Abstract
Background/Aim: Methionine addiction, known as the Hoffman effect, makes cancer cells more sensitive to methionine restriction than normal cells. However, the long-term effects of methionine restriction on cancer and normal cells have not been thoroughly studied. Materials and Methods: HCT-116 human colorectal-cancer cells and Hs27 normal skin fibroblasts were treated with 0-8 U/ml of recombinant methioninase (rMETase) for 12 days. The cells were cultured in Dulbecco’s modified Eagle’s medium in 96-well tissue-culture plates. Results: HCT-116 cells were sensitive to all concentrations of rMETase from 0.125 U/ml to 8 U/ml. After day-8 of treatment, HCT-116 cells were acutely sensitive to rMETase, especially at rMETase concentrations of 0.5 U/ml or higher. Normal Hs27 fibroblasts were much less sensitive to rMETase: In the range of 0.125 U/ml to 0.5 U/ml, rMETase had no effect on Hs27 cells. rMETase concentrations up to 2 U/ml had a slight initial effect on Hs27 cells, whereas at concentrations ranging from 4 U/ml to 8 U/ml, rMETase reduced Hs27 viability over the 12-day test period, with acute loss of viability observed after eight days of exposure. Conclusion: Cancer cells were significantly more sensitive to rMETase than normal cells, with an acute loss of cell viability observed in cancer cells after eight days of treatment at concentrations of 0.5 U/ml or higher. These findings highlight the large difference in sensitivity between cancer and normal cells to rMETase and introduce the phenomenon of acute cell death in methionine restriction, which we term “methionine-depletion catastrophe”.
- Methionine addiction
- Hoffman effect
- methioninase
- colon-cancer cells
- HCT-116
- normal fibroblasts
- Hs27
- methionine restriction
- cell death kinetics
- methionine-depletion catastrophe
Methionine addiction is a general and fundamental hallmark of cancer, termed the Hoffman effect (1). Sugimura et al. (2) discovered in 1959 that removing methionine from the diet slowed rat-cancer growth more than removing other amino acids from the rat diet. Chello and Bertino (3) found leukemic cells arrested at low concentrations of methionine in vitro in 1973. Hoffman found that cancer cells are addicted to methionine in 1976 (4). Wang et al. (5) showed in 2019 that tumor-initiating cells are highly addicted to methionine. Methionine addiction is due to overuse of methionine by cancer cells for transmethylation reactions (6-8). Methylation reactions in cancer cells are elevated (5, 9, 10). Cancer cells arrest in late-S/G2 phase of the cell cycle when deprived of methionine (11, 12) making them more sensitive to chemotherapy (13, 14). However, the susceptibility of cancer and normal cells to recombinant methioninase (rMETase) has not been compared over a long period and over a wide range of concentrations, which the present study does.
Materials and Methods
Cell culture and preparation. The HCT-116 human colorectal-cancer cell line and Hs27 human normal fibroblasts were obtained from The American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY, USA) with 10% fetal bovine serum (GIBCO) and 1 IU/ml Streptomycin Sulfate/Penicillin G Sodium (GIBCO) under a humidified atmosphere of 5% CO2 at 37°C. Both HCT-116 and Hs27 cells were cultured in 100 μl media in 96 well plates.
Recombinant methioninase treatment. Recombinant L-methionine α-deamino-γ-mercaptomethane-lyase (rMETase), a methionine-cleaving enzyme, originally from Pseudomonas putida, was produced in recombinant Escherichia coli by fermentation at AntiCancer Inc. (15, 16). When HCT-116 or Hs27 cells in each well reached confluence, they were treated with rMETase at 0 (control), 0.125, 0.25, 0.5, 1, 2, 4, and 8 U/ml.
Cell viability. Cell viability was measured on days 2, 4, 6, 8, 10, and 12 after rMETase treatment. To measure cell viability, 10 μl WST-8 reagent from a Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) were added to the medium in each well and the resulting optical density was measured at 450 nm using a spectrophotometer (Sunrise™, Tecan Inc. Mannedorf, Switzerland). Cell survival at each rMETase concentration was calculated as the relative survival rate compared to the untreated control group. There were n=6 wells for each rMETase concentration at each time point.
Statistical analysis. Statistical analysis was performed using IBM SPSS Statistics Version 20 (SPSS, Chicago, IL, USA). Continuous values were analyzed using the Mann-Whitney test and p-values ≤ 0.05 were considered to be statistically significant.
Results
Figure 1 shows the survival rates of HCT-116 and Hs27 cells upon treatment for up to 12 days with rMETase at concentrations ranging from 0 to 8 U/ml. The viability of HCT-116 human colorectal-cancer cells decreased over time following treatment with 0.125 U/ml to 8 U/ml of rMETase, and lower survival rates were observed at higher concentrations of rMETase. These results confirmed that HCT116 cells are highly sensitive to rMETase. From day 8 and after of rMETase treatment, the viability of HCT-116 cells sharply declined at rMETase concentrations equal or higher than 0.5 U/ml. In contrast, normal Hs27 cells were more resistant to rMETase than HCT116 cells. At rMETase concentrations equal or lower than 0.5 U/ml, there was no effect on Hs27 cell viability. At 1 and 2 U/ml rMETase concentrations, the viability of Hs27 cells initially decreased but recovered over time. However, at concentrations of 4 U/ml and 8 U/ml, the viability continued to decrease at a high rate over time, with a pattern similar to that observed in HCT-116 cells, termed methionine-depletion catastrophe..
