Abstract
Background/Aim: Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo. Materials and Methods: Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS. Results: A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group. Conclusion: The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS.
Synovial sarcoma (SS) is a rare and malignant form of soft tissue tumor that predominantly affects young adults, accounting for only 5-10% of all soft tissue tumors (1). The primary treatment for SS is surgical removal of the tumor, while chemotherapy and radiotherapy may be used as adjunctive treatments in certain cases. However, the tumor often recurs, making treatment complex. In addition, resistance to chemotherapy is a significant challenge in the treatment of SS (2). Therefore, developing novel therapeutic strategies and preventive measures for SS is crucial to improve patient outcomes.
The redox system is a critical mechanism that regulates various cellular metabolic functions. Reactive oxygen species (ROS) play a significant role in the development of several diseases, including carcinomas and sarcomas (3, 4). Thioredoxin (TXN) plays a vital role in scavenging ROS within the body. TXN reductases (TXNRDs) are enzymes that regulate TXN activity by catalyzing its reduction and have been identified as potential therapeutic targets for the treatment of various types of cancer, including sarcoma (5). Auranofin (AUR), a TXNRD inhibitor approved by the FDA for the treatment of rheumatoid arthritis, has demonstrated potential anticancer properties against different types of cancers, such as osteosarcoma (OS) (6). Celecoxib (CE) has been shown to selectively and effectively inhibit COX-2 activity by binding to its sulfonamide side chains. This inhibition results in the induction of cytotoxicity, formation of ROS, a decrease in mitochondrial membrane potential, lipid peroxidation, and oxidative stress (7). We previously reported that the combination of AUR and CE significantly inhibits the progression of OS (8). However, available literature regarding the efficacy of this combination in treating cancer and sarcoma is limited. Furthermore, there are currently no detailed reports on redox signaling mechanisms in SS. Therefore, this study aimed to investigate the combined effects of AUR and CE on the local progression of SS in vivo.
Materials and Methods
Antibodies and reagents. Antibodies and reagents were obtained from the following commercial sources: AUR (SC-202476; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CE (10008672; Cayman Chemical, Ann Arbor, MI, USA), anti-Ki-67 (NCL-Ki67p; Leica Biosystems, Milton Keynes, UK), peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) polyclonal secondary antibody (#424144; Nichirei Biosciences Inc., Tokyo, Japan).
Animal studies. Six-week-old male BALB/cSLC-nu/nu mice were obtained from Japan SLC (Shizuoka, Japan). The mice were housed in a laboratory environment at a constant temperature of 22±2°C, humidity of 50±10%, and 12-hour light/dark cycle. All animal experiments were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Chiba Cancer Center, Chiba, Japan. The Aska-SS human synovial sarcoma cell line (RRID: CVCL_6C43) was obtained from the RIKEN BioResource Center (Ibaraki, Japan). Aska-SS cells, 2×107 per 300 μl of phosphate-buffered saline (PBS) were implanted subcutaneously into the flanks of BALB/cSLC nu/nu mice (n=15). Primary tumors were allowed to grow for nine days before treatment until they reached a measurable size. Tumor volume was measured using calipers and calculated using the following formula: volume=length × width2/2. The mice were divided into three groups of five mice each, with each group having a similar tumor size distribution. The mice were treated with either 200 μl of vehicle control (40% polyethylene glycol 300+60% sterile PBS), 15 mg/kg of AUR, or a combination of 15 mg/kg AUR and 30 mg/kg CE (AUR-CE). When palpable tumors measuring approximately 50 mm3 were established, injections of control, AUR, and AUR-CE were administered intraperitoneally on a daily basis until the end of the study period. Tumor size measurements and animal weights were documented once per week in an unblinded manner. On the 49th day following the initial injection of vehicle control, AUR, or AUR-CE, the mice were euthanized under anesthesia, and their tumors were excised for tumor weight assessment. Tumor samples were collected for further examination, fixed in formalin, and embedded in paraffin.
