Capsaicin Enhances Temozolomide-Resistant Glioblastoma Cells’ Chemosensitivity and Ferroptosis through FHOD1/IRF2 Downregulation

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Introduction
Glioblastoma (GBM) is the most aggressive brain tumor among malignant brain tumors and has the lowest survival time in patients [1].In the traditional approach to the treatment of GBM, the prognosis is often poor despite surgery and simultaneous radiation therapy and chemotherapy treatments [2].While chemotherapy, particularly utilizing the drug temozolomide (TMZ), has shown some advancements by extending survival rates by approximately 2.5 months, its efcacy is constrained due to the emergence of drug resistance [3].TMZ, an oral alkylating agent, serves as the primary chemotherapy drug used against GBM.Extensive research eforts have been dedicated to unraveling the mechanisms underlying TMZ resistance.Among these, much attention has been directed toward O6-methylguanine-DNA methyltransferase (MGMT) since it directly infuences the cytotoxic efects of TMZ [4].Nevertheless, the role of MGMT alone does not completely explain GBM resistance to TMZ, as over 40% of GBMs with low MGMT levels still demonstrate resistance to the drug [5].
Te formin family, which includes formin homology-2 domain-containing protein-1 (FHOD1), plays a role in modulating the capping and bundling of actin to reshape the actin cytoskeleton [6].Recently, research has highlighted FHOD1's involvement in various processes related to cancer.As an example, in squamous cell carcinoma, increased levels of FHOD1 were associated with the promotion of cancer cell migration and invasion, achieved through the induction of epithelial-mesenchymal transition [7].
Knocking down FHOD1 in the breast cancer cell line, MDA-MB-231 had a notable impact on invasion, migration, and proliferation [8].In addition, in GBM cells, the downregulation of FHOD1 led to a signifcant reduction in cell proliferation and induced cellular sensitivity to ferroptosis [9].Ferroptosis, a recently identifed form of regulated cell death, is known to be initiated by several biological processes [10].Indeed, exploring FHOD1 and its interconnected signaling networks that contribute to cell death mechanisms holds the potential to establish a solid foundation for developing strategies aimed at sensitizing cancer cells to therapies centered around inducing cell death.
Interferon regulatory factors (IRFs) consist of 9 diferent members, and among them, IRF2 plays a role in the regulation of a wide variety of cellular metabolic processes [11].Although some studies have illuminated the association between IRF2 and tumor immunity within gliomas, these investigations have often lacked a comprehensive consideration of additional tumor characteristics [12].Terefore, there is a pressing need to deepen our understanding of IRF2, particularly in the context of its broader implications within tumor biology.
Capsaicin, a lipophilic protoalkaloid renowned for its characteristic pungency, is primarily found in hot peppers (Capsicum annuum L.).It has garnered attention for its utility in alleviating pain associated with neuralgias and neuropathies [13].Beyond its analgesic properties, capsaicin has also been the subject of numerous studies revealing its potential as an agent with anticancer properties [14].Notably, our previous research has highlighted capsaicin's role as a potential inducer of ferroptosis, a type of regulated cell death, and demonstrated its capacity to curtail the proliferation of GBM cells [15].While the roles of FHOD1, IRF2, and capsaicin in tumorigenesis, cancer progression, ferroptosis, and chemoresistance have been increasingly recognized, there remains a notable gap in our understanding regarding their preventive potential in conferring resistance to TMZ chemotherapy in GBM.As of now, no conclusive evidence has emerged to establish whether FHOD1 and IRF2 inhibit TMZ chemoresistance in GBM.In light of this knowledge gap, the current study was undertaken to explore the possibility that capsaicin treatment could regulate the FHOD1/IRF2 signaling pathways.Tis investigation aimed to ascertain whether these pathways exert an infuence on ferroptosis and if capsaicin treatment could potentially prevent TMZ resistance in GBM cells.

Cell Lines and Cell
Culture.U87 and U251 GBM cells were purchased from the American Type Culture Collection.Tese cells were cultured in Dulbecco's modifed Eagle medium (DMEM) containing 2 mM glutamine and 10% fetal bovine serum.Cells were maintained in an incubator with control environmental conditions at 37 °C and 5% CO 2 .
To induce resistance to TMZ in GBM cell lines, we adapted the techniques described by Zhu et al. [16] and Xu et al. [17].U251 and U87 cells (at a density of 1 × 10 5 cells) underwent a 3-week treatment with 200 μM TMZ.Initially, the two cell lines were cultured separately in 12-well plates and allowed to adhere overnight at 37 °C.Subsequently, TMZ treatment was administered every 72 hours for 3 weeks, with a fresh medium containing TMZ provided during each cycle.Te majority of cells died during TMZ treatment, and a small group of cells survived by resisting TMZ.Tese surviving cells were isolated from the culture medium and used to establish TMZ-resistant U251 (U251-R) and U87 (U87-R) cell lines.U251-R and U87-R cells were employed in all subsequent experimental analyses.In a prior investigation, we determined that the cytotoxic concentration of capsaicin in U87 and U251 cells, when treated for 48 hours, was 120 μM [18], and this capsaicin concentration was utilized in other analyses in the present study.