Survival rates of (A) Hs27 normal fibroblasts and (B) HCT-116 colon-cancer cells treated with increasing concentrations of rMETase. Please see Materials and Methods for details.
Individual time-course analysis at each concentration of rMETase with HCT116 cells showed a significant decrease in survival over time as the rMETase concentration increased from 0.125 U/ml to 2 U/ml. In contrast, Hs27 cells exhibited relatively stable survival rates at rMETase concentrations from 0.125 U/ml to 2 U/ml. In this range of rMETase concentrations, the survival rates of HCT-116 were significantly lower than those of Hs27 cells. At the rMETase concentrations of 4 U/ml and 8 U/ml, both HCT-116 and Hs27 cells showed methionine-depletion catastrophe with an increased decline in survival over time (Figure 2).
Cell survival time course according to each rMETase concentration. Red line= HT-116 colon-cancer cells; blue line: Hs27 normal human fibroblasts. *: p<0.05. Please see Materials and Methods for details.
Dose-response analysis of rMETase treatment at each day showed that the difference in survival rates between cancer and normal cells increased over time at low to medium concentrations of rMETase (from 0.125 U/ml to 2 U/ml). At these rMETase concentrations, cancer cells reached methionine-depletion catastrophe, whereas the effect on normal cells was minimal. However, at higher concentrations of rMETase (4 U/ml and 8 U/ml), there was no large difference in survival rates of the cancer and normal cells, both undergoing methionine-depletion catastrophe (Figure 3).
Cell survival rates according to rMETase concentration at each day. Red solid line represents HCT-116 cells and blue solid line represents Hs27 fibroblasts. *: p<0.05. Please see Materials and Methods for details.
Discussion
Methionine addiction is the fundamental and general hallmark of cancer (4-13, 16-34). Although much has been discovered in over a half-century since methionine addiction was discovered, more needs to be learned about the altered transmethylation reactions in methionine-addicted cancer cells, including altered methylation of histone lysine marks in cancer cells (5, 9, 10, 21, 22). The present study showed that cancer cells rapidly undergo loss of cell viability at day-8 when treated with methioninase concentrations equal or greater than 0.5 U/ml, termed methionine-depletion catastrophe. The acute loss of cell viability does not occur in normal cells at these rMETase concentrations.
The present study demonstrating acute loss of HCT-116 cancer-cell viability after day-8 of rMETase treatment, at concentrations equal or greater than 0.5 U/ml, is consistent with the 1980 observation of Hoffman and Jacobsen (11). These results (11) demonstrated that SV-40-transformed cell lines were arrested at the late-S/G2 phase of the cell cycle at day-8 when cultured in homocysteine-containing, methionine-depleted medium. In methionine-depleted, homocysteine-containing medium, no cells were observed at day-8 in the G1 phase of the cell cycle whereas almost all the cells were in late-S/G2 phase by day-8. Thus, the G1 peak completely disappeared, leaving only the late-S/G2 peak on day-8. These results are consistent with the present results of acute decline in cancer-cell viability from day-8 even at low concentrations of rMETase.
At the high rMETase concentrations of 4 U/ml and 8 U/ml, viability of both cancer and normal cells continued to decrease over time, both undergoing methionine-depletion catastrophe. This suggests that severe or complete methionine depletion is detrimental to the survival of not only cancer cells but also of normal cells, but at only high rMETase concentrations. Therefore, it is essential to determine a safe and appropriate rMETase concentration that selectively induces cancer-cell death without affecting the survival of normal cells, as shown in the present in vitro study.
Conclusion
HCT-116 cancer cells were significantly more sensitive to rMETase than Hs27 normal fibroblasts. After 8 days of rMETase treatment at concentrations ranging from 0.5 U/ml to 8 U/ml, cancer cells experienced a pronounced decline in cell viability, termed methionine-depletion catastrophe, which occurred in the normal cells only at 4 U/ml and 8 U/ml. These findings highlight the substantial difference in rMETase sensitivity between cancer and normal cells and introduce a new phenomenon of acute viability loss in cancer cells after 8 days of treatment with rMETase at concentrations of 0.5 U/ml or higher, termed methionine-depletion catastrophe, that occurs in normal cells only at 4 and 8 units of rMETase/ml.
Future experiments will study the molecular changes occurring in cancer cells during the acute phase of methionine depletion which will help to better understand methionine addiction of cancer cells and its exploitation for cancer treatment (1, 4-13, 16-33).
Orally-administered rMETase (o-rMETase) is highly effective in patient-derived orthotopic xenograft (PDOX) mouse models and synergistic with chemotherapy (34-46). o-rMETase is beginning to show clinical promise in individual case studies (14, 47-55).
Acknowledgements
This paper is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R Bertino, MD, J.A.R. Mead, PhD, Eugene P. Frenkel, MD, John Medelsohn, 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, RMH, and QH designed the study. QH produced rMETase. BMK conducted all experiments. BMK wrote the article and RMH revised the article. KM, SM, 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.
- Received November 18, 2024.
- Revision received December 7, 2024.
- Accepted December 10, 2024.
- 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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