Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect (1-5). Methionine restriction was shown to increase the efficacy of chemotherapy drugs on cancer cells almost 40 years ago (2,3). Since then, methionine restriction using recombinant methioninase (rMETase), cloned from Pseudomonas putida, has been shown to increase the efficacy of all cytotoxic chemotherapy drugs tested (2). rMETase targets the methionine addiction of cancer (1-5), and selectively arrests cancer cells in late-S/G₂ phase at which many cytotoxic chemotherapy drugs are active (6-9).

However, rMETase has not been tested in combination with a multiple kinase inhibitor, such as regorafenib. Regorafenib has increased the overall survival of patients with metastatic colorectal cancer (10) and gastro-intestinal stromal tumor (11) with whom first- or second-line therapies have failed. However, the toxicity of regorafenib appears within 3-4 days of the start of treatment and includes severe skin rash, diarrhea, hypertension and fatigue (12). These severe side-effects make it difficult to start regorafenib at the recommended dose of 160 mg/day. We previously reported that 160 mg/day regorafenib increased blood levels of M2 and M5, which are active metabolites of regorafenib, responsible for the severe side-effects. Thus the dose intensity of regorafenib was then reduced, thereby reducing its efficacy (13). Recently, clinical trials showed that starting at a low dose of regorafenib (80 or 120 mg/day) can reduce side-effects and increase dose intensity (14,15). According to these results, the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines^®) recommends a starting dose of 80 mg/day rather than the usual 160 mg/day dose, with dose escalation depending on side-effects (16). However, low-dose regorafenib can also reduce efficacy for some patients.

In the present study, we tested regorafenib in combination with rMETase on a human colon cancer cell line in vitro to determine whether rMETase can reduce the effective dosage of regorafenib.

Cell culture. The HCT-116 human colon cancer cell line was acquired from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in Dulbecco’s minimum essential medium (DMEM) with 10% fetal bovine serum and 100 IU/ml of penicillin/ streptomycin.

rMETase production and formulation. rMETase was produced at AntiCancer Inc. (San Diego, CA, USA) by fermentation of recombinant Escherichia coli transformed with the methioninase gene from Pseudomonas putida. rMETase was purified using a high-yield method, including a 60˚C thermal step, polyethylene-glycol precipitation, and diethylaminoethyl-sepharose fast-flow column chromatography (17).

Cell viability testing. HCT-116 cells were cultivated at subconfluence overnight in Dulbecco’s modified Eagle’s medium in 96-well plates (1.0×10³ cells per well). The following day, HCT-116 cells were treated with concentrations of rMETase ranging from 0.125 to 8 U/ml or regorafenib ranging from 0.25 μM to 16 μM. Regorafenib was purchased from MedChemExpress (Monmouth Junction, NJ, USA). After 72 h of treatment, cell viability was assessed using the Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) with the WST-8 reagent.

ImageJ version 1.53 (National Institutes of Health, Bethesda, MD, USA) was applied to calculate IC₅₀ values and sensitivity curves. After calculating the half-maximal inhibitory concentration (IC₅₀) for rMETase and regorafenib, the IC₅₀ concentrations of both drugs were used to determine the synergistic efficacy of the combination of the drugs. Finally, we repeated the IC₅₀ study for regorafenib on HCT-116 cells treated in combination with the IC₅₀ concentration of rMETase to examine whether methionine restriction increased the efficacy of regorafenib. Each experiment was carried out in triplicate.

Statistics. GraphPad Prism 9.4.0 (GraphPad Software, Inc., San Diego, CA, USA) was used to conduct all statistical analyses. Tukey’s multiple comparison test was performed for the parametric test of comparison between groups. All data are presented as the mean and standard deviation. The significance level was p≤0.05.

Determination of the IC₅₀ of rMETase alone and regorafenib alone and efficacy of their combination on HCT-116 cells in vitro. We first evaluated the sensitivity to rMETase alone and regorafenib alone of HCT-116 cells in vitro, and IC₅₀ values were calculated. The IC₅₀ of rMETase on HCT-116 cells was 0.61 U/ml and the IC₅₀ of regorafenib was 2.26 μM (Figure 1 and Figure 2). The combination of rMETase and regorafenib at their IC₅₀ highly reduced the viability of HCT-116 cells in comparison to either agent alone at its IC₅₀ (Figure 3).

Reduction of the IC₅₀ of regorafenib in combination with the IC₅₀ of rMETase. The IC₅₀ of regorafenib against HCT-116 was reduced from 2.26 μM to 1.46 μM when cells were treated in the presence of rMETase at its IC₅₀ (Figure 4).

Regorafenib is a third-line chemotherapy drug for metastatic colorectal cancer and gastro-intestinal stromal tumor, used after the failure of first- or second-line therapy. The problem with regorafenib is its dose-limiting toxicity. The results of the present study suggest that rMETase, when combined with regorafenib, can reduce the effective dose of regorafenib and thereby also reduce its toxicity. Previously, we showed in an orthotopic mouse model of osteosarcoma that combining rMETase with cisplatinum reduced its effective dose by 50% (18). A similar effect is predicted for the combination of regorafenib and rMETase in future in vivo experiments.

Regorafenib is a multikinase inhibitor of angiogenic, stromal, and oncogenic-receptor tyrosine kinases. In an in vitro study using the human colorectal cancer cell line HT-29, regorafenib increased the intra-cellular S-adenosyl-L-methionine (SAM) level by 34.5% compared to the control (19). SAM is the only provider of methyl groups to DNA, RNA, histones and other proteins. This suggests that regorafenib inhibits transmethylation reactions which use SAM. Methionine depletion reduces SAM levels in cells, further reducing transmethylation reactions which are elevated in cancer cells (4,20-29). Therefore, the combination of rMETase and regorafenib had synergistic efficacy in the present study, giving rise to the possibility of lowering the effective dose of regorafenib in patients, thereby reducing its severe toxicity, and increasing the use of regorafenib in the clinic. rMETase is effective because it targets methionine addiction, the fundamental hallmark of cancer (1-5,20-33).

The Authors declare no competing interests regarding this work.

BBC and YK performed experiments. BBC, YK, and RMH wrote the article. QH provided methioninase. DA, SM, KM, MB, and TT critically reviewed the article.

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.