Introduction

Osteosarcoma (OS) is a prevalent primary malignant bone tumor of mesenchymal origin, predominantly affecting children and adolescents. Epidemiological studies have revealed that OS accounts for 15% of all solid extracranial cancers in adolescents aged 15–19 years1. Current therapeutic approaches for OS primarily revolve around surgical intervention followed by chemotherapy. However, the aggressive nature of OS, coupled with chemotherapy resistance, recurrence, and adverse side effects leading to reduced patient compliance, present significant challenges in achieving satisfactory treatment outcomes. Consequently, there is an urgent need to explore effective and safe agents for the prevention and treatment of OS.

Metabolic reprogramming is a defining hallmark of cancer cells2, and in osteosarcoma (OS), fatty acids and glycerophospholipids emerge as critical metabolic drivers of progression. Fatty acids, central energy sources for tumor cells, fuel OS growth, invasion, and immunosuppressive microenvironment formation3,4. Recent studies show that inhibiting fatty acid β-oxidation—via pharmacological or genetic approaches—disrupts mitochondrial energy production, thereby reducing OS cell viability, migration, and invasion5,6. Beyond de novo fatty acid synthesis mediated by FASN, glycerophospholipids, the most abundant membrane phospholipids, are hydrolyzed by phospholipases (PLs) into bioactive metabolites such as lysophospholipids and fatty acids7,8. These metabolites regulate membrane dynamics, signaling, and proliferation, with aberrant glycerophospholipid metabolism linked to aggressive OS phenotypes. For instance, elevated phospholipase A2 group 16 (PLA2G16) correlates with lung metastasis and poor prognosis9, while phospholipase D (PLD) antagonizes tumor-suppressive miR-638 to drive OS proliferation10. Notably, disrupting phosphatidylcholine (PC) biosynthesis—a major glycerophospholipid subclass—suppresses OS cell proliferation and mineralization capacity11. Clinically, first-line OS chemotherapeutics like cisplatin and doxorubicin act partly by inducing membrane degradation or blocking lipid synthesis12. Collectively, targeting these interlinked metabolic pathways—notably glycerophospholipid metabolism and fatty acid β-oxidation—offers a promising therapeutic strategy to disrupt energy production and membrane synthesis critical for OS progression.

Ganoderma lucidum, a medicinal mushroom with a historical usage spanning over 6800 years13, has been found to influence lipid metabolism. For instance, G. lucidum aqueous extract regulates the biological metabolic process of membrane components in tetrahymena thermophila14 and G. lucidum polysaccharide-cadmium chelate regulates glycerophospholipid metabolism in liver15. Additionally, sporoderm-broken GLS has demonstrated anti-OS effects by inducing apoptosis and inhibiting the STAT3/PD-L1 signaling pathway16,17. However, there have been no reports on the specific role of G. lucidum or its extracts in regulating the metabolic processes of membrane components and phosphoglyceride metabolism to exert its anti-OS effects.

The present study aimed to analyze the effects of sporoderm-removed GLS (RGLS), a highly purified GLS extract18,19 on OS development both in vitro and in vivo, and to elucidate the potential mechanism of RGLS against OS based on the integrative analysis of transcriptomics, proteomics and metabolomics20, which has not been previously reported. Our data indicate that RGLS effectively suppresses OS development by inhibiting proliferation, and inducing cell apoptosis. Additionally, we observed that RGLS promotes PCs metabolism by enhancing endocytosis of macrophage-secreted Pla2g7 by S-180 cells. Furthermore, we found that RGLS regulates fatty acid pool composition and inhibits the conversion of long-chain acylcarnitine to long-chain acyl-CoA in mitochondria, and ultimately leading to fatty acid β-oxidation disorders and intracellular energy insufficiency.

Results

RGLS inhibits S-180 cells growth in vivo

In the present study, we administered RGLS prophylactically to BALB/c nude mice and then injected them subcutaneously with S-180 cells to evaluate RGLS’s tumor-inhibiting effects (Fig. 1A). The results showed that RGLS significantly reduced the weight of S-180 xenograft tumors compared with the model group (Fig. 1B), and the tumor inhibition rate was high at 35.8% (Fig. 1C). In addition, we analyzed the effect of therapeutic administration of RGLS on the growth of S-180 xenograft tumors. As shown in Fig. S1A, in the absence of prophylactic administration, only 7 days of therapeutic administration also reduced the weight of S-180 xenograft tumors (Fig. S1B), but the tumor inhibition rate was lower at 25.8% (Fig. S1C). Moreover, the body weight of mice was not affected by both administration methods of RGLS (Figs. 1D and S1D). These results indicated that RGLS effectively delays the growth of S-180 xenograft tumors.

