Paeoniflorin Regulates NEDD4L/STAT3 Pathway to Induce Ferroptosis in Human Glioma Cells

Background Paeoniflorin is an active component of a widely used traditional Chinese medicine with antitumor activity through ferroptosis induction. It has been reported recently that ferroptosis is emerging in certain types of cancer; however, its relevance in glioma is still not well studied. Methods CCK8 assay was performed for cell proliferation. Expression of mRNA and protein was tested by qPCR and western blot, respectively. Clinical section samples were detected by IHC. The relationship between NEDD4L and STAT3 was validated by a coimmunoprecipitation assay. Apoptosis was identified by TUNEL assay. A xenograft mouse model was utilized to validate the potential of paeoniflorin toward glioma cancer cells. Results The data suggested that paeoniflorin could increase NEDD4L expression in glioma cells. The NEDD4L expression level was lower in glioma cancer tissues compared to adjacent normal tissues, and it correlates with poor prognosis. Meanwhile, NEDD4L mediates the ubiquitination of STAT3. Furthermore, increased NEDD4L significantly inhibited cell viability and induced accumulation of intracellular ROS levels, accompanied by decreased expression of key ferroptosis factors Nrl2 and GPX4, while NEDD4L knockdown had a reverse effect, suggesting that ferroptosis could be involved. NEDD4L-induced ferroptosis could be rescued by forced expression of STAT3. A xenograft nude mouse model showed that paeoniflorin inhibits tumor growth and further sensitizes glioma cells to RSL3, another well-known ferroptosis inducer. Conclusions In summary, this study demonstrated that paeoniflorin might function as an effective drug for glioma by inducing ferroptosis via upregulation of NEDD4L and repression of Nrl2, GPX4, and STAT3.


Introduction
Glioma is the most common primary brain tumor derived from the human central nervous system [1]. Although great eforts have been made to improve the prognosis of glioma in the past 40 years, limited advancement has been achieved, especially in patients with high malignancy [2]. New modalities of cancer therapy targeting glioma has always been essentially needed. Our recent discovery demonstrated that paeoniforin (PF) shows promising antitumor potential in glioma cells [3]. PF is the active ingredient of Paeonia lactifora Pall. (aka P. alba), a traditional Chinese medicine. Recent studies have shown that PF may be an efective antitumor drug for various types of cancers. For example, PF induces cell cycle arrest in HT29 colorectal cancer through p53/14-3-3 zeta pathway [4]. In addition, studies have found that PF can enhance 5-fuorouracil-induced apoptosis in human gastric cancer cells by inhibiting the NF-κB pathway [5].
So far, the mechanism of how PF functions in glioma has not been reported. However, our previous study suggested that the ubiquitination level of STAT3 was elevated in PFtreated glioma cells [3]. Te signal transducer and activator of transcription 3 (STAT3) is well studied as a transcription factor that couples extracellular stimulus signals and gene expression. It is known that STAT3 is involved in multiple vital cellular processes such as cell cycle, angiogenesis, and apoptosis [6,7]. As an important cancer protein during tumorigenesis, the function of STAT3 has been studied extensively [8][9][10]. It has been reported that the expression as well as activation of STAT3 are also associated with the low survival rate and poor prognostic outcome of glioma patients [11,12]. Terefore, STAT3 is apparently an attractive target for the treatment of glioma. It has been demonstrated that inhibition of STAT3 through various approaches can trigger growth arrest as well as apoptosis of glioma cells [13][14][15]. In addition, it has been reported that STAT3 enhances the antioxidant capacity of cells by activating the nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is a transcription factor that prevents oxidative damage by regulating the expression of multiple antioxidant proteins [16]. It has been reported that reactive oxygen species (ROS) can cause aberrant oxidation of molecules such as proteins, lipids, and DNA. Terefore, cancer cells evolved to develop an antioxidant defense system to handle elevated ROS levels in order to successfully establish tumors, which makes the efcient evasion of cell death one of the most important hallmarks of cancer [17][18][19]. As a nonapoptotic form of regulated cell death relative to apoptosis, ferroptosic cells are typically necrosis-like morphologically with the iconic accumulation of lethal levels of iron-dependent lipid ROS biochemically. Multiple oxidative and antioxidant mechanisms control the oxidative damage during ferroptosis. It has been reported that STAT3/Nrf2 signal pathway may be involved in the regulation of ferroptosis [20]. Cells undergoing ferroptosis are diferent from apoptosis, necrosis, and autophagy in terms of cell morphology, protein expression, as well as gene expression levels. Ferroptosis has gradually become a hot spot in scientifc research after the concept was frst proposed in 2012. According to the publications, Erastin and RSL3 are inducers of ferroptosis [21,22]. Glutathione peroxidase 4 (GPX4) is the key regulatory molecule for ferroptosis, and the reduced expression or activity of GPX4 can induce ferroptosis [22]. Terefore, we proposed to investigate whether ferroptosis is involved in PF-triggered cell death in glioma by examining the aforementioned factors.
On the other hand, given that PF can promote the ubiquitination of STAT3 according to our previous study [3], we sought to screen and identify the ubiquitinase (SYVN1, NEDD4L, CBL, and SOCS5) that bind and mediate ubiquitination of STAT3 based on our data analysis result. We found that PF signifcantly promotes the expression of human NEDD4L. As a member of an E3-ubiquitin protein ligase NEDD4 family, NEDD4L is expressed in various types of cancer cells and may have carcinogenic properties [23,24]. By analyzing the cells, array, and RNA sequencing data of TCGA glioma patients, we found that NEDD4L is expressed at a relatively low level in gliomas [25]. In addition, it has been reported that the downregulation of NEDD4L is associated with the progression and malignance of glioma [23]. Furthermore, overexpression of NEDD4L induces glioma cell death [25]. However, the mechanism of how NEDD4L drives these phenotypes are well known. Terefore, this project aims to study the efect and mechanism of PF/NEDD4L/STAT3 on the proliferation of glioma.

