Research ArticleExperimental Studies
Open Access

The Triple Combination of Recombinant Methioninase, Rapamycin, and Chloroquine Synergistically Eradicates HCT116 Colon Cancer Cells

JINSOO KIM, QINGHONG HAN, BYUNG MO KANG, KOHEI MIZUTA, YOHEI ASANO, MICHAEL BOUVET and ROBERT M. HOFFMAN
Anticancer Research July 2025, 45 (7) 2773-2779; DOI: https://doi.org/10.21873/anticanres.17646

Abstract

Background/Aim: The prognosis for advanced/metastatic colon cancer is poor, with low survival rates despite aggressive treatment. Recombinant methioninase (rMETase) targets the methionine addiction of cancer and works synergistically with many anticancer drugs. The present study aimed to determine the synergistic efficacy of the combination of rMETase, rapamycin (RAPA), and chloroquine (CQ) on human HCT116 colon cancer cells in vitro.

Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rMETase, RAPA, and CQ were determined for the human HCT116 cell line in vitro. The synergy of rMETase combined with RAPA; rMETase combined with CQ; and rMETase combined with RAPA and CQ, at their respective IC50 values, was determined on HCT116 cells. Cell viability was measured with the WST-8 reagent.

Results: The IC50 value of rMETase was 0.48 U/ml; for RAPA 19.6 μM; and for CQ 16.5 μM on the HCT116 cell line. Synergy was observed with both the combination of rMETase and RAPA (p=0.02) and the combination of rMETase and CQ (p=0.03). The triple combination of rMETase, RAPA, plus CQ showed the strongest synergistic efficacy, eradicating HCT116 cells (p=0.01).

Conclusion: The triple combination of rMETase, RAPA and CQ shows strong synergistic efficacy on the human HCT116 colon cancer cell line. The results of the present study suggest the potential for future clinical applications of this triple-combination treatment for advanced/metastatic colon cancer.

Keywords:

Introduction

In 1959, Sugimura et al. demonstrated that tumor growth was inhibited in rat models when methionine was excluded from the diet (1). Subsequently, it was observed that cancer cells synthesize normal or greater-than-normal levels of methionine despite their absolute requirement for exogenous methionine (2). These findings led to the concept of methionine addiction in cancer cells, known as the Hoffman effect (3-21). Methionine addiction is due to the excessive utilization of methionine for transmethylation reactions within cancer cells, resulting in their dependency on exogenous methionine (11-17).

Methionine restriction inhibits DNA synthesis by inducing cell-cycle arrest at the late S/G2 phase in cancer cells (18, 19). An effective approach to limiting methionine availability in cancer cells involves the use of methioninase, an enzyme which degrades methionine (20). Our previous investigations have demonstrated the synergistic efficacy of recombinant methioninase (rMETase) in combination with numerous chemotherapeutic agents and targeted therapies in multiple cancer types (22). The selective cell-cycle arrest by rMETase of cancer cells in the S/G2 phase of the cell cycle increases the efficacy of cancer drugs, which also target S/G2.

The mammalian target of rapamycin (mTOR) pathway regulates cell proliferation and protein synthesis in cancer cells (23). Rapamycin (RAPA) is an mTOR inhibitor and has demonstrated clinical efficacy on renal-cell carcinoma and breast cancer (24). We have demonstrated that rMETase and RAPA were synergistic on cancer cells in vitro and in vivo (25-26).

Autophagy degrades damaged organelles and recycles intracellular components to sustain metabolic balance (27). Chloroquine (CQ) inhibits autophagy, leading to apoptosis in cancer cells due to autophagosome accumulation (27, 28). Although CQ is not used as a monotherapy in cancer treatment due to the high dose required for efficacy, its combination with standard therapeutic regimens has been proposed to overcome cancer resistance (29, 30).

Water-soluble derivatives of RAPA (e.g., temsirolimus and everolimus) and CQ (e.g., hydroxychloroquine) had synergistic efficacy on solid tumors and melanoma in phase I clinical trials (31). We have previously demonstrated the synergy of RAPA and CQ in liposarcoma using patient-derived orthotopic xenograft (PDOX) mouse models and in vitro systems (32, 33). We recently demonstrated the synergistic efficacy of the triple combination of rMETase, RAPA, and CQ on osteosarcoma cells in vitro (34).

Building on our previous findings, the present study aimed to investigate whether the triple combination of rMETase, RAPA, and CQ has synergistic efficacy on the HCT116 colon cancer cell line.

Materials and Methods

Cell culture. The HCT-116 human colon carcinoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 with GlutaMAX™ supplement (DMEM/F-12), along with with 10% fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin, at 37°C in a 5% CO2 incubator.

Recombinant methioninase production. rMETase was produced by AntiCancer Inc. (San Diego, CA, USA). The methioninase gene, cloned from Pseudomonas putida, was introduced into Escherichia coli and expressed using fermentation. rMETase purification involved a 60°C thermal step, polyethylene glycol precipitation, and diethylaminoethyl (DEAE)-sepharose ion-exchange chromatography, as previously described (35).

Reagents. RAPA and CQ were obtained from MedChem-Express (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM.

Cell viability assay. Cell viability was assessed using the WST-8 assay (Dojindo Laboratories, Kumamoto, Japan). HCT-116 cells were seeded at 2.0×103 cells/well in 96-well plates and incubated overnight. Cells were treated with varying concentrations of rMETase (0.125-8 U/ml), RAPA, or CQ (1-128 μM) for 72 h. After treatment, 10 μl of WST-8 solution was added to each well, and absorbance was measured at 450 nm using a microplate reader (Sunrise; Tecan, Männedorf, Switzerland). Drug sensitivity curves and half-maximal inhibitory concentration (IC50) values were generated using Microsoft 365 Excel for MacOS (Microsoft, Redmond, WA, USA), ImageJ ver. 1.54g (National Institutes of Health, Bethesda, MD, USA), and GraphPad Prism ver. 10.4.1 (GraphPad Software, Inc., San Diego, CA, USA).