Fig. 1
figure 1

Effect of RGLS on growth of S-180 xenograft tumor in vivo. (A) Experimental protocol. (B–C) Effect of RGLS on tumor size (B) and weight (C). (D) Effect of RGLS on body weight of mice. (E) Effect of RGLS on pathological changes of tumor tissue. (F) Effect of RGLS on Ki-67 expression in tumor tissue. ***P < 0.001 compared with Model group, n = 6.

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Apoptosis is the most classical mode of cell death in tumors. Next, we observed the effects of RGLS on the pathological changes in S-180 xenograft tumors using H&E staining. As shown in Fig. 1E, RGLS greatly induced apoptosis in S-180 cells within xenograft tumors, which was mainly manifested as the loss of intercellular connections, increased cytoplasmic density, reduced cell volume, and nuclear contraction. In addition to apoptosis, inhibiting cell proliferation is also an effective strategy for tumor treatment. Furthermore, we examined the expression of Ki-67, a nuclear cell proliferation marker, in xenograft tumors using IHC staining. The results are shown in Fig. 1F, the expression of Ki-67 was significantly decreased in the RGLS treatment groups compared with the model group. Taken together, RGLS delays S-180 xenograft tumor growth by inhibiting cell proliferation and inducing cell apoptosis.

RGLS alleviates tumor-induced immune deficiency in mice

Immune deficiency is an important factor in tumorigenesis, and in turn, the development of tumors can further exacerbate the immune deficiency. Next, we analyzed the effects of RGLS on internal morphological structure of the thymus using H&E staining. As shown in Fig. S2A, the boundary between cortex and medulla of thymus in control group was distinct and orderly arranged. However, compared to the control group, the cortex and medulla of the thymus in the model group underwent spatial rearrangement, and RGLS exhibited a mitigating effect on this phenomenon.

The thymus is the site of T lymphocyte development, and damage to the thymus inevitably leads to abnormal development of T cells. Further, we analyzed the effects of RGLS on proportions of specific T cell subtypes in peripheral blood, including CD3+, CD3+CD4+, and CD3+CD8+ T cells, relative to total CD3+ T cells. Compared with the control group, the proportion of CD3+ T cells in the model group was significantly reduced, while RGLS increased the proportion of CD3+ T cells compared with the model group. However, the proportions of CD3+CD4+ and CD3+CD8+ T cells did not change significantly in both the model group and the RGLS group (Fig. S2B,C). These results suggest that RGLS ameliorates tumor-induced immune decline, which may be related to the regulation of non-traditional T cell development, such as double negative (DN, CD4−CD8−) iNKT cells21.

RGLS inhibits S-180 cells growth in vitro

To further analyze the anti-OS effects of RGLS, we examined the impact of RGLS on S-180 cells growth in vitro. The CCK-8 assay showed that RGLS decreased the viability of S-180 cells in a dose-dependent manner (Fig. 2A), and the half-maximal inhibitory concentration (IC50) was 2.51 mg/ml. We then examined whether RGLS induced apoptosis in S-180 cells. As shown in Fig. 2B,C, RGLS treatment significantly increased the ratio of apoptotic cells, and the late apoptotic (Annexin V−/PI+) cells were predominant. Specifically, our results show that RGLS treatment significantly reduces the expression levels of pro-Casp3 and Bcl-xL in a concentration-dependent manner, but does not significantly affect the Bax/Bcl-xL ratio (Fig. 2D). Collectively, these findings suggest that RGLS effectively inhibits S-180 cell proliferation by inducing apoptosis, potentially through a mechanism independent of the mitochondrial pathway.

Fig. 2
figure 2

Effect of RGLS on growth of S-180 cells in vivo. (A) Effect of RGLS on S-180 cells proliferation. (B–C) Effect of RGLS on apoptosis rate of S-180 cells. (D) Effect of RGLS on apoptosis-related protein expression in S-180 cells. The representative images of Western blot have been cropped, and the full Western Blot images for (D) are attached in Supplemental Fig. 4A-D. *P < 0.05, ***P < 0.001 compared with Con group, n = 3.

