AUTHOR=Al Awadh Ahmed Abdullah , Sakagami Hiroshi , Amano Shigeru , Sayed Ahmed M. , Abouelela Mohamed E. , Alhasaniah Abdulaziz Hassan , Aldabaan Nayef , Refaey Mohamed S. , Abdelhamid Reda A. , Khalil Heba M. A. , Hamdan Dalia I. , Abdel-Sattar El-Shaymaa , Orabi Mohamed A. A. 

TITLE=In vitro cytotoxicity of Withania somnifera (L.) roots and fruits on oral squamous cell carcinoma cell lines: a study supported by flow cytometry, spectral, and computational investigations

JOURNAL=Frontiers in Pharmacology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2024.1325272

DOI=10.3389/fphar.2024.1325272

ISSN=1663-9812

ABSTRACT=<p>Oral cancer is a severe health problem that accounts for an alarmingly high number of fatalities worldwide. <italic>Withania somnifera</italic> (L.) Dunal has been extensively studied against various tumor cell lines from different body organs, rarely from the oral cavity. We thus investigated the cytotoxicity of <italic>W. somnifera</italic> fruits (W-F) and roots (W-R) hydromethanolic extracts and their chromatographic fractions against oral squamous cell carcinoma (OSCC) cell lines [Ca9-22 (derived from gingiva), HSC-2, HSC-3, and HSC-4 (derived from tongue)] and three normal oral mesenchymal cells [human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF), and human pulp cells (HPC)] in comparison to standard drugs. The root polar ethyl acetate (W-R EtOAc) and butanol (W-R BuOH) fractions exhibited the strongest cytotoxicity against the Ca9-22 cell line (CC<sub>50</sub> = 51.8 and 40.1 μg/mL, respectively), which is relatively the same effect as 5-FU at CC<sub>50</sub> = 69.4 μM and melphalan at CC<sub>50</sub> = 36.3 μM on the same cancer cell line. Flow cytometric analysis revealed changes in morphology as well as in the cell cycle profile of the W-R EtOAc and W-R BuOH-treated oral cancer Ca9-22 cells compared to the untreated control. The W-R EtOAc (125 μg/mL) exerted morphological changes and induced subG<sub>1</sub> accumulation, suggesting apoptotic cell death. A UHPLC MS/MS analysis of the extract enabled the identification of 26 compounds, mainly alkaloids, withanolides, withanosides, and flavonoids. Pharmacophore-based inverse virtual screening proposed that BRD3 and CDK2 are the cancer-relevant targets for the annotated withanolides D (<bold>18</bold>) and O (<bold>12</bold>), and the flavonoid kaempferol (<bold>11</bold>). Molecular modeling studies highlighted the BRD3 and CDK2 as the most probable oncogenic targets of anticancer activity of these molecules. These findings highlight <italic>W. somnifera</italic>’s potential as an affordable source of therapeutic agents for a range of oral malignancies.</p>