The effect of melatonin on mitophagy process and cancer progression in colorectal tumoroid structures composed of colorectal cancer

Abstract

Colorectal cancer (CRC) ranks as the third most prevalent cancer globally, with escalating incidence rates. The current treatments for CRC exhibit limited efficacy, necessitating the exploration of novel therapeutic approaches. Mitophagy, a process involving the removal of damaged mitochondria through autophagy, has emerged as a potential mechanism for cancer treatment due to its considerable impact on specific cancer cell mechanisms. Melatonin, a potent antioxidant hormone renowned for its anti-inflammatory and anti-stress properties, holds promise in reducing the risk and preventing colorectal tumors. Additionally, the multifaceted properties of melatonin suggest its potential modulation of mitophagy. Given the valuable insights gained from tumoroids as research models in comprehending the biological behavior and molecular mechanisms of colorectal cancer, investigating the impact of melatonin on mitophagy in CRC tumoroids holds significant potential for advancing cancer studies. Methods: HT-29 cells were employed in this study. Initially, these cells were cultured in RPMI-1640 medium supplemented with 10% FBS. Upon reaching 70% confluence, three-dimensional tumoroid structures were generated. Subsequently, these structures were treated with melatonin at concentrations ranging from 4 to 10 millimolars. Cell survival and melatonin toxicity on tumoroids were assessed through lactate dehydrogenase assay. Additionally, hematoxylin-eosin staining was applied to analyze the parenchymal structure and uniformity of the tumoroids. Finally, qPCR was employed to quantify the expression levels of crucial genes, including PINK1, OPTN, MFN1, and MFN2, implicated in the mitophagy process. Results: This study investigated the anticancer effects of melatonin on CRC tumoroids over a 72-hour period. A three-dimensional CRC tumoroid model was employed in the laboratory setting, revealing diverse structures within CRC tumoroids. Data analysis identified a central dark region in the tumoroid, indicating necrotic cells, while the peripheral layer exhibited proliferating cells with flattened morphology and specific expression. The study examined the anticancer effect of melatonin on the survival of CRC tumoroids. LDH assay data analysis, focusing on the impact of melatonin on the toxicity or survival of tumoroids, revealed the highest LDH levels at melatonin concentrations of 4 and 10 millimolars. Furthermore, qPCR analysis indicated a dose-dependent increase in the expression levels of PINK1, OPTN, MFN1, and MFN2 genes. Western blot results corroborated a reduction in the protein levels of mitochondrial fusion markers MFN1 and MFN2 and an increase in the protein levels of mitophagy markers PINK1 and OPTN.