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RGLS regulates glycerophospholipid metabolism in S-180 cells

Reprogramming of lipid metabolism is an emerging hallmark that distinguishes tumor cells from normal cells2. To further elucidate the molecular mechanism of RGLS in inhibiting S-180 cell growth, we performed a metabonomics analysis to assess the effects of RGLS on S-180 cell metabolism. Principal component analysis (PCA) revealed that RGLS significantly altered the metabolite composition in S-180 cells (Fig. 3A). Compared with the control group, RGLS upregulated 49 metabolites and downregulated 47 metabolites (Fig. 3B). Following KEGG metabolic pathway enrichment analysis of the differential metabolites (Fig. 3C), nine metabolic pathways, namely Choline metabolism in cancer (ko05231), Efferocytosis (ko040148), Glycerophospholipid metabolism (ko00564), Thermogenesis (ko04714), Sulfur relay system (ko04122), Nucleotide metabolism (ko01232), Zeatin biosynthesis (ko00908), Cysteine and methionine metabolism (ko00270), and ABC transporters (ko02010) were enriched (P < 0.01). Among them, the number of targeted differential metabolites related to glycerophospholipid metabolic pathway is the largest. Focusing on glycerophospholipid metabolic, a heatmap analysis of the total 42 differential metabolites (Fig. 3D) showed the degree of variation with various colors. Specifically, RGLS treatment significantly increased the levels of lysophosphatidylcholine (LysoPC, including LysoPC 16:1, LysoPC 18:2, LysoPC 18:3, and LysoPC 20:2), a metabolite of phosphatidylcholine (PC), which has been shown with anti-tumor activity22,23. In contrast, the RGLS treatment reduced the levels of phosphatidylinositol (PI, including PI 18:0/14:1, PI 16:1/18:2, PI 16:1/18:1, PI 18:2/20:4, PI 18:2/20:3, and PI 18:0/20:4), an important component for cell and organelle membrane biosynthesis and function24, and its metabolite LysoPI (including LysoPI 16:0, LysoPI 17:0, LysoPI 18:3, and LysoPI 20:1), which has been shown with pro-tumor activity25. Taken together, these results indicate that RGLS exerts bidirectional regulation on glycerolphospholipid metabolism to promote anti-tumor activity.

Fig. 3
figure 3

Metabonomics analysis of S-180 cells. (A) Principal component analysis (PCA) of metabonomics data. (B) Differential expression analysis of metabonomics data. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG51) terms enriched by differentially metabolites. (D) Effect of RGLS on phospholipid and lysophospholipid content.

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RGLS regulates glycerophospholipid metabolism by enhancing the interaction between S-180 cells and macrophages

Phospholipases (PLs) are the rate-limiting enzymes in glycerolphospholipid metabolism, including PLA1, PLA2, PLC, and PLD. Among them, glycerolphospholipids are hydrolyzed by PLA1 or PLA2 and then lose a molecule of fatty acid to produce lyso-phospholipids (LP) (Fig. 4A). To further elucidate the regulatory effect of RGLS on PC metabolism, transcriptomic and proteomic analyses were conducted to assess gene and protein expression changes in xenograft tumors. As shown in Fig. 4B, PCA revealed that RGLS significantly altered the gene and protein expression in xenograft tumors. Compared with the control group, RGLS upregulated 1494 genes and 431 proteins, and downregulated 414 genes and 43 proteins. Initially, we verified the role of RGLS in inducing S-180 cell apoptosis. As shown in Fig. S3A, RGLS treatment significantly upregulated the expression levels of apoptosis-related proteins, including the Casp family (Casp1, Casp3, Casp7, Casp8), Bcl-2 family (Bax, Bid), and Aif family (Aif1, Aifm1, Aifm2, Aifm3), suggesting that RGLS induced S-180 cell apoptosis in vivo.

