Rajagopalan, U. | Search bioRxiv

bioRxiv2025

Anti-proliferative and Apoptosis Inducing Effect of Thymoquinone in Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells

Cancer stem cells (CSCs) are key drivers of tumor progression, therapeutic resistance and recurrence. Nigella sativa, a medicinal plant widely used in traditional medicine, has gained significant importance due to its diverse pharmacological properties. Thymoquinone (TQ), a biologically known active compound isolated from N.sativa, has demonstrated anticancer properties in various cancers. However, its effect on CSC-like cells has not been fully elucidated. In the present study, the anti-proliferative and apoptosis inducing properties of TQ was evaluated on human embryonal carcinoma cells (NTERA-2, cancer stem cell like model) and human peripheral blood mononuclear cells (PBMCs) in vitro. Antiproliferative effects of TQ on NTERA-2 cells and PBMCs were evaluated using the Sulforhodamine B (SRB) and WST-1 assays, respectively. The effect of TQ was further evaluated using colony formation assay, cell migration assay, fluorescence microscopy and quantification of caspase 3/7 activities. Oxidative stress markers (reactive oxygen species [ROS]) were also determined in NTERA-2 cells treated with TQ. Thymoquinone revealed promising dose- and time-dependent antiproliferative effects (half-maximal inhibitory concentration [IC50] 1.282, 1.167, and 0.984 g/mL at 24, 48, and 72 h post-treatment) in NTERA-2 cells while exerting a minimal cytotoxic effect in PBMCs. Apoptosis related morphological changes, and increased Caspase 3/7 activities confirmed the pro-apoptotic effects of TQ. Further, NTERA-2 cells treated with TQ expressed a significant increase (P < 0.001) in intracellular ROS activity. Overall results confirm that TQ exerts anti-proliferative and apoptotic effects in a dose- and time-dependent manner. Therefore, TQ can be considered as a potent drug lead for chemotherapy and radiotherapy resistant cancer stem cells.

pharmacology and toxicology↗

bioRxiv2025

A network pharmacology-based approach and molecular docking study to explore the therapeutic potential of a nutraceutical formula (Vernolac) in the treatment of cancer

Vernolac is a commercially available polyherbal nutraceutical capsule comprised of Vernonia zeylanica aerial parts, Nigella sativa seeds, Hemidesmus indicus roots, Leucas zeylanica aerial parts, and Smilax glabra rhizome. Different herbal formulations, organic extracts, and many isolated phytochemicals of the above plants have been reported to exhibit anticancer properties. However, the anticancer mechanisms of action of Vernolac, as a polyherbal formulation, remain unexplored. This study employed an integrative network pharmacology-based approach, complemented by in vitro experiments, to investigate the anticancer potential of Vernolac. Phytochemicals in Vernolac were retrieved from databases, screened for drug-likeness and oral bioavailability using SwissADME, yielding 155 drug-like phytochemicals, and their protein targets were predicted via SwissTargetPrediction. The intersection of targets of phytochemicals and cancer-related targets from GeneCards yielded 137 common targets. Protein-protein interaction analysis in STRING and Cytoscape identified key hub nodes, including AKT1, BCL2, CASP3, CTNNB1, EGFR, ESR1, GAPDH, HSP90AA1, HSP90AB1, IL6, JUN, SRC, STAT3, and TNF. Clustering, topology, and formula-herb-compound-target-disease and target-pathway networks highlighted key phytochemicals, including vernolactone, thymoquinone, quercetin, nigellidine, -hederin, and carvacrol. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that the identified targets are significantly enriched in multiple cancer pathways. Molecular docking and dynamics simulations identified novel target-ligand interactions. Overall, network analysis suggests that Vernolac may exert anticancer effects through apoptosis induction, immune modulation, antioxidant, anti-inflammation, antiproliferative, and chemoradiosensitizing mechanisms. Moreover, Vernolac may exhibit chemoradioprotective potential by alleviating therapy-induced toxicity, supporting its promise as a potential adjunct to conventional cancer treatments. The Sulforhodamine B assay demonstrated selective antiproliferative activity of Vernolac against cancerous cells MCF-7 (IC50 = 54.01 {+/-} 0.02 g/mL), Caco-2 (IC50 = 85.52 {+/-} 0.13 g/mL), NTERA-2 cl.D1 (IC50 = 42.41 {+/-} 0.06 g/mL), and non-cancerous MCF-10A (IC50 = 803.5 {+/-} 0.03 g/mL). Novel target-ligand interactions identified via molecular docking and dynamics simulations, and the underlying mechanisms of Vernolac predicted in this study, require further validation through in vitro and in vivo experiments.

bioinformatics↗