Research ArticleExperimental Studies
Open Access

Combination of Recombinant Methioninase With Rapamycin or Chloroquine Is Synergistic to Highly Inhibit Triple-negative Breast Cancer Cells In Vitro

JINSOO KIM, QINGHONG HAN, BYUNG MO KANG, KOHEI MIZUTA, YOHEI ASANO, MICHAEL BOUVET and ROBERT M. HOFFMAN
Anticancer Research May 2025, 45 (5) 1853-1859; DOI: https://doi.org/10.21873/anticanres.17564

Abstract

Background/Aim: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer with a poor prognosis despite multimodal treatment. New therapeutic approaches for TNBC are necessary. We very recently showed that the triple combination of recombinant methioninase (rMETase), rapamycin (RAPA), and chloroquine (CQ) synergically eradicated osteosarcoma cells in vitro. The present study aimed to determine whether rMETase has synergistic efficacy with either RAPA or CQ on a TNBC cell line.

Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rMETase, RAPA, and CQ were determined on the human MDA-MB-231 TNBC cell line in vitro. The efficacy of rMETase, combined with RAPA or with CQ, at their respective IC50 values, on MDA-MB-231 cell viability was determined using the WST-8 assay.

Results: The IC50 of rMETase was 0.56 U/ml, for RAPA the IC50 was 3.9 μM, and for CQ the IC50 was 5.0 μM. The viability of the MDA-MB-231 cells was significantly decreased after treatment with rMETase plus RAPA or rMETase plus CQ, compared to the control cells or cells treated with one drug only.

Conclusion: rMETase, when combined with either RAPA or CQ, is synergistic on the MDA-MB-231 TNBC cell line. The present findings suggest the potential for future clinical applications of rMETase plus chemotherapy, such as RAPA or CQ, for recalcitrant TNBC.

Keywords:

Introduction

Breast cancer is one of the most common cancers in the United States and the second-leading cause of cancer-related death in women. Among breast cancers, triple-negative breast cancer (TNBC) accounts for about 10 to 20 percent of all cases. TNBC is defined by the lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. TNBC is known to be an aggressive breast-cancer type associated with a high risk of recurrence. Furthermore, treating TNBC is challenging due to the limited availability of targeted therapies and resistance to common chemotherapy. The average 5-year survival rate for patients with TNBC is about 77%, which is 8-16% lower than the average 5-year survival rate for hormone-receptor-positive breast cancer (1, 2).

Methionine addiction of cancer cells is the fundamental hallmark of cancer, known as the Hoffman effect (3-15). It occurs due to the excessive use of methionine in transmethylation reactions within cancer cells, leading to exogenous methionine dependence (8, 16-22). Methionine restriction selectively blocks the late-S/G2 phase of the cell cycle in cancer cells, thereby acting as a DNA synthesis inhibitor (23, 24).

Recombinant methioninase (rMETase), cloned from Pseudomonas putida into Escherichia coli, can degrade methionine to achieve methionine restriction (25, 26). rMETase has shown synergistic efficacy when combined with multiple anticancer drugs or targeted therapies in many cancer types (27). Synergistic efficacy of rapamycin (RAPA) and rMETase has been reported in breast osteosarcoma using the patient-derived orthotopic xenograft (PDOX) mouse model (28), as well as in vitro, on a human colon-cancer cell line (29).

Autophagy is a critical cellular process that maintains homeostasis by degrading damaged organelles to provide metabolic substrates and eliminating cellular signals that could induce apoptosis. Autophagy is inhibited by the overproduction of autophagosomes induced by mTOR inhibitors such as RAPA and further exacerbated by chloroquine (CQ), which prevents autophagosome-lysosome fusion. The inhibition of autophagy can eventually lead to apoptosis due to the accumulation of autophagosomes in cancer cells (30, 31). Consequently, CQ can induce apoptosis in cancer cells as an autophagy inhibitor. We reported that the combination of RAPA and CQ had synergistic efficacy or a PDOX mouse model of dedifferentiated liposarcoma and on an in vitro model of dedifferentiated liposarcoma (32, 33). We have very recently shown that the triple combination of rMETase, CQ, and RAPA synergically eradicated osteosarcoma cells in vitro (34). The present study aimed to determine whether rMETase has synergy with RAPA or with CQ on TNBC.

Materials and Methods

Cell culture. The MDA-MB-231 human triple-negative breast-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), with 10% fetal bovine serum and 100 IU/ml of penicillin/streptomycin.

Recombinant methioninase (rMETase) production. rMETase was obtained from AntiCancer Inc. (San Diego, CA, USA). The methioninase gene, previously cloned from Pseudomonas putida, was transformed into Escherichia coli, which is fermented to produce rMETase. rMETase was purified using a high-yield technique involving a 60°C thermal step, polyethylene glycol precipitation, and diethylaminoethyl-sepharose ion-exchange column chromatography (26).

Reagents. RAPA and CQ were obtained from MedChem Express (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). The stock solution concentration for both RAPA and CQ was 10 mM.

Cell viability test. The viability of MDA-MB-231 cells was assessed using the WST-8 reagent (Dojindo Laboratories, Kumamoto, Japan). Cells were cultured in 96-well plates at 2.0×103 cells/well in DMEM/F-12 overnight at 37°C with 5% CO2. Cells were treated for 96 h with varying concentrations of rMETase (0.125-8 U/ml), or RAPA, or CQ (1-128 μM). After treatment, 10 μl of WST-8 solution was added to each well, and plates were incubated for an additional hour. 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 Excel, ImageJ (ver. 1.54g), and GraphPad Prism (ver. 10.4.1).

