This study aimed to identify a potential native plant for the treatment of colorectal cancer using bioinformatics and in vitro methods. The study involved the investigation of compound and protein libraries, DT network analysis, molecular interaction analysis, cytotoxicity MD simulation analysis, assessment of plant extract, and gene expression analysis. We searched and identified 315 approved native medicinal plants. From these plants, 53 known compounds were found based on the information available in the ChEBI database. Furthermore, 14 target proteins were obtained using PharmMapper and colorectal cancer data.
The construction of a drug-target network provided a holistic perspective, incorporating 47 compounds and 14 protein targets. Network analysis, considering node values and betweenness centrality, facilitated the prioritization of compounds and proteins for further investigation. Four protein targets (ANG, DPP4, INR and MAPK14) were selected based on network analysis. Molecular docking studies were conducted to estimate binding affinities between the selected compounds and protein targets. The top ten compounds exhibiting the highest binding affinity for each protein were identified. Notably, cauferoside, sourced from Ferula gummosa, displayed significant binding affinity to all four proteins. This observation prompted further investigation of the anti-colorectal cancer potential of Ferula gummosa. Molecular dynamics (MD) simulations were used to scrutinize the stability of compound-protein complexes over a 50 ns timeframe. RMSD and RMSF analyses revealed overall structural stability, with slight fluctuations in the INR-cauferoside complex. Average RMSD values provided a quantitative measure of stability across the complexes.
The study progressed to in vitro investigations, evaluating the cytotoxicity of Ferula gummosa extracts using the MTT assay. The IC50 values for leaf and root extracts were determined, indicating a concentration-dependent decrease in cell survival. Importantly, the lower effective concentration of the compound is highlighted for its potential clinical application. Gene expression analysis by qRT-PCR further elucidated the molecular mechanisms underlying the observed cytotoxic effects. Treatment with Ferula gummosa leaf extract significantly down-regulated the expression of the ANG, DPP4, INR, and MAPK14 genes. In particular, INR mRNA levels exhibited the most pronounced reduction. The root extract showed different effects, with increased expression of ANG and minimal impact on other genes.
Angiogenin (ANG) is a protein that plays a crucial role in angiogenesis, the process of forming new blood vessels. In the context of cancer, angiogenin has been extensively studied due to its involvement in tumour growth, invasion, and metastasis. Several studies have shown that angiogenin is overexpressed in various types of cancer, including breast, lung, and colorectal cancer. In addition, angiogenin has been shown to have angiogenesis-independent effects in cancer, such as promoting cancer cell survival and resistance to therapy. The findings highlight the importance of angiogenin as a potential therapeutic target and a biomarker for cancer diagnosis and treatment 28. Dipeptidyl peptidase-4 (DPP4), also known as CD26, is an enzyme that plays a crucial role in various physiological processes, including immune regulation and glucose metabolism. In recent years, there has been a growing interest in understanding the involvement of DPP4 in cancer. Studies have revealed that DPP4 expression is dysregulated in several types of cancers, including breast, prostate, colorectal, and pancreatic cancer. DPP4 has been implicated in cancer progression and metastasis through its effects on tumor cell invasion, migration, and angiogenesis. Furthermore, DPP4 has been explored as a potential biomarker for the prognosis of cancer and as a target for therapeutic intervention 29. The insulin receptor (INSR) is a cell surface receptor that plays a crucial role in mediating the effects of insulin. In recent years, there has been increasing evidence suggesting a link between INSR and cancer. Studies have shown that dysregulation of INSR signaling is associated with tumour growth, progression, and resistance to therapy in various types of cancer, including breast, colorectal, and lung cancer. Aberrant INSR activation can promote cancer cell survival, proliferation, and metastasis by stimulating downstream signaling pathways involved in cell growth and survival 30. MAPK14, also known as p38 MAPK, is a member of the mitogen-activated protein kinase (MAPK) family that plays a crucial role in cellular signaling pathways involved in inflammation, stress responses, and cell proliferation. Over the years, there has been increasing evidence linking MAPK14 to cancer development and progression. Dysregulation of MAPK14 signaling has been observed in various cancer types, including breast, lung, colorectal, and pancreatic cancer. MAPK14 activation has been shown to promote tumor cell survival, proliferation, invasion, and metastasis. The different findings underscore the significance of MAPK14 in cancer and its potential as a therapeutic target 31.
The qRT-PCR results also showed partial inhibition of the the expression of VIM and BRAF genes by plant extracts. BRAF is a proto-oncogene that is usually involved in cancer cell signaling and VIM (vimentin) participates in cancer cell migration and cell adhesion structures. Vimentin is often associated with the epithelial-mesenchymal transition (EMT), a process in which cells lose their epithelial characteristics and gain mesenchymal properties. EMT is involved in cancer progression and metastasis 32,33.