Research ArticleExperimental Studies
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Triple Combination of Recombinant Methioninase and the Anti-parasitic Drugs Ivermectin, and Chloroquine Selectively Eradicates Pancreatic Cancer Cells While Sparing Normal Fibroblasts

YOHEI ASANO, QINGHONG HAN, SHUKUAN LI, KOHEI MIZUTA, BYUNG MO KANG, JIN SOO KIM, YUTA MIYASHI, NORIO YAMAMOTO, KATSUHIRO HAYASHI, HIROAKI KIMURA, SHINJI MIWA, KENTARO IGARASHI, TAKASHI HIGUCHI, SEI MORINAGA, HIROYUKI TSUCHIYA, SATORU DEMURA and ROBERT M. HOFFMAN
Anticancer Research November 2025, 45 (11) 4791-4802; DOI: https://doi.org/10.21873/anticanres.17828

Abstract

Background/Aim: Pancreatic cancer is a recalcitrant disease which often presents with few symptoms in the early stages and is frequently diagnosed with distant metastases, limiting treatment options and resulting in a poor prognosis. Recombinant methioninase (rMETase), which targets cancer-specific methionine addiction, has shown synergistic efficacy with numerous types of chemotherapy against all major cancer types. Ivermectin and chloroquine, anti-parasitic drugs, are showing promise against cancer. The present study investigates in vitro the potential synergy of rMETase combined with ivermectin and chloroquine, two agents with multiple anticancer mechanisms, as a novel treatment strategy for metastatic pancreatic cancer.

Materials and Methods: The human pancreatic-cancer cell line MiaPaCa-2 and normal human fibroblasts Hs27 were cultured in 96-well plates (1×103 cells/well) for 24 h. Cell viability was assessed using the WST-8 reagent following 72-h treatment with rMETase, ivermectin, or chloroquine to determine their 30% inhibitory concentration (IC30) values. To evaluate synergy, cells were treated with each drug alone or double or triple combinations at their respective IC30 concentrations. Additionally, to evaluate the optimal order of the combination therapy, MiaPaCa-2 cells were divided into four groups and sequentially treated for 72 h as follows: (1) untreated control; (2) the triple-drug combination therapy alone; (3) rMETase followed by the triple-drug combination therapy; (4) the triple-drug combination therapy followed by rMETase.

Results: The IC30 value of rMETase was 0.39 U/ml, ivermectin was 4.41 μM, and chloroquine was 3.29 μM on MiaPaCa-2 cells. The triple combination of these agents at their IC30 concentrations significantly inhibited MiaPaCa-2 cell growth compared to monotherapies or dual combinations, indicating a synergistic efficacy. In contrast, the same combination had minimal impact on Hs27 cells at the IC30 values determined for MiaPaCa-2. Regarding the treatment sequence, triple-drug combination therapy and triple-drug combination treatment followed by rMETase treatment significantly inhibited cell proliferation more than rMETase followed by the triple-drug combination treatment.

Conclusion: The combination of rMETase, ivermectin, and chloroquine exhibited a selective cytotoxic synergy against pancreatic-cancer cells while sparing normal fibroblasts. Furthermore, the order of treatment may also affect efficacy. This triple combination treatment may offer a novel and effective first-line therapeutic approach for pancreatic cancer.

Keywords:

Introduction

Pancreatic cancer is a recalcitrant disease and the 7th leading cause of cancer-related deaths worldwide (1). The estimated 5-year survival rate is approximately 10%, which has not significantly changed in decades (1). The poor prognosis is largely attributable to the asymptomatic nature of early-stage disease, leading to delayed diagnosis and a high incidence of recalcitrant distant metastases at initial presentation (2).

Targeting methionine addiction, a fundamental and general hallmark of cancer known as the Hoffman effect, has shown preclinical efficacy and clinical promise (3-14). Previous studies have shown potent efficacy of recombinant methioninase (rMETase) against all major cancer types, including pancreatic cancer, in both in vitro and in vivo models and the clinic (7, 10). Furthermore, rMETase has shown potential for synergy when combined with numerous types of chemotherapy in preclinical studies and clinical case reports of major cancer types (7, 10-14).

Recently, we have combined rMETase with ivermectin, which is conventionally used as an anti-parasitic agent in clinical practice (15-19). Ivermectin also has anti-cancer efficacy through multiple mechanisms, including induction of apoptosis, modulating autophagy, and inhibiting tumor proliferation and metastasis (15, 20-25). A recent in vitro study showed that the combination of rMETase with ivermectin eradicated pancreatic cancer cells (26).

Chloroquine, an anti-malarial drug, has shown efficacy on several malignancies through autophagy inhibition and other mechanisms (27-31). Previous studies have reported the efficacy of chloroquine on pancreatic cancer (32-36). We have previously demonstrated that the combination of rMETase with chloroquine acted synergistically on colorectal cancer cells (37, 38), but its efficacy on pancreatic cancer is unknown.

