Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.


Introduction
Osteosarcoma (OS) is the most frequent bone tumour in children and adolescents, in addition to being the most common aggressive malignancy originating from mesenchymal cells [1,2]. Although neoadjuvant chemotherapy combined with surgery and postoperative chemotherapy has been used to treat OS, the five-year survival rate of OS is not satisfactory on account of development of early metastases and chemotherapy resistance [3,4]. Thus, it is imperative to find new targets and pharmaceuticals to improve the prognosis.
Ferroptosis is a new form of regulated cell death (RCD) that is mediated by intracellular iron and is quite different from other forms of cell death, such as apoptosis, autophagy, or necrosis [5]. Intracellular iron reacts with H 2 O 2 through Fenton reaction (Fe 2+ + H 2 O 2 →Fe 3+ + HO·+ OH -) to gener-ate many reactive oxygen species (ROS) and trigger lipid peroxidation (LP) to induce ferroptosis [6]. Aberrant iron metabolism, ROS generation, and abnormal LP are the hallmarks of ferroptosis [5,7]. Morphologically, ferroptotic cells demonstrate shrunken mitochondria, higher density of the mitochondrial membrane, and reduced or diminished mitochondrial cristae [8]. Additionally, ferroptosis can be inhibited by iron chelators or antioxidants and activated by some small compounds (erastin), or by inhibiting glutathione peroxidase-4 (GPX4) [9]. The cystine/glutamate antiporter system Xc -, which is composed of two subunits, SLC7A11 (light chain) and SLC3A2 (heavy chain), is closely linked to ferroptosis [10]. SLC7A11, which transports cystine into the cells, enhances glutathione (GSH) synthesis, further promoting the inhibition of ferroptosis by GPX4. P53, as an upstream mediator of SLC7A11, mediates the repression of SLC7A11 to initiate ferroptosis in tumour cells [11]. In addition to breast cancer, colorectal cancer, and anaplastic thyroid cancer [12][13][14], ferroptosis also exerts anticancer effects in OS [15,16]. Some clinical drugs including sulfasalazine and sorafenib have proven to induce ferroptosis to trigger anticancer effects [17], via inhibiting the system Xc - [18,19]. Therefore, activating P53 to downregulate SLC7A11/GPX4 to trigger ferroptosis could be a potential method to inhibit progression of OS cells.
Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family and is a vital transcription factor involved in inflammation and tumour progression [20]. Classical STAT3 activation involves the phosphorylation of STAT3 at Tyr705 (p-STAT3 (705)), which interacts with and inhibits P53 [21]. Wang et al. discovered that high expression of STAT3 is relevant to increased malignancy and poor prognosis [22]. As described above, it is critical to inactivate STAT3 to halt tumour progression.
Flavonoids have multiple biological functions, especially anticancer effects induced by the prooxidant activity [23]. Amentoflavone inhibits glioma cells via inducing ROS accumulation and ferroptosis [24]. Robustaflavone also induces ferroptosis by increasing ROS and lipid peroxidation in MCF-7 cells [25]. Bavachin belongs to flavonoids and is a bioactive compound extracted from the fruit of Psoralea corylifolia and displays various functions including anti-inflammatory, lipidlowering, and cholesterol-reducing effects [26]. A previous study showed that bavachin stimulates osteoblast differentiation by activating the Wnt pathway and is considered an estrogen supplement [27]. Recently, it has been shown that bavachin also exerts antitumour effects. Bavachin has been demonstrated to inhibit human hepatocellular carcinoma cells by inducing apoptosis, accompanied by ROS accumulation [28]. It has been reported that bavachin inhibits melanoma cells by downregulating the MAPK signaling pathway [29]. Notably, bavachin was shown to trigger the apoptosis of multiple myeloma cells by inhibiting p-STAT3 (705) and increasing P53 [30]. Although it has been reported that bavachin can exacerbate ROS accumulation and suppress several tumour cells, it is unclear whether ferroptosis could be induced by bavachin, and if so, what is the underlying mechanism?
We hypothesized that bavachin increases intracellular ferrous iron, ROS, and malondialdehyde (MDA) levels and induces ferroptosis by the downregulation of SLC7A11 through inactivating STAT3 to upregulate P53. We hope that our research will provide promising pharmaceutical targets for the treatment of OS.

Cell
Culture. The human OS cell lines MG63 and HOS were purchased from Procell (China). The cells were cultured in a humidified CO 2 incubator at 37°C and 5% CO 2 and grown in DMEM/high-glucose (Gibco, USA) supplemented with 10% foetal bovine serum (MRC, China).

