Evaluating effects of dual mTORC1/2 inhibition on global methylation and histone modification on glioblastoma cells
Project no. 24000 · Status: Kapanmış (Yayın bekleniyor) · 2023
Summary
<p>Previous studies have demonstrated that the PI3K/Akt/mTOR signaling pathway is overexpressed in approximately 88% of glioblastomas. The close relationship between the mTOR pathway and serine–threonine metabolism, together with emerging evidence highlighting correlations between methylation and the mTOR pathway, has driven research over the past three years toward the hypothesis that mTOR can directly and indirectly influence both DNA methylation and histone modifications.</p><p>Palomid 529 is a novel dual mTOR inhibitor that has attracted attention as a promising pharmacological agent for glioblastoma due to its high ability to penetrate the blood–brain barrier. It has been shown to inhibit angiogenesis and tumor growth in U87-MG cells and to cross the blood–brain barrier without restriction by the ABCB1 and ABCG2 transporter cassettes; however, the underlying molecular mechanisms have not yet been elucidated.</p><p>In this study, we primarily aimed to elucidate the epigenetic mechanisms regulated by mTORC1 and mTORC2 in the glioblastoma cell lines U87-MG and T98G, and to evaluate the relationship between the anticancer activity of Palomid 529 and alterations in DNA methylation and histone modifications.</p><p>Accordingly, the cytotoxic effects of Palomid 529 on U87-MG and T98G cells were first assessed using the WST-8 assay, followed by the evaluation of its apoptotic effects using Annexin V and JC-1 assays. Global DNA methylation analysis was performed on DNA samples isolated before and after mTOR inhibitor treatment. Histone modifications were analyzed following histone extraction; protein concentration was determined using the Bradford assay, and antibody-based histone modification analyses were subsequently conducted using a microplate reader. Based on the global methylation results, selected gene expression analyses were performed using qRT-PCR, guided by both the findings of this study and the existing literature.</p><p>As the first study to investigate the effects of mTOR inhibitor treatment on DNA methylation in glioblastoma, this work aims to enable a focus on more specific molecular targets, provide a foundation for future in vivo studies, and contribute to the current literature.</p>