Eleutheroside A inhibits PI3K/AKT1/mTOR-mediated glycolysis in MDSCs to alleviate their immunosuppressive function in gastric cancer

Abstract

Background: An immune-suppressive tumor microenvironment (TME) that encourages tumor growth is a hallmark of gastric cancer (GC), which is implicated in the development, metastasis, and unfavorable prognosis of GC. Acanthopanax senticosus (Rupr.&Maxim.) Harms (AS), also called Siberian Ginseng (Chinese: Ci wu jia), is a commonly used traditional Chinese herbal medicine with immune-enhancing, anti-tumor, anti-fatigue, neuroregulatory, blood circulation-improving, and antioxidant properties. Recently, it has also been demonstrated to improve anti-tumor immunity in GC. Eleutheroside A (EA), one of the primary bioactive saponins of AS, has immunoregulatory functions. Given the immunomodulatory and anti-tumor effects of EA, it is crucial to investigate its regulatory impact on the immune landscape of GC.

Materials and methods: To determine the effects of EA on immune responses in GC, a subcutaneous GC mouse model was established. Tumor growth, body weight changes, and immune responses in the mice treated with EA were measured. The proportion of CD4⁺T, CD8⁺T, B cells, NK cells, TAMs, DCs and MDSCs in the spleens were analyzed using flow cytometry. MDSCs and CD4⁺/CD8⁺ T cell infiltration in tumor tissue were analyzed using immunofluorescence. Bulk RNA sequencing (bulk RNA-seq) data from the Cancer Genome Atlas (TCGA) and two single-cell RNA sequencing (scRNA-seq) datasets (accession numbers GSE183904 and GSE150290) were used to examine changes in MDSCs and T cell infiltration within the TME of GC and to identify MDSCs-related targets. Network pharmacology analysis, protein-protein interaction (PPI) network analysis, dynamics simulations, molecular docking and surface plasmon resonance (SPR) were applied to explore the potential mechanisms underlying EA's intervention in MDSCs. Flow cytometry, qPCR, and western blotting and Seahorse assays were applied for analyzing MDSCs isolated from in vivo and in vitro-induced conditions, aiming to delineate the mechanism of EA on MDSCs glycolysis and immunosuppressive functions mediated by the PI3K/AKT1/mTOR signaling pathway.

Results: In vivo, EA treatment effectively suppressed GC tumor growth and progression in mice, reducing the prevalence of MDSCs and increasing CD4⁺/CD8⁺ T cell levels. In vitro, EA not only decreased the frequency of MDSCs but also alleviated their immune-suppressing capabilities on CD4⁺/CD8⁺ T cells. Network pharmacology, coupled with scRNA-seq analysis, dynamic simulations, and molecular docking studies, suggested that EA might modulate the PI3K/AKT1/mTOR signaling pathway to influence glycolysis in MDSCs. Surface plasmon resonance (SPR) analysis confirmed that EA directly interacts with AKT1. Further validation experiments revealed that in the GC TME, EA treatment decreased the expression of p-PI3K, p-AKT1, p-mTOR, HIF1α, as well as glycolytic genes and glycolytic activity in MDSCs. Additionally, EA led to the downregulation of p-STAT3 and its downstream immunosuppressive factors within these cells. Restoring AKT1 activation could reverse the inhibitory effects of EA on MDSCs glycolysis and the downregulation of immunosuppressive molecules. Moreover, HIF-1α inhibition abolished EA's inhibitory effects on MDSCs.

Conclusion: EA can attenuate the immune-suppressive capacity of MDSCs in GC by inhibiting the PI3K/AKT1/mTOR pathway and suppressing HIF-1α-mediated glycolysis, thereby offering a novel therapeutic approach to targeting the immune-suppressive microenvironment in GC.

Keywords: Eleutheroside a; Gastric cancer; Glycolysis; Myeloid-derived suppressor cells; PI3K/AKT1/mTOR.