Chondrosarcoma represents a malignant bone neoplasm, comprising 20%–30% of all such tumors (1, 2). It affects individuals across all age groups, with a higher prevalence in men (1, 2). The pelvis and long bones, particularly the femur, are the most frequent locations for this tumor (3). Surgical resection remains the standard treatment, with tumor margin excision correlating with improved postoperative survival (3, 4). In contrast, chondrosarcoma exhibits resistance to radiotherapy and chemotherapy (5, 6), primarily due to factors such as limited angiogenesis, an abundant extracellular matrix, restricted cellular proliferation, and slow growth rates (1, 7). The anatomical features and invasive properties of common chondrosarcoma sites complicate effective medical intervention. Furthermore, surgical resection is often not a viable option for patients with metastatic chondrosarcoma (8, 9). Consequently, there is a pressing need for more targeted and effective pharmacological therapies for managing chondrosarcoma.

Andrographolide (Andro), a diterpenoid lactone with the molecular formula C₂₀H₃₀O₅, is the principal bioactive compound derived from Andrographis paniculata (Burm. f.) Nees, a member of the Oleaceae family. Its molecular structure comprises three hydroxyl groups and a lactone ring (10, 11). Andro exhibits a range of biological activities, including anti-cancer, anti-inflammatory, antioxidant, and immune-modulatory effects (10, 11). According to Attri et al., Andro holds promise as a natural anti-cancer agent, capable of overcoming certain limitations inherent in conventional cancer treatments due to its broad-spectrum efficacy and low toxicity (12). The compound exerts anti-cancer actions by disrupting the cell cycle, suppressing cell migration, and inducing apoptosis and autophagic cell death at distinct biological stages (13–15). A previous investigation emphasized Andro’s role as an antiviral agent (16), exploring its activity and that of its derivatives against COVID-19 through systems biology, suggesting its involvement in various biological processes. Furthermore, Wang et al. demonstrated that Andro promoted protective autophagy by targeting DJ-1, triggering reactive oxygen species to induce pancreatic cancer cell death (17). This suggests that autophagy induction is a central mechanism behind Andro’s anti-cancer effects (17). Additionally, previous work has shown that Andro inhibits chondrosarcoma cell proliferation (18), though the mechanisms through which it prevents chondrosarcoma metastasis remain unclear.

The hypothesis that Andro suppresses the migratory and invasive potential of chondrosarcoma cells, with Andro-induced autophagy as a central mechanism, is derived from prior studies. This hypothesis was tested using network pharmacology and in vitro cell experiments to evaluate Andro’s effects on chondrosarcoma cell migration and invasion, as well as to identify the associated mechanisms ( Figure 1 ).

Surgical resection remains limited in the treatment of metastatic chondrosarcoma, often resulting in a poor prognosis (23, 24). Consequently, preventing invasion and metastasis across all pathological stages of chondrosarcoma holds significant clinical relevance. Although Andro exhibits potential anti-cancer properties, its pharmacological effects in chondrosarcoma remain inadequately explored. Previous research has demonstrated that Andro inhibits chondrosarcoma cell proliferation and promotes apoptosis (18). Building upon this evidence, the current study investigates the impact of Andro on chondrosarcoma cell migration and invasion. The findings indicate that Andro diminishes the migratory and invasive capabilities of chondrosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway. These results may contribute to advancing Andro as a therapeutic agent for chondrosarcoma treatment.

