Cancer Diagnosis & Prognosis
Jan-Feb;
5(1):
8-14
DOI: 10.21873/cdp.10405
Received 07 October 2024 |
Revised 19 February 2025 |
Accepted 23 October 2024
Corresponding author
Robert M. Hoffman, Ph.D., AntiCancer Inc, 7917 Ostrow St, Suite B, San Diego, CA, 92111, U.S.A. Tel: +1 6198852284
all@anticancer.com
Abstract
Background/Aim: For second-line chemotherapy of soft-tissue sarcoma, gemcitabine is administered in combination with docetaxel. However, more effective treatments are required for advanced soft-tissue sarcoma, where the efficacy is limited. The purpose of the present study was to compare the efficacy of rMETase and gemcitabine against HT1080 human fibrosarcoma cells and Hs27 normal fibroblasts, as well as to identify and effectively treat HT1080 cells that are resistant to gemcitabine associated with elevated c-MYC. Patients and Methods: Cell viability was measured with the WST-8 reagent. Four groups of in vitro tests were conducted involving HT1080 and Hs27 cells: gemcitabine alone, rMETase alone, and a combination of gemcitabine plus rMETase. Gemcitabine resistant cells (GR-HT1080) were established by culturing HT-1080 cells in increasing concentrations of gemcitabine, ranging from 0.016 nM to 16 nM over five months. Western immunoblotting was performed to measure c-MYC levels in HT1080 and GR-HT1080 cells. Results: Gemcitabine had an IC50 of 12.8 nM against HT1080 cells, 30.8 nM against GR-HT1080 cells, and 4.48 nM against Hs27 cells. The rMETase IC50 value for HT1080 was 0.75 U/ml. The IC50 value of rMETase for GR-HT1080 cells was 0.85 U/ml. The IC50 value for rMETase on Hs27 cells was 0.93 U/ml. Gemcitabine and rMETase demonstrated synergy in killing fibrosarcoma cells, but no synergy was observed on normal fibroblasts. The c-MYC level that was more than 5.1 times higher in GR-HT1080 cells compared to HT-1080 cells. Both the parental HT1080 cells and the GR-HT1080 cells had a similar high sensitivity to rMETase alone. Conclusion: rMETase may be used as a future clinical strategy to overcome gemcitabine resistance in sarcoma.
Keywords: methioninase, Gemcitabine, HT1080, normal fibroblast, efficacy, synergy, fibrosarcoma, gemcitabine-resistance, Methionine restriction, Methionine addiction, Hoffman effect, c-MYC
Gemcitabine is an inhibitor of DNA polymerase (1). Since 2002, gemcitabine has been used in conjunction with docetaxel for the treatment of locally-advanced or metastatic soft-tissue sarcoma. Numerous small studies have investigated this combination therapy over many years, showing only moderate efficacy due to the development of gemcitabine resistance in patients (2-7).
The methioninase gene from Pseudomonas putida was cloned and over-expressed in Escherichia coli, enabling large-scale production of recombinant methioninase (rMETase) (8). Numerous studies have shown that combining chemotherapy with either rMETase, a methionine-free medium, or a diet that is deficient in methionine results in synergistic efficacy with many types of chemotherapy (9-19).
The objective of the present study was to determine the synergy between rMETase and gemcitabine on fibrosarcoma cells compared to normal fibroblasts, as well as investigate the potential of rMETase to reverse c-MYC-associated acquired gemcitabine resistance in fibrosarcoma cells.
Materials and Methods
Cell culture. The HT1080 and Hs27 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml of penicillin and streptomycin was used to cultivate the cells.
Reagents. Gemcitabine was obtained from BluePoint Laboratories (Cork, Ireland). rMETase was produced at AntiCancer Inc. (San Diego, CA, USA). The rMETase production process has been previously described (8).
Establishment of gemcitabine-resistant HT1080 (GR-HT1080) cells. HT1080 cells were cultured with DMEM in progressively higher concentrations of gemcitabine, ranging from 0.016 nM to 16 nM stepwise over five months. This process was carried out in a stepwise manner.
Drug sensitivity assay 1: IC50. The WST-8 reagent (Dojindo Laboratory, Kumamoto, Japan) was used to determine cell viability. Cells (HT1080, GR-HT1080, or Hs27) were cultured in DMEM (100 μl/well) in 96-well plates at a concentration of 3×103 cells per well. The plates were then incubated at 37˚C overnight. Gemcitabine, in concentrations ranging from 4 nM to 64 nM, or rMETase, in concentrations ranging from 0.5 U/ml to 8 U/ml, were administered to the cells for 72 h. After the culture period, 10 μl of the WST-8 solution was added to each well. Then, the plates were subjected to an additional hour of incubation at 37˚C. Absorption of WST-8-treated cells was measured at 450 nM in microplate reader (SUNRISE: TECAN, Mannedorf, Switzerland). The drug sensitivity curves were generated with Microsoft Excel for Mac 2016 version 15.52 (Microsoft, Redmond, WA, USA). Values for the half-maximal inhibitory concentration (IC50) were determined with ImageJ version 1.53k (National Institutes of Health, Bethesda, MD, USA). Each experiment was carried out twice, in triplicate.
