Investigation of endoplasmic reticulum stress in cerulenin treated U87 glioblastoma cell line

Glioblastoma remains as one of the most fatal cancer types due to its high malignancy, insufficient diagnostic methods and poor rate of overall survival. Recent studies indicate that alterations in molecules and/or metabolic pathways during carcinogenesis may come forward as potential targets in regards to prognosis and cure of glioblastoma. Fatty acid synthase, the enzyme which is responsible for converting malonyl coenzyme A to palmitate, and its inhibitor, Cerulenin are one of the most prominent examples of such targets. Cerulenin, an antifungal agent, blocks lipid synthesis pathway via blocking fatty acid synthase and induces cellular stress due to lack of fatty acids. In addition to that, fatty acid deficiency-related stress may cause cells undergo death via activation of death pathways. In this study, MTT assay results revealed that Cerulenin treatment in various concentrations significantly alters U87MG cell viability. Based on the 48 hours of incubation results, one untreated control group and two treated groups were selected for following experiments. One of the two groups was IC20 which represented 20% cytotoxicity, was found as 3.6 μg/mL. The latter group was IC50 that represented 50% cytotoxicity, was determined as 5.55 μg/mL. Western blotting and qPCR were used to analyze changes in protein and gene expressions as a result of Cerulenin-induced ER stress. qPCR data displayed that tmed4 gene expression significantly reduced, with being 0.31 times for IC20 group and 0.48 times for IC50 cells. However, TMED4 protein expression increased 1.26 times and 1.38 times, respectively. In endoplasmic reticulum proteins; IRE1α expression increased 1.04 and 1.22 times, ATF6 expression slightly decreased 0.91 times and 0.84 times, respectively. Phosphorylated PERK, another ER protein, has increased 1.32 and 1.16 times compared to untreated cells. ER stress was visually assessed by using fluoroscent microscopy. A fluoroscent dye named Thioflavin T was used to stain Cerulenin-treated U87MG cells. Microscopy results indicated that dose-dependent inhibtion and fluoroscent signal were positively correlated. Data obtained from the study are expected to contribute in terms of developing novel strategies related to diagnosis and cure of glioblastoma.