Network pharmacology identified PI3K, Akt, and mTOR as central targets of Andro in chondrosarcoma treatment. Numerous studies have established the significant roles of PI3K, Akt, and mTOR in chondrosarcoma metastasis (25–27). Qu et al. (25) demonstrated that PRKCI was overexpressed in osteosarcoma cell lines, and its silencing suppressed the proliferation, migration, and invasion of osteosarcoma cells. Moreover, they identified that PRKCI interacted with SQSTM1, modulating osteosarcoma cell proliferation via the Akt/mTOR signaling pathway. Truong et al. (26) emphasized that the IGF/PI3K/mTOR pathways were crucial in chondrosarcoma, with activation of the PI3K/Akt/mTOR pathway correlating with invasiveness and drug resistance. Additionally, interactions between PRKCI and the Akt/mTOR pathway in osteosarcoma were confirmed (27), highlighting the relevance of this pathway in both osteosarcoma and chondrosarcoma. Given that osteosarcoma and chondrosarcoma are both primary malignant bone tumors (27), they share common biological traits and signaling pathway activations. Collectively, these studies reinforce the pivotal roles of PI3K, Akt, and mTOR in chondrosarcoma metastasis. KEGG pathway enrichment analysis further indicated that the PI3K-Akt signaling pathway was a primary pathway linked to the core target genes. The PI3K/Akt/mTOR pathway is a well-known signaling cascade regulating cell proliferation and apoptosis, playing a critical role in the initiation and progression of various cancers (28, 29). Zhang et al. demonstrated that upon activation, PI3K activated Akt, triggering its translocation to the cell membrane, where it was subsequently activated and underwent auto-phosphorylation (30). Akt thus functions as a key downstream kinase of PI3K (30). Activation of the PI3K/Akt signaling pathway induces the upregulation of intracellular p-Akt (31, 32), which contributes to cell proliferation, apoptosis, transcription, translation, the cell cycle, and vascular growth across various cancers. Moreover, mTOR signaling pathway activation is implicated in the development of multiple malignancies. mTOR is typically upregulated in tumors and suppresses autophagy. Kopecky et al. observed higher mTOR expression in kidney cancer tissues compared to normal kidney tissues (33) and confirmed mTOR as a potential drug target for renal cancer treatment. The PI3K/Akt pathway, a classical apoptotic signaling route, is upstream of mTOR and plays a critical role in its activation (34). The PI3K/Akt/mTOR signaling pathway regulates the initiation and modulation of autophagy; thus, inhibiting this pathway can enhance cellular autophagy (34, 35). Zhu et al. demonstrated that baicalin inhibited the PI3K/Akt/mTOR pathway, inducing apoptosis in human chondrosarcoma cells (36). Similarly, Wu et al. reported that endothelin-1 regulated the FAK/PI3K/Akt/mTOR pathway, promoting MMP-13 expression and migration in human chondrosarcoma cells (37). Furthermore, Kouvaras et al. identified the HIF-1 signaling pathway as a key regulator of chondrosarcoma angiogenesis and malignant progression through VEGF expression, highlighting its potential as a therapeutic target (38). The T-cell receptor signaling pathway also modulates the immune microenvironment and response to chondrosarcoma treatment, suggesting new avenues for immunotherapy (39). These pathways present valuable research prospects for chondrosarcoma treatment (38, 39). Based on network pharmacology findings, drugs targeting the PI3K/Akt/mTOR pathway represent promising therapeutic agents for chondrosarcoma.

Proliferation is a defining feature of tumor cells and a key factor in tumor malignancy. In this study, wound healing and transwell assays demonstrated that Andro significantly reduced the migration and invasion of chondrosarcoma cells in vitro. WB analysis revealed that Andro downregulated vimentin and MMP-9, while upregulating E-cadherin in chondrosarcoma cells. Autophagy inhibitor CQ effectively reversed the inhibitory effects of Andro on cell migration and invasion, highlighting that Andro acts through autophagy modulation. These findings underscore the critical role of autophagy in mediating Andro’s anticancer effects, suggesting that autophagy inhibition is a key mechanism by which Andro suppresses chondrosarcoma progression. Additionally, Andro decreased the p-mTOR/mTOR, p-PI3K/PI3K, and p-Akt/Akt ratios in these cells. Collectively, these results suggest that Andro suppresses the migratory and invasive capabilities of chondrosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway. These results align with those of Zhu et al., who demonstrated that inhibition of the PI3K/Akt/mTOR pathway reduced the metastatic and invasive potential of chondrosarcoma cells (36). Moreover, Zheng et al. reported that curcumin induced autophagy through the inhibition of the PI3K/Akt/mTOR pathway in gastric cancer (40). Moreover, Andro’s dual action of inducing autophagy and inhibiting the PI3K/Akt/mTOR pathway may effectively overcome resistance mechanisms in chondrosarcoma, offering a more comprehensive therapeutic effect than single-target inhibitors.

