The p53/HMGB1 Complex Regulates Cell Autophagy and Apoptosis in Retinoblastoma

Abstract

Background: Retinoblastoma (RB) is a common childhood malignancy and can be fatal if left untreated. However, extensive work is needed to acquire a better understanding of the disease mechanism and identify novel therapeutic options. Therefore, we investigated whether the p53/high mobility group box 1 (HMGB1) complex is involved in regulating autophagy and apoptosis in retinoblastoma cells. Methods: The human RB cell line Y79 was maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The immunoprecipitation approach was used to verify the formation of p53/HMGB1 complex. We induced starvation using Hanks balanced salt solution (HBSS), inhibited p53 with pifithrin-α (PFT-α), inhibited HMGB1 using ethyl pyruvate (EP), inhibited light chain 3 (LC3)-II degradation using bafilomycin A1, induced apoptosis using Adriamycin (ADM) and limited autophagy using 3-methyladenine (3-MA). To determine the effect of altering p53 and high mobility group box 1 (HMB1) on autophagy and apoptosis, different approaches, including RT-qPCR, Western Blot, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), were used. Results: The study revealed that p53 binds with HMGB1 to form a complex in the nuclear region. Furthermore, starvation increased the formation of the p53/HMGB1 complex. Moreover, a decrease in p53 resulted in increased autophagy, while a decrease in HMGB1 led to decreased autophagy. Additionally, a decrease in p53 reduced apoptosis and B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax) expression, while increasing B-cell lymphoma-2 (Bcl-2) expression. We also found that this effect could be reversed by decreased HMGB1, which caused more apoptosis. Finally, we observed that a decrease in autophagy induced more apoptosis. Conclusion: The p53/HMGB1 complex is crucial in regulating autophagy and apoptosis in RB cells. HMGB1 promotes autophagy and limits apoptosis, whereas p53 induces apoptosis and limits autophagy. A decreased level of autophagy can induce apoptosis, suggesting that p53 and HMGB1 may be potential therapeutic targets for treating RB.