*3.3. Cell Death Induced by Scabertopin Treatment Was Not Apoptosis and Ferroptosis*

We utilized a scanning electron microscope to observe the effects of scabertopin treatment on cell morphology. The results showed that the cells without scabertopin treatment had strong three-dimensionality and thick and long pseudopodia; after scabertopin treatment, the cell spreading area increased and the pseudopodia shortened and thinned out (Figure 3A). To preliminarily elucidate the potential mechanism of scabertopin cytotoxicity in bladder cancer cells, we analyzed the effect of scabertopin treatment on the potential disruption of cell cycle phases using flow cytometry. The results showed that scabertopin treatment induced a significant increase in the percentage of cells both in the S and G2/M phases in a concentration-dependent manner (Figure 3B). Meanwhile, we detected the effect of scabertopin on the apoptosis and necrosis of J82 cells using the AnnexinV-FITC/PI double-staining method. The transfer of the cell membrane phospholipid phosphatidylserine from the inner to the outer layer of the plasma membrane is one of the early features of apoptosis. A single positive for AnnexinV-FITC is considered a typical cell in early apoptosis, i.e., the fourth quadrant (Q4), whereas AnnexinV-FITC and PI double-positive cells are considered to be in the end stage of apoptosis, necrosis, or are already dead, i.e., the second quadrant (Q2). The results in Figure 3C showed that, under the treatment with gradient concentrations of scabertopin, the number of early apoptotic cells was relatively small. Under scabertopin treatment, the number of necrosis cells significantly increased in a dose-dependent manner. In addition, water-soluble tetrazolium salts and green-fluorescent calcein-AM were deployed to determine whether scabertopin had an effect on the viability of bladder cancer cells. The results showed that, in contrast to the control treatment, scabertopin increased cytotoxicity in a concentration- and time-dependent manner, thus resulting in cell death (Figure 3D). However, scavenging of ROS with NAC significantly rescued scabertopin-induced cell death (Figure 3E). To reveal the mechanism of scabertopin-induced J82 cell death, we verified apoptosis-related caspase proteins Bcl-2 and Bax (Figure 3F), on the one hand, and ferroptosis-related GPX4 and pyroptosis-related GSDMD, on the other hand, using WB (Figure 3G). The results showed that scabertopin-treated J82 cells did not die by apoptosis, ferroptosis, or pyroptosis.

#### *3.4. Necroptosis Is a Type of Cell Death Induced by Scabertopin and can Be Inhibited by NAC*

To further investigate the extent to which cell organelles were altered by scabertopin treatment and to further determine the manner in which scabertopin induces death in bladder cancer cells, we performed TEM. The results showed that, in contrast to the control group cells, the scabertopin-treated cells were swollen with vacuolated cytoplasm, had severely swollen mitochondria, had a lack of membrane blebbing, had partially dissolved or absent organelles, and showed perforation of cell membranes (Figure 4A). These morphological changes are consistent with the characteristics of necroptosis [32]. We examined the expression of necroptosis-related proteins using Western blotting, and the expressions of phosphorylated RIP1, RIP3 and MLKL significantly increased in the J82 cells treated with scabertopin (Figure 4B). NAC significantly inhibited the expressions of phospho-RIP1, phospho-RIP3, and phospho-MLKL (Figure 4C).

#### *3.5. Scabertopin Inhibits the Migration and Invasion of Bladder Cancer Cells*

We performed wound healing and transwell assays to characterize how scabertopin affected the migratory and invasive abilities of bladder cancer cells. To this end, 24 h treatment with scabertopin inhibited the wound healing ability of J82 cells in a dosedependent manner (Figure 5A). Similarly, the results of the transwell assays also showed that, after scabertopin treatment, the invasive ability of cells significantly reduced. This effect was negatively correlated with the concentration of scabertopin (Figure 5B). In addition, we further detected the expressions of motor-related molecules p-FAK, FAK, p-PI3K, PI3K, p-AKT, AKT, and MMP-9 after scabertopin treatment by performing Western blot analysis. The results showed that the expressions of phospho-FAK (Tyr397), phospho-PI3K (Tyr607), phospho-AKT (Ser472, Ser473, Ser474), and MMP-9 decreased significantly after scabertopin treatment in a dose-dependent manner (Figure 5C). Therefore, we suggest that scabertopin can downregulate the expression of MMP-9 by inhibiting the activation of the FAK/PI3K/Akt signaling axis and, ultimately, inhibit the invasiveness of bladder cancer cells. Similarly, we sought to understand the role of ROS in this process. We found that the expressions of phospho-FAK, phospho-PI3K, phospho-Akt, and MMP-9 were significantly inhibited by NAC (Figure 5D). This indicated that ROS played a very important role in scabertopin-mediated invasion and metastasis of J82 cells. These results suggested that scabertopin may be a multifunctional inhibitor for the treatment of bladder cancer.