In this study, in vitro experiments demonstrated that Andro significantly induced autophagy in chondrosarcoma cells, providing novel insights into its anti-chondrosarcoma mechanisms. Autophagy, a process of cellular self-degradation, preserves homeostasis by degrading damaged organelles and protein aggregates (41). The expression of autophagy-related proteins p62, Beclin-1, and LC3A/B was assessed through WB. Results revealed that Andro treatment notably reduced p62 expression while increasing Beclin-1 levels and the LC3A/B-II to LC3A/B-I ratio. Given that p62 functions as a selective autophagy receptor (42), its reduction suggests enhanced autophagic degradation. Beclin-1, a critical initiator of autophagy, shows increased expression during autophagy activation (43). An elevated LC3A/B-II to LC3A/B-I ratio reflects increased autophagosome formation. Furthermore, Andro treatment significantly boosted autophagic flux, as evidenced by a marked increase in the red/green fluorescence ratio, indicating that Andro promotes both autophagosome formation and substrate degradation. Previous studies have also suggested that promoting autophagy can hinder the pathological progression of chondrosarcoma (44, 45). Sun et al. (44) reported that GANT-61 inhibited chondrosarcoma growth by inducing autophagy through suppression of the RNAP III pathway and tRNA-Gly-CCC synthesis, involving ULK1 and MAPK pathways. Pang et al. (45) demonstrated that vitamin E subtypes (AnTT, γ-T3, and δ-T3) triggered autophagy in chondrosarcoma cells by activating pathways such as ER stress and the unfolded protein response, thereby inhibiting cell proliferation and tumor development. As a key regulator of autophagy, the PI3K/Akt/mTOR pathway generally suppresses autophagy when activated (46, 47), whereas its inhibition promotes autophagy. Our results demonstrate that Andro significantly induces autophagy in chondrosarcoma cells, which is associated with the inhibition of cell migration and invasion. However, it should be noted that autophagy is a context-dependent process. While autophagy can promote tumor survival under certain stress conditions, as reported in some studies, our findings suggest that Andro-induced autophagy exerts a tumor-suppressive effect in chondrosarcoma cells. This discrepancy may be attributed to the specific signaling pathways modulated by Andro. In particular, Andro inhibits the PI3K/Akt/mTOR signaling pathway, which is known to suppress autophagy when activated. By downregulating this pathway, Andro promotes autophagy, leading to the degradation of pro-metastatic proteins such as vimentin and MMP-9, and the upregulation of E-cadherin, thereby inhibiting the metastatic potential of chondrosarcoma cells. Therefore, the autophagy induced by Andro appears to be beneficial in the context of chondrosarcoma treatment, highlighting the importance of understanding the context-dependent outcomes of autophagy in different tumor settings. Our findings suggest that Andro’s inhibition of the PI3K/Akt/mTOR pathway may offer a potential strategy to address metastasis in chondrosarcoma, a tumor type resistant to conventional therapies. These results provide a rational basis for exploring Andro’s clinical relevance in improving patient outcomes.

This study presents several limitations that warrant consideration. Firstly, our study’s findings are currently limited to in vitro experiments, and the link between Andro-induced autophagy and migration inhibition lacks direct in vivo validation. Future work will include autophagy inhibitor experiments and knockout models to directly demonstrate this link, as well as in vivo studies using genetic or chondrosarcoma-specific mouse models to further elucidate Andro’s mechanisms. Secondly, the databases employed for network pharmacology are continually updated, implying that the bioinformatics analysis is based on the current data available. Thirdly, the KEGG pathway enrichment analysis identifies multiple pathways (such as the HIF-1 and FoxO signaling pathways) beyond the PI3K/Akt signaling pathway, necessitating further validation of these pathways as a key area for future research. Finally, the use of inhibitors or activators to target core signaling pathways, followed by a comparison of their effects with those of Andro, could clarify Andro’s mechanism and efficacy. This approach is essential for advancing future research, particularly in vitro studies.

This study demonstrates that Andro can inhibit chondrosarcoma cell migration and invasion, likely through the suppression of the PI3K/Akt/mTOR pathway, alleviation of autophagy inhibition, and activation of autophagy. This discovery provides a novel molecular mechanism underlying Andro’s anti-chondrosarcoma effects and supports the development of autophagy-targeted cancer therapies. Future research should evaluate Andro’s in vivo antitumor efficacy and explore its potential synergistic effects with other anticancer agents. Further investigation into Andro’s long-term influence on autophagy and its role within the tumor microenvironment will contribute to a deeper understanding of its clinical application potential.