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
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
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 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.experiments showed that BBR treatment did not significantly reduce tumor volume or we
Abstract
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 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.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 CD8CD3and CD69CD8activated T cells was significantly increased, the expression level of granzyme B in CD8T cells was notably elevated, and the number of regulatory T cells (Tregs, CD4CD25Foxp3) 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.