Fig. 4
figure 4

RGLS enhances PC metabolism. (A) Schematic diagram of phospholipid metabolic sites. (B) PCA analysis of transcriptomics and proteomics data. (C–D) Gene Ontology Biological Process (GOBP) terms enriched by upregulated genes (C) and proteins (D). (E) Effect of RGLS on the expression of phospholipase in tumor tissue. (F) Combined proteomic and transcriptomic analysis. (G) GOBP terms enrichment of genes and proteins upregulated by RGLS simultaneously. (H) Effect of RGLS on the expression of ABC families in tumor tissue. (I) Model diagram of co-culture of S-180 cells with Raw264.7 cells. (J) Effect of RGLS on the expression of Pla2g7 in S-180 cells co-cultured with Raw264.7 cells. (K) Effect of RGLS on the expression of Pla2g7 in Raw264.7 cells. The representative images of Western blot have been cropped, and the full Western Blot images for (K,J) are attached in Supplemental Fig. 4E–F. **P < 0.01, ***P < 0.001 compared with Con group, n = 3.

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Next, gene ontology (GO) enrichment analysis showed that the upregulated genes and proteins are mainly enriched in cellular compartment terms related to membrane, such as lysosomal membrane (GO:0098574), microvillus membrane (GO:0031528), endosome membrane (GO:0010008), actin cytoskeleton (GO:0015629), mitochondrial inner membrane (GO0005743), and mitochondrial membrane (GO:0031966) (Fig. 4C,D). This suggests that lysosome may be involved in the process of RGLS regulating PC metabolism26. Indeed, we found that RGLS significantly increased autophagy levels in S-180 cells in vitro, including increasing the number of autophagosomes (Fig. S3B), upregulating LC3B protein levels and decreasing p62 protein levels (Fig. S3C), and enhancing lysosomal acidity (Fig. S3D). In addition, RGLS upregulated the expression levels of lysosomal functional proteins (Lamp1 and Lamp2) (Fig. S3E) and lysosomal cathepsin (Fig. S3F).

Furthermore, we analyzed the effect of RGLS on PLA expression. As shown in Fig. 4E, we found that RGLS upregulated the expression levels of multiple LPA members in S-180 xenograft tumors, especially Plaa (a PLA2 activating protein) and Pla2g7 (the seventh member of the PLA2 family), a secreted PL that metabolizes PC to LysoPC under the action of lysophosphatidylcholine acyltransferase (Lpcat) in the lysosome27. However, Pla2g7 is usually derived from macrophages28. Indeed, transcriptome data showed that S-180 xenograft tumor did not express Pla2g7 and RGLS did not affect Pla2g7 mRNA expression levels (Fig. S3G). These results suggest that RGLS may promote PC metabolism by enhancing the interaction between S-180 cells and macrophages.

Endocytosis is an important pathway for cellular uptake of biological macromolecules. Combined transcriptome and proteome analyses revealed that 46 regulatory factors, such as Smap1, Unc119 and Ubaln2, were upregulated simultaneously at both gene and protein levels (Fig. 4F). Further GO enrichment analysis revealed a strong association between co-upregulated genes/proteins and regulation of clathrin-dependent endocytosis (GO:2000369) (Fig. 4G). We also identified a series of ABC superfamily members whose expression levels were upregulated by RGLS (Fig. 4H). Thus, we hypothesized that RGLS might enhance the uptake of Pla2g7 secreted by macrophages in S-180 cells. To test this hypothesis, we performed co-culture of S-180 cells with Raw264.7 cells (Fig. 4I). Compared to S-180 cells cultured alone, the Pla2g7 protein levels (Fig. 4J), but not mRNA levels (Fig. S3G), were significantly higher in S-180 cells co-cultured with Raw264.7 cells. However, it was disappointing that RGLS (2 mg/ml) treatment did not further increase significantly the levels of Pla2g7 protein and mRNA (Figs. 4J and S3G), in co-cultured S-180 cells. This phenomenon may be explained by the Pla2g7 protein in S-180 cells in the form of enzyme–substrate binding or the Pla2g7 protein released by Raw264.7 cells reaching its limit. In addition, at non-toxicity concentrations (2 mg/ml) (Fig. S3H), RGLS decreased significantly Pla2g7 protein levels in Raw264.7 cells (Fig. 4K). Interestingly, RGLS did not reduce pla2g7 mRNA expression levels in Raw264.7 cells (Fig. S3I). These results showed that RGLS promoted the release of Pla2g7 protein in Raw264.7 cells and the phagocytosis of Pla2g7 protein in S-180 cells, thus enhancing PC metabolism.