Immunoprecipitation (IP) Detection.
Te total proteins were grouped to incubate with 1 μg of Rabbit-IgG (Sc-2027, Santa Cruz Biotechnology) and 1 μg of IP-indicated antibody at 4°C overnight, while untreated proteins were used as an input control, followed by incubation of Protein A/G PLUS-Agarose for 2 hours at room temperature to form an immune complex. After centrifuging for 4 min at 3000 rpm at 4°C, 1 ml of lysis bufer was added to wash the Protein A/G Plus-Agarose beads 3 times, and appropriate protein loading bufer was added and boiled for 5 min to elute the immunoprecipitates. Following centrifugation 3000 rpm for 1 min, the supernatant was collected for western blot analysis. Te antibodies of anti-NEDD4L (Ab46521, Abcam) and anti-STAT3 (Ab32500, Abcam) were applied for IP detection, while anti-NEDD4L (13690-1-AP, Proteintech) and anti-STAT3 (10253-2-AP, Proteintech) were used in western blot analysis.

Western Blot Analysis.
To determine protein level, the sample cell lysates were separated with SDS polyacrylamide gels. Proteins of interest were transferred to polyvinylidene fuoride membranes which were then blocked with 5% nonfat milk for at least one hour. Te membranes were then blotted with optimized primary antibodies overnight at 4 C and secondary antibodies for one hour at room temperature, respectively. Protein abundance was assessed using a chemiluminescent imaging system (Tanon 5200, Shanghai, China). Anti-SYVN1 (Ab170901), anti-CBL (Ab32027), anti-SOCS5 (Ab97283), anti-NEDD4L (Ab46521), anti-GPX4 (Ab125066), anti-STAT3 (Ab68153), anti-p-STAT3 (Ab76315) supplied from Abcam, and anti-Nrf2 (16396-1-AP) and anti-GAPDH (60004-1-1G) obtained from Proteintech were used.  Journal of Oncology permeabilized with 3% H 2 O 2 (10011218, Shanghai Sinopharm) for 10 min, then were blocked in 1% bovine serum albumin (A8010, Solarbio) for one hour at room temperature. Afterwards, the tissue slices were incubated with primary antibodies against NEDD4L (13690-1-AP, Proteintech) and STAT3 (Abcam, ab68153) in a humidifying box for 1 hour at room temperature. Te tissue slices were rinsed with PBST thrice to remove any residual antibodies. Next, the tissue slides were incubated in HRP-labeled secondary antibodies (D-3004, Changdao) for 30 min at room temperature. Te slides were stained with DAB (FL-6001, Changdao), and then rinsed with tap water to remove the staining solution. Te slides were counterstained with hematoxylin (714094, BASO) for 3 min, followed by incubation with 1% hydrochloric acid for alcohol diferentiation. Te slides were then washed with tap water for 10 min and then subsequently subjected to a grill, transparent and closed, after which the slices were imaged under a microscope (ECLIPSE Ni, NIKON). Te IHC results were accessed by two experienced pathologists independently. Te staining signal was graded as 0, negative; 1, weakly positive; 2, moderate positive; and 3, strong positive. Te percentage of positive cells was scored as 0, < 5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; and 4, > 75%. Te staining index was defned as follows: staining index = staining intensity × percentage of positive staining cells. Te sample was categorized as a high expression if the staining index was higher than 3.
2.11. In Vivo Experiments. U251 cells (6 × 10 6 ) were inoculated into the fanks of 4-to 5-week-old athymic nude mice (Shanghai Laboratory Animal Company, Shanghai, China) subcutaneously to generate a subcutaneous xenograft tumor model. After 2 weeks, the tumor model was successfully constructed, the mice were treated single and combined with 100 mg/kg RSL3 (2 times/week) and 1.0 g/kg/ days PF. Tumor volumes were measured every 4 days to draw the growth curve. Mice were sacrifced 4 weeks after cell injection. Tumor xenografts were collected, photographed, and weighed and the tumor apoptosis was analyzed by Tunel staining. All care and experiment of the laboratory animals were performed following protocols approved by Huzhou Cent Hospital, Afliated Cent Hospital Huzhou University (Zhejiang, China).