In the present study, we aimed to determine the synergistic efficacy of a triple-drug combination of rMETase, ivermectin, and chloroquine against a pancreatic-cancer cell line compared to normal fibroblasts.

Materials and Methods

Cell culture. The human pancreatic-cancer cell line MiaPaCa-2 and normal human fibroblasts Hs27 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and maintained at 37°C in a humidified atmosphere containing 5% CO2.

Recombinant methioninase (rMETase) and drugs. rMTEase was produced as previously reported (39) at AntiCancer Inc (San Diego, CA, USA). Ivermectin and chloroquine diphosphate were obtained from MedChemoExpress (Monmouth Junction, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

Cell viability assay and 30% inhibitory concentration (IC30) determination. MiaPaCa-2 and Hs27 cells were seeded into 96-well plates at a density of 1×103 cells per well in 100 μl DMEM and cultured for 24 h. After confirming cell adhesion and proliferation, the cells were treated with rMETase at concentrations ranging from 0.0625 to 8 U/ml, and with either ivermectin or chloroquine at concentrations ranging from 1 to 128 μM for 72 h. Subsequently, to measure cell viability; the WST-8 reagent (10 μl) (Dojindo Laboratories, Kumamoto, Japan) was added to each well, followed by incubation for 1 h. Absorbance was then measured at 450 nm using a microplate reader (Sunrise, Tecan, Männedorf, Switzerland).

Based on the absorbance data, drug-sensitivity curves for rMETase, ivermectin, and chloroquine were generated for both MiaPaCa-2 and Hs27 cells using Microsoft Excel for Mac 2024 (ver. 16.89.1; Microsoft, Redmond, WA, USA) and ImageJ (ver. 1.54g; National Institutes of Health, Bethesda, MD, USA). The 30% inhibitory-concentration (IC30) values were subsequently calculated from these curves. In addition, the half-maximal inhibitory concentration (IC50) of rMETase was also determined for use in treatment-sequence experiments.

Evaluation of synergy between rMETase, ivermectin, and chloroquine on MiaPaCa-2 and Hs27 cells. MiaPaCa-2 and Hs27 cells were seeded into 96-well plates and cultured for 24 h following the procedure described above. Subsequently, the cells were divided into eight groups and treated for 72 h. Each treatment of MiaPaCa-2 cells and Hs27 fibroblasts was performed using the IC30 value previously determined for each agent on MiaPaCa-2 cells. The experimental groups were as follows: 1) control (DMEM), 2) rMETase alone (IC30), 3) ivermectin alone (IC30), 4) chloroquine alone (IC30), 5) rMETase + ivermectin (each at IC30), 6) rMETase + chloroquine (each at IC30), 7) ivermectin + chloroquine (each at IC30), 8) rMETase + ivermectin + chloroquine (each at IC30). In addition, Hs27 fibroblasts were also treated with each agent at the IC30 values for Hs27 fibroblasts. After treatment, absorbance was measured, and the relative cell viability of each treatment group was calculated compared to the control.

Determination of optimal order of the triple-combination and rMETase alone. MiaPaCa-2 cells were seeded and cultured in 96-well plates following the procedure as described above. The cells were then divided into four groups and treated for 72 h each, as follows: 1) control (DMEM), 2) triple-drug combination (each at IC30), 3) rMETase (IC50) followed by the triple-drug combination (each at IC30), 4) the triple-drug combination (each at IC30) followed by rMETase (IC50). At the time of medium exchange after the first 72-h treatment, the cells were washed with phosphate-buffered saline (PBS). After 144 hours of total treatment, the WST-8 reagent was added, absorbance was measured as described above, and the relative cell viability was calculated for each treatment group compared to the control group.

Statistical analysis. All experiments in the present study were performed in triplicate and independently repeated twice. All data are presented as the mean±standard deviation (SD). Statistical comparisons between the treatment groups were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R. A p-value of ≤0.05 was considered statistically significant.

Results

IC30 of rMETase, ivermectin, and chloroquine οn MiaPaCa-2 pancreatic-cancer cells and Hs27 normal fibroblasts. Drug-sensitivity curves of rMEase, ivermectin, and chloroquine for MiaPaCa-2 and Hs27 cells were generated based on cell-viability assays and their IC30 values were determined (Figure 1). The IC30 of rMETase was 0.39 U/ml, ivermectin 4.41 μM, and chloroquine 3.29 μM οn MiaPaCa-2 cells. The IC30 of rMETase was 0.50 U/ml, ivermectin was 4.62 μM, and chloroquine was 3.58 μM οn Hs27 fibroblasts. In addition, the IC50 of rMETase on MiaPaCa-2 cells was 0.58 U/ml (Table I).