Efficacy of the combination of rMETase plus RAPA or rMETase plus CQ. MDA-MB-231 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 with as follows: control DMEM/F-12 only; rMETase at the IC50 concentration (0.56 U/ml); RAPA at the IC50 concentration (3.9 μM); CQ at the IC50 concentrations (5.0 μM); rMETase plus RAPA a the IC50 concentrations (0.56 U/ml and 3.9 μM, respectively); rMETase plus CQ at the IC50 concentration (0.56 U/ml and 5.0 μM, respectively). After 96 h treatment, cell viability was determined using the WST-8 reagent. Experiments were performed in triplicate.

Statistical analysis. Data are presented as mean±standard deviation. One-way analysis of variance (ANOVA) was used to compare data among the three groups. Tukey’s multiple comparison test was used for intergroup comparisons. Statistical analyses were performed using GraphPad Prism (ver. 10.4.1, GraphPad Software Inc., La Jolla, CA, USA), with p-values ≤0.05 considered statistically significant.

Results

Determination of IC50 values. The IC50 values for MDA-MB-231 cells were 0.56 U/ml for rMETase, 3.9 μM for RAPA, and 5.0 μM for CQ. (Figure 1).

Figure 1.
Figure 1.

Determination of the half-maximal inhibitory concentration (IC50) of recombinant methioninase (rMETase), rapamycin, (RAPA) and chloroquine (CQ) for MDA-MB-231 cells in vitro. Cell viability was measured using the WST-8 reagent. Data are presented as the mean±standard deviation.

Synergistic efficacy of rMETase plus RAPA or rMETase plus CQ. All drugs significantly reduced the viability of the MDA-MB-231 cells compared to the control group. Compared to the control cells, treatment with rMETase (0.56 U/ml) reduced the viability of MDA-MB-231 cells by approximately 55.8% (p< 0.0001); RAPA (3.9 μM) by 21.9% (p<0.0001); and CQ (5.0 μM, IC50) by 15.6% (p=0.0014). The combination of rMETase plus RAPA reduced MDA-MB-231 cell-viability by 77.0% (p<0.0001, compared to the control); and rMET plus CQ reduced the viability of MDA-MB-231 cells by 74.3% (p< 0.0001), compared to the control. The combination of rMETase plus RAPA or rMETase plus CQ synergically reduced MDA-MB-231 cell viability compared to RAPA, CQ, or rMETase alone (p<0.001 for all comparisons) (Figure 2).

Figure 2.
Figure 2.

Cell viability of MDA-MB-231 cells treated with the combination of recombinant methioninase (rMETase) plus rapamycin (RAPA) or rMETase plus chloroquine (CQ), using the half-maximal inhibitory concentration (IC50) for each agent on MDA-MB-231 cells. Data are presented as the mean±standard deviation. **p=0.0014, ***p=0.0004, ****p<0.0001.

Discussion

Our laboratory first identified methionine addiction in cancer cells, a phenomenon known as the Hoffman effect (3-24, 35-41). To target methionine addiction, we developed rMETase (26). Since first showing the synergy of methionine restriction and chemotherapy (9), we have demonstrated synergy of methionine restriction and rMETase with many chemotherapy regimens and targeted therapies on multiple cancer types (27).

TNBC is a highly aggressive and heterogeneous breast cancer subtype with a poor prognosis, despite multimodal treatments (1, 2). In the present study, we demonstrated that rMETase combined with either RAPA or CQ is synergistic to inhibit MDA-MB-231 cell viability.

RAPA targets mTOR kinase, thereby inhibiting the PI3K/AKT signaling pathway. rMETase decreases intracellular adenosylmethionine (SAM) concentrations, inhibiting SAMTOR binding and subsequently inhibiting mTOR activity (42).

CQ, initially developed as an antimalarial agent, inhibits autophagy in cancer cells, leading to autophagosome accumulation and apoptosis (30, 31). Although the anticancer efficacy of CQ in combination with chemotherapy has already been demonstrated (33, 34), its synergistic interaction with methionine restriction is important and can be immediately applied to the clinic. Previous studies have shown that rMETase can promote autophagy (43). Thus, the interaction of rMETase and CQ needs further study.

Mouse models of TNBC will be used in the future to determine if by combining RAPA or CQ with rMETase, the effective concentrations of these agents can be lowered with less toxicity.

The main limitation of our study is its in vitro design, which does not fully replicate the complexities of the tumor environment in vivo. Further studies will be performed on mouse models of cancer and in the clinic. Nevertheless, the present study demonstrated that rMETase is synergistic with either RAPA or CQ on TNBC. These results can be immediately applied to the clinic as RAPA and CQ are approved drugs and rMETase is administered clinically as a dietary enzyme supplement (44, 45).

Conclusion

The combination of rMETase with either RAPA or CQ demonstrated synergy on MDA-MB-231 TNBC cells. Future in vivo experiments will involve applying these combinations in mouse models of TNBC to evaluate their efficacy and toxicity.

Acknowledgements

This paper is dedicated to the memory of A.R. Moossa, MD, Sun Lee, MD, Richard W. Erbe, MD, Professor Gordon H. Sato, 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

    All Authors have no conflicts of interest or financial ties to disclose related to this study.

  • Received March 11, 2025.
  • Revision received April 2, 2025.
  • Accepted April 7, 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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Combination of Recombinant Methioninase With Rapamycin or Chloroquine Is Synergistic to Highly Inhibit Triple-negative Breast Cancer Cells In Vitro
JINSOO KIM, QINGHONG HAN, BYUNG MO KANG, KOHEI MIZUTA, YOHEI ASANO, MICHAEL BOUVET, ROBERT M. HOFFMAN
Anticancer Research May 2025, 45 (5) 1853-1859; DOI: 10.21873/anticanres.17564
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