Abstract
Background/Aim: Docetaxel combined with gemcitabine is a second-line treatment for soft-tissue sarcoma; however, its effectiveness is limited because of docetaxel resistance. The objective of the present study was to determine the potential of recombinant methioninase (rMETase) to enhance the efficacy of docetaxel on high-docetaxel-resistant human fibrosarcoma cells in vitro. Materials and Methods: Docetaxel-resistant HT1080 (DTR-HT1080) human fibrosarcoma cells were established by culturing them in by progressively increasing concentrations of docetaxel from 0.02 to 9 nM in vitro. The IC50 values for docetaxel and rMETase, as well as the efficacy of their combination, in inhibiting HT1080 human fibrosarcoma cells, DTR-HT1080 cells, and Hs27 normal human fibroblasts were determined. Four experimental groups were examined in vitro: control group without treatment; docetaxel alone; rMETase alone; docetaxel combined with rMETase. Results: The IC50 of docetaxel for DTR-HT1080 cells was 7.57 nM, compared to the parental HT1080 cells with an IC50 of 1.68 nM, a 4.5-fold increase. The IC50 of docetaxel on Hs27 fibroblasts was 4.46 nM. The IC50 of rMETase on HT1080 cells was 0.75 U/ml (data from [6]). The IC50 of rMETase on DTR-HT1080 cells was 0.55 U/ml. The IC50 of rMETase on Hs27 fibroblasts was 0.93 U/ml (data from [6]). Docetaxel (1.68 nM [IC50]) plus rMETase (0.75 U/ml [IC50]) synergistically reduced the viability of HT1080 cells (p<0.05). In contrast, docetaxel (4.46 nM) plus rMETase (0.93 U/ml) did not reduce the viability of Hs27 fibroblasts, compared to either agent alone. The combination of rMETase (0.55 U/ml [IC50]) and docetaxel (1.68 nM [IC50 of the parental cells]) overcame docetaxel resistance of DTR-HT1080 cells, resulting in an inhibition of 48.1% compared to docetaxel alone (6.8%) or rMETase alone (37.5%) (p<0.05). rMETase thus increased the efficacy of docetaxel 7-fold on docetaxel-resistant human fibrosarcoma cells. Conclusion: The combination of docetaxel and rMETase was synergistic on HT1080 fibrosarcoma cells, but not normal fibroblasts. rMETase plus docetaxel synergistically reduced the high docetaxel resistance of DTR-HT1080 cells. The present results indicate the clinical potential of rMETase to reduce docetaxel resistance in soft-tissue sarcoma patients in the future.
- Docetaxel resistance
- human fibrosarcoma
- human fibrobalsts
- recombinant methioninase
- synergy
- methionine addiction
A promising therapeutic approach for advanced soft-tissue sarcoma has been the concurrent administration of docetaxel and gemcitabine as second-line therapy (1). However, efficacy is limited by the onset of docetaxel resistance. One large clinical trial involving the combination therapy of gemcitabine and docetaxel demonstrated an overall response rate of only 18.4% and a median overall survival period of only 12.1 months (2).
Methionine addiction of cancer, also known as the Hoffman Effect is a fundamental and universal hallmark of cancer (3, 4). Multiple studies have shown that due to their synergistic efficacy, recombinant methioninase (rMETase), or a methionine-free medium, or a diet low in methionine, all of which target methionine addiction of cancer, improve the efficacy of chemotherapy of major cancer types, both in vitro, in mouse models, and in clinical settings (5-23).
The present study determined whether rMETase could enhance the efficacy of docetaxel on highly-docetaxel-resistant (DTR) HT1080 fibrosarcoma cells.
Materials and Methods
Cell culture. HT1080 human fibrosarcoma cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HT1080 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with the addition of 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin (10-013-CV; Corning, Corning, NY, USA).
Reagents. Docetaxel was obtained from Accord Healthcare Inc. (Durham, NC, USA). Recombinant methioninase (rMETase) was produced by AntiCancer Inc. (San Diego, CA, USA) as previously described (24).
Establishment of docetaxel-resistant HT1080 cells (DTR-HT1080). HT1080 human fibrosarcoma cells were cultured stepwise in progressively higher concentrations of docetaxel, ranging from 0.02 to 9 nM over a period of five months.
