Panaxadiol inhibits the proliferation and immune evasion of esophageal squamous cell carcinoma cells by suppressing HIF-1α/STAT3/PD-L1 pathway

Background: Several malignancies, including esophageal squamous cell carcinoma (ESCC), depend on immune evasion for growth and proliferation. This study aimed to determine whether panaxadiol can inhibit immune evasion and, hence, have anti-cancer effects in ESCC.

Methods: Human ESCC cell lines (EC109 and EC9706) were exposed to 3 or 10 μM panaxadiol dissolved in dimethyl sulfoxide. Colony formation and ethynyl deoxyuridine assays were performed to measure ESCC cell proliferation. After ESCC and CD8⁺T cell coculture, crystal violet staining and Lactate Dehydrogenase (LDH) assays were used to detect ESCC cell viability and death. An enzyme-linked immunosorbent assay was performed to measure the levels of inflammatory cytokines (interferon (IFN)-γ, interleukin (IL)-4, and IL-10). To establish a xenograft mouse model, 3 × 10⁵ EC109 cells treated with or without panaxadiol were subcutaneously injected into mice. Western blotting was conducted to assess the protein expression of proliferation markers (cyclin D1, c-Myc, and vascular endothelial growth factor), HIF-1α, programmed cell death 1 ligand 1 (PD-L1), and STAT3 phosphorylation in ESCC cells or mouse tumors.

Results: Both 3 and 10 μM panaxadiol significantly inhibited ESCC cell proliferation. After coculture with CD8⁺T cells, ESCC cell viability was markedly reduced, whereas IFN-γ, IL-10, and IL-4 levels were prominently elevated. LDH assays revealed that panaxadiol enhanced CD8⁺T cell-induced cytotoxicity toward ESCC cells. Moreover, panaxadiol effectively suppressed xenograft tumor growth in ESCC. The activities of STAT3, PD-L1, and HIF-1α in ESCC cells and mouse tumor tissues were significantly suppressed by panaxadiol. Additionally, FG-4592 (a factor that can stabilize HIF-1α) reversed the suppression of 10 μM panaxadiol-induced HIF-1α/STAT3/PD-L1 activity in ESCC cells.

Conclusion: Panaxadiol suppresses cell proliferation, immunological evasion, and tumor formation in ESCC by inhibiting PD-L1 expression through the suppression of HIF-1α and STAT3.

ESCC; HIF-1α; Immune evasion; PD-L1; Panaxadiol; STAT3.