RGLS regulates acylcarnitine and fatty acid metabolism

Fatty acids are intermediate metabolites of glycerolphospholipid, releasing ATP through β-oxidation. Surprisingly, treatment with RGLS significantly reduced intracellular ATP content in S-180 cells (Fig. 5A). To investigate whether β-oxidation is compromised by RGLS, we analyzed its role in regulating fatty acid metabolism. It is well established that short- and medium-chain fatty acids can enter the mitochondria directly through diffusion, whereas long-chain fatty acids are converted to fatty acyl-CoA by long-chain acyl-CoA synthetase (Acsl) before entering the mitochondria. As shown in Fig. 5B, RGLS treatment increased the expression levels of Acsl family members, particularly Acsl3. Subsequently, we analyzed the effect of RGLS on carnitine-acyl transferase 1 (Cat1), which facilitates the binding of acyl-CoA to carnitine to form acylcarnitine, thereby enabling entry into the mitochondria. As shown in Fig. 5C, RGLS treatment significantly increased the protein expression level of carnitine-palmitoyl transferase 1a (Cpt1a), a member of the Cat1, suggesting that RGLS may promote the entry of long-chain fatty acids into the mitochondria.

Fig. 5
figure 5

RGLS inhibits fatty acid metabolism. (A) Effect of RGLS on ATP content in S-180 cells. (B–D) Effect of RGLS on the expression of Acsl families (B), Cpt1a (C), and Cat2 (D) in tumor tissue. (E) Effect of RGLS on L-carnitine content in S-180 cells. (F–G) Effect of RGLS on long-chain Acylcarnitine (F) and short-, and medium-chain Acylcarnitine (G) content in S-180 cells. (H) Effect of RGLS on the composition of fatty acid pool in S-180 cells. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Con or Model group, n = 3.

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In the mitochondria, long-chain acylcarnitine reconverted to long-chain acyl-CoA under the action of Cat2 and L-carnitine is released. As shown in Fig. 5D, RGLS treatment significantly decreased the protein expression level of Cat2, and reduced the L-carnitine content in S-180 cells (Fig. 5E), suggesting that RGLS may inhibit long-chain acylcarnitine metabolism. Next, we analyzed the changes of acylcarnitine levels in S-180 cells. As illustrated in Fig. 5F,G, RGLS significantly increased the levels of long-chain acylcarnitine (C 16:1 and C16:0) and slightly decreased the levels of short- and medium-chain acylcarnitine (C 4:0, C 5:0, and C 9:1). Further, we analyzed the effect of RGLS on fatty acid composition in S-180 cells. As shown in 5H, RGLS treatment significantly reduced the levels of short- and medium-chain fatty acids while increasing the levels of various long-chain fatty acids. These results suggest that RGLS compromises energy supply by increasing the proportion of long-chain fatty acids and inhibiting long-chain acylcarnitine metabolism, thereby inhibiting fatty acid β-oxidation and promoting S-180 cell death.

Discussion

Among the myriad of TCM remedies, G. lucidum renowned for its health-promoting and life-extending properties, has shown considerable promise in managing cardiovascular diseases and brain injury29,30. While G. lucidum has been recognized for its significant therapeutic benefits, its potential in osteosarcoma prevention and treatment has remained relatively underexplored. Limited studies have been conducted to investigate the anti-OS effects of G. lucidum or its active component16,17.

In our research, we evaluated the anti-OS effects of RGLS and its mechanisms (as summarized in Fig. 6). Encouragingly, our results showed that RGLS has a good inhibitory effect on tumor growth. This observation supports the notion that RGLS possesses a strong potential for tumor prevention and treatment. The efficacy of RGLS might be attributed to its ability to target specific molecular pathways or cellular processes involved in tumor initiation and progression. However, to fully comprehend the clinical applicability of RGLS in tumor prevention, further research is essential. Robust pre-clinical and clinical studies are necessary to validate the efficacy, safety, and dosage regimen of RGLS in preventing various types of tumors. Additionally, exploring the mechanistic basis of RGLS’s tumor-preventive effects would provide critical insights into its mode of action and potential targets.

