml, full restoration of cell proliferation was observed only when methionine was replenished after two days of treatment. Attempted rescue by methionine replenishment after four or six days resulted in only partial recovery, characterized by a cessation of proliferation without overt cell death (Figure 1B).
Notably, exposure to a higher concentration of rMETase (6 units/ml) induced a more pronounced cytotoxic effect. Under these conditions, removal of rMETase and replenishment of methionine on days 2 and 4 led to irreversible proliferative arrest, and rescue on day 6 was ineffective, with cells continuing to undergo cell death despite the re-introduction of methionine (Figure 1C).
Discussion
In the present study, we investigated the reversibility of rMETase-induced suppression of proliferation and viability of HCT-116 human colon cancer cells by evaluating their ability to resume proliferation following the removal of rMETase and methionine replenishment. After growth suppression by rMETase, methionine replenishment resulted in three outcomes: resumption of proliferation; continued growth arrest; or cell death. Low-dose treatment (1 unit/ml) of rMETase resulted in complete recovery of proliferative capacity regardless of the timing of methionine replenishment. In contrast, intermediate dosing (3 units/ml) of rMETase permitted full recovery only when rescue was initiated early (day 2), while delayed rescue (days 4 or 6) led to a non-lethal continued proliferation arrest. Treatment with 6 units/ml rMETase led to irreversible proliferation arrest when rescue was performed on days 2 and 4, and to cell death when rescue was delayed until day 6, suggesting a dose- and time-dependent loss of recovery capacity. These findings help clarify and extend the understanding of methionine addiction in cancer cells – a metabolic vulnerability in which cancer cells, although capable of making methionine themselves, still require an external source to support their increased transmethylation and continuous growth (4).
Recombinant methioninase (rMETase) exploits the cancer-cell addiction to extracellular methionine, thereby disrupting methylation-dependent processes and inducing cancer-selective cytostatic or cytotoxic responses depending on the intensity and duration of treatment (18-21). The differential responses observed in the present study suggest the existence of a threshold beyond which methionine depletion causes irreversible metabolic damage, termed methionine-depletion catastrophe (15). At lower doses of rMETase or shorter exposures, cells retain the ability to re-enter the cell cycle upon methionine replenishment. However, prolonged or higher-dose rMETase treatment appears to disrupt crucial cellular processes, thereby rendering rescue ineffective. The non-lethal arrest observed following intermediate-dose-time rMETase treatment may represent a quiescent or senescence-like state in which cells remain viable but are unable to proliferate. This state could be therapeutically significant, offering a window to suppress tumor growth without inducing collateral toxicity. Importantly, the cell death observed with high-dose and long-duration rMETase treatment supports the potential of rMETase as an effective cancer therapeutic under optimized conditions. The inability of methionine repletion to rescue cells in this setting suggests the induction of permanent metabolic collapse or activation of apoptotic pathways, methionine-depletion catastrophe (15). One mechanism of proliferation arrest or cell death may be the lability of certain methylated lysine histone marks when cancer cells, unlike normal cells, are treated with rMETase (9-11, 22). An additional mechanism of proliferation arrest or cell death upon methionine restriction is loss of methylation of a critical protein involved in RNA splicing (23). Also, the lowering of the S-adenosylmethionine/S-adenosylhomocysteine ratio during methionine restriction may permanently arrest or kill the cancer cells (5). These studies show the linkage of methionine addiction, altered methylation and malignancy (1-32).
Future studies are warranted to elucidate the specific molecular mechanisms underlying irreversible proliferation arrest or cell death induced by rMETase, including changes in methylation status, metabolic reprogramming, and cell-cycle control.
We previously showed that cancer cells in medium with methionine replaced by its immediate precursor homocysteine (Met−-Hcy+) could be rescued by very-low amounts of methionine (4). High amounts of methionine, such as the ones used in the present study, could rescue cells in Met−-Hcy+ over long periods of time (20). In contrast, rMETase treatment in the present study could reach the point of no return.
Conclusion
The present` findings demonstrate that the efficacy of rMETase treatment is tightly linked to the balance between methionine-depletion intensity and duration. These insights contribute to a more comprehensive understanding of methionine addiction and support the rationale for timing- and dose-optimized rMETase regimens as a strategy to selectively target methionine-addicted cancer.
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, John W. Littlefield, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, PhD. Eugene P. Frenkel, MD, 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
BMK and RMH designed the study. QH and SL produced rMETase. BMK conducted all experiments. BMK wrote the article and RMH revised the article. KM, JSK, YA, and MB critically read the manuscript.
Conflicts of Interest
The 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 May 7, 2025.
- Revision received May 16, 2025.
- Accepted May 19, 2025.
- Copyright © 2025 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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