Abstract
Background/Aim: Methionine restriction selectively arrests cancer cells during the S-phase of the cell cycle. We hypothesized that DNA damage may occur in S-phase in cancer cells during methionine restriction. To determine if this occurs, we used MiaPaCa-2Tet-On 53BP1-green fluorescent protein (GFP) pancreatic cancer cells, which report GFP fluorescence in real time after DNA-damage response (DDR) in these cells. We also determined whether a chemotherapy drug in combination with methionine restriction increases the rate of DNA damage. Materials and Methods: MiaPaCa-2Tet-On 53BP1-GFP cells were used for in vitro experiments. The 25% and 50% inhibitory concentrations (IC25 and IC50, respectively) of recombinant methioninase (rMETase) and paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP pancreatic cancer cells were determined. Cell viability and DDR with rMETase alone, paclitaxel alone, and their combination were measured in MiaPaCa-2Tet-On 53BP1-GFP cells. Results: The IC25 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 1.66 U/ml. The IC25 for paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP cells was 3.31 nM. The combination of rMETase and paclitaxel synergistically reduced the viability of MiaPaCa-2Tet-On 53BP1-GFP cells. The IC50 of paclitacel on MiaPaCa-2Tet-On 53BP1-GFP cells was 5.1 nM. The IC50 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 2.3 U/ml. The combination of rMETase (IC50) plus paclitaxel (IC50) on MiaPaCa-2Tet-On 53BP1-GFP cells also caused more DNA damage than either agent alone. Conclusion: The present study suggests the synergy of methionine restriction and chemotherapy is due, at least in part, to DNA damage of cancer cells.
We discovered methionine addiction, which is a fundamental and general hallmark of cancer (1). Methionine addiction of cancer is termed the Hoffman Effect (2). Our laboratory has developed a recombinant methioninase (rMETase) to specifically restrict the availability of methionine and target the methionine addiction of cancer (3). Multiple studies have shown that combining chemotherapy with methionine restriction, achieved through the use of either rMETase, a methionine-free medium, or a methionine-depleted diet, results in a synergistic effect (4).
Methionine restriction selectively arrests cancer cells in the S-phase of the cell cycle during DNA replication (5, 6). We hypothesized that during selective S-phase arrest of cancer cells by methionine restriction, DNA damage and repair may occur.
Efimova et al. (7) linked the green fluorescent protein (GFP) with the DNA damage-response (DDR) protein 53BP1 to establish a reporter for live-cell imaging of DDR. In the MiaPaCa-2Tet-On 53BP1-GFP pancreatic-cancer cell line, GFP expression in the cells reports DNA damage and repair in real time.
Paclitaxel is widely used in oncology and it induces mitotic arrest in cancer cells by interfering with the normal functioning of microtubules (8).
The present study aimed to demonstrate if rMETase alone or paclitaxel alone or their combination are synergistic on MiaPaCa-2Tet-On 53BP1-GFP cells to induce DNA damage and repair, as well as reduce cell viability.
Materials and Methods
Cell culture. The MiaPaCa-2Tet-On 53BP1-GFP cell line (Clontech, Carlsbad, CA, USA) was previously genetically modified by introducing a fusion of 53BP1 and GFP using a lentiviral vector (Clontech) which is controlled by a tetracycline- or a doxycycline-responsive promoter (7). MiaPaCa-2Tet-On 53BP1-GFP cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with high glucose (Thermo Fisher Scientific, Waltham, MA, USA) and fetal bovine serum-tet-system approved (Thermo Fisher Scientific) (10%) that is compatible with tet-responsive promoters (7).
Reagents. Paclitaxel was obtained from FRESENIUS KABI (Lake Zurich, IL, USA). rMETase was produced in AntiCancer Inc. (San Diego, CA, USA). The method for producing rMETase has been previously reported (9).
Determination of IC25 and IC50 of rMETase and paclitacel on MiaPaCa-2Tet-On 53BP1-GFP cells. Cell viability was assessed using the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). MiaPaCa-2Tet-On 53BP1-GFP cells were cultured in 96-well plates (3×103 cells/well) in DMEM (100 μl/well) and incubated at 37°C overnight. MiaPaCa-2Tet-On 53BP1-GFP cells were treated with increasing concentrations of paclitaxel alone, between 0.5 nM and 8 nM for 72 h; or MiaPaCa-2Tet-On 53BP1-GFP cells were treated with increasing concentrations of rMETase alone between 0.5 U/ml and 8 U/ml for 72 h. At the end of the culture period, 10 μl of the WST-8 solution was added to each well and the plate was additionally incubated for 1 h at 37°C. Absorption was measured with a microplate reader (Sunrise; Tecan, Mannedorf, Switzerland) at 450 nm. Drug-sensitivity curves were obtained with Microsoft Excel ver. 15.52 (Microsoft, Redmond, WA, USA) and one-quarter- and half-maximal inhibitory concentration (IC25 and IC50, respectively) values were calculated using ImageJ ver. 1.53 (National Institutes of Health, Bethesda, MD, USA). Experiments were performed twice, each in triplicate.
Determination of synergy to reduce cell viability. MiaPaCa-2Tet-On 53BP1-GFP cells were seeded at 3×103 cells/well in 96-well plates. Twenty-four hours later, four treatment groups were established Control (DMEM); paclitaxel (3.31 nM [IC25]); rMETase (1.66 U/ml [IC25]); and paclitaxel (3.31 nM) plus rMETase (1.66 U/ml). Seventy-two hours later, cell viability was measured in triplicate as described above. In the present study, we defined synergy as an effective combination greater than either component alone.