**Figure 3.** Cell death induced by scabertopin treatment can be rescued by NAC. (**A**) SEM images show that, in J82 cells treated for 24 h with 10 μM of scabertopin, the cell spreading area increases and the cell pseudopods shorten and thin out. The 24 h scabertopin treatment induces cell cycle arrest (**B**) and death (**C**) in J82 cells in a concentration-dependent manner (the black arrowheads in Figure 3B indicate the values of the peaks). The live/death cell assay shows scabertopin inhibits the viability of J82 cells after 24 and 48 h of treatment (**D**), and this effect can be reduced by NAC treatment (**E**,**F**). The expression levels of the apoptosis-related proteins caspase-9, caspase-8, caspase-3, Bax, and Bcl-2 in J82 cells do not significantly change after 24 h of scabertopin treatment. (**G**). Scabertopin does not significantly alter the expression of the ferroptosis- and pyroptosis-related proteins GPX4 and GSDMD, respectively, in J82 cells after 24 h treatment. A total of 5 μM of NAC was used in the experiments, \* means nonspecific bands. Data represent the mean ± s.e.m. of the three independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, ns: no significance vs. 0 μM scabertopin-treated group. The grayscale values are normalized to GAPDH. Bax: Bcl-2-associated X; Bcl-2: B cell lymphoma-2; GPX4: glutathione peroxidase 4; GSDMD: Gasdermin-D; NAC: *N*-acetylcysteine; PE-A: phycoerythrin area; PerCP-Cy5-5-A: Peridinin-Chlorophyll-Protein Complex-Cyanine5.5 area; SA: scabertopin.

**Figure 4.** Scabertopin-treated J82 cells undergo necroptosis, which can be rescued by NAC. (**A**) TEM images are shown for the control group (CON) and the J82 cells treated with 10 μM of scabertopin (SA) for 24 h. The image on the right is an enlarged image of the left image, and the red arrow points to the perforation on the cell membrane during necroptosis; at least three independent samples were observed in each group. (**B**) Scabertopin can significantly increase the phosphorylation of the necroptosis-related proteins RIP1, RIP3, and MLKL (n = 3) after 24 h of treatment; (**C**) Scabertopininduced phosphorylation of RIP1, RIP3, and MLKL can be reversed by NAC in the group treated with 10 μM of scabertopin for 24 h. A total of 5 μM of NAC was used in the experiments. The grayscale values are normalized to GAPDH. Data represent the mean ± s.e.m. of the three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, ns: no significance vs. 0 μM scabertopin-treated group. MLKL: mixed lineage kinase like; NAC: N-acetylcysteine; p-: phosphorylated; RIP1: receptorinteracting protein.

**Figure 5.** Scabertopin treatment inhibits the migratory and invasive ability of J82 cells by inhibiting the FAK/PI3K/Akt/MMP-9 signaling pathway, which can be rescued by NAC. Scabertopin significantly inhibits the migration ability (difference in area reduction between red lines) (n = 3) (**A**) and invasion of (n = 5) (**B**) of J82 cells treated with gradient concentrations of scabertopin for 24 h. (**C**) Phospho-FAK, phospho-PI3K, phospho-Akt, and MMP-9 expression levels significantly decrease in J82 cells after 24 h of scabertopin treatment. (**D**) Scabertopin-induced phosphorylation of p-FAK, p-PI3K, p-Akt, and MMP-9 expression can be reversed by NAC. A total of 5 μM of NAC was used in the experiments. The grayscale values are normalized to GAPDH. Data are presented as the mean ± s.e.m. of the three independent experiments (n = 3). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, ns: no significance vs. 0 μM scabertopin-treated group. AKT: Akt-protein kinase B; CON: control group; FAK: focal adhesion kinase; MMP-9: matrix metalloprotease-9; NAC: N-acetylcysteine; p-: phosphorylated; PI3K: phosphoinositide 3-kinase; SA: scabertopin.

