INTERNATIONAL CONGRESS OF
CancerGenetics
Effects of Anticancer Activity of Interferon Gamma on Colon Cancer Cell Lines: based on Network Pharmacology Analysis
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Narjes Akbari,1,* Reza Heidari,2
1. National Institute of Genetic Engineering and Biotechnology
2. Imam Khomeini International University, Qazvin
- Introduction: IFNs, especially IFN-γ, are secreted by immune cells such as monocytes and lymphocytes and are commonly used to treat viral diseases and some cancers. These glycosylated proteins are of great importance for clinical use in the treatment of a wide range of viral diseases, especially COVID-19, and various types of cancer due to their antiviral and antitumor activities (Myasnikov et al., 2021). Colorectal cancer (CRC) is one of the most common cancers in humans, occurring in the large intestine or rectum and affecting this organ in the digestive tract. In 2022, the number of new cases diagnosed with CRC and the number of deaths due to CRC were reported to be approximately 1.93 million and 903,859, respectively. It is the third most common cancer in terms of incidence (9.6% of all cancers) and the second most common cancer in terms of mortality (9.3% of all cancers) among men and women worldwide in 2022. By 2030, colorectal cancer cases are expected to increase to 2.2 million new cases and 1.1 million deaths (Bray, F et al., 2024). IFN-γ regulates the activity of the immune system and is able to enhance antitumor activity. Therefore, it is an important part of cancer immunotherapy. IFN-γ inhibits tumor cell proliferation by inhibiting angiogenesis, sensitizes tumor cells to programmed cell death, increases the expression of major histocompatibility complex (MHC) class I and II, and stimulates antitumor immune activity (Miller, Maher and Young, 2009). Several studies have demonstrated the inhibitory role of Ad-IFNγ in prostate cancer cell growth (Zhao et al., 2007), nasopharyngeal carcinoma (Zuo et al., 2011), and pancreatic cancer (Xie et al., 2013). Several studies have demonstrated the toxic effect of IFN-γ against various cancer cell lines. Breast cancer cell lines (T47D and MCF-7) treated with hIFN-γ showed a dose-dependent decrease in cell viability (Widowati et al., 2016; Widowati et al., 2018).
- Methods: In this study, E. coli strain Rosetta, pET-28a(+) expression vector, and pET-IFN and pET-IFN-ELP recombinant expression vectors were used to produce recombinant protein. Purification of recombinant proteins was performed by affinity chromatography using the HisPurTM Cobalt Purification kit. Determination of the concentration of extracted protein samples was performed using the Bradford method (Bradford, 1976). The quality of protein samples was examined on a 12.5% polyacrylamide gel according to the Laemmli method (Laemmli et al., 1976). Indirect ELISA test and confirmation of protein samples were performed using Western blotting and dot blotting. Vero cells from African green monkey kidney tissue (Cercopithecus aethiops) and a colon cancer cell line (HCT116) were used in this study. Before investigating the anticancer properties, the cytotoxic effects of hIFN-γ on Vero cells were first investigated using an MTT-based colorimetric assay. The anticancer biological activity of recombinant hIFN-γ protein on HCT116 cells was investigated using an MTT-based colorimetric assay (Rezaei-Moshaei et al., 2021). To analyze the effect of hIFN-γ on clonal cancer (COAD), gene expression data were obtained from the Gene Expression Omnibus (GEO) database. RNA-seq data with accession number GSE135458 were downloaded from the GEO database registered by Klatt et al. (2020). RNA-seq data included 4 samples of human HCT116 cell line treated with hIFN-γ for 24 h and control sample, each with two replicates. Raw data were based on GPL18573 platform (H. sapiens) and sequenced on Illumina NextSeq 500. PPI network consisting of DEGs was constructed by Cytoscape v3.7.0 software (Utleg et al., 2003) and evaluated and identified to identify hub genes in PPI network using CytoNCA plugin (Tang et al., 2015).
- Results: The biological function of recombinant hIFN-γ-ELP protein was investigated by examining its anticancer activity on colon cancer cell line (HCT116) using MTT-based colorimetric assay. The obtained results showed that recombinant hIFN-γ-ELP protein inhibited the growth and proliferation of cancer cells and the highest level of cell proliferation inhibition was observed at a concentration of 32.00 pg/ml hIFN-γ after 72 hours of incubation. A protein-protein interaction (PPI) network was constructed using the expression data obtained from the HCT116 cancer cell line treated with hIFN-γ, and about 370 nodes were identified and considered as hub genes. Functional enrichment analysis revealed that hub genes are active in pathways such as cytoplasmic ribosome, cell cycle, ribosome biogenesis in eukaryotes, and oxidative phosphorylation. Furthermore, most hub genes were downregulated after treatment with hIFN-γ, suggesting their potential role in inducing tumor cell growth and proliferation, and confirming the cytostatic, antiproliferative, and proapoptotic functions of IFN-γ. Based on network pharmacology, several survival-affecting genes, including nop9, mphosph10, nifk, nup214, pdcd11, and utp14A, were identified in CRC cell lines after treatment with hIFN-γ. These genes may be considered as drug targets, and targeting them may improve the treatment of CRC patients. Furthermore, analysis of Bax, p53, and Bcl-2 gene expression confirmed the proapoptotic role of hIFN-γ in HCT116 cell line.
- Conclusion: The biological activity study showed that the recombinant hIFN-γ proteins (with or without the ELP sequence) were fully biologically active and were able to inhibit the growth and proliferation of colon cancer cells (HCT116). Therefore, it is possible that the hIFN-γ protein could be biologically active even without undergoing the N-glycosylation process. Also, the attachment of the recombinant hIFN-γ protein to the ELP sequence, in addition to enhancing the function, did not interfere with the structure and biological activity of the recombinant protein. A PPI network was constructed using the 4733 DEGs identified from the hIFN-γ-treated samples versus the control samples. About 1038 genes out of the original 4733 genes were removed from the network, thus creating a network with 3695 nodes. After network analysis using Cytoscape software, 3695 nodes and 43090 edges were obtained. DEG enrichment analysis was performed by filtering pathways based on FDR ≤ 0.05 using the KEGG database. The enrichment analysis indicated that DEGs with increased expression were involved in the endocytosis process, while the important pathway for DEGs with decreased expression was the ribosome synthesis and assembly process.
- Keywords: Colon cancer, Human interferon-γ, Network pharmacology
