A Metabolomic Investigation of Eugenol on Colorectal Cancer Cell Line HT-29 by Modifying the Expression of APC, p53, and KRAS Genes

Abstract

Colorectal cancer is one of the most lethal cancers with a high mortality rate. Chemotherapy results in drug resistance in some cases; hence, herbal medicines are sometimes used in adjunct with it. Eugenol has been reported to have anti-inflammatory, antioxidant, and anticancer properties. Metabolomics is a study of metabolic changes within an organism using high-throughput technology. The purpose of this research was to investigate the anticancer effects of eugenol and variations in p53, KRAS, and APC gene expression and metabolic changes associated with the abovementioned gene expressions using 1HNMR spectroscopy. The MTT method was used to determine cell viability and its IC50 detected. After treating HT-29 cells with IC50 concentration of eugenol, RNA was extracted and cDNA was obtained from them and the expression of p53, KRAS, and APC genes was measured using the qRT-PCR technique. Metabolites were extracted using the chloroform-ethanol method, lyophilized, and sent for 1HNMR spectroscopy using the 1D-NOESY protocol. Chemometrics analysis such as PLS-DA was performed, and differentiated metabolites were identified using the Human Metabolome Database. Integrated metabolic analysis using the metabolites and gene expression was performed by the MetaboAnalyst website. The observed IC50 for eugenol was 500 μM, and the relative expression of APC and p53 genes in the treated cells increased compared to the control group, and the expression of KRAS oncogene gene decreased significantly. The crucial changes in convergent metabolic phenotype with genes were identified. The results indicate that eugenol exhibits its antitumor properties by targeting a specific biochemical pathway in the cell’s metabolome profile due to changes in genes involved in colon cancer.

Comparison of Phytochemical Composition and Untargeted Metabolomic Analysis of an Extract from Cnidoscolus aconitifolius (Mill.) I. I. Johnst and Porophyllum ruderale (Jacq.) Cass. and Biological Cytotoxic and Antiproliferative Activity In Vitro

“…Colon cancer is a multistage disease characterized by successive changes in various genes, including Apc and Kras , leading to changes in CRC signaling pathways such as the APC Wnt/β-catenin and K-RAS [ 43 ]. As Apc and Kras are frequently mutated in CRC, targeting these genes could provide insights into the effects of bioactive compounds in CRC treatment [ 44 ].…”

Eugenol Induces Apoptosis in Tongue Squamous Carcinoma Cells by Mediating the Expression of Bcl-2 Family

“…Based on the intensity of the colored formazan product, which has an absorbance near 570 nm, the assay enabled us to assess the viability of cells treated with various concentrations of Eug. The concentrations used in this study (0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM) were chosen based on a comprehensive review of the currently available literature [ 27 , 28 , 29 , 30 ]. Several in vitro studies have evaluated eugenol’s cytotoxicity in blood, breast, skin, cervical, and colon cell lines via various mechanisms of action [ 31 ].…”

“…These findings are aligning with the available literature on this topic. Ghodousi-Dehnavi E. et al showed that Eug has the ability to increase the expression of the p53 tumor suppressor gene and decrease the expression of the KRAS oncogene gene in colon adenocarcinoma cells [ 30 ]. Moreover, Fathy M. and colleagues showed that Eug produces a decrease in the expression of the Bcl-2 gene, while increasing the expression of the Bax gene in HeLa cells [ 43 ].…”

Sishen Pill and its active phytochemicals in treating inflammatory bowel disease and colon cancer: an overview

“…In addition, the regulatory effect of linalool on the oxidative response was beneficial for the treatment of colon cancer; it was confirmed that cellular apoptosis was induced by promoting the production of hydroxyl radicals and 4-HNE (a marker of oxidative stress due to increased lipid peroxidation) ( Iwasaki et al, 2016 ). Moreover, some studies focused on the inhibitory effects of eugenol, isoeugenol, and dihydrodiiisoeugenol on colon cancer cells, showing that their anti-cancer mechanisms involve the regulation of metabolic pathways, apoptosis/metastasis-related gene expression, and the activation of endoplasmic reticulum stress-induced inhibition of autophagy ( Li et al, 2021a ; Ghodousi-Dehnavi et al, 2021 ; Bilgin et al, 2023 ). In above studies, Iwasaki et al ( Iwasaki et al, 2016 ) and Li et al ( Li et al, 2021b ) used the tumor xenograft model in their experiments, while evidence from other studies mainly came from cell experiments.…”