Drug sensitivity assay 1: Determination of the IC50 of docetaxel and rMETase on HT1080 cells, DTR-HT1080 cells, and Hs27 normal fibroblasts. Cell viability was determined with the WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan). HT1080, DTR-HT1080 cells, and Hs27 normal fibroblasts were grown in 96-well dishes, with 3×103 cells per well, in Dulbecco’s modified Eagle’s medium (DMEM) (100 μl per well), and were incubated for several hours. rMETase (0.5 U/ml to 8 U/ml) or docetaxel (1 nM to 16 nM) were added to the cells for 72 h. At the end of the culture period, 10 μl of the WST-8 solution was added to each well. Subsequently, the dish was incubated for 1 h at 37°C. Absorption was measured at 450 nm with a microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland). Microsoft Excel for Mac 2016 version 15.52 (Microsoft, Redmond, WA, USA) was used to generate drug-sensitivity curves. ImageJ version 1.53k (National Institutes of Health, Bethesda, MD, USA) was used to determine half-maximal inhibitory concentration (IC50) values. Each experiment was conducted twice in triplicate.
Drug sensitivity assay 2: Determination of synergy of docetaxel and rMETase on HT1080 human fibrosarcoma cells, DTR-HT1080 cells, and Hs27 normal fibroblasts. HT1080, DTR-HT1080 cells or Hs27 fibroblasts were seeded in 96-well plates at a density of 3×103 cells per well. Each cell line was treated with single agents as follows 24 h later: untreated control (DMEM); docetaxel alone (1.68 nM) for HT1080 cells and DTR-HT1080 cells or 4.46 nM for Hs27 fibroblasts; rMETase alone (0.75 U/ml) for HT1080 cells, 0.55 U/ml for DTR-HT1080 cells or 0.93 U/ml for Hs27 fibroblasts. For the drug combination experiments the cells were treated as follows: docetaxel (1.68 nM) for HT1080 cells or DTR-HT1080 cells or 4.46 nM for Hs27 fibroblasts, plus rMETase (0.75 U/ml) for HT1080 cells, 0.55 U/ml for DTR-HT1080 cells or 0.93 U/ml for Hs27 fibroblasts. The viability of the cells was evaluated 72 h later, as described above.
Statistical analysis. EZR software (Jichi Medical University, Saitama, Japan) was used for statistical analyses (25). Tukey–Kramer analysis was used to analyse the relationship between variables. p≤0.05 was considered statistically significant.
Results
IC50 of docetaxel alone and rMETase alone on HT1080 cells, DTR-HT1080 cells, and Hs27 normal fibroblasts. The IC50 value of docetaxel alone on HT1080 cells was 1.68 nM. The IC50 of docetaxel alone on DTR-HT1080 cells was 7.57 nM, a 4.5-fold increase of resistance. The IC50 of docetaxel alone on Hs27 fibroblasts was 4.46 nM. The IC50 for rMETase alone on HT1080 cells was 0.75 U/ml [data from (6)] and the IC50 of rMETase alone on DTR-HT1080 cells was 0.55 U/ml. The IC50 for rMETase alone on Hs27 fibroblasts was 0.93 U/ml [data from (6)]. These results demonstrate the efficacy of rMETase alone on DTR-HT1080 was similar to that on HT1080 cells (Figure 1).
Docetaxel and rMETase sensitivity of HT1080 fibrosarcoma cells, docetaxel-resistant HT1080 (DTR-HT1080) cells, and Hs27 normal fibroblasts (mean±standard deviation). A) Sensitivity of HT1080 cells to docetaxel. B) Sensitivity of HT1080 cells to rMETase [data from (6)]. C) Sensitivity of DTR-HT1080 cells to docetaxel. D) Sensitivity of DTR-HT1080 cells to rMETase. E) Sensitivity of Hs27 cells to docetaxel. F) Sensitivity of Hs27 cells to rMETase [data from (6)].
Synergy of rMETase plus docetaxel on HT1080 fibrosarcoma cells and DTR-HT1080 cells, but not on Hs27 fibroblasts. Docetaxel [1.68 nM (IC50 on HT1080 cells)] plus rMETase [0.75 U/ml (IC50 on HT1080 cells)] synergistically reduced the viability of HT1080 cells (p<0.05). In contrast, docetaxel [4.46 nM (IC50 on Hs27 cells)] plus rMETase [(0.93 U/ml (IC50 on Hs27 cells)] did not reduce the viability of Hs27 fibroblasts, compared to either agent alone. The combination of rMETase [0.55 U/ml (IC50 on DTR-HT1080 cells)] and docetaxel [1.68 nM (IC50 on HT1080 cells)] reduced the docetaxel resistance of DTR-143B cells, resulting in an inhibition of 48.1% compared to docetaxel alone (6.8%) or rMETase alone (37.5%) (p<0.05). rMETase thus increased the efficacy of docetaxel 7-fold on DTR-HT1080 cells by reducing docetaxel resistance of DTR-HT1080 (Figure 2).