Detection of Apoptosis.
To detect apoptotic cells, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin Nick end labelling (TUNEL) method was conducted. In brief, specimens were frst dewaxed and rehydrated, then incubated in proteinase K (40 μg/ml) at 37°C for one hour. Te slides were then treated with 3% H 2 O 2 (diluted in methanol) for 30 minutes at room temperature. After washing with PBS, sections were incubated with equilibration bufer for fve minutes at room temperature, then incubated with the terminal deoxynucleotidyl transferase enzyme at 37°C for two hours. Te sections were then incubated with 100 ul of stop bufer for 30 minutes at 37°C. Te slides were covered in antidigoxigenin peroxidase for 30 minutes at 37°C. Slides were then visualized with diaminobenzene and counterstained with hematoxylin. 500 cells in total were counted for each section. Te apoptotic index was calculated as a ratio of apoptotic cell number/total cell number times 100. Tumor specimens were categorized according to TUNEL staining into ≤10% or >10% stained cells.
2.13. Statistical Analysis. All statistical analyses in the current project were conducted in GraphPad Prism 7.0 software (San Diego, CA, USA). At least three independent repetitive experiments were performed for each result. Te data were presented as a mean value ± SD. One-way analysis of variance (ANOVA) with Tukey's posthoc tests were applied for the comparison of mean values. p < 0.05 was regarded as statistically signifcant.

Treatment of PF Signifcantly Increases NEDD4L Expression in Glioma Cells.
Our previous study demonstrated that PF can promote the ubiquitination of STAT3 [3]. To identify the potential ubiquitinase that carries out the STAT3 ubiquitination, at frst, we selected 4 candidates (SYVN1, NEDD4L, CBL, and SOCS5) that can bind with STAT3 through data analysis. And then the expression of these 4 candidates in U251 cells in response to PF treatment were examined at both mRNA and protein levels. We noticed that PF only promotes the expression of NEDD4L signifcantly in a dose-dependent manner without afecting the other 3 ubiquitinases (Figures 1(a) and 1(b)). To verify this observation, we examined the expression level of NEDD4L in another glioma cell line U87 upon the treatment of PF. In U87 cells, the expression of NEDD4L is increased gradually as the dosage of PF increases (Figures 1(c) and 1(d)). In addition, PF promotes the expression of NEDD4L in a time-dependent manner in glioma cells as well (Figures 1(e) and 1(f )). Taken together, our data suggest that PF may induce STAT3 ubiquitination by promoting the expression of NEDD4L.

NEDD4L is Lowly Expressed in Glioma and Associated with the Prognosis of Patients.
In order to explore the potential roles of NEDD4L in glioma, we frst compared the expression of NEDD4L in 168 cancer samples and 5 normal tissue from the TCGA database. Te data showed that NEDD4L is signifcantly downregulated in gliomas (Figure 2(a)). In addition, we carried out a gene set enrichment analysis (GSEA) to identify the signaling pathways that are signifcantly altered along with the level of NEDD4L. It turned out that NEDD4L negatively regulates the activation of the STAT3 pathway (Figure 2(b)). In order to verify the expression levels of NEDD4L and STAT3 in glioma tissue, we performed immunohistochemical staining of NEDD4L and STAT3 on a commercially available glioma tissue chip, and found that NEDD4L expression level negatively correlates with STAT3 expression in glioma tissues, while the expression levels of these two genes are comparable in normal tissue (Figure 2(c) left panel). In addition, Kaplan-Meier analysis showed that patients with NEDD4L-low-expressing glioma exhibit a worse prognosis compared with patients with NEDD4L-high-expressing glioma (Figure 2(c) right panel), which demonstrates the potential of NEDD4L function as a biomarker to predict the clinical outcome of glioma patients.