Fig. 6
figure 6

Working model of molecular mechanisms by which RGLS exerts its anticancer activity in osteosarcoma S-180 cells and xenograft tumors. On the one hand, RGLS promotes S-180 uptake of pla2g7 protein released by macrophages, which promotes the metabolism of PCs to produce LysoPCs with cytotoxicity. On the other hand, RGLS restructures fatty acid composition and compromises energy supply by inhibiting β-oxidation of long-chain fatty acid in S180 cells.

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Phospholipids are the material basis of cellular and organismal life activities, and they are not only the main components of cell and organelle membranes, but also involved in the regulation of countless signal pathways31. In tumor cells, the content of phospholipids, especially PC, PI and sphingomyelin, is sharply increased32, which suggests that phospholipid metabolism as a target may be an effective strategy for tumor treatment33. In our study, we found that RGLS significantly increased the content of LysoPCs. As an intermediate product of PC metabolism, LysoPCs are not only closely involved in atherosclerosis and diabetes31,32 but also inhibits the growth of a variety of tumor cells, including bladder cancer, lung cancer, and melanoma22,23,34 and are used to predict the efficacy and favorable prognosis of lenvatinib combined with programmed death-1 (PD-1) inhibitor in the clinical treatment of unresectable hepatocellular carcinoma (uHCC)35. In addition, LysoPC has been shown to continuously increase intracellular calcium ion concentration by activating calcium-permeable channels and contribute to of cell cytoskeletal36. Correspondingly, our study also found that RGLS regulates the biological processes of the cytoskeleton (GO: 0005856) (Fig. 4C), which may be a potential mechanism by which RGLS regulates PC metabolism to exert anti-tumor effects.

Lysosomes are essential for maintaining cell homeostasis and are involved in a variety of biological processes, such as immunity and intracellular pH regulation37. Recent studies have shown that lysosomes also participate in glycerophospholopid metabolism27. PLA2 is a ubiquitous enzyme uniquely characterized by a sub-cellular localization to the lysosome and late endosome38, which hydrolyzes PC to produce LysoPC and causes lysosome destabilization39. In our research, RGLS significantly increased Pla2g7 protein levels but not mRNA levels in S-180 cells, suggested that Pla2g7 protein in S-180 cells might be exogenous. Indeed, Pla2g7 is highly expressed in immune cells, such as B cells and macrophages28. Our study also found that RGLS increases the expression of CD47 (Fig. S3J), an immune checkpoint that interacts with SIRPα expressed by macrophages40. These phenomena indicate that RGLS enhances the interaction between S-180 cells and macrophages. Our previous studies have shown that polysaccharide derived from RGLS inhibits the growth of hepatocellular carcinoma cells by promoting macrophages to M1 type polarization and inflammatory cytokines secretion such as TNF-α, IL-1β, IL-641. However, studies have shown that in the course of COVID-19 infection, Pla2g7 is primarily produced by pro-inflammatory (M1 type) macrophages42, and macrophages with high expression of PLA2 showed a profound immunosuppressive phenotype43. The above background suggests that RGLS regulation of Pla2g7 release may be a novel mechanism to promote macrophage-mediated immunity, and the regulation of endocytosis of tumor cells on Pla2g7 may be related to lysosomal-dependent cell death induced by RGLS44, these speculations are worthy of further study.

Fatty acids are important energy sources for tumor cells, and targeting fatty acid metabolism is considered an effective strategy for tumor (including cancer) management2. Fatty acid utilization is a delicately regulated process. Short- and medium-chain fatty acids can enter the mitochondria directly, but long-chain fatty acids require conversion to long-chain acyl-CoA and transport by L-carnitine45. However, this is not the end of the process: once inside the mitochondria, long-chain acyl-CoA must be re-converted to long-chain acylcarnitine to generate energy through β-oxidation46. If any of these stages are obstructed, the metabolism of fatty acids is compromised. In our study, RGLS promotes the entry of long-chain fatty acids into mitochondria by upregulating expression levels of Acsl family members and ABC transport families. However, RGLS treatment did not increase intracellular ATP levels. In contrast, RGLS treatment significantly increased the content of long-chain acylcarnitine and decreased the L-carnitine content. These results suggest that RGLS inhibits the re-conversion of acylcarnitine to acyl-CoA and the renewal of L-carnitine, which prevents new acylcarnitine from entering mitochondria and exacerbates mitochondrial metabolic disorders. Interestingly, in the fatty acid pool, RGLS significantly increased the content of long-chain fatty acids, which means that RGLS interferes with fatty acid metabolism by increasing the source of long-chain fatty acids and inhibiting the utilization of long-chain fatty acids. In addition, the depletion of short- and medium-chain acylcarnitine and accumulation of long-chain acylcarnitine are considered markers of mitochondrial stress or damaging to fatty acid metabolism47.

