Abstract
Background/Aim: Ivermectin is a widely-used anti-parasitic agent and has shown early promise as an anticancer agent. Recombinant methioninase (rMETase) is a methionine-depleting enzyme targeting the methionine addiction of cancer and has broad efficacy against all tested cancer types. However, the combination efficacy of ivermectin and rMETase on breast cancer cells remains unexplored. The present study aimed to determine the synergistic efficacy of ivermectin and rMETase on MCF-7 human breast cancer cells in vitro. Materials and Methods: The IC10 of ivermectin and IC50 of rMETase were determined on MCF-7 cells using the WST-8 reagent to measure cell viability in vitro. MCF-7 cells were treated with four groups: untreated control; ivermectin alone (4.89 μM, IC10); rMETase alone (2.75 U/ml, IC50); and a combination of ivermectin (4.89 μM) and rMETase (2.75 U/ml). Cell viability was assessed 72 hours after treatment with the WST-8 reagent. Results: Treatment with ivermectin (4.89 μM) did not significantly reduce the viability of MCF-7 cells. rMETase (2.75 U/ml) alone significantly reduced MCF-7 cell viability compared to the control group. The combination of ivermectin and rMETase resulted in a significantly greater reduction in cell viability than either agent alone, including a 9.9-fold greater efficacy than ivermectin alone, demonstrating synergistic efficacy (p<0.05). Conclusion: The combination of ivermectin and rMETase had synergistic efficacy against MCF-7 breast cancer cells in vitro. The present findings suggest that the combination of ivermectin and rMETase is a promising strategy for breast cancer requiring further preclinical and clinical evaluation.
- Ivermectin
- recombinant methioninase
- synergy
- in vitro
- breast cancer cells
- methionine addiction
- Hoffman effect
Breast cancer remains the most common malignancy among women worldwide, accounting for a significant proportion of cancer-related morbidity and mortality (1). The development of effective therapeutic strategies targeting breast-cancer cells is critical for improving patient outcomes. The MCF-7 cell line, representing hormone-receptor-positive breast cancer, serves as a widely-used model for discovering novel therapeutic strategies (2).
Ivermectin, a macrocyclic lactone, is generally used as an antiparasitic agent. The Nobel prize was awarded to Satoshi Omura and William C. Campbell for its use against parasitic roundworms (3). Ivermectin has recently been found to have anticancer properties (4). Several studies have demonstrated its ability to induce cell-cycle arrest, apoptosis, and autophagy in various cancer cell lines, including breast cancer (5, 6).
Recombinant methioninase (rMETase), an enzyme that degrades extracellular methionine, targets the methionine addiction of cancer, termed the Hoffman effect (7, 8). Previous studies have demonstrated the efficacy of rMETase in preclinical models and patients with breast cancer (9-14).
We have previously shown synergy of ivermectin and rMETase in a pancreatic-cancer cell line (15). The present study investigated the efficacy of ivermectin and rMETase, individually and in combination, on MCF-7 human breast-cancer cells in vitro to determine their potential synergistic interaction.
Materials and Methods
Cell culture. MCF-7 breast-cancer cells were from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin (Corning, Corning, NY, USA) for all experiments.
Reagents. Ivermectin was from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant methioninase (rMETase) was produced by AntiCancer Inc. by fermentation of recombinant E. coli transformed with the Pseudomonas putida methioninase gene, following a previously-established process (16).
Drug sensitivity assay 1: Determination of the IC10 of ivermectin and IC50 of rMETase on MCF-7 breast cancer cells. To determine the IC10 of ivermectin and IC50 of rMETase, MCF-7 cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). MCF-7 cells (3×103 cells per well) were seeded in 96-well plates containing 100 μl of DMEM per well. After incubation, rMETase at concentrations ranging from 0.5 U/ml to 8 U/ml or ivermectin at concentrations from 5 μM to 80 μM were added to the wells for 72 h. Following the 72 h incubation at 37°C, WST-8 solution (10 μl) was then added to each well, and the plates were incubated for an additional hour at 37°C. Absorbance at 450 nm, which correlates with cell viability, was measured using a microplate reader (SUNRISE, TECAN, Mannedorf, Switzerland). Drug-sensitivity curves were generated using Microsoft Excel (Microsoft, Redmond, WA, USA). The IC50 of rMETase and IC10 of ivermectin were calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA). Each experiment was conducted twice in triplicate.