Cell Counting Assay for Cell Viability.
Te viability analysis of cells was evaluated using the Cell Counting Kit-8 (CCK-8, Cat no.E-CK-A362) assay.Cells (1 × 10 5 per well) were cultured in 96-well plates.After a 24-hour adaptation period, cells were exposed to TMZ at concentrations ranging from 0 to 2000 μM for 48 hours.Following this, the cells were incubated with 10 μl of the CCK-8 solution for 2 hours.Subsequently, a microplate reader (BioTek) was used to measure cell viability at a wavelength of 490 nm.

Cell Proliferation Assay.
Cell proliferation was assessed utilizing the 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit (2750, Sigma-Aldrich), adhering to the prescribed guidelines from the manufacturer.Te BrdU incorporation method stands as a widely employed assay for the quantifcation of in vitro cell proliferation.Initially, cells (1 × 10 5 per well) were incubated in 96-well plates and analyses were performed according to the manufacturer's instructions.Subsequently, the cells were subjected to varying concentrations of capsaicin over a 48-hour period.Te absorbance values of the samples were subsequently gauged at a wavelength of 450 nm.

Cell Cycle
Analysis.TMZ-resistant cells were seeded in 12-well plates (1 × 10 5 per well).Subsequently, these cells underwent a 48-hour exposure to both TMZ and capsaicin.Following the treatment interval, cellular fxation was achieved by using a 4% paraformaldehyde solution, ensuring the preservation of cellular structures and preventing further alterations.Te fxed cells underwent staining with propidium iodide (PI) to visualize distinct phases of the cell cycle, encompassing G1 (gap phase 1), S (DNA synthesis phase), G2 (gap phase 2), and M (mitosis phase).Te fuorescence intensity emitted by the stained cells was quantifed by employing fow cytometry, specifcally by utilizing the Muse ® fow cytometry system.2.5.Biochemical Analysis.FHOD1, IRF2, intracellular levels of ferrous (Fe 2+ ), glutathione peroxidase 4 (GPX4), malondialdehyde (MDA), cellular total oxidant status (TOS), and reduced glutathione (GSH) levels were assessed using enzyme-linked immunosorbent assay (ELISA) kits (MBS9322747, MBS166682, MBS2000338, MAK025, MAK085, Rel Assay Diagnostics, and MBS727656, respectively) by following the manufacturer's instructions.To carry out these analyses, U251-R and U87-R cells were seeded in 96-well plates at a density of 1 × 10 5 cells and then treated with 120 μM of capsaicin for 48 hours.According to the manufacturer's instructions, the absorbance value of each sample was analyzed with a microplate reader.
2.6.Cell Migration Assay.U251-R and U87-R cells were seeded into a 6-well plate and allowed to grow until they reached a confuence level of over 90%.To commence the scratch assay, the aseptic technique was employed with a sterile pipette tip, creating a straight vertical line along the center of the plate.Following the creation of the scratch, the cells were treated with capsaicin.Images of the scratch area were captured by using an inverted microscope at two distinct time points: immediately after creating the scratch (0 hours) and 24 hours after capsaicin treatment.To quantify the extent of scratch closure, ImageJ software was utilized.

Quantitative Reverse-Transcription Polymerase Chain
Reaction and Western Blotting Assay.TMZ-resistant cells (1 × 10 5 per well) were cultured in 96-well plates.Following this, the cells underwent treatment with capsaicin for 48 hours.Te transcription levels of FHOD1 and IRF2 were assessed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR).To initiate this process, total RNA was extracted from FHOD1 and IRF2 using TRIzol ® Reagent (Invitrogen, 12594025).Te extracted RNA was then subjected to cDNA synthesis by using the SuperScript ™ IV One-Step kit (Invitrogen, 12594025).For the qRT-PCR analysis, the StepOnePlus ™ Real-Time PCR System was employed in conjunction with SYBR Green Master Mix.Te relative mRNA levels of FHOD1 and IRF2 were determined using the 2 −ΔΔCT method, with β-actin used as an endogenous control.Te qRT-PCR was carried out with the following conditions: an initial preincubation step for 15 min at 60 °C, followed by polymerase activation for 10 min at 95 °C.Tis was succeeded by 45 cycles, each consisting of denaturation at 95 °C for 10 s, and annealing and extension at 60 °C for 40 s.Te primer sequences were as follows: FHOD1 forward 5′-CCT CAG CTG ACA CCT CCA GC-3′, FHOD1 reverse 5′-CAG CGC AAC CTG CTT CTC-3′, IRF2 forward 5′-CGA CCG ATC GCT CGG GAC-3′, IRF2 reverse 5′-GCT GCA GAG TGG GCC ATG-3′, β-actin forward 5′-GCC ATG GCC ATC ATG AAG-3′, and β-actin reverse 5′-GTC GTA CGG AGA TGC CCA ACG-3′.
FHOD1 and IRF2 protein levels in U251-R and U87-R cells were assessed using Western blot analysis.Tis process was executed step by step following the methodology described in our previous study.Te antibodies used for analysis were as follows: FHOD1 antibody, diluted to 1 : 1000 (PA5-115233, Invitrogen), and IRF2 antibody, diluted to 0.2 μg/mL (PA5-79515, Invitrogen).To visualize the protein bands, we employed an enhanced chemiluminescence kit (34579, Termo Scientifc).Te images were analyzed using ImageJ.

