This study investigated the role of human β-defensin-3 (HBD-3) in osteosarcoma and its underlying molecular and immunological mechanisms. HBD-3 was overexpressed or knocked down in MG63 and MNNG/HOS cells, and cell proliferation, apoptosis, and cell cycle were detected. PI3K/AKT agonists (740Y-P, SC79) were used for mechanism exploration. In vivo, MG63-bearing nude mouse models were used to monitor tumor growth and survival, and LM8-bearing C57BL/6 models to evaluate immune responses. Flow cytometry was used to detect the proportion and functional status of specific T cells and dendritic cells (DCs) in spleen and tumor tissues. HBD-3 was highly expressed in MG63 and MNNG/HOS cells. Functional assays showed that HBD-3 overexpression significantly promoted osteosarcoma cell proliferation and suppressed apoptosis, while HBD-3 knockdown induced G0/G1 phase cell cycle arrest, inhibited cell proliferation, and enhanced apoptosis. Mechanistically, HBD-3 overexpression activated the PI3K/AKT signaling pathway and increased the phosphorylation level of FOXO3A. In contrast, HBD-3 silencing attenuated PI3K/AKTactivation, reduced FOXO3A phosphorylation, and facilitated its nuclear translocation—indicating that the regulatory effect of HBD-3 on osteosarcoma cells was partly dependent on the PI3K/AKT pathway. Furthermore, HBD-3 knockdown promoted DC maturation and enhanced CD8 T cell activation in the tumor microenvironment, thereby reshaping the anti-tumor immune response. Collectively, our findings demonstrated that HBD-3 exerts its oncogenic functions partly via the PI3K/AKT/FOXO3A pathway to drive malignant progression. Moreover, HBD-3 participates in regulating the tumor anti-tumor immune response. These results identify HBD-3 as a potential novel therapeutic target for osteosarcoma treatment, and targeted inhibition of HBD-3 may become a promising strategy for osteosarcoma immunotherapy. Graphical Abstract Core mechanism summary. Overexpressed HBD-3 activated the PI3K/AKT signaling pathway, enhancing AKT-mediated phosphorylation of FOXO3A. This phosphorylation event downregulated the expression of cell cycle regulators p21 and p27, facilitating the transition of MG63 and MNNG/HOS cells from G0/G1 phase to S phase and promoting cell proliferation. Meanwhile, phosphorylated FOXO3A inhibited the transcription of pro-apoptotic genes Bim and PUMA, suppressing osteosarcoma cell apoptosis. Collectively, HBD-3 promoted the proliferation of MG63 and MNNG/HOS cells through the PI3K/AKT/FOXO3A axis by regulating cell cycle progression and inhibiting apoptosis.

β-defensin-3 promoted osteosarcoma progression via activating the PI3K/AKT pathway and regulated tumor immune response

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发表日期：

2026年4月15日

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Kang Zou,
Ke Wang

摘要

This study investigated the role of human β-defensin-3 (HBD-3) in osteosarcoma and its underlying molecular and immunological mechanisms. HBD-3 was overexpressed or knocked down in MG63 and MNNG/HOS cells, and cell proliferation, apoptosis, and cell cycle were detected. PI3K/AKT agonists (740Y-P, SC79) were used for mechanism exploration. In vivo, MG63-bearing nude mouse models were used to monitor tumor growth and survival, and LM8-bearing C57BL/6 models to evaluate immune responses. Flow cytometry was used to detect the proportion and functional status of specific T cells and dendritic cells (DCs) in spleen and tumor tissues. HBD-3 was highly expressed in MG63 and MNNG/HOS cells. Functional assays showed that HBD-3 overexpression significantly promoted osteosarcoma cell proliferation and suppressed apoptosis, while HBD-3 knockdown induced G0/G1 phase cell cycle arrest, inhibited cell proliferation, and enhanced apoptosis. Mechanistically, HBD-3 overexpression activated the PI3K/AKT signaling pathway and increased the phosphorylation level of FOXO3A. In contrast, HBD-3 silencing attenuated PI3K/AKTactivation, reduced FOXO3A phosphorylation, and facilitated its nuclear translocation—indicating that the regulatory effect of HBD-3 on osteosarcoma cells was partly dependent on the PI3K/AKT pathway. Furthermore, HBD-3 knockdown promoted DC maturation and enhanced CD8 T cell activation in the tumor microenvironment, thereby reshaping the anti-tumor immune response. Collectively, our findings demonstrated that HBD-3 exerts its oncogenic functions partly via the PI3K/AKT/FOXO3A pathway to drive malignant progression. Moreover, HBD-3 participates in regulating the tumor anti-tumor immune response. These results identify HBD-3 as a potential novel therapeutic target for osteosarcoma treatment, and targeted inhibition of HBD-3 may become a promising strategy for osteosarcoma immunotherapy.

Graphical Abstract

Core mechanism summary. Overexpressed HBD-3 activated the PI3K/AKT signaling pathway, enhancing AKT-mediated phosphorylation of FOXO3A. This phosphorylation event downregulated the expression of cell cycle regulators p21 and p27, facilitating the transition of MG63 and MNNG/HOS cells from G0/G1 phase to S phase and promoting cell proliferation. Meanwhile, phosphorylated FOXO3A inhibited the transcription of pro-apoptotic genes Bim and PUMA, suppressing osteosarcoma cell apoptosis. Collectively, HBD-3 promoted the proliferation of MG63 and MNNG/HOS cells through the PI3K/AKT/FOXO3A axis by regulating cell cycle progression and inhibiting apoptosis.