Figure 1.
Figure 1.

Drug-sensitivity curves and 30% inhibitory concentration (IC30) of recombinant methioninase (rMETase), ivermectin (IVM), and chloroquine (CQ) on MiaPaCa-2 and Hs27 cells.

View this table:
Table I.

The 30% inhibitory concentration (IC30) values of recombinant methioninase (rMETase), ivermectin (IVM), and chloroquine (CQ) on pancreatic cancer cells (MiaPaCa-2) and normal fibroblasts (Hs27).

Synergy of triple combination of rMETase, ivermectin, and chloroquine on MiaPaCa-2 cells and Hs27 fibroblasts. MiaPaCa-2 pancreatic cancer cells and Hs27 fibroblasts were treated with each drug at its specific IC30 concentration, either as monotherapy or in combination. The triple combination of rMETase, ivermectin, and chloroquine demonstrated synergistic efficacy against MiaPaCa-2 cells compared to each monotherapy or dual combination using the MiaPaCa-2 IC30 values for each agent (Figure 2A). In contrast, this triple combination did not show significant synergistic efficacy on Hs27 cells using the Hs27 IC30 values for each agent (Figure 2B). Moreover, when Hs27 cells were treated using the IC30 values determined for MiaPaCa-2 cells, minimal cytotoxicity was observed (Figure 2C and Table II), further suggesting the selective efficacy of the combination on the cancer cells.

Figure 2.
Figure 2.

Treatment with ivermectin, chloroquine and recombinant methioninase on MiaPaCa-2 pancreatic cancer cells and normal Hs27 fibroblasts. (A) Viability of MiaPaCa-2 cells treated with recombinant methioninase (rMETase, at IC30 for MiaPaCa-2 cells), ivermectin (IVM, at IC30 for MiaPaCa-2 cells), chloroquine (CQ, at IC30 for MiaPaCa-2 cells), rMETase plus IVM (at each IC30 for MiaPaCa-2 cells), rMETase plus CQ (at each IC30 for MiaPaCa-2 cells), IVM plus CQ (at each IC30 for MiaPaCa-2 cells), and rMETase plus IVM plus CQ (at each IC30 for MiaPaCa-2 cells). (B) Viability of Hs27 cells treated as in (A) but using the IC30 value of each agent on Hs27 fibroblasts. (C) Viability of Hs27 cells treated as in (A), using the IC30 for each agent determined for MiaPaCa-2 cells. *p-Value <0.05, ns: Not significant.

View this table:
Table II.

Cell-proliferation inhibitory rate of recombinant methioninase (rMETase), ivermectin (IVM), chloroquine (CQ) on MiaPaCa-2 and Hs27 cells treated with each drug alone and in combination.

Optimal order of the triple combination therapy. Among the treatment groups (Figure 3), continuous administration of the triple combination from the beginning resulted in the greatest inhibition of MiaPaCa-2 cell viability. The group treated with the triple drug combination followed by rMETase showed the second-highest inhibitory effect. There was no significant difference between these two groups (p=0.26). Both of these groups demonstrated significantly greater cell-proliferation inhibition compared to the group treated with rMETase followed by the triple-drug combination therapy (Figure 4, both p<0.05).

Figure 3.
Figure 3.

Determination of the optimal order of combination therapy (Comb) with recombinant methioninase (rMETase) alone, ivermectin (IVM), and chloroquine (CQ) on MiaPaCa-2 cells. rMETase also was administered at its IC50 for MiaPaCa-2 cells, while the triple-drug combination of rMETase, ivermectin and chloroquine was applied using the IC30 of each drug against MiaPaCa-2 cells. Each treatment was carried out for 72 h, and cell viability was subsequently assessed as described in the Materials and Methods. IVM: Ivermectin; CQ: chloroquine.

Figure 4.
Figure 4.

Efficacy of treatment sequence of recombinant methioninase (rMETase) alone and the triple-drug combination (Comb) of rMETase, ivermectin and chloroquine on the viability of MiaPaCa-2 cells. Please see Figure 3 for treatment schema. *p-Value <0.05, ns: Not significant.

Discussion

The present results demonstrated that the triple combination of rMETase, ivermectin, and chloroquine exerts selective synergistic cytotoxic efficacy on pancreatic cancer cells (MiaPaCa-2) in contrast to normal fibroblasts.

rMETase targets methionine addiction, a fundamental and general hallmark of cancer (3-6). Its efficacy has been demonstrated in both in vitro and in vivo studies on all major cancer types, including pancreatic cancer (7, 10). rMETase selectively arrests the cancer cell cycle at the S/G2 phase (40, 41). Consequently, rMETase has been shown to act synergistically with chemotherapeutic agents targeting the S-phase of the cell cycle, suggesting its potential as additive therapy to enhance the efficacy of chemotherapy (3, 7, 10, 42-47), especially in malignancies with poor prognosis such as metastatic pancreatic cancer.