In summary, our study has provided compelling evidence that RGLS effectively inhibits OS cell growth and induces apoptosis by targeting glycerophospholipid metabolism and fatty acid β-oxidation. These findings shed light on the intricate molecular mechanisms through which RGLS exerts its anti-cancer effects against OS. Importantly, our study offers a strong rationale for the potential clinical application of RGLS as a therapeutic agent for OS treatment.

Materials and methods

Materials

Shouxiangu® sporoderm-removed G. lucidum spores were obtained from Jinhua Shouxiangu Pharmaceutical Co., Ltd (Wuyi, Zhejiang, China). Cell counting Kit-8 (CCK-8) was purchased from MedChemExpress Technology (Princeton, New Jersey, USA). Polyclonal Bax, Bcl-xL, LC3B, p62, β-Actin antibodies and HRP-conjugated goat anti-rabbit IgG (H + L) were purchased from Diagbio Biotechnology Co., Ltd (Hangzhou, Zhejiang, China). Polyclonal Casp3, Ki-67 antibodies were purchased from CST (Boston, USA). The Pla2g7 antibody was purchased from Proteintech (Wuhan, Hubei, China). The mouse osteosarcoma S-180 cells was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the mouse macrophage Raw264.7 were purchased from American Type Culture Collection (ATCC, VA, USA).

Xenograft mouse model

The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the NIH guidelines. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Research Institute of Traditional Chinese Medicine (Approved Number: 20190003). The specific pathogen-free (SPF) male BALB/c nude mice (18–20 g), provided by the Hangzhou Medical College, were raised in the Zhejiang Research Institute of Traditional Chinese Medicine.

The mice were randomly divided by weight into three groups: con group (distilled water, n = 6), model group (distilled water, n = 12), RGLS group (0.7 g/kg, n = 12, the dosage, based on clinical use, was weekly adjusted to match the mice’s weight changes). Continuous administration for 45 days and S-180 cells (4 × 106 cells in 200 μL PBS) were subcutaneously transplanted into mice on day 38 of administration, and all mice were sacrificed seven days later by inhaling CO2. We first excised tumors from six mice for each group, and these tumors were stored immediately in liquid nitrogen for omics study (three for proteomics and three for transcriptomics study). For another six mice in each group, we weighed body and tumor weight. Then, three tumors per group were placed in formalin solution for pathological detection, and another three per group were placed in 4% formaldehyde for hematoxylin and eosin (H&E) staining.

Tumor tissue histological examination

The harvested tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Then, the sections were stained with H&E according to the previous description48. The morphological changes in tumor tissues were observed, and images were randomly captured by a microscope (DM4000, Leica Biosystems).

Immunohistochemical analysis of tumor tissues

The expression level of Ki-67 in tumor tissues was detected by immunohistochemistry (IHC) referred to previous reports49. The images were randomly captured by a microscope (DM4000, Leica Biosystems).

Preparation of RGLS solution for in vitro experiments

RGLS was dissolved in DMEM as stock solution of 10 mg/ml, and then passed through 0.22-μm filter and diluted to required concentrations.

Cell culture

Mycoplasma-free S-180 cells and Raw264.7 were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Bio-channel, Nanjing, China) supplemented with 10% fetal bovine serum (FBS, Genom, Hangzhou, China) and 1% penicillin–streptomycin (Bio-channel, Nanjing, China), and maintained in a humidified atmosphere of 5% CO2 at 37 °C.

Cell viability assay

S-180 and Raw264.7 cells were seeded into 96-well plates at a density of 1 × 104 cells per well and incubated in DMEM medium containing 10% FBS until cells reach 50% confluence. Cells well then treated with different concentration of RGLS for 24 h. Then 10% CCK-8 solution was added and incubated for an additional 2 h. After incubation, the absorbance was measured at 450 nm using a multi-well plate reader (Molecular Devices Instrument Inc., California, USA). The relative cell viability was calculated according to the formula: relative cell viability (%) = (ARGLS − Ablank)/(ACon − Ablank) × 100%, where A is the absorbance.