#### **4. Discussion**

Despite extensive advances in the treatment of bladder cancer, it remains one of the most recurring and life-threatening tumors. Natural herbs are an important source of potential anticancer compounds in the field of drug discovery and development [33]. Natural compounds themselves contain unique structurally diverse molecules with multiple targets, making them ideal candidates for drug discovery and development.

Scabertopin, a sesquiterpene compound extracted from *E. scaber L*, has attracted interest because of its promising antitumor effects. In the present study, the anti-proliferative ability of scabertopin against various bladder cancer cell lines and human ureteral epithelial immortalized cells (SV-HUC-1) was assessed using the CCK-8 assay. Our results showed that scabertopin significantly inhibited the viability of bladder cancer cells in a dose-dependent manner but had a weak effect on the viability of noncancerous SV-HUC-1 cells. The above results suggested that scabertopin may be a potentially useful agent for bladder tumor treatment. In addition, we also found that J82 cells treated with scabertopin displayed a decrease in ΔΨ and an increase in superoxide anion production. ROS are a different class of molecular oxygen derivatives produced during normal aerobic metabolism. They include peroxides, superoxides, singlet oxygen, and free radicals. ROS levels are higher in different types of cancer cells than in normal cells. However, further elevation of ROS levels increases the susceptibility of cancer cells to oxidative stress-induced cell death [34,35]. Natural active ingredients and their derivatives are one of the main sources of antitumor drugs [7], and studies have shown that natural products can exert antitumor effects by increasing ROS levels. For example, isoalantolactone induces the elevation of

ROS levels in human pancreatic ductal epithelial carcinoma PANC-1 cells, arrests these cells in the S phase, thereby inhibiting cell proliferation and inducing apoptosis [36]. We used flow cytometry to detect the effect of scabertopin on the cell cycle of J82 cells. Our results demonstrated that scabertopin could induce cell cycle arrest at the S and G2/M phases in a concentration-dependent manner. In addition, ROS can act as signaling molecules and play a key role in the drug-induced inhibition of tumor invasion and metastasis. In the present study, we likewise observed that scabertopin significantly inhibited cell migration and invasive ability. Cell migration and invasion are key phenotypes that affect the metastasis of tumors. FAK is a cytoplasmic nonreceptor protein tyrosine kinase, a member of the adhesion patch complex family, and an important regulator that mediates cell adhesion to the extracellular matrix (ECM) [37]. The upregulation and hyperphosphorylation of FAK expression have been shown to increase the invasive capacity of several malignancies, including gastric and breast cancers, whereas the inhibition of FAK activity significantly reduces the migration capacity of breast cancer cells [38]. Tyr397 is the major phosphorylation site of FAK and phosphorylation of FAK leads to tumor metastasis and disease progression by promoting migration and invasion [39]. We found that scabertopin could inhibit the expression of MMP-9 in J82 cells by inhibiting the FAK/PI3K/Akt signaling pathway, and the inhibition could be rescued by NAC. MMP-9 is a member of the MMP family and a key enzyme necessary for the degradation of the ECM. The ECM is the first barrier that restricts tumor cells from undergoing migration. Activated MMP-9 can degrade the ECM and basement membrane components, allowing tumor cells to break through the primary site and become invasive and metastatic [40]. Therefore, we propose that scabertopin inhibits the pathway of FAK/PI3K/Akt/MMP-9 axis, which in turn inhibits cell migration and invasion.

