Research ArticleExperimental Studies
Open Access

Selective Synergy of Ivermectin Combined With Recombinant Methioninase Against Colon-Cancer Cells in Contrast to Normal Fibroblasts

YOHEI ASANO, QINGHONG HAN, SHUKUAN LI, KOHEI MIZUTA, BYUNG MO KANG, JIN SOO KIM, NORIO YAMAMOTO, KATSUHIRO HAYASHI, HIROAKI KIMURA, SHINJI MIWA, KENTARO IGARASHI, TAKASHI HIGUCHI, SEI MORINAGA, HIROYUKI TSUCHIYA, SATORU DEMURA and ROBERT M. HOFFMAN
Anticancer Research June 2025, 45 (6) 2257-2263; DOI: https://doi.org/10.21873/anticanres.17600

Abstract

Background/Aim: Recent studies have shown that ivermectin, developed as an anti-parasitic drug, has efficacy against several cancer types. Methionine restriction, including the use of recombinant methioninase (rMETase), has been developed to target methionine addiction, a fundamental hallmark of cancer, termed the Hoffman effect. Metastatic colorectal cancer (CRC) is a recalcitrant disease that requires novel and disruptive treatment approaches. The present study aimed to determine the efficacy of ivermectin in combination with rMETase on a CRC cell line compared to normal fibroblasts.

Materials and Methods: The human CRC cell line HCT-116 and normal human fibroblasts Hs27 were seeded at a density of 1,000 cells per well in 96-well plates and cultured overnight at 37°C. After treatment with ivermectin (0.5 μM to 128 μM) or rMETase (0.0625 U/ml to 8 U/ml) for 72 h, cell viability was assessed using the WST-8 reagent to determine the half-maximal inhibitory concentration (IC50) values for ivermectin and rMETase on both cell lines. Using these IC50 values, cell-viability assays for ivermectin alone, rMETase alone, and the combination of ivermectin and rMETase were performed on both cell lines to determine their synergy.

Results: The IC50 value for ivermectin alone was 4.81 μM and rMETase alone was 0.61 U/ml against HCT-116 colon-cancer cells. The IC50 value for ivermectin alone was 8.67 μM and rMETase alone was 0.67 U/ml against Hs27 normal fibroblasts. In HCT-116 cells, treatment with the combination of ivermectin and rMETase resulted in greater reduction of cell proliferation, compared to treatment with each drug alone; however, no synergy was observed against Hs27 cells.

Conclusion: rMETase and ivermectin showed selective synergistic anti-cancer efficacy against colon-cancer cells, indicating the clinical potential of the combination for metastatic CRC.

Keywords:

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with over 1.9 million new cases and approximately 935,000 deaths reported in 2022 (1). Targeted therapy and immunotherapy have been used for CRC (2,3), but metastatic CRC (mCRC) is still a recalcitrant disease due to frequent resistance to standard treatment, which limits the efficacy of current therapeutic strategies.

Ivermectin belongs to the class of 16-membered macrocyclic lactones and has been widely used as an anti-parasitic agent (4-8). It has been shown to induce apoptosis to inhibit cancer-cell proliferation in breast, lung, colon, and biliary cancers (4, 9-13). Ivermectin also inhibits cancer-cell proliferation by inducing autophagy mediated by p21-activated kinase (PAK1) as well as caspase-dependent apoptosis (14).

Our laboratory has developed recombinant methioninase (rMETase) to target methionine addiction in cancer, a phenomenon known as the Hoffman effect (15-19). Previous studies have demonstrated the therapeutic efficacy of methionine restriction, including rMETase on CRC (20-23). Furthermore, we have shown that the combination of rMETase and ivermectin was synergistic on breast and pancreatic cancer cells in vitro (24, 25).

In the present study, we focused on the combination of rMETase and ivermectin as a novel treatment strategy for CRC. The aim of the present study was to determine the synergy of rMETase and ivermectin on a human CRC cell line in vitro compared to normal fibroblasts.

Materials and Methods

Cell culture. The human HCT-116 colon-cancer cell line and Hs27 normal human fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA, USA) at 37°C in a 5% CO2 incubator.