Western blotting

Total protein of S-180 and Raw264.7 cells were extracted using ice-cold RIPA buffer (Beyotime, Beijing, China), and total protein concentrations were measured by BCA protein assay kit (Thermo Scientific, Waltham, Massachusetts, USA), and the expression levels of Casp3, Bax, Bcl-xL, LC3B, p62, Pla2g7, and β-Actin were detected according to previous reports49. The antibodies concentrations as listed in Table S1, and the semi-quantitative analysis of optical density was done using ImageJ 1.41 software (Bethesda, MD, USA).

RNA isolation and quantitative real-time PCR

Total RNA isolation was performed using EASYspin reagent Kit (Aidlab Biotech, Beijing, China) according to the manufacturer’s protocol. Both the quality and quantity of total RNA were analyzed by the NanoDrop™ one spectrophotometer (Thermo Scientific, Grand Island, NY, USA). First-strand cDNA was synthesized from 1 µg of total RNA using iScript Reverse transcription supermix (Vazyme Biotech, Nanjing, China). qRT-PCR analysis was performed using Faststart Essential DNA Green Master (Roche, Basel, Swit) on LightCycler96 Real-Time PCR system (Roche). Primers were designed with open-sourced software Primer3Plus (Cambridge, MA, USA). GAPDH was used as the reference gene. The relative expression of mRNA was calculated by following the previous literature49. qRT-PCR primer sequences are shown in Table S2.

Flow cytometric analysis of apoptosis

S-180 cells were seeded into 6-well plates at a density of 2 × 105 cells per well and treated with different concentrations of RGLS for 24 h. Cells were then washed with PBS, harvested by trypsinization and washed twice with PBS, and cells were stained with propidium iodide (PI) and annexin V-FITC using Annexin V-FITC apoptosis detection kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. The stained cells were analyzed by C6 plus flow cytometer (BD, New Jersey, USA), and the percentage of early apoptosis (annexin V+/PI−), late apoptosis (annexin V+/PI+), and necrosis (annexin V−/PI+) cells were analyzed. Data was represented as rate of total apoptosis cells with both early and late apoptotic rate indicated.

ATP content detection

Intracellular ATP content detection was using ATP content assay kit (Solarbio life sciences, Beijing, China) according to the manufacturer’s instructions.

Label-free proteomic analysis

Total protein of tumor tissue was extracted, enzymatically digested, and desalted before separation using nanoliter reversed-phase liquid chromatography and detection by mass spectrometry. Protein abundance was quantified using a Proteome Discoverer library search. Before performing differential protein expression analysis, we used the mice (v3.16) package to perform multiple imputations and fill in missing values. Next, we took the logarithm of the protein expression matrix. We then used the edgeR (v3.42) package to identify differential proteins with adjusted P-values less than 0.05. Finally, we conducted functional enrichment using the clusterProfiler (v4.8) package.

Transcriptomics analysis

Total RNA of tumor tissue was extracted, and libraries were constructed and sequenced to obtain mRNA sequence information. The sequences were aligned to the mouse mm10 genome using Bowtie2 and GENCODE database, and gene expression was quantified using HTSeq. Gene expression values were then normalized using the TMM method. Differential gene expression analysis was performed using edgeR, with genes having a P-value less than 0.05 and an absolute fold change greater than two considered as differentially expressed.

Metabonomics analysis

Total metabolite of S-180 cells (about 1 × 107 cells) was extracted by 1000 μl methanol: acetonitrile: H2O (2:2:1), sonicated at 4 °C for 10 min, incubated at − 40 °C for 60 min and centrifuged at 4000×g for 15 min at 4 °C. The supernatant was collected and filtered through 0.22 μm PVDF membrane. Next, the detection, identification, and data analysis of metabolites in S-180 cells were referred to previous reports50.

Statistical analysis

All data are presented as the mean ± standard error (SE) of at least three independent experiments. For comparisons between the two conditions' a student t-test was used. And for multiple comparisons, one-way analysis of variance (ANOVA) with Tukey’s test was used. All analyzed were performed using Graphpad Prism 8.0 software (Graphpad Software, Inc., USA), and a value of P < 0.05 was a considered to be statistically significant difference.