NEDD4L Binds to STAT3 and Induces its Ubiquitination in Glioma Cells.
In order to determine the interaction between NEDD4L and STAT3 in glioma cells, we engineered glioma U251 cells to overexpress NEDD4L, which was validated by both qPCR and western blot (Figures 3(a) and 3(b)). Ten, it turned out that forced expression of NEDD4L signifcantly inhibits the protein levels of the total as well as phosphorylated STAT3 (Ser727) without afecting the level of STAT3 mRNA (Figures 3(c) and 3(d)). On the other hand, this process is dramatically inhibited by the treatment of MG132, suggesting that NEDD4L could downregulate the expression level of STAT3 through the protein degradation pathway (Figure 3(e)). We then carried out a coimmunoprecipitation (Co-IP) assay to determine whether STAT3 interacts with NEDD4L in glioma U251 cells. As we expected, the binding between NEDD4L and STAT3 was elucidated by both forward and reverse IP (Figure 3(f )). Furthermore, forced expression of NEDD4L signifcantly increases the ubiquitination of STAT3 (Figure 3(g)).

Forced Expression of NEDD4L Signifcantly Inhibits Proliferation and Increases Intracellular ROS Levels in Glioma Cells, Whereas NEDD4L Downregulation has a Reverse Efect.
As suggested by our previous data that a high level of NEDD4L improves the overall survival of glioma patients, we proposed to investigate whether the manipulation of NEDD4L afects glioma cell proliferation. We generated a U251 NEDD4L-overexpression (oeNEDD4L) cell line as mentioned above and performed a CCK8 assay to examine the cell proliferation once every 12 hours for 48 hours. It turns out that forced expression of NEDD4L prohibits cell proliferation signifcantly 24 hours after transfection (Figure 4(a)). In addition, the intracellular ROS levels were increased (Figure 4(b)). Meanwhile, the expression of Nrf2 and GPX4, two well-known key factors of a ferroptosis Journal of Oncology 5 signaling pathway, was suppressed (Figure 4(c)). Meanwhile, the levels of total and phosphorylated STAT3 are dramatically decreased. In order to further validate this observation, we generated NEDD4L-knockdown (shNEDD4L) U87 glioma cell lines using three independent shRNAs targeting NEDD4L. As shown in Figures 4(d) and 4(e), the expression of NEDD4L is signifcantly prohibited at both mRNA and protein levels. Contrary to the oeNEDD4L cells, proliferation is promoted in the NEDD4L-knockdown glioma cells (Figure 4(f )). Likewise, the intracellular ROS levels were signifcantly decreased after knocking down NEDD4L (Figure 4(g)). Te expressions of Nrf2, GPX4, STAT3, as well as phosphorylated STAT3 all increased, while the NEDD4L expression decreased after shRNA knocking down (Figure 4(h)). Tese data suggest that NEDD4L negatively regulates key ferroptosis factors as well as STAT3.

Upregulation of STAT3 Signifcantly Reversed NEDD4L-Induced Proliferative Inhibition and Intracellular ROS Levels
Elevation in Glioma Cells. In order to investigate whether NEDD4L regulates glioma cell proliferation through manipulating STAT3, we generated STAT3-overexpressing U251 glioma cells, and the overexpression of STAT3 was validated by both qPCR and western blot (Figures 5(a) and  5(b)). Furthermore, we induced overexpression of STAT3, either alone (oeSTAT3) or together with NEDD4L overexpression (oeSTAT3-oeNEDD4L). Interestingly, we noticed that the suppression of cell proliferation induced by oeNEDD4L is rescued by cooverexpression of STAT3 ( Figure 5(c)). Consistent with this observation, the elevated intracellular ROS levels induced by NEDD4L overexpression were also rescued by forced expression of STAT3 ( Figure 5(d)). Likewise, NEDD4L-induced decreased Nrf2 and GPX4 expression were restored to the normal or even higher levels after overexpression STAT3 ( Figure 5(e)). Te protein levels of STAT3 and phosphorylated STAT3 are all increased upon the overexpression of STAT3 even in the oeNEDD4L background ( Figure 5(e)). Taken together, these data imply that NEDD4L carries out its efect on glioma by manipulating STAT3, which appears to be the downstream factor for the signaling pathway.

