Abstract
Background/Aim: Colorectal cancer (CRC) is the third-leading cause of death in the world. Although the prognosis has improved due to improvement of chemotherapy, metastatic CRC is still a recalcitrant disease, with a 5-year survival of only 13%. Irinotecan (IRN) is used as first-line chemotherapy for patients with unresectable CRC. However, there are severe side effects, such as neutropenia and diarrhea, which are dose-limiting. We have previously shown that methionine restriction (MR), effected by recombinant methioninase (rMETase), lowered the effective dose of IRN of colon-cancer cells in vitro. The aim of the present study was to evaluate the efficacy of the combination of low-dose IRN and MR on colon-cancer in nude mice. Materials and Methods: HCT-116 colon-cancer cells were cultured and subcutaneously injected into the flank of nude mice. After the tumor size reached approximately 100 mm3, 18 mice were randomized into three groups; Group 1: untreated control on a normal diet; Group 2: high-dose IRN on a normal diet (2 mg/kg, i.p.); Group 3: low-dose IRN (1 mg/kg i.p.) on MR effected by a methionine-depleted diet. Results: There was no significant difference between the control mice and the mice treated with high-dose IRN, without MR. However, low-dose IRN combined with MR was significantly more effective than the control and arrested colon-cancer growth (p=0.03). Body weight loss was reversible in the mice treated by low-dose IRN combined with MR. Conclusion: The combination of low-dose IRN and MR acted synergistically in arresting HCT-116 colon-cancer grown in nude mice. The present study indicates the MR has the potential to reduce the effective dose of IRN in the clinic.
- Colon cancer
- HCT-116
- nude mice
- irinotecan
- methionine restriction
- combination therapy
- synergy
- dose reduction
- methionine addiction
- Hoffman effect
Colorectal cancer (CRC) is the fourth most common cancer and the third-leading cause of death in the world (1). Approximately 40%-54% of patients diagnosed with CRC develop unresectable metastatic disease, especially liver metastases (2). The prognosis for CRC with distant metastases is approximately 30 months (3) with a 5-year survival of 10.5-13% (4, 5), despite the improvement brought by multidisciplinary treatment, including chemotherapy (6).
Irinotecan (IRN) has been used as first-line chemotherapy for CRC patients with unresectable distant metastases and has improved the prognosis of CRC patients with overall survival ranging approximately from 5 months to 33 months (7, 8). However, IRN has severe toxicity such as neutropenia and diarrhea, which is dose-limiting (9, 10).
Methionine addiction is a fundamental and general hallmark of cancer known as the Hoffman effect (11-14). Methionine addiction of cancer cells is at least partly due to excessive transmethylation reactions, resulting in a significantly greater need for exogenous methionine compared to normal cells, despite normal or greater than normal endogenous synthesis of methionine in cancer cells (15-19). Consequently, cancer cells are unable to survive without external methionine, even though cancer cells produce methionine at a high rate (11, 17-19).
Methionine restriction (MR) including diet and/or recombinant methioninase (rMETase) selectively arrests the cell cycle of cancer cells in late-S/G2 phase (20, 21). Since S-phase is the main target of cytotoxic chemotherapy, MR is synergistic with cytotoxic chemotherapy. There are numerous reports for the past 4 decades that MR has synergistic efficacy with many chemotherapy drugs without side effects (22).
We have previously shown that MR with rMETase lowered the effective dose of IRN in an vitro study (23). The aim of the present study was to evaluate the efficacy of combination of low-dose IRN and MR against colon-cancer nude mice.
Materials and Methods
Cell culture. The HCT-116 human-colon cancer cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum and 100 IU/ml of penicillin/streptomycin (Thermo Fisher Scientific).
Mice. Athymic nu/nu nude mice (AntiCancer, Inc., San Diego, CA), which were 4-6 weeks old, were used in the present study. All mice were housed in a controlled environment with a high efficiency particulate air (HEPA)-filtered rack, at a regular 12-hour light/dark cycle. The animal experiments were carried out with an AntiCancer Institutional Animal Care and Use Committee (IACUC)-protocol approved for this study. The principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals were followed. HCT116 cells were cultured and harvested by trypsinization. HCT-116 cells (1.0×106) in 100 μl PBS were injected subcutaneously into the right flank of nude mice. Anesthesia consisted of 0.02 ml of a solution containing 20 mg/kg of ketamine, 15.2 mg/kg of xylazine, and 0.48 mg/kg of acepromazine maleate, as previously described (24).