Reagents. rMETase used in the present study was produced at AntiCancer Inc. (San Diego, CA, USA) by fermentation of recombinant Escherichia coli transformed with the Pseudomonas putida methioninase gene as described previously (26). Ivermectin was obtained from MedChemoExpress (Monmouth Junction, NJ, USA).

Cell viability assay. Cell viability assays were performed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan) to determine the half-maximal inhibitory concentration (IC50) of ivermectin and rMETase on the HCT-116 and Hs27 cell lines. Both cell lines were seeded in 100 μl DMEM at 1,000 cells/well in 96-well plates and incubated overnight. After confirming the proliferation of these cells, they were treated with rMETase at concentrations ranging from 0.0625 U/ml to 8 U/ml, or with ivermectin at concentrations ranging from 0.5 μM to 128 μM for 72 h. Then, 10 μl of WST-8 solution was added to each well. After 1 h, the absorbance at 450 nm was measured using a microplate reader (Sunrise, Tecan, Mannedorf, Switzerland). Drug-sensitivity curves were generated using Microsoft Excel for Mac 2024 ver. 16.89.1 (Microsoft, Redmond, WA, USA), ImageJ ver. 1.54g (National Institutes of Health, Bethesda, MD, USA), and GraphPad Prism 10.4.1 (GraphPad Software, Inc., San Diego, CA, USA). The IC50 was then calculated. These experiments were performed twice, each in triplicate.

Synergy determination of the combination of rMETase and ivermectin. To evaluate the synergy of the combination of rMETase and ivermectin, HCT-116 and Hs27 cells were seeded in 96-well plates and assigned to one of four treatment groups: (i) control (DMEM); (ii) rMETase (IC50); (iii) ivermectin (IC50); or (iv) the combination of rMETase and ivermectin (each at IC50). After 72 h treatment, cell viability was assessed using the WST-8 assay, and absorbance was measured to determine cell viability.

Statistical analysis. All experiments in the present study were performed twice, each in triplicate. All data are presented as mean±standard deviation. Comparisons between treatment groups were calculated using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Statistical analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), with p≤0.05 considered statistically significant.

Results

Determination of the IC50 of ivermectin and rMETase on HCT-116 and Hs27 cells. Drug sensitivity curves of HCT-116 and Hs27 treated with rMETase and ivermectin were generated (Figure 1, Figure 2). For HCT-116 colon-cancer cells, the IC50 value of rMETase was 0.61 U/ml and the IC50 of ivermectin was 4.81 μM. For normal Hs27 fibroblasts, the IC50 values of rMETase and ivermectin, were 0.67 U/ml and 8.67 μM, respectively.

Figure 1.
Figure 1.

Drug-sensitivity curves and half-maximal inhibitory concentrations (IC50) for (A) ivermectin and (B) recombinant methioninase (rMETase) on HCT-116 colon-cancer cells. Please see the Materials and Methods for details.

Figure 2.
Figure 2.

Half-maximal inhibitory concentration (IC50) for (A) ivermectin and (B) recombinant methioninase (rMETase) on Hs27 normal human fibroblasts. Please see the Materials and Methods for details.

Determination of synergy of the combination of rMETase and ivermectin on HCT-116 and Hs27 cells. The IC50 concentrations determined from the drug-sensitivity curves were used to assess viability of HCT-116 and Hs27 cells treated with rMETase alone; ivermectin alone; or a combination of rMETase and ivermectin. The combination resulted in significantly lower cell viability of the HCT-116 cell line, compared to rMETase and ivermectin monotherapy (both p<0.05, Figure 3A). In contrast, although rMETase alone and ivermectin alone inhibited Hs27 cells, their combination did not show enhanced efficacy (Figure 3B).

Figure 3.
Figure 3.

Synergy determination of ivermectin and recombinant methioninase (rMETase) on (A) HCT-116 colon-cancer cells and (B) Hs27 normal human fibroblasts treated with ivermectin (IC50) alone, rMETase (IC50) alone, and their combination. Please see the Materials and Methods for details.

