Identification of baicalein as a ferroptosis inhibitor by natural product library screening

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Abstract

Ferroptosis, a novel form of regulated cell death, is characterized by oxidative injury from iron accumulation and lipid peroxidation. In a natural product library screening for ferroptosis inhibitor, we found that baicalein is a potent inhibitor of erastin-induced ferroptosis in pancreatic cancer cells. Baicalein (also termed 5,6,7-trihydroxyflavone) is a flavonoid originally obtained from the roots of Scutellaria baicalensis and Scutellaria lateriflora. We showed that baicalein exhibits remarkable anti-ferroptosis activity compared with well-known ferroptosis inhibitors such as ferrostatin-1, liproxstatin-1, deferoxamine mesylate, and β-mercaptoethanol. At the biochemistry level, baicalein limits erastin-induced ferrous iron production, glutathione depletion, and lipid peroxidation. At the protein level, baicalein suppresses erastin-mediated degradation of glutathione peroxidase 4, a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. Thus, baicalein enhances cellular anti-ferroptosis capacity and could be a potential therapeutic agent for ferroptosis-associated tissue injury.

Introduction

It is well-known that both serious deficiencies and large excesses of various elements can cause diseases. Iron is an essential nutrient for various species, including mammals. Iron deficiency leads to anemia, which is associated with chronic infectious diseases, whereas excess iron increases the risk for liver disease, heart disease, diabetes, and cancer [1]. Growing pathological evidence indicates that iron is an important initiator and mediator of regulated cell death by activation of oxidative injury [2]. In particular, ferroptosis is a recently identified iron-dependent form of regulated cell death in cancer cells [3]. Interestingly, the accumulation of other metals fails to induce ferroptosis [3]. In addition to cancer death [4], [5], [6], [7], impaired ferroptosis contributes to multiple pathological processes such as neurotoxicity [8], acute renal failure [8], [9], [10], drug-induced hepatotoxicity [11], tissue ischemia/reperfusion injury [9], [10], and T cell death [12]. Thus, pharmacologic inhibition of ferroptosis may provide protection from inflammation-associated injury.

The classical chemical trigger of ferroptosis is erastin, which was originally identified from high-throughput screening of oncogene RAS-selective drugs to treat cancer [13]. We now know that erastin induces ferroptosis in both an RAS-dependent and -independent manner, which is dependent on tumor type and downstream signal [6], [14], [15], [16]. Different from apoptosis and necrosis, the activation of caspase, as well as adenosine triphosphate depletion, are not observed in ferroptosis [3]. Excess iron causes ferroptosis due to Fenton reaction-mediated production of reactive oxygen species (ROS), which eventually forms hydroxyl radicals from superoxide or hydrogen peroxide [3]. In addition, lipid peroxidative products such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal are increased in iron overload. Several molecules have recently been identified to regulate ferroptosis by directly or indirectly targeting pathways of iron metabolism or lipid peroxidation [17], [18]. Among them, glutathione peroxidase 4 (GPX4) displays strong anti-ferroptosis activity by inhibition of lipid peroxidation [4]. Thus, inhibition of GPX4 activity or expression contributes to ferroptosis.

Natural products and their derivatives play a critical role in drug discovery [19]. They have been the most successful source of new drugs; more than 60% of the drugs on the market are derived from natural sources. In this study, we performed a small natural product library screening and identified baicalein as an inhibitor of erastin-induced ferroptosis in pancreatic cancer cells. Furthermore, we demonstrated that baicalein limits erastin-induced iron accumulation and lipid peroxidation in ferroptosis. Baicalein inhibits lipid peroxidation partly through preventing glutathione (GSH) depletion and GPX4 degradation. These findings confirm novel pharmacological activity of baicalein in the protection against cellular ferroptosis.

Section snippets

Regents

The antibodies to NRF2 and SLC7A11 were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody to GPX4 was obtained from Abcam (Cambridge, MA, USA). The antibody to tubulin was obtained from Sigma Aldrich (Milwaukee, WI, USA). Erastin, baicalein, staurosporine, Z-VAD-FMK, ferrostatin-1, liproxstatin-1, and the natural product library were purchased from Selleck Chemicals (Houston, TX, USA). Deferoxamine mesylate and β-mercaptoethanol were purchased from Sigma Aldrich

Identification of baicalein as a cellular inhibitor of ferroptosis

To identify the inhibitor of erastin-induced ferroptosis, we screened a small natural product library from Selleck Chemicals with 143 compounds in the human pancreatic cancer cell line PANC1. Cell viability was measured using CCK-8 in PANC1 cells following treatment with 20 μM erastin at 24 h in the absence or presence of 10 μM natural product compound. Among all 143 compounds, baicalein exhibited the strongest protection against erastin-induced cell death in PANC1 cells (Fig. 1). Baicalein

Discussion

In the current study, we identified baicalein as a novel ferroptosis inhibitor using natural product library screening. The anti-ferroptosis activity of baicalein is involved in the modification of iron accumulation and lipid peroxidation. In particular, baicalein prevents erastin-induced GPX4 degradation, which impairs lipid peroxidation.

The Nomenclature Committee on Cell Death, led by Dr. Guido Kroemer, proposes several cell death classification criteria, including morphological appearance,

Conflict of interest

The authors declare no conflicts of interest or financial interests.

Acknowledgments

We thank Christine Heiner (Department of Surgery, University of Pittsburgh) for her critical reading of the manuscript. This work was supported by grants from the National Institutes of Health of USA (R01GM115366 and R01CA160417 to D.T.), the National Natural Science Foundation of China (31171229 and U1132005, to X.S.), the National Natural Science Foundation of Guangdong (D.T.), and a Science of Guangzhou Key Project (201508020258, 201400000003-4, and 201400000004-4 to X.S.).

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