Study design. Two weeks after HCT-116 cell implantation, when the average tumor size reached approximately 100 mm3, 18 mice were randomized into the following 3 groups; Group 1: untreated control with a normal methionine (MET)-containing diet (n=6), no treatment); Group 2: high-dose IRN with a normal methionine-containing diet (n=6). IRN was administered at 2 mg/kg, intraperitoneally (i.p.), once a week; Group 3: low-dose IRN with MR (n=6). IRN was administered at 1 mg/kg, i.p., once a week with a methionine-choline-deficient diet; the mice in group 3 also received L-methionine (2 mg in 200 μl PBS, i.p., daily) (Figure 1). Treatment duration was two weeks. The mice were followed for an additional week. Tumor size was measured twice a week. Tumor volume was calculated using the following formula: tumor volume (mm3)=length (mm) × width (mm) × width (mm) × 1/2. All mice were humanely sacrificed after three weeks, or when the estimated tumor volume exceeded 2,000 mm3.
Study design schema. Group 1 (G1): control mice on a normal methionine (MET)-containing diet, no treatment; Group 2 (G2): mice were treated with high-dose irinotecan (IRN) on a MET-containing diet (2 mg/kg, i.p., weekly, for two weeks); Group 3 (G3): mice treated with low-dose IRN (1 mg/kg, i.p., weekly, for two weeks) and methionine restriction (MR) with a methionine-deficient diet supplemented with 2 mg L-methionine/mouse in 200 μl PBS i.p., daily.
Methionine restricted diet. A methionine-deficient diet (TD.90262, Inotiv, Inc., West Lafayette, IN, USA) was used in the present study. Two mg of L-methionine in 200 μl PBS per mouse, were administered intraperitoneally daily to the mice on the methionine-choline-deficient diet as a maintenance dose.
Statistical analysis. Statistical analyses were performed using GraphPad Prism 10.0.3 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was defined as p≤0.05.
Results
Although high-dose IRN alone suppressed HCT-116 colon-cancer growth in nude mice compared to the control group, the difference was not statistically significant (p=0.10). The combination of low-dose IRN and a MR diet arrested tumor growth (p=0.03) (Figure 2, Figure 3). At one-week after the end of treatment, the body weight of all groups of mice was normal or returned to normal (Table I).
Comparison of the efficacy between control on a normal diet; high-dose irinotecan (IRN) on a normal diet; and low-dose IRN with a methionine restriction (MR) diet. There was not a significant difference between control and high-dose IRN. However, the combination of low-dose IRN and the MR diet arrested colon-cancer growth (p=0.03).
Efficacy of treatment. A) control on a normal diet; B) high-dose Irinotecan (IRN) on a normal diet; C) low-dose IRN with a methionine restriction (MR) diet.
Mouse body weight at the start and end of the experiment. The mean value±SD (standard deviation).
Discussion
The present study demonstrated that the combination of low-dose IRN and an MR diet arrested colon-cancer growth in a nude-mouse model. Decreasing the dose of IRN by half, combined with a MR diet, was more effective than the high-dose IRN without MR. The present study suggests the possibility of low-dose IRN combined with MR to treat metastatic colon cancer without severe neutropenia and diarrhea in patients with CRC.
Topoisomerase I is a DNA-unwinding protein, which functions during active DNA replication and transcription (25, 26). Inhibition of topoisomerase I by IRN results in the arrest of the cell cycle in the S-phase and the induction of apoptosis (27-29). MR also selectively arrests the cell cycle of cancer cells at the late S/G2 phase (20, 21), which leads to a strong synergistic effect with IRN, even at the low-dose used in the present study.
We have previously shown low-dose IRN combined with MR effected by rMETase was synergistic in vitro. We have also shown that MR effected by rMETase combined with cisplatinum was effective against colon-cancer in nude mice (30) rMETase in combination with oxaliplatinum and 5-fluorouracil was effective against a colon-cancer peritoneal carcinomatous model in nude mice (31). We have also shown that MR effected by rMETase is effective in patients with CRC (32, 33).
Conclusion
In the present study, low-dose IRN combined with MR was effective against CRC in nude mice. Even at half the dose of IRN with MR, the efficacy was higher than high-dose IRN without MR. The present study indicates the future possibility of reducing the effective dose of IRN in the clinic. MR is synergistic with chemotherapy because it targets the fundamental basis of cancer, methionine addiction (11-17, 20-23, 34-50).
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, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R Bertino, MD, and J.A.R. Mead, PhD.
Footnotes
Authors’ Contributions
Conception and design MS, KM, YK, and RMH. MS, KM, RM, AB, and KK performed experiments. MS and RMH wrote the article. MS, KM, RM, OH, KK, DA, YK, QH, AB, SM, BMK, MB, NK, YI, AN, and RMH critically reviewed the article.
Conflicts of Interest
The Authors declare no competing interests regarding this work.
Funding
The Robert M. Hoffman Foundation for Cancer Research provided funds for this study.
- Received January 18, 2024.
- Revision received February 21, 2024.
- Accepted February 22, 2024.
- Copyright © 2024 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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