Elsevier

Gene

Volume 816, 30 March 2022, 146172
Gene

In vitro anti-synovial sarcoma effect of diallyl trisulfide and mRNA profiling

https://doi.org/10.1016/j.gene.2021.146172Get rights and content

Highlights

  • The development of new therapeutic drug for SS treatment is urgent medical need.

  • Apoptosis, cycle arrest, intracellular ROS, mitochondria and autophagy were detected to identify effect of DATS on SS.

  • High-throughput RNA-sequencing analysis was performed to clarify a comprehensive molecular portrait for DATS-induced transcriptional regulation.

  • Go and KEGG analysis were performed to reveal the related molecular events induced by DATS pharmacological properties.

  • PPI network was conducted to identify hub genes and protein feature.

Abstract

Objective

Synovial sarcoma (SS) is a malignant soft tissue sarcoma and its natural history is a long, indolent clinical course followed by high rate of local recurrence and distant metastasis. Current therapies are still limited in increasing satisfactory of 5-year survival, especially for patients with recurrence and metastasis. Accordingly, finding new therapeutic drug for SS treatment is clinically urgent need. Diallyl trisulfide (DATS), a bioactive compound derived from garlic, is reported as a promising anti-cancer agent for various carcinomas. However, its effect on anti-SS remains unknown. This study investigated the anti-SS effect of DATS in human synovial sarcoma SW982 cells.

Methods

CCK-8 assay were used to examine the cell viability. High-content Imaging System was used to examine the apoptosis, intracellular ROS and autophagy. Flow cytometry was used to detect cell cycle. qPCR and Western blot were used to examine the expression of related mRNA and protein. High-throughput RNA-sequencing and bio-information analysis were used to investigate the mRNA profiling.

Results

The results showed a suppressive effect of DATS on tumor biology of SW982 cells including inducing apoptosis, triggering G2/M cell cycle arrest, elevating intracellular ROS and damaging mitochondria. Further high-throughput RNA-sequencing analysis clarified a comprehensive molecular portrait for DATS-induced transcriptional regulation. Besides, protein–protein interaction (PPI) analysis demonstrated that a network consisted of FOXM1, CCNA2, CCNB1, MYBL2, PLK1 and CDK1 might be response for DATS-induced G2/M cell cycle arrest and increased intracellular ROS. Notably, protein feature analysis revealed structure enrichment in microtubule network like kinesin motors domain, and tubulin domain. Molecular function analysis suggested that DATS-induced dysfunction of microtubule network might be the major cause for its effect on cell cycle arrest and successive apoptosis. Furthermore, 28 hub genes (including KIF2C, PLK1, CDK1, BIRC5, CCNB2, CENPF, TPX2, TOP2A and so on) were determined. Finally, pathway analysis showed that DATS-induced differentially expressed genes were mainly involved in cell cycle.

Conclusion

Collectively, our findings for the first time provided the DATS-induced cellular response and transcriptional profiling of SW982 cells, which proposes that suppression of DATS on SS is multi-targeted and represent a therapeutic evidence for SS.

Introduction

Synovial sarcoma (SS), one of the most common soft tissue sarcomas, is a malignant mesenchymal tumor with a significant increase in the incidence over the past twenty years (Aytekin et al., 2020, Bessen et al., 2019). Although SS can affect patients of any age, it occurs predominantly in adolescents and young adults (Corey et al., 2014). SS frequently arises in the extremities with asymptomatic and slow growth, which resulting in a delayed diagnosis and therapy even for longer than 10 years (Chambers and Lesher, 2018, Severino and Severino, 2017, Imaizumi et al., 2002, Xia et al., 2020). Besides, SS can be aggressive for its local recurrence and metastasis which occur after 3–5 years and the related incidence rates are up to 50% (Krieg et al., 2011). Currently, surgery, radiotherapy or their combination are still main treatment for SS. However, five-year survival ranges from 24% to 71%, which determined by many prognostic factors such as age, location, tumor size and histological grade (Riedel et al., 2018). More than a quarter of patients succumb to this cancer in the first 5 years after diagnosis despite the best available management (Yang et al., 2018). Although SS is relatively chemosensitive compared with other soft-tissue sarcomas, the role of adjuvant chemotherapy is still controversial for its limited success in increasing satisfactory of five-year survival (Vining et al., 2017, Minami et al., 2020, Khan et al., 2019). Besides, recurrence, metastases and potential dysfunction of the affected limbs or tissue organs after surgical operation and chemotherapy are still torment for patients. Therefore, the development of new therapeutic drug for SS treatment is urgent medical need.