Treatment of PF Inhibits Cell Proliferation and Increases
Intracellular ROS Levels in Glioma Cells by Regulating NEDD4L Expression. As mentioned above, our previous study implied the relationship between PF's antitumor effects and STAT3 ubiquitination. In order to further elucidate the mechanism involved in this process, we frst checked the intracellular ROS levels in response to the PF treatment. As shown in Figure 6(a), the intracellular ROS levels increase  gradually as the PF dosage increases. In order to determine if ferroptosis is afected by PF treatment, we examined the expressions of STAT3/p-STAT3, Nrf2, and GPX4 in PFtreated glioma U87 cells, and found out that all these factors are inhibited at the protein level in a dosage-dependent manner ( Figure 6(b)). Moreover, treatment of PF signifcantly inhibited cell proliferation (Figure 6(c)) and increased the levels of MDA (Figure 6(d)) and Fe 2+ (Figure 6(e)) in glioma cells, whereas ferroptotic inhibitors DFO could partly reverse the efect of PF, suggesting that PF treatment can induce ferroptosis in glioma cells. To test this hypothesis that PF might function through the elevation of NEDD4L, we knocked down the NEDD4L in U87 glioma cells for 24 hours before subjecting these cells to PF treatment. As we speculated, following ablation of NEDD4L, the promoted intracellular ROS levels in the cells with PF treatment was decreased (Figure 6(f )), consistent with intracellular ROS levels, ablation of NEDD4L attenuated the growth inhibitory efect of PF (Figure 6(g)). Likewise, the expression levels of Nrf2, GPX4, STAT3, and p-STAT3 (Figure 6(h)), as well as the levels of MDA (Figure 6(i)) and Fe 2+ (Figure 6(j)) were restored close to normal after receiving PF treatment in the absence of NEDD4L. Taken together, our data suggest that PF carries out its antitumor efect by regulating the expression of NEDD4L, which could further manipulate ferroptosis in glioma cells.

PF Inhibits Tumor Growth by Increasing Ferroptosis of Tumor Cells In Vivo and In
Vitro. Next, we sought to assess the therapeutic efect of PF in the U251 xenograft tumor model. In general, U251 cells were implanted into nude mice subcutaneously, and the mice bearing established tumor 2 weeks after injection and they were subjected to one of the four following treatments: vehicle; RSL3 (100 mg/kg, twice/ week), an experimentally verifed drug for inducing ferroptosis [26]; PF (1 g/kg/day); concomitant administration of RSL3 and PF. Te tumor growth was closely monitored. Our results showed that both monotherapies signifcantly attenuated tumor growth. Furthermore, the combination of RSL3 and PF results in signifcantly decreased tumor burden and prolonged progression compared to RSL3 or PF monotherapy (Figures 7(a) and 7(b)). Te mice were sacrifced 28 days after receiving treatment. Tumor tissues were collected afterwards and subjected to a TUNEL assay to assess the apoptosis status. Apparently, the PF treatment substantially induces apoptosis which leads to suppressed tumor growth eventually, and enhanced the apoptosis induced by RSL3, which could further sensitize glioma cells to  RSL3 treatment (Figure 7(c)). Consistent with the in vivo results, the in vitro results showed that the combination of RSL3 and PF was superior to RSL3 or PF monotherapy and more signifcantly inhibited cell proliferation (Figure 7(d)) and increased MDA (Figure 7(e)) and Fe 2+ (Figure 7(f )) levels in gliomas. Together, our data suggest that the combination of PF and RSL3 may be benefcial to inhibit glioma tumors by inducing ferroptosis.