Discussion

rMETase targets methionine addiction, a fundamental hallmark of cancer cells (15-21, 27-37). Methionine restriction selectively arrests cancer cells at the late S/G2 phase of the cell cycle (35, 36). rMETase has been demonstrated to enhance the efficacy of numerous chemotherapy drugs that target cells in S-phase (18, 20-22, 38-41).

Ivermectin belongs to the class of 16-membered macrocyclic lactones and has been widely used as an anti-parasitic agent (4-8). Ivermectin has been shown to induce caspase-dependent apoptosis in cancer cells and to inhibit cancer-cell proliferation by autophagy via PAK1 (4, 9-14). The present study demonstrated promising selective and synergistic efficacy of the combination of ivermectin and rMETase against colorectal-cancer cells (HCT-116) but not normal human fibroblasts (Hs27). The present results suggest a critical difference between cancer cells and normal cells, possibly related to the methionine addiction of cancer cells.

Ivermectin and methionine restriction, including rMETase, inhibit the S-phase of the cell cycle of cancer cells (11, 35, 36, 42, 43), which may account for their synergy. Previous in vitro studies have shown the efficacy of ivermectin against gemcitabine-resistant cholangiocarcinoma cells and paclitaxel-resistant non-small cell lung cancer cells (11, 43), as well as breast and lung cancer (9, 10, 12, 13). However, few studies have been conducted on the combination of ivermectin with other drugs to determine anti-cancer efficacy. The present study is the first to show that ivermectin in combination with rMETase may have synergistic efficacy on colorectal-cancer cells.

Our previous in vitro studies have reported the synergistic efficacy of rMETase and ivermectin on breast and pancreatic cancers (24, 25). As mentioned above, ivermectin can induce autophagy and apoptosis in cancer cells (4, 9-14). Future experiments will investigate the efficacy of rMETase combined with ivermectin on apoptosis and autophagy of cancer cells compared to normal cells.

Limitations of the study include its in vitro design and the use of a single cancer cell line. In addition, the present study is focused on the effect or rMETase and ivermectin on cell proliferation, leaving uninvestigated the underlying mechanisms of these effects. Further studies that will be performed on a larger number of in vitro and in vivo models of colorectal cancer, are necessary to confirm our promising findings.

Conclusion

The combination of ivermectin and rMETase enhanced the anti-cancer efficacy of these agents against colon-cancer cells. The synergy was selective for cancer cells. The results of the present study can be readily translated to the clinic for recalcitrant cancer, as ivermectin is an FDA approved drug and rMETase is used as a dietary enzyme supplement for cancer patients (38, 39).

Acknowledgements

This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Philip Miles, Richard W. Erbe, MD, Professor Milton Plesur, Professor Gordon H. Sato, John W. Littlefield, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, PhD. Eugene P. Frenkel, MD, John Mendelsohn, 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

    YA and RMH designed the study. QH provided rMETase. YA performed experiments and formal analysis. YA was the major contributor to writing – original draft and RMH revised the manuscript. QH, KM, BMK, JSK, NY, KH, HK, ShM, KI, TH, SeM, HT, and SD critically read and approved the final manuscript.

  • Conflicts of Interest

    The Authors have no conflicts of interest.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received April 2, 2025.
  • Revision received May 5, 2025.
  • Accepted May 12, 2025.

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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Selective Synergy of Ivermectin Combined With Recombinant Methioninase Against Colon-Cancer Cells in Contrast to Normal Fibroblasts
YOHEI ASANO, QINGHONG HAN, SHUKUAN LI, KOHEI MIZUTA, BYUNG MO KANG, JIN SOO KIM, NORIO YAMAMOTO, KATSUHIRO HAYASHI, HIROAKI KIMURA, SHINJI MIWA, KENTARO IGARASHI, TAKASHI HIGUCHI, SEI MORINAGA, HIROYUKI TSUCHIYA, SATORU DEMURA, ROBERT M. HOFFMAN
Anticancer Research Jun 2025, 45 (6) 2257-2263; DOI: 10.21873/anticanres.17600
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