Germinated Seeds of Hordeum vulgare Target Extrinsic Pathway of Apoptosis in Triple-Negative Breast Cancer Cells

Background: The absence of receptors, lack of specific treatment regimen, and emergence of resistance against various currently available anticancer drugs have led to the development of lead molecules from botanicals for the mainstay treatment of triple-negative breast cancer (TNBC). Hordeum vulgare, commonly known as barley, has been reported to have many traditional uses, and the main alkaloid in its germinated seeds, hordenine, has been evaluated for many pharmacological properties. Objectives: The present study aimed to evaluate the anticancer activity of methanol extract of germinated seeds of H. vulgare (MGHV) against MDA-MB-231 TNBC cells, quantify hordenine in MGHV, and derive the probable target of action of the extract and hordenine. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, morphological evaluation, acridine orange/ethidium bromide (AO/EB) staining, Hoechst 33258 staining, JC-1 staining, comet assay, and Western blot were conducted to evaluate the anticancer effect. High-performance liquid chromatography (HPLC) analysis was conducted to detect and quantify the presence of hordenine in the extract. Stimulation of extrinsic pathway of apoptosis was evaluated by studying the interaction of hordenine with Caspase-8 using in silico methods. Results: MTT assay revealed significant concentration-dependent cytotoxicity. AO/EB staining exhibited yellow-green fluorescence indicative of early apoptosis. Hoechst 33258 staining showed nuclear marginalization and fragmentation. The results of JC-1 staining showed a combination of red and green fluorescence indicative of partial reduction in mitochondrial membrane potential. Comet assay revealed that the extract did not produce deoxyribo nucleic acid damage. Western blotting analysis did not show any change in the expression of Bcl-2, whereas significant upregulation of Caspase-8 by 2.8 folds was noticed indicative of extrinsic pathway of apoptosis. HPLC analysis detected the presence of hordenine as the major constituent in the extract. Morphological assessment of MDA-MB-231 cells treated with hordenine showed cytotoxic changes similar to that of the extract. In silico studies with hordenine also substantiated the results of Western blot. Conclusion: Thus, it was concluded that MGHV targeted extrinsic pathway of apoptosis due to the presence of hordenine and hence hordenine could evolve as a potent anticancer molecule against TNBC.