CS has favorable demonstrates potent inhibitory activity against various cancer cells and possesses anti-tumor properties. Its complex chemical composition and diverse pharmacological effects primarily consist of flavonoids, triterpenes, sterols, and anthraquinone compounds, with flavonoids, including isoflavones, dihydroflavonoids, and chalcones, being the most abundant. Previous studies have reported [
12] that flavonoids in CS extracts effectively inhibited the proliferation and growth of human lung carcinoma, colorectal cancer and other tumor cells. Liu B et al. [
13] also observed the inhibitory effect of various solvents extracts of CS on the growth of human OS Saos-2 cells, aligning with our own findings. In this study, through MTT assay results, revealed that the inhibition rates of Saos-2 and U2OS increased proportionally with the concentration of CS extract and duration of treatment. Moreover, the IC
50 of CS extracts for OS cells decreased over time. Furthermore, research on CS extracts [
14] have also documented their capacity to impede tumor activity, influence the cell cycle, and suppress tumor metastasis in transplanted Lewis lung cancer mouse models. Clinical pharmacological investigations [
15] have highlighted the anti-thrombotic effects of CS in addition to its anticancer properties, with reported inhibition of induced metastasis [
16]. CS extracts have been shown to effectively inhibit colorectal cancer invasion and metastasis by inhibiting ADP-induced platelet aggregation in vivo and in vitro, thereby further validating the impact of CS extracts on OS metastasis and invasiveness. This study demonstrated that CS extracts effectively reduced the abnormally high expression of CXCR4 in OS cells and effectively suppressed Saos-2 and U2OS migration, and invasiveness.
The metastasis of OS encompasses a diverse array of biological processes, including oncogenes and cell invasiveness and metastasis. Circulating tumor cells (CTCs) detached from the primary tumor site and adhered to the local migration matrix via stromal tissue, which is closely associated with cell signaling and cell–matrix interactions [
17,
18]. CXCR4, a molecule known to be upregulated in a variety of cancers, hampers cancer cell angiogenesis and metastasis [
19,
20]. In vitro studies have shown that CXCR4 inhibitors induce apoptosis and inhibit proliferation of OS cells [
21]. Oda et al. [
22] observed high expression of CXCR4 in metastatic and primary sites, which correlated with the activation of the vascular endothelial growth factor signaling pathway. This study revealed that CXCR4 was highly expressed in OS cells and inhibiting CXCR4 through inhibitors or CXCR4 gene knockout effectively impeded OS cell growth and metastasis. Additionally, CS extracts have shown efficacy in inhibiting OS cell migration and invasiveness by specifically regulating CXCR4. However, the precise mechanism by which CXCR4 modulates OS cell metastasis requires further elucidation, particularly regarding downstream signaling pathways. Tumor invasiveness and metastasis involve a network of interconnected signaling pathways, among which the PI3K/AKT and ERK1/2 axis have been demonstrated to play a pivotal role in the invasiveness and metastasis of solid tumors. Leelawat et al. [
23] were the first to discover that the interaction between CXCR4 and chemokine SDF-1 could activate ERK1/2 and PI3K axis, inducing invasiveness in cholangiocarcinoma cells. Jiang et al. [
24] showed that interfering with CXCR4 expression using siRNA, induced apoptosis in human OS cells, and the down-regulation of CXCR4 was achieved by inhibiting PI3K/Akt/NF-κβ axis, highlighting the significance of CXCR4 in the clinical treatment of OS. Further study revealed that, CXCR4/SDF-1 stimulated the phosphorylation of Akt, JNK and c-Jun, while the CXCR4 inhibitor AMD3100 reduced the phosphorylation activity in OS cells [
25]. This study provided the first findings demonstrating that SDF-1 promoted the survival and metastasis of CXCR4-mediated OS by activating the Akt and JNK axis. AMD3100 counteracted the effects and downstream pathways influenced by CXCL12/CXCR4 interactions in the regulation of OS survival and metastasis. In recent times, an increasing number of CXCR4 inhibitors have entered clinical trials, demonstrating their pivotal role in treating patients unresponsive to checkpoint inhibitors, or those with cancer metastasis or recurrence. For instance, mavorixafor, an orally bioavailable CXCR4 antagonist, has shown promise in modulating immune cell trafficking in melanoma patients [
26]. Balixafortide (BLX), a protein epitope mimetic inhibitor of CXCR4, has been shown to enhance docetaxel-mediated antitumor activity in PCa bone metastases [
27]. Additionally, a novel CXCR4 inhibitor, modified with a picolinamide scaffold (CPZ1344), has entered pre-clinical research, demonstrating potential as a novel therapeutic agent against glioblastoma [
28].
Our research demonstrates that CS extracts effectively inhibit the PI3K/AKT signaling pathway in OS cells, and overexpressing CXCR4 effectively prevents the inactivation of the PI3K/AKT signal caused by CS extracts in OS cells. Therefore, the impact of CS extracts on OS growth and metastasis is achieved by targeting CXCR4 to regulate the PI3K/AKT axis.