80P - Suppression of glioblastoma progression by FDA-approved central nervous system-accumulating drugs via autophagy modulation and ER stress-induced apoptosis

Background

The standard treatment for glioblastoma multiforme involves surgery followed by temozolomide (TMZ) treatment. However, many patients develop TMZ resistance either due to autophagic clearance of unwanted materials or reversal of TMZ-induced DNA alterations by methyl guanine o-methyl transferase. Additionally, other drugs are often ineffective because they cannot cross the blood-brain barrier (BBB). Hence, our objective is to repurpose FDA-approved drugs capable of crossing BBB and regulating autophagic pathways.

Methods

We identified differentially expressed genes regulating cellular processes in GBM, and used the Connectivity-MAP (C-Map) database to identify potential drugs, capable of reversing differentially expressed autophagy-associated genes. Following in-silico screening, we conducted cytotoxicity assays with selected drugs to evaluate IC50 values of drugs in 2-D and 3-D models. Additionally, immunoblotting and IF were performed to examine protein expression. Furthermore, we employed MDC staining to identify autophagic vacuoles, Annexin-PI to quantify apoptotic cells.

Results

Analysing GEO dataset, we identified top 10 dysregulated genes. Most downregulated genes were calcium signaling, while upregulated ones were PI3-Akt pathway. After identifying key genes and associated pathways, we used C-Map to identify top 5 drugs with high connectivity scores and BBB penetrance. They were assessed in vitro for their potential against GBM. Among them, Doxylamine and Gemfibrozil exhibited the best cytotoxicity, surpassing the standard drug TMZ. These drugs also hindered autophagic flux, verified by increased p62 and LC3B II proteins. Overexpression of PERK and CHOP confirmed ER stress-induced apoptosis. Furthermore, inhibition of Calmodulin, Calreticulin, and Calnexin post-drug-stress influenced the calcium signaling pathway. AnnexinV-PI assay demonstrated that increased ER stress and subsequent restriction of autophagic flux led to cell death.

Conclusions

Our research highlighted that doxylamine and gemfibrozil could serve as potential alternatives to TMZ for inducing apoptosis, by inhibiting autophagy, inducing ER stress, and generating ROS.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

The authors.

Funding

The Indian Council of Medical Research (ICMR).

Disclosure

All authors have declared no conflicts of interest.