Berberine induces PD-L1 degradation via the autophagy-lysosome pathway through the PI3K-Akt pathway and enhances immunogenic cell death in triple-negative breast cancer

Objective This study aimed to investigate the regulatory effect and molecular mechanism of Berberine (BBR) on programmed death-ligand 1 (PD-L1) expression in Triple-Negative Breast Cancer (TNBC) cells. Methods In vitro cellular experiments (using MDA-MB-231 and BT-549 cell lines) and mouse models (Balb/c-nu and Balb/c mice) were performed. Combined with techniques including Western blot and immunofluorescence assay, the effect of BBR on PD-L1 expression was evaluated. Results BBR reduced PD-L1 protein expression in TNBC cells in a time- and dose-dependent manner, decreased the membrane localization of PD-L1, and inhibited interferon-γ (IFN-γ)-induced PD-L1 upregulation. Mechanistically, BBR activated autophagy by inhibiting the PI3K-Akt pathway, leading to PD-L1 degradation primarily mediated by the autophagy-lysosome pathway, with minimal contribution from the proteasome under the conditions tested. Cycloheximide (CHX) pulse-chase assay confirmed the degradation kinetics of PD-L1. Experiments evaluating lysosomal function excluded the influence of abnormal lysosomal function, demonstrating that this degradation was independent of the ubiquitin-proteasome system and did not affect lysosomal function. Furthermore, BBR increased the expression and release of immunogenic cell death (ICD)-associated markers in TNBC cells, including calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release. In vivo experiments showed that BBR treatment did not significantly reduce tumor volume or weight in Balb/c-nu mice, but effectively decreased tumor volume and weight in the 4T1 Balb/c mouse model without obvious systemic toxicity. In the tumor tissues of 4T1 Balb/c mice, the proportion of CD8 + CD3 + and CD69 + CD8 + activated T cells was significantly increased, the expression level of granzyme B in CD8 + T cells was notably elevated, and the number of regulatory T cells (Tregs, CD4 + CD25 + Foxp3 + ) was significantly reduced. Conclusion BBR can regulate PD-L1 degradation and induce ICD through the PI3K-Akt-autophagy pathway. Its mode of immune checkpoint degradation and immunogenic activation provides experimental evidence for the immunotherapy of TNBC.