Immunohistochemical staining. Hematoxylin and eosin (H&E) staining was performed as previously described (8). In addition, immunohistochemical staining was conducted on formalin-fixed, paraffin-embedded, 3 μm sections of tumor samples. For immunostaining, the tumor sections were incubated overnight at 4°C with a primary antibody against Ki-67 (dilution, 1:2,000). Subsequently, the tissue sections were incubated for 30 min at 20°C with a peroxidase-conjugated anti-rabbit IgG polyclonal secondary antibody. The percentage of Ki-67-positive cells was determined by counting the number of stained cells in five randomly selected fields per slide (200× magnification).
Statistical analysis. The experimental data are presented as the mean±standard deviation (SD). Tukey-Kramer honest significance testing after one-way ANOVA was performed for multiple comparison testing. The level of statistical significance was set at p<0.05. All statistical analyses were performed using the SAS software, version 14.2 (SAS Institute, Inc., Cary, NC, USA).
Results
Combined therapy with AUR and CE synergistically inhibits the local progression of SS. During the conduct of the study, no statistically significant differences were observed in the body weights of mice among the groups administered with vehicle, AUR, or AUR-CE (Figure 1A). However, the size of the SS was suppressed in the AUR- and AUR-CE-treated groups compared with that in the vehicle-treated group over the course of the study (Figure 1B). Furthermore, at the time of excision, the tumor size and weight were significantly smaller in the AUR- and AUR-CE-treated groups compared to that of the vehicle-treated group (Figure 1C-D). The AUR-CE-treated group showed synergistic inhibition of local tumor growth. Histopathological analysis of H&E-stained local SS tumors revealed significantly reduced cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to the vehicle-treated group (Figure 2A). Additionally, immunohistochemical staining revealed a significant decrease in the number of Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group (Figure 2A and B).
The combination of auranofin and celecoxib suppresses local progression of synovial sarcoma (SS). (A) Graph representing the differences in body weights of mice in the vehicle, auranofin (AUR), and AUR-celecoxib (AUR-CE)-treated groups throughout the course of the study. (B) Graph representing the SS tumor size in the vehicle, AUR-, and AUR-CE-treated groups over the course of the study. (C) Representative images demonstrating differences in tumor size in the vehicle, AUR-, and AUR-CE-treated groups at the time of excision. (D) Graph representing the differences in tumor weight in the vehicle, AUR-, and AUR-CE-treated groups at the time of excision. n=5 for each group; n.s., not statistically significant. Data are presented as the mean±SD. Tukey-Kramer honest significance testing after one-way ANOVA was performed for multiple comparisons testing. *p<0.05.
Histopathological analysis of primary tumors of synovial sarcoma (SS). (A) Left panel: immunohistochemistry of hematoxylin and eosin (H&E) staining of the local SS tumor sections representing cell density and nuclear deformation in the vehicle, auranofin (AUR), and AUR-celecoxib (AUR-CE)-treated groups. Right panel: immunohistochemical staining for Ki-67. (B) Quantification of Ki-67 staining represented in (A). Scale bars=50 μm; n.s., not statistically significant. Data are presented as the mean±SD. Tukey-Kramer honest significance testing after one-way ANOVA was performed for multiple comparisons testing. *p<0.05.
Discussion
The use of chemotherapy as a potential treatment option for SS is an area of ongoing research. For patients with a high risk of cancer metastasis or advanced disease, chemotherapy may be considered a potential treatment approach. Treatment with neoadjuvant chemotherapy has proven beneficial for high-risk patients with grade 3 SS (9). Cytotoxic chemotherapy, particularly with anthracyclines and ifosfamide, is currently the preferred treatment method (10). Although these treatment strategies may cause adverse effects (11), their impact on improving outcomes in patients with SS is limited. Further research is essential to enhance the long-term survival rate of patients with SS. Pazopanib and trabectedin are considered second-line therapies for the treatment of soft tissue sarcomas. Carroll et al. performed a meta-analysis to assess the efficacy of these drugs in patients with metastatic SS who previously received treatment (12). This study revealed that these drugs did not yield satisfactory outcomes, emphasizing the need for novel treatments to improve the clinical outcomes of metastatic SS. Furthermore, chemotherapy-resistant SS poses a significant challenge and necessitates the development of novel agents (2).