Diallyl trisulfide (DATS) is a bioactive compound which is derived from garlic oil (accounting for 38.93% of garlic oil). As an organosulfur phytochemical, DATS has been confirmed to have a multitude of pharmacological properties including antioxidant (Hsieh et al., 2020, Silva-Islas et al., 2019), anti-inflammation (Liang et al., 2019, Leung et al., 2020) and regulating metabolism (Jeremic et al., 2020). Accumulating in vitro data have revealed that DATS modulates multiple cancer hallmark pathways including cell cycle, apoptosis, cancer stem cell, angiogenesis, invasion and metastasis (Li et al., 2018, De Greef et al., 2021, Tao et al., 2017). In vivo studies also report that DATS treatment significantly retards not only tumor growth but also tumor progression to invasion and metastasis without causing weight loss and any side effects (Xiao et al., 2006, Singh et al., 2008). In addition, one five-years-follow-up clinical study reports that 200 mg synthetic DATS treatment offers protection for males from gastric cancer with no harmful side effect (Li et al., 2004). These data support the concept that DATS is a promising agent potentially useful for treatment and/or prevention of cancer. Accordingly, these findings prompt us to investigate whether DATS has similar suppressive effect on the pathology of SS.

Here, we aimed to investigate the effect of DATS on anti-SS. First, we detected the cellular response of human synovial sarcoma SW982 cells to DATS through examining its cell viability, apoptosis, intracellular ROS, mitochondria and autophagy. Furthermore, mRNA profiling was explored by high-throughput RNA sequencing. GO enrichment, protein–protein interaction, and KEGG pathway enrichment analyses were applied to identify biological process, molecular function, protein feature, hub gene and pathway of DATS-regulated genes. Our work highlighted the regulatory feature of the intrinsic mRNA expression of SW982 cells by DATS and provided further evidence for the clinical development of DATS as chemopreventive drug for SS treatment.

Section snippets

Cell culture

Human synovial sarcoma SW982 cells purchased from SIBCB (Shanghai, China) were grown in L-15 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and incubated in a humidified atmosphere at 37 °C. The cell lines were identified by DNA profiling (short tandem repeat) and tested negative for mycoplasma contamination with the MycoAlert mycoplasma detection kit (Lonza, Rockland, ME, USA).

Cell viability assay

For cell viability assay, cells

DATS inhibited the survival of cultured human synovial sarcoma SW982 cells

To investigate the effect of DATS on the survival of human synovial sarcoma SW982 cells, CCK-8 assay were carried out to examine the cell viability. After the treatment with DATS for 24 h, the cell viability of SW982 reduced in dose-dependent manner (Fig. 1. A and B). Then, the reduced survival of SW982 cells caused by DATS treatment lead us to investigate the effect of DATS on apoptosis of SW982 using annexinV-FITC/PI staining. As demonstrated by HCIS analysis, the percentage of early

Discussion

Clinically, therapeutic drug for SS, especially for advanced SS, remains insufficient. Despite considerable research of DATS on its effective anti-cancer activity against many kinds of cancer cell lines such as breast cancer cell, anaplastic thyroid carcinoma cell (Zheng et al., 2019) and osteosarcoma cells(Wang et al., 2016), its effect on SS, one of the major malignant mesenchymal tumor, is still unknown. Here, we demonstrated the effect of DATS on cell viability, cell cycle, intracellular

Conclusion

In conclusion, our study clarified a comprehensive molecular portrait of DATS regulation for its suppressive effect on tumor biology of SW982 cells like inducing apoptosis, triggering cell cycle arrest and elevating intracellular ROS. These results pave the way toward the clinical development of DATS as chemopreventive drug for SS treatment.

CRediT authorship contribution statement

Sheng-li Xia: Data curation, Funding acquisition, Investigation, Project administration, Writing-original draft, Resources. Zi-yuan Ma: Investigation, Formal analysis, Investigation, Writing-original draft. Bin Wang: Investigation, Methodology. Feng Gao: Investigation, Visualization, Validation. Cun-guo Yi: Methodology, Visualization. Xiao-xiao Zhou: Investigation, Formal analysis, Funding acquisition, Resources. Sheng-yang Guo: Investigation, Formal analysis. Li Zhou: Conceptualization,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by grants from The Training Planned Fund of Academic Leaders, Shanghai Pudong New Area Health System (PWR 12018-09), National Natural Science Foundation of China (NSFC No. 82072902), The Featured Clinical Discipline Project of Shanghai Pudong (PWY ts2018-2), Key Subject Construction Project of Shanghai Health System (ZK2019B05).

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