Lee, Dahae Yu, Jae Sik Ha, Ji Won Lee, Seoung Rak Lee, Bum Soo Kim, Jin-Chul Kim, Jung Kyu Kang, Ki Sung Kim, Ki Hyun 2024-01-19T11:03:54Z 2024-01-19T11:03:54Z 2022-10-11 2022-09 2076-3921 https://pubs.kist.re.kr/handle/201004/114578 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 Withania somnifera against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. W. somnifera (Solanaceae), well-known as 'ashwagandha', is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of W. somnifera root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active n-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (1-5), including one new metabolite, withanoside XIII (1), 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 (1a) was identified as the new aglycone (1a) of 1. Isolated withanolide glycosides 1-5 and la were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (3) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 mu M. 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 (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers. English MDPI AG Antitumor Potential of Withanolide Glycosides from Ashwagandha (Withania somnifera) on Apoptosis of Human Hepatocellular Carcinoma Cells and Tube Formation in Human Umbilical Vein Endothelial Cells Article 10.3390/antiox11091761 1 Antioxidants, v.11, no.9 Antioxidants 11 9 Y scie scopus 000859810000001 Biochemistry & Molecular Biology Chemistry, Medicinal Food Science & Technology Biochemistry & Molecular Biology Pharmacology & Pharmacy Food Science & Technology Article CYTOTOXIC WITHANOLIDES ROOTS ANGIOGENESIS ACTIVATION MAPK Withania somnifera withanolide glycosides hepatocellular carcinoma HepG2 cells apoptosis angiogenesis