Preclinical studies have identified several actionable targets in SS, including the WNT-b-catenin and AKT-mTOR pathways, ALK, MET, and the cyclin D1-CDK4/6-Rb axis (13). Limited information is available regarding redox regulation in SS. Epinecidin-1-mediated cytotoxicity in SS is linked to calcium overload-induced calpain activation, oxidative stress, and mitochondrial dysfunction (14). Takeuchi et al. revealed that blue light triggers apoptosis through the ROS-mitochondrial signaling pathway, while blue light also activates autophagy to protect SS cells from apoptosis (15).
Our previous study showed that AUR, a TXNRD inhibitor, induces apoptosis in OS cells by activating the oxidative stress-mitogen-activated protein kinase (MAPK)-caspase-3 pathway (6). Furthermore, AUR has demonstrated potential efficacy against various types of cancers and sarcomas (16). We previously reported that AUR effectively inhibits the local progression of rhabdomyosarcoma (RMS) through the oxidative stress-apoptosis pathway using a patient-derived xenograft (PDX) model (17). Currently, research on the effectiveness of the combination of AUR and CE as a treatment for cancer is limited. Han et al. reported that this combination could lead to severe oxidative stress and disrupt mitochondrial redox homeostasis, ultimately resulting in a significant reduction in ATP generation and anticancer effects in colon cancer (18).
In our previous report, we emphasized the promising potential of AUR monotherapy and the combination of AUR and CE as treatment options for patients with OS (6, 8). Our previous and present studies have demonstrated the efficacy of AUR in treating OS, RMS, and SS. This suggests that AUR may be a potential therapeutic option for various types of bone and soft tissue sarcomas.
Drug repositioning is a promising strategy to accelerate drug discovery and development (19). Yoon et al. suggested that repurposed drugs have the potential to manage pre-existing diseases and prevent cancer (20). By capitalizing on established bioavailability, pharmacokinetics, efficacy, and safety, existing approved drugs can be repurposed for novel medical applications. Owing to their FDA approval as antirheumatic and analgesic agents, AUR and CE exhibit potential for early drug repositioning in SS.
In our previous study, although AUR monotherapy failed to demonstrate a significant advantage in controlling the local progression of OS, it was effective in controlling the local progression of RMS (6, 17). Previous findings have also indicated the limited effectiveness of AUR monotherapy in other types of cancers (21, 22). However, in this study, a significant difference was observed in the control of local progression of SS between the AUR monotherapy and control groups. Further studies are required to assess the efficacy of AUR monotherapy in various sarcomas.
Despite its significant contributions, this study is not without limitations. Firstly, as an in vivo study, detailed evaluation of intracellular signaling was limited. Further investigation related to the involvement of the MAPK pathway in AUR-mediated anticancer effects in SS is needed. Secondly, the findings of the study were tested in human cell lines and mouse models, which limits their generalizability. Therefore, it is crucial to incorporate the PDX models in future studies.
Conclusion
The present study assessed the efficacy of combined therapy with two FDA-approved drugs, AUR and CE, in significantly impeding the local progression of SS. Both drugs are effective anti-rheumatic and analgesic agents. These findings suggest that AUR and CE may be considered as early treatment options for SS.
Acknowledgements
This work was supported by JSPS KAKENHI grants (23K14608), The Mother and Child Health Foundation, Chiba Foundation for Health Promotion and Disease Prevention, Kato Memorial Bioscience Foundation, The Hamaguchi Foundation for the Advancement of Biochemistry, Children’s Cancer Association of Japan, and Funds for Gold Ribbon Network.
Footnotes
Authors’ Contributions
H.K and S.K designed and performed experiments, analyzed data, and wrote the article; H.K, Y.H, S.O, and T.Y gave conceptual advice.
Conflicts of Interest
The Authors have no conflicts of interest directly relevant to the content of this article.
- Received March 7, 2024.
- Revision received April 8, 2024.
- Accepted April 17, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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