Evaluation of Entospletinib as a Novel Therapeutic Strategy Targeting Spleen Tyrosine Kinase in Retinoblastoma Treatment: An In-vitro Study

Abstract

Introduction: Retinoblastoma (RB) is the most common paediatric intraocular malignancy. The Spleen Tyrosine Kinase (SYK) acts as a proto-oncogene crucial for RB cell survival and is absent in the normal retina, making it a valuable therapeutic target. Aim: Entospletinib, a selective oral SYK inhibitor currently in phase 2 trials for chronic lymphocytic leukaemia, was evaluated for its activity on the RB cell line Y-79. Materials and Methods: The effects of Entospletinib on cell proliferation and death of the human RB Y79 cells were assessed by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, phase contrast microscopy and AO/EtBr dual labeling. The Annexin-V kit was used to quantify the cell mortality following treatment using flow cytometry. The mRNA levels of caspase-3, BAX, BCL-2, SYK, p53, TNF-and NF-κB were measured using RT-PCR to better understand the mechanism of action of this drug. Western blot was used to further validate the data. Results: Entospletinib treatment led to a time- and dose-dependent reduction in cell viability, with significant cytotoxicity observed at an IC50 of 61.3 μM/ml. Annexin V/Propidium Iodide (PI) staining demonstrated a dose-dependent increase in apoptosis. The treatment upregulated mRNA levels of Caspase-3, Bax, and p53 while downregulating SYK, TNF-α, and Bcl-2. RT-PCR and Western blot analyses confirmed a dose-dependent decrease in NF-κB/p65 expression. Conclusion: The study findings suggest that Entospletinib inhibits cell proliferation and induces apoptosis in RB cells by downregulating SYK/TNF-α/NF-κB/p65, highlighting its potential as a promising therapeutic candidate for RB.

Keywords

• di-methylthiazolyl di-phenyltetrazolium assay
• nuclear factor-kappa of activated b cells
• reverse transcriptase-polymerase chain reaction
• tumor necrosis factor-alpha
• y79 cell line