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.
Highlights
Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, with a higher prevalence in women [1]
Cell Culture. e epithelial HT-29 colorectal cancer cell line (NCBI code C154 and ATCC number HTB-38) was obtained from the Cell Bank of Pasteur Institute of Iran. e cells are routinely karyotyped and examined for mycoplasma contamination. e cells were defrosted under sterile conditions and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium containing 10% FBS and 1% v/v c penicillin-streptomycin and incubated at 37°C with 5% CO2 and 95% humidity. e culture medium was changed every 3 days, and when the cell density at the bottom of the flask reached 80%, trypsin/ EDTA solution was added to detach them and they were divided into two flasks after centrifugation at 5000g at 4°C
In order to evaluate the performance of the primers, the melting curve diagram for each gene was examined to confirm the accuracy of the desired gene peak and the lack of dimer primers. e quantitative real-time polymerase chain reaction was performed in 3 replicates [9]. reshold cycle (CT) for the internal control gene of GAPDH mRNA and
Summary
Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, with a higher prevalence in women [1]. Cancer progression is associated with oncogenic mutations and tumor suppressor genes such as APC, KRAS, and p53, which lead to metabolic disorders [2]. Despite numerous treatments such as advanced surgery and combination methods such as chemotherapy and radiotherapy, the 5-year survival rate of patients rarely reaches over 60%. Understanding the molecular functions of cancer and the use of Evidence-Based Complementary and Alternative Medicine natural resources such as plants and their derivatives can be considered a new solution in the prevention and treatment of this cancer [3]
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