Behnoosh Rafienia - Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Elham Hoveizi - Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Ali Shahriari - Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz,Ahvaz, Iran

Background and Aim: Colorectal cancer (CRC) constitutes a significantportion of mortality and morbidity due to cancer around the world. Theregulation of autophagy has emerged as a promising new target in cancertreatment. Previous studies have reported that long unsaturated fattyacids exhibit antiproliferative effects through induction of oxidative stressand possess the potential to cure cancers. Similarly, the GW9508 smallmolecule is an unsaturated free fatty acid receptor agonist. In this study,we investigated the autophagic and anticancer effects of GW9508 on theHT29 tumor cells cultured in the 3D fibrin scaffold.Methods: Fibrin hydrogel was prepared by polymerization of fibrinogen(3 mg/mL) by thrombin (120 U/mL). HT29 cells were seeded at a densityof 50x10 into 24-well plate in 3D fibrin hydrogel and then wereexposed to various concentrations of GW9508. MTT assay was used tomeasure cell viability. Also, Acridine Orange/Ethidium Bromide (AO/EB),DAPI, and monodansylcadvarine (MDC) stainings were used to detectautophagy. Moreover, the expression levels of LC3-II, AKT and mTORgenes were examined by qRT-PCR.Results: Our results revealed a decrease in cell viability of treated cellswith IC50 concentration of GW9508 to the extent of 52%,10.5% and 6% after 1, 3, and 5 days respectively, in a dose and timedependentmanner. Morphological observation, AO/EB and DAPI stainingconfirmed autophagic morphology in treated cells. Also, the activation ofautophagy was determined through changes in the number of autophagyvesicles (AVs) infected with MDC. Furthermore, the expression levels of AKT and mTOR were down-regulated whereas, LC3-II expression was increased after GW9508 treatment compared with control samples.Conclusion: Therefore, we suggest that GW9508 exerts anticanceractions, cytotoxic effects and promoting autophagy and can be used as anew therapeutic target for colorectal cancer treatment.

Autophagy; Colorectal cancer; Fibrin hydrogel scaffold; GW9508; 3D culture