Cell Viability.
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) (APExBIO, China). According to the protocol, the absorbance values of the samples were read at 450 nm using a fluorescence microplate reader (Varios-kan&LUX, Thermo Fisher, China). Subsequently, the cell death ratio was calculated using the formula: cell death ratio ð%Þ = ðA sample − A blank Þ/ðA control − A blank Þ × 100.

Transmission Electron Microscope (TEM)
. MG63 and HOS cells were collected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) at room temperature for 24 h. Then, the cells were fixed in 2% osmium tetroxide in PBS at room temperature for 1 h. Next, the samples were dehydrated with a graded series of ethanol solutions (50% to 100%). Subsequently, the samples were embedded in epoxy resin and sectioned into ultrathin sections (60 nm). Finally, the ultrathin sections were steeped with uranyl acetate (1%) and lead citrate (0.1%) and observed and photographed using an H-7500 TEM (Hitachi, Japan).

Mitochondrial Membrane Potential (MMP) Detection.
MMP was observed using JC-1 assay kit (Beyotime, China). According to the protocol, the cells were stained with the JC-1 probe and observed under a fluorescence microscope (DMIL4000, Leica, China). Green fluorescence indicates a low MMP, whereas red fluorescence indicates a high MMP.
2.6. Ferrous Iron Assay. Cellular ferrous iron levels were detected using FerroOrange (Dojindo, China). According to the protocol, the cells were incubated with FerroOrange for 0.5 h. Fluorescence intensity (Ex: 543 nm, Em: 580 nm) was assessed using a fluorescence microplate reader (Varios-kan&LUX, Thermo Fisher, China). The ferrous iron levels were finally expressed as a ratio to the fluorescence intensity value of the control.
2.7. ROS Assay. Cellular ROS levels were measured using a commercial ROS assay kit (Beyotime, China). According to the protocol, the cells were loaded with a 2,7-dichlorodihydrofluorescein diacetate probe (10 μM). The cells were then incubated at 37°C for 20 min. Finally, the fluorescence intensity (Ex: 488 nm, Em: 525 nm) was assessed using a fluorescence microplate reader (Varioskan&LUX, Thermo Fisher, China).  3 Oxidative Medicine and Cellular Longevity for 5 min. Each protein sample (40 μg) was loaded onto an SDS-PAGE (10%-12%) gel and transferred to PVDF membranes. The membranes were blocked with NcmBlot Blocking Buffer (NCM Biotech, China) and incubated with the primary antibodies at 4°C overnight. The membranes were subsequently incubated with the secondary antibody at room temperature for 1.5 h. Finally, the bands were developed using a HyperSignal ECL kit (4A Biotech, China).

Cell Transfection.
For evaluating the overexpression of STAT3 and SLC7A11, an overexpressing plasmid along with a negative control (NC) plasmid was purchased from Vigene Biosciences (China). Briefly, cells were transfected with the plasmids using Lipofectamine 2000 (Invitrogen, China). After 48 h, the cells were subjected to subsequent experiments.
2.12. Statistical Analysis. Data are presented as means ± SD (n = 3). One-way ANOVA and Tukey's multiple comparisons test were used to analyze the data. All statistical analyses were performed using GraphPad Prism 7 (https://www .graphpad.com/). Statistical significance was set at p < 0:05. respectively. Therefore, 40 μM of bavachin concentration was selected for the following experiments. Morphological changes in MG63 and HOS cells following bavachin treatment were observed. Light microscopy images showed that the shape of the cells became round, and there was an increase in cell shrinkage and cell death with increasing concentration of bavachin (Figure 1(c)).    (Figure 3(a)). Moreover, in fluorescence images of JC-1 staining, green fluorescence was observed in the bavachin (40 μM for 24 h) group and red fluorescence in the control group (Figure 3(b)). Bavachin treatment reduced the MMP of OS cells compared with that of the control group. Finally, to clarify whether bavachin induced ferroptosis, OS cells were pretreated with ferroptosis inhibitors Fer-1 (5 μM), Lip-1 (2 μM), and Vit E (150 μM) for 1 h. As shown in Figure 3(c), the results of the CCK8 assay showed that Fer-1, Lip-1, and Vit E rescued bavachin-induced OS cell death.

