The Cytotoxic Effect of Cobalt Oxide Nanoparticle Conjugated by Menthol on Colorectal Cancer Cell Line and Evaluation of the Expression of CASP8 and FEZF1-AS1

Abstract

Colorectal cancer (CRC) poses a significant health challenge, driving the search for novel treatments, and nanoparticles have been introduced as a practical component for better patient outcomes. This study explored the cytotoxic effects of cobalt oxide nanoparticles combined with menthol (Co₃O₄@Glu-Menthol) on CRC cells by assessing the expression of caspase-8 (CASP8) and FEZF1-AS1 genes. Co₃O₄@Glu-Menthol nanoparticles were synthesized by mixing cobalt nitrate with sodium hydroxide and were surface functionalized with glucose and menthol coating. Characterization of nanoparticles was assessed using Fourier Transform Infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Dynamic Light Scattering (DLS), Energy-Dispersive X-ray Spectroscopy (EDS), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis/necrosis evaluation via Annexin V/Propidium Iodide assay, and caspase-8 (CASP8) and FEZF1-AS1 genes expression by qRT-PCR after RNA extraction of CRC cell line, cDNA synthesis, and primer design from both treated and untreated cells. Also, Hoechst staining was performed to characterize the anticancer mechanism of Co₃O₄@Glu-Menthol NPs. The synthesized NPs were spherical, within a 30–60 nm size range, and without impurities. The particles’ DLS size and zeta potential were 175 nm and − 41.1 mV, respectively. MTT assay showed that the IC₅₀ values of NP were 149 µg/mL and 87 µg/mL for 24 and 48 h, respectively, for CRC cell line and 320 µg/mL for normal cell line. Flow cytometry showed significant differences in apoptosis and necrosis between treated and untreated groups. Gene expression analysis revealed a significant increase in CASP8 gene expression (2.4 fold) and a decrease in FEZF1-AS1 gene expression (0.4 fold), indicating apoptotic effects (P < 0.001). Also, Caspase 3 activity was significantly increased in treated cells (6.2 fold) (P < 0.05). The study suggested that Co₃O₄@Glu-Menthol nanoparticles possess potent anticancer properties against CRC cells, potentially through induction of apoptosis and modulation of gene expression related to apoptosis pathways.