Abstract
Background/Aim: The present study aimed to determine if oral methioninase (o-rMETase) combined with oxaliplatinum (OXA) plus 5-fluorouracil (5-FU) increases survival of mice with peritoneal-carcinomatosis formed from HCT-116 green fluorescent protein (GFP)-expressing colon-cancer cells implanted intra-peritoneally in nude mice. Materials and Methods: HCT-116-GFP human colon-cancer cells (2×106) were injected intraperitoneally in athymic nude mice. Forty-five HCT-116-GFP colon-cancer peritoneal-carcinomatosis nude-mouse models were divided into the following groups: untreated control; combination of 5-FU (50 mg/kg, once a week), plus OXA (6 mg/kg, once a week); combination of 5-FU + OXA + o-rMETase (100 unit/day). Tumor growth was followed weekly using non-invasive GFP imaging for 3 weeks. At necropsy, tumor tissue was obtained. Frozen sections were made for fluorescence imaging. Tumor tissues were also stained with hematoxylin and eosin. The date of death of all mice was recorded. Results: o-rMETase combined with 5-FU + OXA significantly reduced peritoneal growth of the HCT-116 tumor compared to the untreated control or the combination 5-FU and OXA group. Histological analysis revealed extensive necrosis induced by the o-rMETase + 5-FU + OXA combination. The combination of 5-FU plus OXA and o-rMETase achieved significantly longer survival of the mice with peritoneal carcinomatosis compared to the control or combination of 5-FU plus OXA treatments. Conclusion: o-rMETase shows future clinical promise for increasing the survival of patients with peritoneal metastasis of colon cancer when combined with first-line treatment of this recalcitrant disease.
- Colon cancer
- peritoneal metastasis
- HCT-116
- GFP
- methionine addiction
- Hoffman effect
- methioninase
- oral administration
- 5-FU
- oxaliplatinum
- combination
- synergy
- Hoffman protocol
Peritoneal metastasis of colon cancer is a recalcitrant disease, generally resistant to chemotherapy (1). Because of its poor prognosis, colon-cancer peritoneal carcinomatosis is a pre-terminal condition suitable only for palliative treatment. Currently first-line chemotherapy of colon-cancer peritoneal carcinomatosis comprises the combination of 5-fluorouracil (5-FU) together with oxaliplatinum (OXA).
The elevated methionine (MET) requirement of cancer cells is referred to as MET addiction or the Hoffman effect (2), which is analogous to the Warburg effect of excess glucose consumption by cancers (3). The Hoffman effect (2) is more pronounced than the Warburg effect (3) as seen by comparison of radioactive MET and deoxy-glucose positron emission tomography (PET) imaging (4). Methionine addiction is due to overuse of methionine for transmethylation reactions (5-11).
Recombinant methioninase (rMETase) (12) greatly inhibits the growth of all cancer cell types tested both in vitro and in vivo (12-18). Methionine restriction (MR) (19) is synergetic with chemotherapy such as 5-FU (20, 21) and cisplatinum (12, 22) due to selective cell-cycle arrest of methionine-restricted cancer cells in S/G2 (17-19).
rMETase administered parenterally is highly effective on recalcitrant cancer (23-30). Oral rMETase (o-rMETase) is also highly effective, including when combined with 5-FU plus OXA on a colon-cancer patient-derived orthotropic xenograft (PDOX) mouse model and a peritoneal-surface metastatic mouse model to inhibit tumor growth (31-33).
The present report demonstrates the survival efficacy of o-rMETase combined with 5-FU plus OXA on a colon-cancer peritoneal-metastasis nude-mouse model.
Materials and Methods
Mice and cells. Four to six-weeks-old athymic nu/nu nude mice were obtained from AntiCancer Inc. (San Diego, CA, USA). HCT-116 cells expressing green fluorescent protein (GFP) (AntiCancer Inc.) were thawed and cultured for one week and 2×106 cells in 100 μl phosphate-buffered saline (PBS) (Mediatech, Inc., Manassas, VA, USA) were injected to the right-lower-quadrant intraperitoneal space of each mouse. All animal experiments were conducted under National Institutes of Health (NIH) Assurance Number A3873-1 (33).
Production of rMETase. L-methionine-α-deamino-γ-mercaptomethane lyase (rMETase) (EC 4.4.1.11) was produced by fermentation of recombinant E. coli containing the METase gene from P. putida (AntiCancer, Inc.) and purified using a modified previous procedure (34). Pure methioninase was dissolved in PBS or water at 5 mg/ml.
Study design for treatment of colon-cancer peritoneal metastasis. Two weeks after cancer-cell intraperitoneal injection, colon-cancer peritoneal-metastasis models were randomized into three groups of fifteen mice. The first group served as a negative control and did not receive treatment. Mice in the second group were treated intraperitoneally once a week with 50 mg/kg 5-fluorouracil (5-FU), and 6 mg/kg oxaliplatinum (OXA). Mice in the third group received 5-FU, OXA, as in the second group, and 100 units/day of o-rMETase by gavage. All mice in the second and third groups were treated until they died (Figure 1).
Treatment schema. Group 1, Untreated control; Group 2, treatment with 5- fluorouracil (5-FU) + oxaliplatinum (OXA); Group 3, treatment with 5-FU+OXA + oral recombinant methioninase (o-rMETase).
Fluorescence imaging of peritoneal tumors. Mice were imaged non-invasively, weekly for peritoneal colon cancer. Fluorescence intensity of HCT-116-GFP tumors was measured and calculated using the UVP ibox® (Analytik Jena, Berlin, Germany) (Figure 2).