In the present study, we found that scabertopin could induce the production and accumulation of ROS in J82 cells, which was identified as superoxide anion-dominated mitochondrial ROS (Figure 2). ROS is one of the important mechanisms that cause ferroptosis in cells, and the level of intracellular ROS accumulation is often positively correlated with the severity of ferroptosis [41]. However, in this study, although we found an increase in ROS, there was no difference in the expression of ferroptosis-related molecules (Figure 3). This phenomenon may be related to the type of ROS. They can cause cell death by damaging DNA, RNA, and lipid molecules [42]. During ferroptosis, the accumulation of lipid peroxides, especially phospholipid peroxides, is considered to be a landmark event of ferroptosis and also a prerequisite for ferroptosis [43]. Accordingly, some scholars refer to lipid peroxides that can specifically cause ferroptosis, such as arachidonoyl (AA)-phosphatidylethanolamine (PE) and adrenoyl (AdA)-PE, as ferroptosis-specific lipid peroxidation [44]. However, this is not rigid, because hydrogen peroxide in the presence of iron ions can be converted into hydroxyl radicals through the Fenton reaction, which in turn oxidizes lipids to form lipid peroxides [45]. While levels of iron, ironcontaining proteins [46], and lipid peroxides [47] also promote necroptosis, GPX4 can prevent RIP3-dependent necroptosis in erythroid precursor cells by avoiding lipidic ROS accumulation [48]. Another major difference between ferroptosis and necroptosis is that the cellular morphology of ferroptosis is very unique. Unlike apoptosis or necroptosis, the morphological features of ferroptosis are mainly changes in mitochondrial structure without the shrinkage, rupture, and perforation of the plasma membrane [32]. In tumor cells, the original level of ROS is higher than that of ordinary cells, but abnormally high levels of ROS can also induce different forms of cell death [49]. The occurrence of necroptosis is closely related to the overproduction of ROS [44]. Although the mechanism of ROS in necroptosis is not fully understood, there is a lot of evidence that ROS play a crucial role in many drugs-induced necroptosis [50,51], which is accompanied by mitochondrial injury and decreased expression of MMPs [52]. The key molecules in necroptosis, RIP1 and RIP3, are most closely related to mitochondrial ROS. For example, mitochondrial ROS can mediate the autophosphorylation of RIP1, which subsequently induces necroptosis by recruiting and promoting the phosphorylation of RIP3, suggesting that mitochondrial

ROS are the initiators of necroptosis [28]. On the other hand, it has been observed in hepatic stellate cells that the activation of RIP1/RIP3 not only promotes necroptosis, but also increases ROS production via a positive feedback loop [22]. Mediating necroptosis by drug-induced ROS has emerged as a potential approach for tumor therapy because the occurrence of necroptosis not only bypasses apoptosis [53,54], but also causes death in apoptosis-resistant cancer cells. Second, this mechanism of necroptosis induced by ROS can occur in most common cancers. This is consistent with the phenomena we observed, such as membrane perforation and mitochondrial shrinkage, in the scabertopin-treated J82 cells using TEM. Flow cytometry showed that the treatment of scabertopin could increase the number of late apoptotic, necrotic, and dead J82 cells in the Q2 region. However, the expression of apoptosis-related caspase protein, Bcl-2, and Bax displayed no significant difference, which was the same as for ferroptosis. However, necroptosis-related phospho-RIP1, phospho-RIP3, and phospho-MLKL were significantly upregulated by scabertopin treatment and could be inhibited by NAC. Therefore, we believe that scabertopin can promote the increase of mitochondrial ROS by causing a decrease in mitochondrial membrane potential, thereby stimulating the phosphorylation and activation of RIP1/RIP3/MLKL and, finally, triggering necroptosis (Figure 6).

**Figure 6.** Mechanism of scabertopine inhibiting invasion and promoting necroptosis in human bladder cancer J82 cells. Scabertopin can reduce mitochondrial membrane potential and stimulate mitochondrial ROS production, thereby activating RIP1/RIP3/MLKL phosphorylation, mediating J82 cell necroptosis, and inhibiting the FAK/PI3K/Akt/MMP-9 signaling pathway. This in turn inhibits the migration and invasive potential of J82 cells. Akt: Akt-protein kinase B; FAK: focal adhesion kinase; MLKL: mixed lineage kinase like; MMP-9: matrix metalloprotease 9; NAC: *N*-acetylcysteine; PI3K: phosphoinositide 3-kinase; p-: phosphorylated; RIP: receptor-interacting protein.

