**1. Introduction**

Bladder cancer is one of the most common malignant tumors of the urinary system. The cancer statistics released in 2022 state that, in the United States, bladder cancer has an estimated incidence of 81,800 cases and a mortality rate of 17,100 cases [1]. These

**Citation:** Gao, Y.; Nie, Z.; Cao, H.; Huang, D.; Chen, M.; Xiang, Y.; Yu, X.; Zhang, S. Scabertopin Derived from *Elephantopus scaber* L. Mediates Necroptosis by Inducing Reactive Oxygen Species Production in Bladder Cancer In Vitro. *Cancers* **2022**, *14*, 5976. https://doi.org/10.3390/ cancers14235976

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 30 September 2022 Accepted: 1 December 2022 Published: 2 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

rates are slightly lower than those reported in 2021 [2]. In China, bladder cancer had an incidence of approximately 80,500 and a mortality rate of 32,900 in 2015 [3]. Uroepithelial cancer is the main pathological type of bladder cancer. Approximately 75% and 25% of the cases of this type of cancer are non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), respectively. The current clinical treatment of bladder cancer remains based on surgery combined with postoperative radiotherapy [4,5]. Mitomycin C, epirubicin, adriamycin, methotrexate, vincristine, pirarubicin, and cisplatin are used as the first-line chemotherapeutic agents for bladder cancer in clinical practice [6]. However, the use of these drugs is greatly limited by their unsatisfactory efficacy, the widely reported drug tolerance of bladder cancer, and their adverse effects. Although the rapid development of molecular biology and genetics research has provided new therapeutic tools for the clinical development of immunotherapy, gene therapy, and targeted therapy, these approaches fail to exert an ideal treatment effect on bladder cancer because they use a large number of chemically synthesized drugs. This approach results in high treatment costs, drug side effects, hepatotoxicity, and drug resistance. Therefore, the search for new drugs with low toxicity and side effects has been a hot issue in tumor treatment.

In recent years, many natural compound products have played an important role in the treatment of tumors. Finding compounds with significant tumor-inhibiting activity from natural products is an important approach and means for the research and development of anticancer drugs [7]. Scabertopin is a sesquiterpene lactone compound that is mainly derived from the chemical constituents of *Elephantopus scaber* L. It is a kind of herbaceous plant belonging to the phylum Angiosperm, class Dicotyledonous, order Campanulaceae, family Asteraceae, and genus Elephantopus [8], which is widely distributed worldwide, particularly in East Asia, Southeast Asia, Africa, Australia, India, and South America, and has been reported to have various pharmacological properties, such as anti-bacterial, antidiabetic, anti-inflammatory, and anti-cancer efficacy [9,10]. An increasing number of studies have shown that the antitumor activity of *Elephantopus scaber* L. is attributed to sesquiterpene lactones, which execute significant antitumor activities in nasopharyngeal [11,12], cervical [13], breast [14], colon [15,16], and hepatocellular carcinomas [17]. For example, in HCT116 and RKO cells, deoxyelephantopin (DET), another sesquiterpene lactone compound derived from *Elephantopus scaber* L. can significantly increase reactive oxygen species (ROS) levels, thereby activating the JNK signaling pathway and triggering cell death. In the mouse breast cancer cell line TS/A, DET can inhibit TNF-α-induced NF-κB activity and down-regulate the NF-κB regulated gene products of matrix metalloproteases (MMP)-2 and MMP-9, thereby inhibiting the migration and invasion in vitro and in vivo [14]. In addition, sesquiterpenoids exhibit different cytotoxicities toward tumor cells and normal cells, whereby they selectively target tumor cells and show little systemic toxicity [18,19]. However, the research on the anticancer effect and mechanism of scabertopin in bladder cancer has not been reported.

Necroptosis is a unique form of programmed cell death, which is characterized by caspase-independent activation and is morphologically accompanied by the swelling of organelles and the rupture of cell membranes, resulting in the spillage of the cellular contents into the surrounding cells and, consequently, triggering inflammatory response [20,21]. Various drugs and active sites of traditional Chinese medicine have been confirmed to exert their anticancer activity through necroptosis [22,23]. Receptor-interacting protein (RIP)1, RIP3, and mixed lineage kinase-like (MLKL) are three key factors in the regulation of necroptosis; RIP3 has been well-established to recruit MLKL and induce its phosphorylation. Phosphorylated-MLKL (p-MLKL) then undergoes oligomerization and translocates to the plasma membrane to execute cellular necrosis [24–26]. However, the pathway through which RIP1 undergoes autophosphorylation remains controversial. ROS have been recently reported to act as upstream cell signaling molecules to activate and drive necroptosis [27]. RIP1 can sense ROS by modifying three key cysteine residues (C257, C268, and C586) and, subsequently, induces their autophosphorylation at the S161 site, which in turn results in the recruitment of RIP3 to form a necrosome [28]. Previous studies have also shown

a correlation between necroptosis and drug-induced ROS. For example, artesunate can inhibit the proliferation of renal cancer cell lines in vitro by inducing elevations in ROS levels and, thus, promoting RIP1-dependent necrotic apoptosis [29]. In addition, emodin promotes the occurrence of necrotic apoptosis in renal cell carcinoma (RCC) cell lines, thereby restricting tumor development by inducing the production of ROS that promote increases in the levels of RIP1 and MLKL phosphorylation [30]. In conclusion, the positive correlation of necroptosis with ROS implies that regulating ROS to induce necroptosis in tumor cells is a potential strategy for cancer therapy.

In this study, we aimed to evaluate the inhibitory effects and potential molecular mechanisms of scabertopin on bladder cancer cells in vitro. Scabertopin is one of the four sesquiterpene lactones with the highest content in *Elephantopus scaber* L. Since DET has shown a good anti-tumor effect in a variety of tumor cell lines, we speculated that scabertopin would also have similar efficacy. Therefore, we determined our research objective was to study the effect of scabertopin on bladder cancer cell lines and explore its way to promote the death of bladder cancer cell lines. Our results showed that scabertopin with antitumor properties inhibited the growth, migration, and invasion of the bladder cancer cell line J82. Further in-depth studies demonstrated that the above effects of scabertopin may be related to the promotion of ROS generation, the induction of focal adhesion kinase (FAK) phosphorylation, and the activation of necroptotic signaling pathways.