Efficacy of treatment on colon-cancer peritoneal metastasis in nude mice implanted intraperitonelly with HTC-116-GFP human colon-cancer cells. Images were obtained 21 days after the start of treatment. (A) Green fluorescent protein (GFP) fluorescence image of an untreated control mouse. (B) GFP fluorescence image of a mouse treated with 5-fluorouracil (5-FU) + oxaliplatinum (OXA) + oral recombinant methioninase (o-rMETase).
Tumor histology. At necropsy, tumor tissue was obtained, and frozen sections were made and observed under a confocal laser microscope (FV1000, Olympus Corp., Tokyo, Japan). The excitation wavelength for GFP fluorescence was 395 nm. Tumor tissues were embedded in melted paraffin wax and cut into thin sections (5 μm). The slides were stained with hematoxylin and counterstained with eosin.
Statistical analysis. Significant differences for continuous variables were determined using the Student’s t-test. p-Values of ≤0.05 were considered statistically significant. Mortality differences were evaluated by Kaplan-Meier analysis.
Results
Efficacy of treatment on colon-cancer peritoneal metastasis. HCT-116-GFP tumor growth was measured by GFP fluorescence intensity over 21 days. The GFP fluorescence of the untreated HCT-116 tumor growing on the peritoneum was bright and extensive (Figure 2A) compared to mice treated with the combination of o-rMETase, 5-FU plus OXA (Figure 2B).
The fluorescence intensity of the combination of o-rMETase with 5-FU + OXA-treated mice was statistically-significantly lower than the control and 5-FU plus OXA treated mice over a 21-day treatment period (p<0.05) (Figure 3).
Time-course of the tumor fluorescence intensity relative values of the treatment and control groups. 5-FU: 5-fluorouracil; OXA: oxaliplatinum; o-rMETase: oral recombinant methioninase. *p<0.05.
Tumor histology. Intense GFP fluorescence was observed in frozen sections of tumor from the peritoneum of an untreated mouse (Figure 4A). Hematoxylin and eosin (H&E) staining showed that the tumor tissue of the untreated control tumor mainly comprised viable carcinoma cells (Figure 4B). Only the combination of 5-FU, OXA and rMETase induced tumor necrosis (Figure 4C).
Tumor histology. Tissue sections were obtained at necropsy. (A) Confocal-laser microscope image of a frozen section of the HCT-116-green fluorescent protein (GFP) tumor growing on the peritoneal surface of an untreated mouse. (B) Hematoxylin and eosin (H&E)-stained image from the HCT-116-GFP tumor growing on the peritoneum of an untreated mouse. (C) H&E-stained image from the HCT-116-GFP tumor growing on the peritoneum of a mouse treated with the combination of 5-fluorouracil (5-FU) + oxaliplatinum (OXA) + oral recombinant methioninase (o-rMETase).
Survival. Kaplan-Meier analysis showed that survival of mice treated with the combination of o-rMETase with 5-FU + OXA was significantly extended compared to that of control or 5-FU + OXA-treated mice (p<0.05) (Figure 5). The mean survival time for all groups was: Group 1 (untreated control): 20.4 days; Group 2 (5-FU + OXA): 39.5 days; Group 3 (5-FU + OXA + rMETase): 44.6 days. Maximum survival for untreated control mice was 22 days; for mice treated with 5-FU + OXA, 47 days; and for the mice treated with o-rMETase + 5-FU + OXA, 60 days.
Kaplan-Meier survival curves for overall survival of the treatment and control groups. 5-FU: 5-fluorouracil; OXA: oxaliplatinum; o-rMETase: oral recombinant methioninase. *p<0.05.
Discussion
All tested cancer types have been shown to be methionine addicted (15), and thus require a large amount of methionine (5-6, 35). Methionine restriction targets the methionine addiction of cancer cells and METase is very effective for methionine restriction (36). We originally discovered that methionine restriction combined with chemotherapy was synergistic (37), and is termed the Hoffman protocol (38). Our previous studies showed that o-rMETase combined with 5-FU plus OXA was effective on the growth of colon-cancer liver metastasis and peritoneal metastasis (31-33). In the present study, o-rMETase used in combination with 5-FU and OXA increased survival of a massive colon-cancer peritoneal-carcinomatosis HCT-116 nude-mouse model.
Clinical studies of o-rMETase both with and without chemotherapy have shown an effect on rectal cancer (39), pancreatic cancer (40) and prostate cancer (41-44) and breast-cancer metastasis (45). o-rMETase has little side effects. rMETase therapy has future clinical cancer use in combination with 5-FU plus OXA for recalcitrant colon-cancer peritoneal metastasis, because o-rMETase targets the fundamental basis of malignancy (5-11, 35, 46-51).
Acknowledgements
This paper is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Joseph R. Bertino, MD, and Jack Geller, MD.
Footnotes
Authors’ Contributions
Conception and design: MJK, JHP and RMH. QH provided the methioninase. Acquisition of data: MJK, JHP, QH and MB. Analysis and interpretation of data: MJK, QH, MB and JHP. Writing, review and revision of the manuscript: MJK, JHP and RMH.
Conflicts of Interest
RMH is an unsalaried associate of AntiCancer. QH is an employee of AntiCancer. This research was supported in part by Grant no.2019-13 from the Kangdong Sacred Heart Hospital Fund and the Robert M. Hoffman Foundation for Cancer Research.
- Received August 9, 2022.
- Revision received November 6, 2022.
- Accepted November 15, 2022.
- Copyright © 2023 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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