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

Abstract

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 1a 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 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 (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.

Withanolides: Promising candidates for cancer therapy

“…Both the in vitro and in vivo investigations illustrated that withano- Hsu et al, 2012;Lee et al, 2017;Lee et al, 2022;Ma et al, 2016;Mondal et al, 2010;Rao et al, 2016;Stan et al, 2008;Wu et al, 2012;Yu et al, 2017;Zhao et al, 2017) cytostatic autophagy (Ma et al, 2017;Rah et al, 2015;Xu et al, 2017;Yu et al, 2017), whereas our group previously found that the cytoprotective autophagy was initiated by physalin A (He, Zang, Feng, Wang, et al, 2013). Intriguingly, withaferin A is quite distinctive since the autophagy induced by withaferin A was cytoprotective in prostate cancer cells , and in breast cancer cells (Muniraj et al, 2019).…”

“…Notably, withaferin A (Das et al, 2016), 4β-hydroxywithanolide E (H-C Wang, Hu, et al, 2019, and withanolide D (Sarkar et al, 2014) (Xue & Hemmings, 2013). 3-azidowithaferin A (Rah et al, 2015) and withagenin A diglucoside are reported to inhibit the phosphorylation of mTOR (Lee et al, 2022). Furthermore, mTORC1 regulates cell growth by phosphorylating p70S6 kinase 1 (p70S6K1) and eIF-4E-binding protein 1 (4EBP1).…”

“…Activation of BAX and BAK can lead to the formation of pores in the outer membrane of mitochondria, which can touch off the cytochrome c release. Withaferin A, physagulide P, withametelin, withanolide D, 4β‐hydroxywithanolide E, physapubescin, physapubescin B, physapubenolide, physalin A, physalin B, physalin F, and withagenin A diglucoside can initiate intrinsic mitochondrial apoptosis by up‐regulating BAX (Chen, Xia, Qiu, et al, 2016; He, Zang, Feng, Chen, et al, 2013; Hsu et al, 2012; Lee et al, 2017; Lee et al, 2022; Ma et al, 2016; Mondal et al, 2010; Rao et al, 2016; Stan et al, 2008; Wu et al, 2012; Yu et al, 2017; Zhao et al, 2017) to trigger apoptosis. Recently, using small RNA next‐generation sequencing (NGS) and qRT‐PCR technology, Shuaib et al found that the co‐treatment strategy of withaferin A and miR‐181c‐5p mimic showed a pronounced anticancer effect by inducing the intrinsic apoptosis pathway in TNBC cells by upregulating the caspase‐8 and its downstream targets (Shuaib et al, 2022).…”

“…Phosphorylated Akt has been linked to cell growth, proliferation, and motility, as a result of its activation of signals that impede normal regulatory mechanisms, ultimately giving rise to mTOR activation (Xue & Hemmings, 2013). 3‐azidowithaferin A (Rah et al, 2015) and withagenin A diglucoside are reported to inhibit the phosphorylation of mTOR (Lee et al, 2022). Furthermore, mTORC1 regulates cell growth by phosphorylating p70S6 kinase 1 (p70S6K1) and eIF‐4E‐binding protein 1 (4EBP1).…”

Datura stramonium Flowers as a Potential Natural Resource of Bioactive Molecules: Identification of Anti-Inflammatory Agents and Molecular Docking Analysis

“…They are present in several genera of Solanaceae such as Acnistus , Datura , Dunalia , Jaborosa , Physalis, and Withania [ 14 ]. In recent years, withanolides were devoted substantial consideration due to their versatile biological properties such as their antitumor [ 15 ], anti-inflammatory [ 16 ], immunosuppressive [ 17 ], and chemoprevention [ 18 ] properties.…”