Abstract
Background/Aim: Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN. Materials and Methods: Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×103 cells per well in Dulbecco’s modified Eagle’s medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC50) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC50 concentration of rMETase, we determined the IC50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.. Results: The IC50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction. Conclusion: rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN.
- Colon cancer
- irinotecan
- toxicity
- methionine
- combination therapy
- synergy
- IC50
- methionine addiction
- Hoffman effect
Irinotecan (IRN) is a prodrug of SN-38 and exerts its efficacy by inhibiting topoisomerase I, which is required for DNA replication (1,2). IRN has been used as first- or second-line chemotherapy for over 20 years for a variety of cancers, including gastrointestinal, gynecological, and respiratory cancer (3). In particular, IRN has become first-line treatment for colorectal cancer with unresectable distant metastases (4). However, toxicity such as neutropenia and diarrhea, is a dose-limiting factor of IRN. Appropriate management of adverse effects, including dose reduction, is essential for the use of IRN, but reduces efficacy (3, 5).
All cancer cell types are addicted to methionine (6, 7) due at least partially to elevated levels of abnormal transmethylation (8-10). Methionine addiction, termed the Hoffman effect (9), is much stronger than the Warburg effect for glucose addiction (11). Methionine restriction (MR) has been observed to selectively arrest the cell cycle of cancer cells, specifically in the late-S/G2 phase (12, 13), a target of most chemotherapy drugs (14-17). However, methionine is present in many types of food and its restriction through dietary means alone is a challenge (18-21). Our laboratory has developed recombinant methioninase (rMETase), an enzyme that degrades methionine. Methioninase inhibits tumor growth in all malignancies in vitro and in vivo (22-27). Oral administration of rMETase (oral-rMETase) is effective in inhibiting tumor growth without side effects and has shown synergy with many chemotherapy drugs (27-35).
In the present study, we determined the synergistic efficacy of the combination of rMETase and SN-38, the active form of IRN, and whether the effective dose of IRN could be potentially reduced in order to eliminate severe side effects.
Materials and Methods
Cell culture. The HCT-116 human colon cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 IU/ml of penicillin/streptomycin.
Recombinant methioninase (rMETase) production. Recombinant l-methionine α-deamino-γ-mercapto-methane lyase [recombinant methionase (rMETase)] (EC 4.4.1.11) from Pseudomonas putida was produced in recombinant Escherichia coli (AntiCancer, Inc., San Diego, CA, USA) as described previously (22, 34, 35).
Cell viability. Cell viability was assessed by cultivating HCT-116 cells at subconfluence overnight in 96-well plates, seeded at a density of 1.0×103 cells per well in DMEM. On the subsequent day, HCT-116 cells were treated with increasing concentrations of SN-38 ranging from 0.5 nM to 32 nM, and/or rMETase, increasing from 0.125 to 8 U/ml. SN-38 was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Cell viability was determined after treatment for 72 h, using the Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) with the WST-8 reagent.
Determination of IC50 values. The software ImageJ version 1.54 (National Institutes of Health in Bethesda, MD, USA), was utilized for the computation of IC50 values and the construction of drug-sensitivity curves. Following the determination of the half-maximal inhibitory concentration (IC50) values for SN-38 alone and rMETase alone, the synergistic efficacy of their combination was assessed using the IC50 concentration of rMETase to determine its effects on the IC50 of SN-38. IC50 values were calculated using nonlinear regression.
Statistical analysis. Statistical significance was defined as p≤0.05. Statistical analyses were performed using GraphPad Prism 10.0.3 (GraphPad Software, Inc., San Diego, CA, USA).
Results
The IC50 of IRN (SN-38) alone was 3.50 nM and the IC50 of rMETase alone was 0.55 U/ml on HCT-116 cells (Figure 1 and Figure 2). Next, we investigated the efficacy of the combination of IRN (SN-38) and rMETase on HCT-116 cells in vitro. The reduction of the IC50 of IRN, in combination with the IC50 of rMETase, was 15-fold, from 3.50 nM to 0.232 nM (p<0.0001) (Figure 3).
Efficacy of SN-38 on HCT-116 cells in vitro. Cell viability was determined using the WST-8 reagent. The concentration axis uses a logarithmic base 2 scale. IC50: Half-maximal inhibitory concentration.
Efficacy of recombinant methioninase (rMETase) on HCT-116 cells in vitro. Cell viability was determined using the WST-8 reagent. The concentration axis uses a logarithmic base 2 scale. IC50: Half-maximal inhibitory concentration.
Recombinant methioninase (rMETase) lowered the half-maximal inhibitory concentration (IC50) of SN-38 on HCT-116 cells in vitro from 3.50 nM to 0.232 nM (p<0.001). Cell viability was determined using the WST-8 reagent. The concentration axis uses a logarithmic base 2 scale.
Discussion
The present study demonstrated a 15-fold reduction in the IC50 of IRN, by combining IRN with rMETase on HCT-116 colon cancer cells.
IRN (SN-38) binds and inhibits topoisomerase I, which plays a crucial role in DNA unwinding during replication and transcription. Inhibition of topoisomerase I leads to the arrest of the cell cycle in S-phase and the induction of apoptosis (36-38). The proximity of IRN (SN-38)’s action point to that of rMETase, which selectively arrests the cell cycle of cancer cells at the late S/G2 phase (12, 13), results in the very strong synergistic efficacy on HCT-116 colon cancer cells observed in the present study. rMETase acts synergistically with many other cytotoxic chemotherapy drugs (33).
A recent study showed greater efficacy of the combination of cytotoxic chemotherapy drugs with targeted drugs, as compared to cytotoxic–cytotoxic chemotherapy combinations. Particularly, one of the most effective combinations was between IRN (SN-38) and targeted agents, which act on the cell cycle (39).
Oral rMETase is showing promise in the clinic, especially in combination with chemotherapy (40-47).
Recently we have shown that rMETase greatly lowers the effective dose of regorafenib (48).
Conclusion
In the present study, the combination of IRN (SN-38) and rMETase had a synergistic effect, lowering the IC50 of SN-38 by 15-fold on colon cancer cells. The present study indicates the possibility of reducing the dose of IRN, without attenuating its efficacy, by combining it with rMETase, and provides an improved therapeutic opportunity for patients with colon cancer. rMETase is effective since it targets the fundamental and general hallmark of cancer, methionine addiction (6-10, 49-60).
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. The Robert M Hoffman Foundation for Cancer Research provided funds for this study.
Footnotes
Authors’ Contributions
Conception, design and experiments MS, YK and RMH. MS, KM and AB. MS and RMH wrote the article. QH provided methioninase. MS, YK, QH, AB, KM, SM, MB, NK, YI, AN, BMK and RMH critically reviewed the article.
Conflicts of Interest
The Authors declare no competing interests regarding this work.
- Received November 15, 2023.
- Revision received December 4, 2023.
- Accepted December 5, 2023.
- Copyright © 2024 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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