Efficacy of the combination of docetaxel plus rMETase on HT1080 fibrosarcoma cells, Hs27 normal human fibroblasts, and DTR-HT1080 cells. A) HT1080 cells: Control (DMEM); docetaxel [1.68 nM (IC50 for HT1080 cells)]; rMETase [0.75 U/ml (IC50 for HT1080 cells)]; docetaxel [1.68 nM (IC50 for HT1080 cells)] plus rMETase [0.75 U/ml (IC50 for HT1080 cells)]. B) Hs27 normal human fibroblasts: Control (DMEM); docetaxel [4.46 nM (IC50 for Hs27 fibroblasts)]; rMETase [0.93 U/ml (IC50 for Hs27 fibroblasts)]; docetaxel [4.46 nM (IC50 for Hs27 fibroblasts)] plus rMETase [0.93 U/ml [IC50 for Hs27 fibroblasts)]. C) DTR-HT1080 cells: Control (DMEM); docetaxel [1.68 nM (IC50 for HT1080 cells)]; rMETase [0.55 U/ml (IC50 for DTR-HT1080 cells)]; docetaxel [1.68 nM (IC50 for HT1080 cells)] plus rMETase [0.55 U/ml (IC50 for DTR-HT1080 cells)].
Discussion
In a clinical study of docetaxel alone on anthracycline- and ifosfamide-resistant advanced soft tissue sarcoma, median progression-free survival and overall survival were 2.4 and 7.7 months, respectively (26). In another study of docetaxel alone for soft tissue sarcoma, resistant to at least one anthracycline-containing regimen, the median progression-free survival was 1.4 months, while the median overall survival was 11.7 months (27). Though the combination of docetaxel and gemcitabine has been used for second-line chemotherapy of soft-tissue sarcoma, the effect is limited due to the onset of docetaxel resistance. In clinical studies of the combination of docetaxel and gemcitabine for advanced soft-tissue sarcoma, the median overall survival was only 10.3-14.2 months and progression-free survival was only 3-3.3 months (1, 28).
In the present study, the combination of rMETase and docetaxel resulted in a 7-fold increase in efficacy on DTR-HT1080 cells compared to docetaxel alone. The combination of rMETase and docetaxel has clinical potential for soft tissue sarcoma patients.
We have previously shown that rMETase sensitized osteosarcoma to methotrexate, docetaxel, and cisplatinum in patient-derived orthotopic xenograft (PDOX) mouse models (29-31), and sensitized soft tissue sarcoma to doxorubicin in PDOX mouse models (32-38). We have also recently shown that rMETase sensitized drug-resistant soft-tissue sarcoma cells to eribulin, trabectedin, and doxorubicin (12, 14, 15). Recently we showed that rMETase sensitized androgen-resistant prostate carcinoma cells to docetaxel (39).
It is a challenge to minimize damage of chemotherapy on normal cells and kill chemotherapy-resistant cells (40). In the present study, the combination of docetaxel plus rMETase had synergy on HT1080 and DTR-HT1080 cells but not Hs27 normal fibroblasts. We have recently shown that the combination of rMETase plus docetaxel could overcome docetaxel resistance of osteosarcoma cells (41).
The present results suggest that rMETase in combination with docetaxel has clinical potential for soft-tissue sarcoma patients who have developed docetaxel resistance, a major clinical problem.
rMETase is effective because it targets the fundamental basis of cancer, methionine addiction, known as the Hoffman Effect (42-64).
rMETase is synergistic with chemotherapy (13) because it selectively arrests cancer cells in the late S/G2-phase of the cell cycle, which chemotherapy targets (65-67).
rMETase is now showing early clinical potential (8, 16, 18-23).
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, PhD, Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, Joseph Leighton, MD and John Mendelsohn, MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for the present study.
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, 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 September 22, 2024.
- Revision received October 13, 2024.
- Accepted October 14, 2024.
- Copyright © 2024 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).