DNA damage-response assay. MiaPaCa-2Tet-On 53BP1-GFP cells were used to report nuclear DNA-damage response treated either with paclitaxel alone, rMETase alone, or their combination. The cells were seeded at 6×105 cells per well in 6-well plates. To observe DNA damage response with GFP, 1mg/ml of doxycycline was added to the culture wells in all groups. Treatments were as following: control (DMEM); paclitaxel (5.1 nM [IC50]); rMETase (2.3 U/ml [IC50]); and paclitaxel (5.1 nM [IC50]) plus rMETase (2.3 U/ml [IC50]). After 48 hours, the cells were examined for GFP fluorescence using an IX71 fluorescence microscope (Olympus, Tokyo, Japan) at a magnification of ×200. DNA-damage response was visualised by GFP fluorescence and was quantified per visual field for six separate fields.
Statistical analysis. All statistical analyses were conducted using the EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan). Associations between variables were tested using Tukey-Kramer analysis. Results with p-values ≤0.05 were considered statistically significant.
Results
Determination of synergy of paclitaxel and rMETase to reduce viability of MiaPaCa-2Tet-On 53BP1-GFP cells. The IC25 value of paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP cells was 3.31 nM. The IC25 for rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 1.66 U/ml (Figure 1). The combination of paclitaxel (3.31 nM) and rMETase (1.66 U/ml) was synergistic to reduce the viability of MiaPaCa-2Tet-On 53BP1-GFP cells to a greater extent than either agent alone (p<0.05) (Figure 2).
Paclitaxel and recombinant methioninase (rMETase) treatment of MiaPaCa-2Tet-On 53BP1-GFP cells. (A) Sensitivity of MiaPaCa-2Tet-On 53BP1-GFP cells to paclitaxel. (B) Sensitivity of MiaPaCa-2Tet-On 53BP1-GFP cells to rMETase (mean±standard deviation, n=3).
Efficacy of the combination of paclitaxel and rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells. The combination of recombinant methioninase (rMETase) (1.66 U/ml [IC25]) and paclitaxel (3.31 nM [IC25]) was synergistic for MiaPaCa-2Tet-On 53BP1-GFP cells. n=3 *p<0.05 using Tukey-Kramer analysis.
Determination of synergy of paclitaxel and rMETase to cause DNA damage response in MiaPaCa-2Tet-On 53BP1-GFP cells. The combination of paclitaxel (5.1 nM [IC50]) plus rMETase (2.3 U/ml [IC50]) caused significantly more DNA-damage response, as observed by GFP fluorescence in MiaPaCa-2Tet-On 53BP1-GFP cells, than either agent alone (p<0.05) (Figure 3).
Effect of paclitaxel alone, recombinant methioninase (rMETase) alone and their combination on DNA damage in MiaPaCa-2Tet-On 53BP1-GFP cells. Cells were untreated (Control) or treated with 5.1 nM (IC50) paclitaxel alone, 2.3 U/ml (IC50) rMETase alone or their combination. (A) Images of DNA damage and repair in MiaPaCa-2Tet-On 53BP1-GFP cells expressing green fluorescent protein (GFP). Scale bars: 100 μm. (B) Quantification of DNA damage in treated cells. GFP fluorescence was observed with an IX71 fluorescence microscope (Olympus, Tokyo, Japan). *p<0.05 (n=6) using Tukey-Kramer analysis.
Discussion
Genomic instability is a fundamental feature of cancers and is linked to an increased susceptibility to accumulate DNA damage (10, 11).
rMETase specifically targets the methionine addiction of cancer cells, by methionine restriction. Methionine restriction leads to the selective arrest of cancer cells in the late-S/G2 phase of the cell cycle while leaving normal cells unaffected (3, 5, 6). Methionine restriction and chemotherapy are synergistic due to the selective S-phase arrest by methionine restriction which chemotherapy targets (4-6). Examples of the synergy of methionine restriction and chemotherapy include rMETase combined with oxaliplatinum plus 5-fluorouracil or ethionine or rapamycin or irinotecan or eribulin (12-16). We hypothesized that DNA damage is occurring during S-phase arrest in cancer cells induced by methionine restriction. The selection of paclitaxel and METase was based on the ability of METase to arrest cancer cells in the late-S/G2 phase, and if they attempt to enter mitosis, they become susceptible to paclitaxel, an antimitotic agent. The G2/M checkpoint becomes significantly more crucial in cancer cells after S-phase arrest, due to increased DNA damage. Sufficient DNA damage can surpass the threshold at which cancer cells can survive, even when the G2/M checkpoint is functioning properly (11).
In the present study, the combination of rMETase and paclitaxel was synergistic to induce DNA damage response on MiaPaCa-2Tet-On 53BP1-GFP cells resulting in loss of cell viability. We have previously shown that rMETase and taxanes are synergistic in both mouse models of cancer and in the clinic (17-20).
We recently showed that methionine restriction induced an alternate DNA repair enzyme in BRACA1-mutant breast cancer cells (21). 53BPI mediates and effects the response to double-strand breaks (DSB) of DNA (10, 11) suggesting DSBs occur during S-phase-induced cell-cycle arrest of cancer cells by methionine restriction, enhanced by chemotherapy. Further studies are warranted to determine the role of DNA damage in the synergistic efficacy of rMETase and chemotherapy.
rMETase is effective because it targets the fundamental hallmark of cancer, methionine addiction, termed the Hoffman effect (1-3, 5, 6, 22-43).
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, John Mendelsohn, MD, and Joseph Leighton, MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.
Footnotes
Conflicts of Interest
The Authors declare that there are no competing interests.
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, MS, MB, NY, KH, HK, SM, KI, TH, HT and SD critically read the manuscript.
- Received July 14, 2024.
- Revision received August 10, 2024.
- Accepted August 17, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
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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