Osteosarcoma is a highly malignant primary bone tumor. Despite the level of clinical treatment constantly improving, the survival rate of OS patients is still unsatisfactory, especially in patients with lung metastases [19, 20]. Currently used anti-OS drugs such as ifosfamide, methotrexate, and cisplatin are prone to resistance. What's worse, although these drugs have an anti-osteosarcoma effect, most of them are highly cytotoxic to normal cells and can cause liver toxicity and kidney dysfunction. [21–23] Therefore, it is necessary to explore safe and effective drugs for the treatment of OS with relatively low side effects.
Psoralidin (PSO) is the main extract of psoralen. [8] In addition to its antioxidant, antibacterial, and anti-inflammatory effects, PSO also has significant inhibitory effects on various cancers. [9–14, 24, 25] In this study, we first determined that PSO can inhibit the proliferation, migration, and invasion of OS cells and promote the apoptosis of OS cells in vitro and inhibit the tumor growth of OS cells in vivo.
The malignant proliferation and excessive migration and invasion of osteosarcoma cells are the main reasons for the poor prognosis of the tumor. [26] We found that PSO significantly inhibited the proliferation of OS cells in a dose-dependent manner by crystal violet assay, CCK-8 assay, and EdU staining assay. PCNA is closely related to DNA synthesis and plays an important role in the initiation of cell proliferation.[27] In this study, we found that PSO significantly downregulated PCNA in a dose-dependent manner. In addition, we found that the arrest of OS cells by PSO mainly occurred in G0/G1 phase from flow cytometry analysis. This further confirmed that PSO played an anti-proliferative role in osteosarcoma cells.
Tumor metastasis constitutes one of the most significant biological characteristics of malignant tumors and an important cause of death in tumor patients. Therefore, the key to the treatment of tumors is limiting metastasis. [28, 29] We demonstrated that PSO inhibited the migration and invasion of OS cells by scratch wound healing assays and transwell assays. Epithelial-mesenchymal transition (EMT) is an important process in tumorigenesis, which is closely related to tumor invasion, metastasis, and prognosis. Snail is a key transcription factor in EMT, and regulates the connections between cells.[18, 30] Matrix metalloproteinases (MMP) can destroy the matrix-degrading balance, and promote cancer invasion through the histological barrier (constituted by the basement membrane and ECM) into the surrounding tissues and metastasis to distant tissues.MMP-2 and MMP9 are the most essential for tumor metastasis and invasion.[31, 32] We found that MMP-2, MMP-9, and Snail were down-regulated after PSO treatment. These findings suggest that PSO may inhibit OS cell migration and invasion by blocking the EMT process and inhibiting MMP function.
Apoptosis is an important homeostatic mechanism, which involves the activation, expression, and regulation of a series of genes, and has potential therapeutic significance.[33] Therefore, inducing tumor cell apoptosis is one strategy to develop anticancer drugs. We confirmed that PSO significantly increased the apoptosis rate of OS cells by flow cytometry. Caspase3 is the main executor in the process of apoptosis. and plays a role in the early stage of apoptosis.[34] Bcl-2 family proteins are key regulators of mitochondria-mediated apoptosis and include antiapoptotic members such as Bcl-2 and proapoptotic members such as Bax. [35] In this study, we found that PSO significantly increased the apoptosis rate of OS cells, promoted the protein levels of Bax, and cleaved caspase-3, while reducing and downregulating Bcl-2 levels. These results further indicated that PSO could affect OS cells through the apoptotic pathway.
To explore the potential mechanism of the antitumor effect of PSO in OS cells, we performed RNA sequence analysis, which showed significant changes in PI3K/Akt and FAK signaling in PSO-treated 143B, MG63 cells. Further GESA and KM analysis revealed important DEGs, such as ITGB1, PIK3CD, MAPK3 and PRKCA, which are often associated with tumors. Many studies have confirmed that ITGB1 is responsible for Focal adhesion kinase (FAK) activation and Integrin ligand adhesion triggers an increase in FAK tyrosine (Tyr) 397 phosphorylation and FAK is a marked effect on tumor cell survival, migration, invasion, angiogenesis, and metastasis. [36, 37] In addition, after ITGB1 activates Tyr397 phosphorylation, FAK can form a complex with Src, which binds to the P85 subunit of PI3K, which can further activate downstream Akt and regulate cell proliferation and cycle. [38–40] Therefore, it is necessary to inhibit the expression of ITGB1. Several studies have revealed that repression of ITGB1 expressions can attenuate Metastasis and invasiveness of tumor. [41–43] In this study, we confirmed that PSO inhibited FAK expression and downstream PI3K/Akt phosphorylation. Further molecular docking assays demonstrated that PSO targeted ITGB1 for decreasing FAK activation. Our Western blot results supported the conclusion of our RNA sequence analysis, showing that PSO treatment reduced the expressed level of ITGB1, FAK and p-FAK and ratio levels of p-PI3K /PI3K, and p-Akt/Akt in OS cells. Therefore, our study suggests that PSO therapy may inhibit the growth and progression of osteosarcoma by down-regulating ITGB1 expression via the FAK and PI3K/Akt signaling pathways.