Combination treatment. HCT116 cells were seeded in 96-well plates at a density of 2.0×103 cells/well in DMEM/F-12. After 24 h, the cells were treated as follows: 1) control (DMEM/F-12 only); 2) rMETase at its IC50 concentration (0.48 U/m); 3) RAPA at its IC50 concentration (19.6 μM); 4) CQ at its IC50 concentration (16.5 μM); 5) rMETase (0.48 U/ml) plus RAPA (19.6 μM); 6) rMETase (0.48 U/ml) plus CQ (16.5 μM); and 7) rMETase (0.48 U/ml) plus RAPA (19.6 μM) plus CQ (16.5 μM). After 72 h treatment, cell viability was determined with the WST-8 reagent. Experiments were performed in triplicate.

Statistical analysis. Data are presented as mean±standard deviation. Groups were compared using one-way analysis of variance (ANOVA). Statistical significance was evaluated using Tukey’s multiple comparison test, with p-values ≤0.05 considered significant.

Results

Determination of IC50 values of rMETase, RAPA, and CQ on HCT116 cells. The IC50 value of rMETase was 0.48 U/ml; RAPA was 19.6 μM; and CQ was 16.5 μM for HCT116 cells (Figure 1).

Figure 1.
Figure 1.

Determination of the half-maximal inhibitory concentration (IC50) of recombinant methioninase (rMETase), rapamycin (RAPA), and chloroquine (CQ) on HCT116 cells in vitro. Cell viability was measured using the WST-8 assay. Data are shown as the mean±standard deviation. Please see the Materials and Methods for details.

Synergy of rMETase, RAPA, and CQ on HCT116 cells. All agents alone significantly reduced the viability of the HCT116 cells compared to the control cells: rMETase alone (0.48 U/ml, IC50) reduced the viability of HCT116 cells by approximately 37.5% (p=0.03). RAPA alone (19.6 μM, IC50) reduced the viability of HCT116 cells by 52.4.0% (p=0.04). CQ alone (16.5 μM, IC50) reduced the viability of HCT116 cells by 42.1% (p=0.05). rMETase plus RAPA reduced the viability of cells by 82.2% (p= 0.02). rMETase plus CQ reduced the viability of HCT116 cells by 66.7% (p=0.03). rMETase plus RAPA plus CQ essentially eradicated HCT116 cells by 95.2% (p= 0.01). The triple combination of rMETase, RAPA and CQ caused significantly greater reduction in the viability of HCT116 cells compared to monotherapies or the dual combinations rMETase plus RAPA or rMETase plus CQ (p< 0.001; Figure 2).

Figure 2.
Figure 2.

Synergy of the combination of recombinant methioninase (rMETase), rapamycin (RAPA), and chloroquine (CQ) at their IC50 values on HCT116 colon cancer cells. Data are shown as the mean±standard deviation. Please see the Materials and Methods for details. Significantly different at: *p<0.05, **p<0.01, ***p<0.001.

Discussion

Colorectal cancer is the third most common malignancy and the second leading cause of cancer-related mortality worldwide (36). Although surgery, chemotherapy, and radiation have improved the prognosis of localized colorectal cancer with an approximately 65% 5-year overall survival rate, the survival rate for metastatic/advanced disease is only approximately 14% (37). Therefore, new treatment strategies are needed for the treatment of metastatic colorectal cancer (38).

Based on our previous demonstrations of synergy of rMETase and chemotherapy drugs in colon cancer in vitro and in mouse models (39-42), the present study demonstrated that the triple combination of rMETase, RAPA, and CQ was highly synergistic in HCT116 colon cancer cells. Methionine restriction reduces the intracellular S-adenosylmethionine (SAM) concentration, which in turn inhibits SAMTOR binding and mTOR activity (6, 43). This may explain the synergy of rMETase and RAPA. Methionine restriction also affects autophagy, which may explain the synergy of rMETase and CQ (44, 45). We recently reported the synergy of rMETase, RAPA, and CQ on the 143B osteosarcoma cell line (34). The present study shows that HCT116 cells are more sensitive to the double combinations of rMETase plus RAPA and rMETase plus CQ than 143B osteosarcoma cells determined previously (34).

The triple combination of rMETase, RAPA, and CQ will be tested on colon-cancer mouse models and in the clinic in the future.

Conclusion

The triple synergistic effect of rMETase, RAPA, and CQ on HCT116 colon cancer cells can be readily translated to the clinic as RAPA and CQ are approved drugs and rMETase is given to cancer patients as a dietary enzyme supplement (46, 47, 48).

rMETase is effective as it targets the fundamental hallmark of cancer, methionine addiction (2-21).

Acknowledgements

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

    JK and RMH designed the study. QH produced rMETase. JK conducted all experiments and wrote the article. RMH revised the article. BMK, KM, YA, and MB critically read the manuscript.

  • Conflicts of Interest

    The Authors have no conflicts of interest or financial ties to disclose related 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 April 8, 2025.
  • Revision received April 28, 2025.
  • Accepted April 29, 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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The Triple Combination of Recombinant Methioninase, Rapamycin, and Chloroquine Synergistically Eradicates HCT116 Colon Cancer Cells
JINSOO KIM, QINGHONG HAN, BYUNG MO KANG, KOHEI MIZUTA, YOHEI ASANO, MICHAEL BOUVET, ROBERT M. HOFFMAN
Anticancer Research Jul 2025, 45 (7) 2773-2779; DOI: 10.21873/anticanres.17646
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