Drug sensitivity assay 2: Determination of synergy of ivermectin and rMETase on MCF-7 breast cancer cells. To determine the combined efficacy of ivermectin and rMETase on MCF-7 cells, the cells were seeded in 96-well plates at a density of 3×103 cells per well. After 24 h, the cells were treated as follows: control (DMEM); ivermectin alone (4.89 μM, [IC10]); rMETase alone (2.75 U/ml, [IC50]); and a combination of ivermectin (4.89 μM, [IC10]) and rMETase (2.75 U/ml, [IC50]). After 72 h of treatment, cell viability was measured as described above.
Statistical analysis. All statistical analyses were performed using EZR software (Jichi Medical University, Saitama, Japan). The Tukey–Kramer test was used to evaluate relationships between groups, with a significance level set at p≤0.05.
Results
Determination of IC10 of ivermectin alone and IC50 of rMETase alone on MCF-7 breast-cancer cells. The IC10 of ivermectin alone on MCF-7 cells was 4.89 μM. The IC50 for rMETase alone on MCF-7 cells was 2.75 U/ml (Figure 1).
Determination of the IC10 of ivermectin and IC50 of recombinant methioninase (rMETase) on MCF-7 breast cancer cells in vitro. A) IC10 of ivermectin on MCF-7 cells. B) IC50 of rMETase on MCF-7 cells. Please see Materials and Methods for details.
rMETase sensitized MCF-7 breast cancer cells to ivermectin. Compared to the control cells, treatment with ivermectin alone (4.89 μM, IC10) reduced the viability of MCF-7 cells by approximately 7.1 % (p>0.05), rMETase alone (2.75 U/ml, IC50) reduced the viability by 46.3 % (p<0.05), and the combination of ivermectin (4.89 μM, IC10) plus rMETase (2.75 U/ml, IC50) reduced the viability of MCF-7 cells by approximately 70.6%; p<0.05 compared to ivermectin alone or rMETase alone (Figure 2). Thus, rMETase incrased the sensitivity of MCF-7 cells to ivermectin 9.9 fold.
Recombinant methioninase (rMETase) synergistically sensitized MCF-7 breast cancer cells to low-dose ivermectin in vitro. Control; DMEM; ivermectin (4.89 μM, [IC10 for MCF-7 cells]); rMETase (2.75 U/ml, [IC50 for MCF-7 cells]); ivermectin (4.89 μM, [IC10 for MCF-7 cells]) plus rMETase (2.75 U/ml, [IC50 for MCF-7 cells]). Please see Materials and Methods for details.
Discussion
rMETase targets methionine addiction, a fundamental and general hallmark of cancer (7, 8). rMETase has demonstrated efficacy in preclinical models of all types of malignancies, including breast cancer (9-14, 17-18). Additionally, rMETase sensitized cancer cells to all major chemotherapy drugs (18-22). By targeting a global metabolic vulnerability of cancer cells, rMETase represents a promising strategy for enhancing the effectiveness of existing therapies.
The present study demonstrates, for the first time, the synergistic efficacy of ivermectin and rMETase against breast-cancer cells in vitro. Both rMETase and ivermectin arrest cancer cells during the S-phase of the cell cycle (23-27), which may account for their synergy.
We have previously shown that ivermectin and rMETase were synergistic against pancreatic-cancer cells (15). The present study demonstrated the synergy of ivermectin and rMETase on breast-cancer cells, even though the breast-cancer cells were not sensitive to ivermectin alone. Further studies are necessary in mouse cancer models and clinical settings to determine the mechanism underlying the synergistic efficacy of ivermectin and rMETase. Other studies have also shown surprising efficacy of ivermectin. In a study of cholangiocarcinoma (bile duct) cancer cells in vitro, ivermectin overcame gemcitabine resistance (25). Ivermectin also overcame paclitaxel resistance in non-small-cell lung-cancer cells by abolishing p-glycoprotein expression (26).
Methionine restriction is effective because it targets methionine addiction, the fundamental hallmark of cancer (7, 8, 27-44).
rMETase is showing clinical promise in breast cancer (11, 12), as well as pancreatic cancer (45, 46), rectal cancer (47, 48), prostate cancer (49-52) and leukemia (53), which can be combined with ivermectin for all cancer types.
Acknowledgements
This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, Ph.D., Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, Joseph Leighton, MD and John Mendelsohn, MD.
Footnotes
Authors’ Contributions
SM, RMH, and QH designed the study. QH provided rMETase. SM performed experiments. SM was the major contributor to writing the article and RMH revised the article. KM, BMK, CH, MB, NY, KH, HK, SM, KI, TH, HT, and SD critically read the manuscript.
Conflicts of Interest
The Authors have declared that there are no competing interests in relation to this study.
- Received December 25, 2024.
- Revision received January 12, 2025.
- Accepted January 13, 2025.
- Copyright © 2025 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).