The effect of metformin on induction of autophagy in tumoroid structures of colorectal cancer

Abstract

Colorectal cancer is a type of gastrointestinal cancer accompanied by complex genetic and molecular issues. Autophagy is a crucial cellular mechanism regulating cell renewal and death. Metformin (MTF) is an anti-diabetic drug utilized for type 2 diabetes treatment, with a potential impact on autophagy suggested by studies. This study focuses on the effect of MTF on inducing autophagy in colorectal cancer tumoroids. Methods: The HT-29 cell line will be employed in this research. Initially, these cells are cultured in RPMI-1640 medium containing 10% FBS. After covering 70% of the petri dish surface, three-dimensional structures (spheroids) are formed. These structures are then treated with MTF at doses ranging from 0.5 to 2 micromolar. Cell viability is assessed using the lactate dehydrogenase assay, and formed tumoroids are analyzed through hematoxylin-eosin staining. Finally, key proteins involved in autophagy like Beclin1, microtubular-associated protein 1 light chain 3 (LC3), and SQSTM1/p62 will be measured using Western blotting. Results: This study investigates the anticancer effects of MTF on colorectal cancer tumoroids over 72 hours. A three-dimensional model of colorectal cancer tumoroids is employed, showing diverse structures. Data analysis indicates central and dark regions in the nodule pointing to necrotic cells while proliferating cells with specific morphology and expression are observed in the outer layer. The effect of MTF on CRC tumoroid survival was examined, but no significant impact was observed. Some tumoroids treated with MTF were analyzed using the LDH assay, revealing no significant cellular toxicity difference between groups. Furthermore, the effect of MTF on tumoroids size was investigated, demonstrating an increase with MTF dose, eventually approaching the control size. The examination of MTF's impact on autophagy status via Western blotting illustrates its ability to influence autophagy in CRC tumoroids, aiding in controlling this mechanism. Additionally, QPCR analysis demonstrates MTF's effects on autophagy-related gene expression, varying proportionally with different concentrations of MTF. This study contributes to a better understanding of MTF's effect on the autophagy process in CRC tumoroids.