Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and β-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of β-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.

Cysteine desulfurase IscS is required for cellular iron-sulfur protein maturation in eukaryotes and prokaryotes. In this study, we examined the effect of dietary iron intake on the expression in rat skeletal muscle of IscS in relation to 2 iron-sulfur proteins, cytosolic aconitase (c-aconitase) and mitochondrial aconitase (m-aconitase). Three groups of male weanling Wistar rats were used; 1 group was fed an iron-deficient diet (D), and the other 2 groups were pair-fed (P) or freely fed (C) a control (35 mg Fe/kg diet) diet for 1 or 2 wk. At the end of wk 1 and 2, the mitochondrial IscS protein levels in the skeletal muscle of iron-deficient rats had decreased to 45 and 50% of those of the control and pair-fed rats, respectively, whereas the IscS mRNA levels did not differ among the 3 groups, indicating that iron deficiency affected the expression of IscS protein at the post-transcriptional level. Iron deficiency caused a 55–76% reduction in c-aconitase activity and an ∼50% reduction in the c-aconitase protein level. The m-aconitase activity and protein level in iron-deficient rats also declined to 50 and 58–64% of the control levels, respectively. Our results indicate that dietary iron modulates mitochondrial IscS protein and aconitase at the post-transcriptional level, and mitochondrial IscS may be associated with this regulation of aconitase in skeletal muscle.