| Summary: | Hepatocellular carcinoma (HCC) is the fastest-growing tumor capable of spreading to other organs via blood vessels formed by endothelial cells. Apoptosis and angiogenesis-targeting therapies are attractive for cancer treatment. In this study, we aimed to study the in vitro cytotoxicity of <i>Withania somnifera</i> against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. <i>W. somnifera</i> (Solanaceae), well-known as ‘ashwagandha’, is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of <i>W. somnifera</i> root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active <i>n</i>-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (<b>1</b>–<b>5</b>), including one new metabolite, withanoside XIII (<b>1</b>), aided by liquid chromatography–mass spectrometry-based analysis. The new compound structure was determined by 1D and 2D nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectroscopy, electronic circular dichroism, and enzymatic hydrolysis. In addition, withanoside XIIIa (<b>1a</b>) was identified as the new aglycone (<b>1a</b>) of <b>1</b>. Isolated withanolide glycosides <b>1–5</b> and <b>1a</b> were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (<b>3</b>) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 μM. WAD cytotoxicity was mediated by both extrinsic and intrinsic apoptosis pathways. Treatment with WAD increased protein expression levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bcl-2-associated X protein (Bax), and cleaved poly(ADP-ribose) polymerase (cleaved PARP) but decreased expression levels of B-cell lymphoma 2 (Bcl-2). Moreover, WAD inhibited tubular structure formation in human umbilical vein endothelial cells (HUVECs) by inhibiting the protein expression of vascular endothelial growth factor receptor 2 and its downstream pathways, including extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). These effects were also enhanced by co-treatment with ERK and PI3K inhibitors. Overall, these results indicate that WAD (<b>3</b>) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.
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