Identification of oncogenes associated with colorectal cancer mortality and the effect of cinnamon-conjugated magnetic nanoparticles on their expression

Abstract

Finding the molecular targets involved in the severity and drug resistance of Colorectal cancer (CRC) and applying targeted treatments against them is a promising approach. In this study, some candidate oncogenes related to disease severity and mortality were identified by extracting bioinformatics data, and the effect of Fe₃O₄@Glu-Cinnamon NPs on the survival of CRC cells (SW480) and the expression of these oncogenes was investigated. The NPs were characterized by FT-IR, XRD, DLS and zeta potential measurement, TEM and SEM imaging, EDS-mapping and VSM analysis. Cytotoxicity of the NPs was evaluated by the MTT assay and a flow cytometry analysis was done to investigate cell apoptosis/necrosis levels and cell cycle analysis of cancer cells. The Fe₃O₄@Glu-Cinnamon NPs with spherical morphology were correctly synthesized, containing no elemental impurities, with a size range of 26.8-60.2 nm, DLS of 213 nm, zeta potential of -15.4mV and maximum magnetization level of 20.33emu/g. Treatment of cancer cells with the NPs elevated primary and late apoptosis and cell necrosis levels to 20.85, 16.83 and 9.56% and treated cells were mainly arrested at the S and G2/M phases. The expression level of the oncogenes associated with mortality, SNAI1, THBS2 and INHBA reduced to 0.74, 0.66 and 0.7 folds, respectively. The magnetic properties of Fe₃O₄ NPs enable their potential use in targeted drug delivery, allowing for site-specific accumulation within tumors. This could minimize systemic toxicity while enhancing treatment efficacy.

Keywords: Cinnamon; Colorectal cancer; Iron oxide nanoparticles; Oncogene.

Fig. 1

The flowchart that illustrates the overall steps of the study.

Fig. 2

Differential expression analysis of multiple genes in Colorectal Cancer. The results revealed that a total of 1052 genes were significantly upregulated in cancer samples compared to normal ones and 2107 genes showed significant downregulation.

Fig. 3

Relation between the expression of the identified genes and patient survival rates. A total number of 53 genes were identified as having both significant upregulation and association with poor prognosis, while 115 genes were downregulated and associated with a good prognosis.

Fig. 4

Association of candidate genes with malignancy pathways. Enrichment analysis of the 53 upregulated genes indicated that many of them are involved in cellular processes such as metastasis, angiogenesis, hypoxia, and inflammation (A) and the 115 downregulated genes associated with a good prognosis suggested that some are involved in pathways like fatty acid metabolism, propanoate metabolism, and the citrate cycle (B).

Fig. 5

Identification of hub genes using the PPI network for the upregulated genes. For the 53 upregulated genes associated with poor prognosis, 18 genes were found to be interconnected, among which the SERPINE1, SPP1, SNAI1, THBS2, and INHBA had the highest degree of interaction (degree > 7).

Fig. 6

Identification of hub genes using the PPI network for the downregulated genes. For the 115 downregulated genes associated with a good prognosis, 36 genes were identified as interconnected. The PXMP2, ACOX1, ACO2, ACSS2, PPARGC1A, and CLCA1 had the most interactions with other genes.

Fig. 7

FT-IR of Fe₃O₄ NPs (a), Cinnamon (b) and Fe₃O₄@Glu-Cinnamon NPs (c).

Fig. 8

XRD spectrum of Fe₃O₄@Glu-Cinnamon NPs.

Fig. 9

(a) TEM and (b) SEM images and (c) EDS-mapping analysis of Fe₃O₄@Glu-Cinnamon NPs. The NPs contained no elemental impurity and were in a size range of 26.8–60.2 nm.

Fig. 10

(a) DLS and (b) zeta potential analyses of Fe₃O₄@Glu-Cinnamon NPs. The surface charge and zeta potential of the NPs were 213 nm and − 15.4 mV.

Fig. 11

Magnetization curve of Fe₃O₄@Glu-Cinnamon NPs. The maximum magnetization level of the NPs was 20.33 emu/g, which was observed at an applied field of 14,000 Oe.

Fig. 12

Cell viability assay. Viability percentage of the normal (a) and CRC (b) cell lines treated with different concentrations of Fe₃O₄@Glu-Cinnamon NPs. (c) Viability of CRC cells treated with different concentrations of cinnamon. The IC₅₀ value of Fe₃O₄@Glu-Cinnamon NPs for normal and CRC cells were 340 and 127 µg/mL, respectively and IC₅₀ value of cinnamon alone for CRC cells was 362 µg/mL.

Fig. 13

Flow cytometry analysis of CRC cells. (a) control, (b) Treated with Fe₃O₄@Glu-Cinnamon NPs. Exposure to the NPs elevated primary and late apoptosis and cell necrosis levels. Q1; healthy cells, Q2: cell necrosis, Q3; late apoptosis and Q4; primary apoptosis.

Fig. 14

Cell cycle analysis of CRC cells. (a) control, (b) Treated with Fe₃O₄@Glu-Cinnamon NPs. Exposure to the NPs caused cell cycle arrest mainly at the S and G2/M phases.

Fig. 15

Hoechst staining of CRC cells. (a) control, (b) Treated with Fe₃O₄@Glu-Cinnamon NPs. Exposure to the NPs caused nuclear alterations such as chromatin condensation and fragmentation.

Fig. 16

The mRNA expression levels of the SNAI1, THBS and INHBA oncogenes in the CRC cells treated with Fe₃O₄@Glu-Cinnamon NPs relative to control cells. Exposure to the NPs reduced the mRNA levels of all oncogenes.