Bavachin Induces GSH Depletion and LP Accumulation
in OS Cells. The levels of GSH, ROS, and MDA were measured to determine whether bavachin induces oxidative stress in OS cells. It was found that the level of GSH declined gradually as bavachin concentration (10 μM, 20 μM, and 40 μM) increased over 24 h (Figures 4(a) and 4(b)). However, ROS levels were remarkably upregulated by bavachin stimulation (Figures 4(c) and 4(d)). Moreover, ROS levels reached their highest values at 24 h of 40 μM bavachin stimulation ( Supplementary Figure 1(c) and 1(d)). As illustrated in Figures 4(e) and 4(f), bavachin markedly elevates the MDA level, which is a marker of LP, in a concentrationdependent manner for 24 h. To further analyze the molecular mechanism of bavachin-induced LP in OS cells, western blotting was performed to detect the expression of related proteins. The results demonstrated that bavachin inhibited GPX4, SLC7A11, and p-STAT3 expressions and increases P53 expressions in OS cells (Figures 4(g) and 4(h)).

P-STAT3 Activation Downregulates P53 Expression to
Rescue Bavachin-Induced Ferroptosis in OS Cells. To evaluate whether STAT3 inhibits P53 expression to alleviate bavachin-induced ferroptosis in OS cells, OS cells were transfected with a STAT3 overexpressing plasmid, followed by treatment with 40 μM bavachin for 24 h. As shown in Figures 7(a) and 7(b), compared with that in the NC group, p-STAT3 was activated, although the p-STAT3/STAT3 ratio was unchanged. p-STAT3 activation inhibited the upregulation of P53 expression induced by bavachin, while increased GPX4 expression suppressed by bavachin. p-STAT3 activation recovered GSH depletion (Figure 7(c)) and decreased     Oxidative Medicine and Cellular Longevity the accumulation of ROS and MDA, which is stimulated by bavachin (Figures 7(d) and 7(e)). Moreover, p-STAT3 upregulation rescued bavachin-induced ferroptosis in OS cells (Figure 7(f)). As described above, p-STAT3 activation inhibits P53 expression and rescues bavachin-induced ferroptosis in OS cells.

Discussion
Our study revealed that bavachin could induce OS cell death, which was reversed by iron chelator (DFO) and ferroptosis inhibitors (Fer-1, Lip-1, and Vit E). Moreover, bavachin reduced the MMP and led to mitochondrial shrinkage, increased mitochondrial membrane density, and reduced mitochondrial cristae in OS cells. Furthermore, bavachin elevated intracellular ferrous iron levels by increasing TFRC and DMT1 expression and decreasing FTH and FTL expressions. Bavachin also reduced SLC7A11 and GPX4 expression and promoted ROS and MDA accumulation by downregulating p-STAT3 to upregulate P53 expression (Figure 8).
As a new form of RCD, ferroptosis significantly differs from other types of cell death, which is due to characteristics of iron-dependence, GPX4 inhibition, and abnormal LP [5]. Moreover, ferroptosis is rescued by iron chelators and antioxidants [31,32]. Our study consistently showed that bavachin increases ferrous iron levels and induces OS cell death, which can be rescued by Fer-1, Lip-1, Vit E, and DFO. After treatment with bavachin, morphological changes in OS cells are consistent with the classical ferroptosis morphology, which is characterised by a smaller mitochondria, higher mitochondrial membrane density, and reduced mitochondrial cristae [8]. These findings indicate that bavachin induces ferroptosis in OS cells.
Aberrant intracellular iron metabolism, especially ferrous iron overloading, is an initiating factor for ferroptosis. TFRC and DMT1 mediate ferrous iron formation and transport [33]. Previous studies suggest that downregulation of TFRC expression inhibits ferroptosis [34], and overexpression of TFRC could increase iron levels to increase ferroptosis sensitivity [35]. Similar to TFRC, DMT1 is also a positive regulator of iron accumulation, leading to ferroptosis [36]. Consistent with previous studies, our findings show that TFRC and DMT1 expressions are positively related to ferroptosis induced by bavachin. Additionally, ferritin, composed of FTL and FTH, also serves as a critical mediator of iron storage and a regulator of iron homeostasis. Previously published studies have revealed that ferritin has a two-sided effect on ferroptosis. For example, overexpression of ferritin inhibits ferroptosis in PC-12 cells via ferritinophagy [37], whereas ferroptosis occurs in glioma cells by upregulating FTL and FTH expression [38]. Although the expression of FTH and FTL in ferroptosis is diverse, our results demonstrate that bavachin downregulates FTL and FTH expressions to promote ferroptosis, which can probably be ascribed to ferritinophagy-degrading ferritin to release Fe 2+ that is involved in ferroptosis progression [9].
Triggering LP, which is described as a process by which ROS attacks polyunsaturated fatty acids to cause dehydrogenation, is an essential event that drives ferroptosis [39]. Both ROS, generated from iron in the Fenton reaction, and MDA, the final product of LP, are contributing factors for ferroptosis activation [40]. Bavachin has been demonstrated to promote ROS generation via suppressing the MMP to trigger HepG2 cell apoptosis [28]. However, whether bavachin induces MDA has not been reported in the literature. Similarly, our results suggest that bavachin elevates the level of ROS in a dose-dependent manner and suppresses the MMP. Moreover, we found that bavachin distinctly increased the MDA content to promote ferroptosis.
The SLC7A11/GPX4 axis efficiently protects cells from ferroptoss [41]. SLC7A11, a key subunit of the system Xc-, is responsible for transporting cystine, which is reduced to cysteine for synthesis of GSH. As a vital nonenzymatic antioxidant, GSH not only scavenges free radicals but also acts as a