Ivermectin is a 16-membered macrocyclic lactone that has been used clinically as an anti-parasitic agent (15-19). However, recent studies have reported its anti-cancer properties through multiple mechanisms, including the induction of caspase-dependent apoptosis, inhibition of cance-cell proliferation via p21-activated kinase 1 (PAK1)-mediated autophagy, cell-cycle arrest in the S phase, and reversal of chemo-resistance (15, 20-25, 48). In vitro efficacy of ivermectin has been demonstrated in various cancers, including cholangiocarcinoma (22), lung cancer (49), breast cancer (21, 23), colorectal cancer, and leukemia (50, 51).

Similarly, chloroquine, originally developed as an anti-malarial drug, exerts diverse anti-cancer mechanisms. Previous studies have shown that chloroquine induces G1 cell-cycle arrest by inhibiting autophagy, promotes apoptosis via activation of the caspase cascade, and suppresses tumor invasion and metastasis by normalizing the structure and function of tumor vasculature (28, 30, 31, 52, 53) demonstrated in multiple malignancies such as primary effusion lymphoma (29), lung cancer (54), pancreatic cancer (32-36), colorectal cancer (37, 38, 52, 53, 55), breast cancer (56), and osteosarcoma (57). Especially, chloroquine has been used in combination with chemotherapy for pancreatic cancer both pre-clinically and clinically (32-36).

The combination of rMETase with ivermectin previously showed synergy on pancreatic, breast, and colorectal cancer cells in vitro (26, 51, 58), and the combination of rMETase with chloroquine showed synergy on breast cancer, osteosarcoma, and colorectal cancer cells in vitro (37, 56, 57). In the present study, we provide the first evidence that the triple combination of these agents exerts selective synergistic efficacy on pancreatic-cancer cells, in contrast to normal fibroblasts.

Both rMETase and ivermectin target the S-phase of the cell cycle, suggesting the potential for enhanced S-phase inhibition when used in combination. Moreover, since chloroquine targets the G1-phase, the triple combination may exert broad cell cycle inhibition, which could contribute to the observed synergy. Both ivermectin and chloroquine are known to induce apoptosis through modulation of autophagy (15, 21, 23, 27, 29, 32). Methionine restriction itself has also been suggested to influence autophagy (59, 60), and the impact of this triple combination on autophagic pathways may be another possible mechanism underlying the synergy.

Although the synergy of rMETase in combination with chemotherapeutic agents has been demonstrated in various studies, the optimal order of such combination treatments remains largely unexplored. In a previous study investigating the sequential administration of rMETase and rapamycin in colorectal cancer HCT-116 cells, the cells that received continuous combination treatment had the greatest inhibition of proliferation. The group treated with rapamycin followed by rMETase showed the second highest inhibitory effect (61), consistent with the present findings. While the underlying mechanisms of treatment sequence–dependent efficacy were not elucidated in the present study, our results suggest that initiating triple combination therapy as a first-line treatment may be effective in the clinical setting. Additionally, in cases where the combination therapy must be discontinued due to adverse effects, continuing rMETase mono-therapy may still have therapeutic benefit. The present findings may contribute to the development of novel therapeutic strategies for pancreatic cancer.

The present study is an in vitro investigation focusing on the inhibition of pancreatic cancer cells compared to normal-fibroblast proliferation through the synergistic action of the three agents only on the pancreatic-cancer cells in contrast to normal cells. Further studies are needed to elucidate the underlying mechanisms, particularly regarding cell-cycle dynamics and autophagy. Although the present results suggest that the triple combination has minimal effects on normal cells, future clinical applications will require comprehensive in vivo and clinical evaluation to confirm the selective efficacy and safety of this approach.

Conclusion

The combination of rMETase, ivermectin, and chloroquine demonstrated selective synergistic efficacy against pancreatic cancer cells, which was not observed on normal cells. Furthermore, this combination therapy may be most effective when initiated early in the course of treatment. The present findings suggest the potential of this triple combination as a targeted therapeutic strategy for pancreatic cancer. However, further studies using in vivo models are necessary to evaluate the efficacy and safety of this combination treatment in selectively targeting pancreatic cancer.

Methionine restriction is effective because it targets a fundamental hallmark of cancer (3-9, 62-75).

Acknowledgements

This article is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD,, Professor Philip Miles, Richard W. Erbe, Professor Milton Plesur, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Joseph R. Bertino, MD, Shigeo Yagi, PhD, J.A.R Mead, PhD, Eugene P. Frenkel, 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, SL, KM, BMK, JSK, YM, 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 to declare in relation to this study.

  • 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 July 29, 2025.
  • Revision received August 18, 2025.
  • Accepted August 19, 2025.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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