In fact, previous studies have reported the phenomenon of sesquiterpenoid-induced ROS increase. For example, Verma et al. found that isodeoxyelephantopin and deoxyelephantopin can inhibit the activation of NF-κB by inducing the production of ROS and inhibit the growth of breast cancer [55]. Xanthatin, a sesquiterpenoid derived from Xanthium strumarium L, may induce the elevation of ROS, mitochondrial injury, and apoptosis in non-small cell lung cancer (NSCLC) [56]. The ability of sesquiterpenes to induce ROS generation may stem from the fact that they both have α-methylene-γ-lactone and [57] a cyclopentenone ring-like structure (butenolide in scabertopin) (Figure 1A) [58]. The

most direct mechanism of sesquiterpenoid-induced ROS elevation may be related to its ability to change the mitochondrial ΔΨ [59]. In fact, there are a variety of ROS inducers that directly act on the mitochondrial voltage-dependent anion channel, resulting in a change in ΔΨ, a decrease in glycolysis, and an increase in ROS [60,61]. From this, we speculate that these unique structures of sesquiterpenes, namely α-methylene-γ-lactone and cyclopentenone ring-like structures, may affect the changes in mitochondrial ΔΨ and cause the increase in ROS. Scabertopin not only has the capacity to trigger this mechanism owing to its unique structure but also meets the rules of five in terms of pharmacokinetics. Since the molecular weight (MW) of SA is 358.4 Da, the logarithm of lipid water partition coefficient (LogP) is 2.6, the hydrogen bond donor count is 0, the hydrogen bond acceptor count is 6, and the rotatable bond count is 3 [62]. These characteristics of SA well meet the Lipinski rules, which requires the MW to be less than 500 Da, the LogP to be between −2 and 5, the hydrogen bond donor count to be less than 5, and the count of hydrogen bond acceptor and the rotatable bond to be less than 10 [63]. In our study, we found that scabertopin significantly reduced ΔΨ, promoted ROS generation, and increased intracellular ROS accumulation by depleting GSH in J82 cells. ROS-induced necroptosis is likely to be associated with decreased GSH [64]. The decrease in GSH, the most important antioxidant-reduction factor in cells, is the result of ROS-induced GSH oxidation, and ROS production is further enhanced by depleting GSH [65]. NAC can supplement GSH by donating cysteine, thereby exerting anti-ROS effect [66]. NAC-treated cells also promote mitochondrial integrity through multiple mechanisms, maintain mitochondrial function, and reduce ROS production, thereby protecting cells from necroptosis [67].

However, we did not evaluate the therapeutic efficacy of scabertopin in vivo, and its pharmacokinetics and pharmacodynamics in vivo remain unclear. Therefore, further studies are needed to provide additional evidence for the use of scabertopin as a chemotherapeutic agent or adjuvant in the chemotherapy of bladder cancer.

#### **5. Conclusions**

We demonstrate that scabertopin can deplete GSH in bladder cancer cells and cause ROS elevation and accumulation by reducing ΔΨ. It also inhibits bladder cancer cell migration and invasion by targeting the FAK/PI3K/Akt/MMP-9 axis. Furthermore, scabertopin can mediate necroptosis of bladder cancer cells by activating the RIP1/RIP3/MLKL pathway through phosphorylation and inhibit the proliferation and viability of bladder cancer cells. Using the ROS scavenger NAC can not only reduce cell death caused by scabertopin but also rescue J82 cells from scabertopin-mediated necroptosis and limit their invasive and migratory ability by inhibiting the phosphorylation activation of the two abovementioned signaling pathways. In conclusion, our study provides new insights into the anti-bladder cancer mechanisms of scabertopin, suggesting that scabertopin may be a potential alternative drug for bladder cancer therapy.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14235976/s1, File S1: Original blots.

**Author Contributions:** Experiments and data collection were performed by Y.G., Z.N. and H.C. Data analysis was performed by Y.G., Z.N., H.C., M.C., Y.X. and D.H. The study was designed by Y.G., Z.N., X.Y. and S.Z. The manuscript was written by Y.G. and Z.N. All authors revised the previous versions of the manuscript. Y.G. and Z.N. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Finance Science and Technology Project of Hainan Province (grant numbers 820QN423, 821QN424, and ZDYF2021SHFZ096), the National Nature Science Foundation of China (grant No. 82160531), and the Health Industry Scientific Research Project of Hainan Province (grant numbers 21A200412 and 21A200149).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data can be shared upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.