Drug sensitivity assay 2: Synergy. HT1080 or Hs27 cells (3×103) were seeded in 96-well plates. After 24 h, the cells were treated as follows: 1) Control (DMEM); 2) Gemcitabine (12.8 nM or 4.48 nM, respectively); 3) rMETase (0.75 U/ml or 0.93 U/ml, respectively); and 4) a combination of gemcitabine (12.8 nM or 4.48 nM, respectively) and rMETase (0.75 U/ml or 0.93 U/ml, respectively). The viability of the cells was evaluated after 72 h. Cells were assessed using the method of Drug Sensitivity Assay 1, which was performed three times.
Western immunoblotting. RIPA Lysis buffer and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) were used for protein extraction. Protein samples were loaded onto 10% SDS-PAGE gels. Subsequently, the samples were transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA). Bullet Blocking One for Western Blotting (Nakalai Tesque, Inc., Kyoto, Japan) was used for membrane blocking. Anti-c-MYC antibody (1:2,000, #10828-1-AP, Proteintech, Rosemont, IL, USA) and β-Actin (20536-1-AP, 1:1,000) were from Proteintech. Secondary antibodies were horseradish-peroxidase-conjugated anti-rabbit IgG (1:5,000, #SA00001-2, Proteintech). UVP ChemStudio (Analytik Jena, Upland, CA, USA) and the Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) were used to scan the western blot. The experiments were carried out three times to ensure accuracy.
Statistical analysis. EZR software (Jichi Medical University, Saitama, Japan) was used for statistical analyses (20). Tukey–Kramer analysis and the Welch’s t-test were used to determine the relationship between variables. For statistical significance, p-values less than 0.05 were considered adequate.
Results
Generation of gemcitabine-resistant HT1080 cells. Gemcitabine- resistant cells (GR-HT1080) were established by cultivating HT1080 cells in DMEM in progressively increasing concentrations of gemcitabine, ranging from 0.016 nM to 16 nM. GR-HT1080 cells were 2.4 times more resistant to gemcitabine than the parental HT1080 cells.
Drug sensitivity assay 1: IC50 of gemcitabine alone and rMETase alone on HT1080, GR-HT1080, and Hs27 cells. The IC50 of gemcitabine for HT 1080 cells was 12.8 nM; and 30.8 nM for GR-HT1080 cells. The IC50 of gemcitabine for Hs27 cells was 4.48 nM. The IC50 of rMETase for HT1080 cells was 0.75 U/ml [data from (10)], and 0.85 U/ml for GR-HT1080 cells. The IC50 of rMETase for Hs27 cells was 0.93 U/ml [data from (10)] (Figure 1).
Drug sensitivity assay 2: Synergy of gemcitabine and rMETase. The combination of gemcitabine [12.8 nM (IC50)] plus rMETase [0.75 U/ml (IC50)] had synergist efficacy on HT1080 cells (p<0.05). In contrast, gemcitabine [4.48 nM (IC50)] plus rMETase (0.93 U/ml [IC50]) did not show synergy in Hs27 cells (Figure 2A and B).
Western blotting of c-MYC. There was a 5.1-fold increase in the expression of c-MYC in GR-HT1080 cells compared to HT1080 cells (p<0.05) (Figure 3 and Figure 4).
Discussion
Patients with metastatic soft-tissue sarcoma participated in a randomized phase II study, in which results showed that the median progression-free survival was 6.2 months for gemcitabine plus docetaxel and 3.0 months for gemcitabine alone. Additionally, the median overall survival was 17.9 months for gemcitabine-docetaxel and 11.5 months for gemcitabine alone (21). Among patients with previously untreated advanced unresectable or metastatic soft-tissue sarcomas, a randomized controlled phase III trial revealed that the group receiving gemcitabine plus docetaxel had a median progression-free survival of only 5.9 months (22). Therefore, significantly improved therapy is necessary.
Methionine addiction of cancer was first identified by one of the authors (RMH) in 1976 (23-26). To target methionine addiction, our research group developed rMETase. rMETase was found to be effective by oral administration (27).
It has been demonstrated that a wide range of combinations of rMETase and chemotherapy drugs are synergistic (9-19). The present study demonstrated that the combination of rMETase and gemcitabine was synergistic effect on HT1080 cells and reversed the high gemcitabine resistance of GR-HT1080 cells. Methionine depletion causes cancer cells to enter a reversible arrest in the late S/G2-phase (28), which is the phase that gemcitabine targets (1). Our previous research demonstrated that gemcitabine and rMETase were synergistic in the mouse models of an orthotopic pancreatic cancer cell-line and a patient-derived orthotopic xenograft (29,30). In contrast, Hs27 normal fibroblasts did not demonstrate synergy with the combination of gemcitabine and rMETase. This is due to the fact that rMETase does not arrest normal cells in the S-phase of the cell cycle (31,32).
c-MYC is one of the most frequently activated oncogenes in human cancer (33,34). It is responsible for promoting cell proliferation and growth. A correlation has been observed between increased c-MYC expression and elevated chemo-resistance (35-37). c-MYC levels were shown to be elevated in methotrexate-resistant cells in our previous study (37). In the present study, the expression of c-MYC was 5.1 fold higher in GR-HT1080 cells than in parental HT1080 cells.
rMETase is effective because it targets the fundamental cancer hallmark of methionine addiction (23,24-26,28,31,32,38-49). The present study suggests that rMETase and gemcitabine synergy has the potential to be used in the treatment of drug-resistant soft tissue sarcoma in future clinical trials.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
Authors’ Contributions
SM, HQ, KM, BMK, MB, NY, KH, HK, SM, KI, TH, HT SD, and RMH designed the study. SM performed experiments. SM was a major contributor to writing the manuscript and RMH revised the paper. All Authors read and approved the final manuscript.
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