Discussion
Finding active components of traditional Chinese medicine and discovering the underlying mechanism have become a hot topic in medicine studies. As an active chemical compound, it is one of the major constituents of Paeonia lactifora Pall. (a.k.a P. alba), paeoniforin (PF) exhibits great potential antitumor efects in various types of cancer by   Journal of Oncology targeting diferent cell events such as cell cycle arrest, apoptosis, migration, and epithelial-mesenchymal transition (EMT) [27][28][29]. In addition, the anticancer efects of PF have been reported in the context of glioblastoma [30][31][32]. Our previous study showed that PF inhibits human glioma cell proliferation by inhibiting the STAT3 pathway via regulating its turnover through the ubiquitination pathway [3].
In this study, we pushed forward our knowledge about the therapeutic value of PF and the mechanism that renders PF's antitumor capability. Our data suggest that PF can induce STAT3 ubiquitination by promoting the expression of NEDD4L. As a member of an E3 ligase NEDD4 family, NEDD4L not only targets membrane proteins including ion channels and transporters, but also triggers the degradation of certain proteins involved in cancer signaling pathways such as Dvl2, SMAD2, and SMAD7 [33,34]. It has been demonstrated that NEDD4L suppresses ferroptosis by mediating the degradation of lactotransferrin (LTF) protein which functions as an activator of ferroptosis [35]. Ferroptosis is defned as a type of oxidative iron-dependent lipid peroxidation-induced programmed cell death. One of the most essential characteristics of ferroptosis is the rapid accumulation of reactive oxygen species (ROS) induced by high levels of cellular labile iron [36]. And the role of ROS in the context of cancer has been well studied. For instance, it has been well established that elevated levels of ROS render disruptive efects on functions and structures of cells which leads to oxidative stress and subsequently to the development of various pathologies including infammatory, agerelated disorders, and cancer [37,38]. And it is known that ROS play vital roles in mitogenic signaling cascades by prolonging the activation of growth factors and boosting cellular signaling factors [39,40]. However, the exact role of NEDD4L in glioma is still not fully elucidated. In this study, we noticed that the expression of NEDD4L is upregulated by PF in a dosage-and timedependent manner. We then found out that NEDD4L is downregulated in glioma cancer tissue compared to normal tissue, which is consistent with published data [25], and the expression level of NEDD4L correlates with prognosis in cancer patients. We also provide solid evidence to show that NEDD4L negatively regulates STAT3 level by mediating its ubiquitination, which bridged the gap between PF and STAT3 as reported in the previous study. Te oncogenic roles of STAT3 have been studied extensively in the past few decades [8][9][10]. It has been reported that STAT3 could be a promising target for the treatment of glioma [41]. It also has been reported that the expression as well as activation of STAT3 are associated with the low survival rate and poor prognosis of glioma patients [11,12]. In addition, STAT3 has been suggested to participate in glioma cell progression by shaping the immune microenvironment [42]. On the other hand, it has been suggested that STAT3 plays key roles in ferroptosis which demonstrates a new approach to attenuate drug resistance in osteosarcoma [20]. Taken together, these data endorsed the importance of tackling down STAT3 pathway in the battle of fghting glioma.
In addition, our data demonstrated that forced expression of NEDD4L inhibits glioma cell growth probably by inducing ferroptosis, characterized by intracellular ROS accumulation and suppressed expression of the key factors in ferroptosis such as Nrf2 and GPX4. It has been reported that Nrf2 is required for glioma stem cell self-renewal [43][44][45]. GPX4 is also involved in the proliferation, migration, and apoptosis of glioma cells [46]. Terefore, the reduced expression of Nrf2 and GPX4 driven by oeNEDD4L might contribute to the suppressed glioma cell growth by multiple means. In addition, NEDD4L drives the ubiquitination of STAT3, which also plays critical roles in ferroptosis as previously discussed. Furthermore, our xenograft data demonstrated that PF can suppress glioma tumor growth by activating NEDD4L/STAT3/Nrf2/GPX4 signal axis which eventually triggers ferroptosis. And the antitumor efect could be enhanced by the concomitant administration of PF and RSL3.
It is known that as a ferroptosis inducer, RSL3 inhibits tumor growth by targeting GPX4. Ten, PF could enhance the RSL3 treatment by either thoroughly inhibiting GPX4 or inducing both ferroptosis and apoptosis. Terefore, more studies need to be performed to explore the specifc underlying mechanism.

Conclusions
Taken together, this study demonstrates that PF can function as an antitumor agent for glioma treatment by targeting NEDD4L-dependent STAT3 ubiquitination as well as by regulating the Nrf2/GPX4 signaling axis, which might trigger ferroptosis. Furthermore, PF could beneft glioma patients by enhancing antitumor efect of other ferroptosis inducers such as RSL3, which could pave the road for new therapeutic modalities.

Data Availability
Te data supporting the current study are given in the article.

Ethical Approval
All care and experiment of the laboratory animals were performed following protocols approved by Huzhou Cent Hospital, Afliated Cent Hospital HuZhou University (Zhejiang, China).

Disclosure
Xiao-Hu Nie is the frst author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.