Apigetrin Promotes TNFα-Induced Apoptosis, Necroptosis, G2/M Phase Cell Cycle Arrest, and ROS Generation through Inhibition of NF-κB Pathway in Hep3B Liver Cancer Cells

Abstract

Apigetrin (7-(β-D-glucopyranosyloxy)-4′,5-dihydroxyflavone), a glycoside bioactive dietary flavonoid derived from Taraxacum officinale and Teucrium gnaphalodes, is known to possess anticancer, antioxidant, and anti-inflammatory effects on numerous cancers. In the present study, we examined the effect of apigetrin in Hep3B hepatocellular cancer cell line (HCC). Apigetrin inhibited cell growth and proliferation of Hep3B cells, as confirmed by MTT and colony formation assay. We used apigetrin at concentrations of 0, 50, and 100 µM for later experiments. Of these concentrations, 100 µM of apigetrin showed a significant effect on cell inhibition. In apigetrin-treated Hep3B cells, cell cycle arrest occurred at the G2/M phase. Apoptosis and necroptosis of Hep3B cells treated with apigetrin were confirmed by Annexin V/propidium iodide (PI) staining and flow cytometry results. Morphological observation through 4′,6-diamidino-2-phenylindole (DAPI) staining showed intense blue fluorescence representing chromatin condensation. Hematoxylin staining showed necroptotic features such as formation of vacuoles and swelling of organelles. Apigetrin increased reactive oxygen species (ROS) levels in cells, based on fluorescence imaging. Furthermore, the underlying mechanism involved in the apoptosis and necroptosis was elucidated through western blotting. Apigetrin up-regulated TNFα, but down-regulated phosphorylation of p-p65, and IκB. Apigetrin inhibited the expression of Bcl-xl but increased Bax levels. Up-regulation of cleaved PARP and cleaved caspase 3 confirmed the induction of apoptosis in apigetrin-treated Hep3B cells. Additionally, necroptosis markers RIP3, p-RIP3, and p-MLKL were significantly elevated by apigetrin dose-dependently, suggesting necroptotic cell death. Taken together, our findings strongly imply that apigetrin can induce apoptosis and necroptosis of Hep3B hepatocellular cancer cells. Thus, apigetrin as a natural compound might have potential for treating liver cancer.

Necroptosis: A Pathogenic Negotiator in Human Diseases

“…Rubiarbonol-B (Ru-B), a natural arborinane triterpenoid extracted from Rubia philippinesis , switches apoptosis to necroptosis by upregulation of RIPK1 phosphorylation and NOX-1-derived ROS generation in colorectal cancer cells [ 269 ]. Bhosale et al [ 270 ] reported that administration of apigetrin, a glycoside dietary bioactive flavonoid, triggered both apoptosis and necroptosis in human Hep3B hepatocellular cancer cells via inhibition of NF-κB subunit phosphorylation (p-p65) and the IκB pathway. In addition, protein-bound polysaccharides (PBPs) have shown anti-tumor activity in MCF-7 breast cancer cells and SKMel-188 melanoma by ROS-mediated necroptosis [ 271 ].…”

A holistic comparison of flavor signature and chemical profile in different harvesting periods of Chrysanthemum morifolium Ramat. based on metabolomics combined with bioinformatics and molecular docking strategy

“… 46 Apigenin possessed anticancer, antioxidant, and anti-inflammatory effects, including inhibiting cell growth and proliferation in Hep3B cells. 47 It was reported that diosmetin could inhibit the progression of cancer. Diosmetin also exhibited antitumor effects on HCC cells by inhibiting cell proliferation via cell cycle arrest and interfering with lipid metabolism.…”

Ferroptosis, Necroptosis, and Pyroptosis in Gastrointestinal Cancers: The Chief Culprits of Tumor Progression and Drug Resistance

“…The findings of these studies suggested a promising therapeutic application of these molecules in the treatment of GI cancers. [135] The overexpression of heterogeneous nuclear ribonucleoprotein A1 (HNRNP A1) has been observed to enhance the proliferation, migration, and invasion of HCC cells, promoting tumor growth, and delaying senescence. As evaluated through mechanistic tests, silence of HNRNP A1 has been found to promote the expression of P16INK4, causing cell cycle arrest.…”

“…The findings of these studies suggested a promising therapeutic application of these molecules in the treatment of GI cancers. [ 135 ]…”