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Oxidative Medicine and Cellular Longevity cofactor of the GPX4-mediated reduction reaction of lipid hydroperoxides (LOOH). Inhibition of SLC7A11/GPX4, which is the most important antioxidant enzyme in the body, can trigger ferroptosis, while activation of SLC7A11/GPX4 facilitates the suppression of ferroptosis [42]. Shi et al. reported that tirapazamine induced ferroptosis in OS cells via downregulation of SLC7A11 and GPX4 expression, whereas the phenotype of cells induced by tirapazamine was reversed after overexpression of SLC7A11 [43]. Lin et al. also found that EF24, a synthetic drug, triggered ferroptosis in OS cells via inhibiting GPX4 [16]. Similarly, our results showed that bavachin led to ferroptosis in OS cells with accumulation of ROS and MDA, GSH depletion, and downregulation of GPX4 expression, while overexpression of SLC7A11 rescued OS cells from ferroptosis caused by bavachin. Therefore, we believe that bavachin triggers OS cell ferroptosis via the SLC7A11/GPX4 axis.
P53 is known as a tumour suppressor protein and can inhibit tumour cells by triggering ferroptosis [44]. Jiang et al. discovered that P53 induced ferroptosis by negatively regulating SLC7A11 to inhibit cystine transport [11]. However, a few studies have reported a contrasting view about the function of P53 in ferroptosis. In colorectal cancer cells, P53 inhibited LP and ferroptosis by binding to DPP4, while the loss of P53 led to the recovery of DPP4 to increase cell ferroptosis [45]. Tarangelo

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Oxidative Medicine and Cellular Longevity some cells [46,47]. Therefore, the two-sided functions and mechanisms of P53 in ferroptosis need to be further explored in diverse extracellular environments and cell lines.
Our study showed that bavachin increased P53 expression and downregulated SLC7A11 expression to induce ferroptosis in OS cells. However, in OS cells pretreated with PFT-α, a P53 inhibitor, these effects were reversed. These findings suggest that bavachin triggers ferroptosis in OS cells by activating P53 to inactivate SLC7A11. STAT3 inactivation is also associated with ferroptosis. It has been reported that STAT3 inhibits P53 to facilitate tumour progression by binding to the promoter of p53, which is an important regulator of ferroptoss [21,48]. Qiang et al. found that increasing the expression of p-STAT3 could alleviate ferroptosis via SLC7A11 in MLE12 cells by regulating SLC7A11 [49]. In OS cells, overexpression of STAT3 aggravated ROS accumulation and ferroptosis via the Nrf2/GPX4 axis [50]. According to these studies, STAT3 appears to be a potential pharmacological target. Although bavachin was verified to inactivate p-STAT3 and increase P53 expression in multiple myeloma cells, the regulatory relationship between STAT3 and P53 has not yet been explored [30]. Our study displayed that bavachin triggers ferroptosis by inhibiting p-STAT3 but not STAT3 and increases P53 expression; however, after OS cells were transfected with the STAT3 overexpression plasmid, which simultaneously increased STAT3 and p-STAT3 expressions, bavachin no longer induced ferroptosis. It has been suggested that bavachin enables ferroptosis by inhibiting STAT3 to upregulate P53 in OS cells.
Taken together, our study displayed that bavachin triggers ferroptosis in OS cells by increasing intracellular ferrous iron levels and inhibiting the STAT3/P53/SLC7A11 axis. It suggests that bavachin could be a promising pharmaceutical for the treatment of OS.

Data Availability
The data of this study is included within the article. The data is available from the first author (royalman1984@sina.cn) upon request.