Statistical Analysis.
Statistical analysis was carried out using GraphPad Prism 8 (GraphPad Inc., USA).All experiments were performed with three replicates in three independent experiments.Te data were presented as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) was used to compare three or more groups, followed by Tukey's post hoc test for multiple-group comparisons.Statistical signifcance was determined when the P value was less than 0.05.
Moreover, in order to illustrate the substantial infuence of capsaicin on TMZ resistance in U87-R and U251-R cells, we examined the reaction of these cells to TMZ treatment.As depicted in Figure 1, the inclusion of capsaicin in U87-R and U251-R cells led to a substantial decrease in cell viability following TMZ treatment.U87-R and U251-R cells treated with 120 μM showed no viability at 2 mM TMZ exposure.U87-R cells, subjected to 120 μM capsaicin and exposed to 200 μM, 400 μM, 800 μM, and 1 mM TMZ for 48 hours, manifested a reduction in cell viability by 18.1%, 41.7%, 83.5%, and 92.8%, respectively, compared to the control group (P < 0.0001; Figure 1(b)).In addition, the treatment of U251-R cells with 200 μM, 400 μM, 800 μM, and 1 mM TMZ resulted in a decrease in cell viability by 24.6%, 49.2%, 88.5%, and 98.1%, respectively, compared to the control group (P < 0.0001).Consistent with the CCK-8 results following the capsaicin treatment, the IC 50 TMZ values for U87-R and U251-R cells were determined as 522.6 μM and 407.2 μM, respectively.

Journal of Food Biochemistry
In Figure 1(c), to evaluate the impact of capsaicin on TMZ resistance in U87-R and U251-R cells, we examined cell proliferation in both TMZ-treated and untreated cells.An intermediate concentration of 650 μM TMZ was selected for treating U87-R and U251-R cells based on CCK-8 analysis for BrdU analysis and subsequent biochemical analysis.Te proliferation of U87-R and U251-R cells treated with TMZ did not exhibit a statistically signifcant decrease compared to the control groups (P > 0.05).However, subsequent to capsaicin treatment, the proliferation of U87-R and U251-R cells decreased by 7.1% and 9.3%, respectively, compared to the control group (P < 0.05).Furthermore, the proliferation of U87-R and U251-R cells cotreated with capsaicin and TMZ experienced a reduction of 42.7% and 55.1%, respectively, compared to the control group (P < 0.0001).Our fndings suggest that capsaicin treatment diminishes cell viability and proliferation by mitigating TMZ resistance in U87-R and U251-R cells.

Capsaicin Specifcally Induced Cell Cycle Arrest in U87-R and U251-R Cells.
As seen in Table 1, TMZ treatment did not cause a signifcant change in the cell cycle in TMZ-resistant cells (U87-R and U251-R).However, capsaicin treatment 4 Journal of Food Biochemistry caused an increase in the number of U87-R and U251-R cells in the G0/G1 phase.Furthermore, TMZ + capsaicin treatment reduced the number of U87-R and U251-R cells in the G2/M phase.Tus, simultaneous treatment of TMZ and capsaicin regulates the cell cycle of U87-R and U251-R cells, causing an arrest in the G0/G1 phase of these cells.

Efects of Capsaicin on
Biomarkers in U87-R and U251-R Cells.Te fndings presented in Figures 2(a)-2(g) indicate that capsaicin treatment had a signifcant impact on various factors in U87-R and U251-R cells.In response to capsaicin treatment, observable changes occurred in FHOD1, IRF2, MDA, GPX4, intracellular iron, TOS, and GSH levels.However, no statistically signifcant diferences were observed in these levels between the control group and cells treated with TMZ (P > 0.05).In both U87-R and U251-R cells, capsaicin led to a reduction in FHOD1, IRF2, GSH, and GPX4 levels while causing an enhancement in intracellular iron, TOS, and MDA levels.ELISA analysis results revealed specifc percentage changes in these factors.
Remarkably, the combined administration of capsaicin and TMZ had a signifcant impact on various biochemical biomarkers in both TMZ-resistant U87-R and U251-R cells.
In U87-R cells, TMZ + capsaicin treatment resulted in a 37.3% reduction in FHOD1, a 29.2% reduction in IRF2, a 40.6% reduction in GSH, and a 35.8% reduction in GPX4 levels (P < 0.0001 vs. control).Conversely, this treatment led to a 24.7% increase in intracellular iron, a 42.1% increase in TOS, and a 30.5% increase in MDA levels (P < 0.0001).Likewise, TMZ + capsaicin treatment in U251-R cells caused a 40.4% reduction in FHOD1, a 35.3% reduction in IRF2, a 52.3% reduction in GSH, and a 47.2% reduction in GPX4 levels (P < 0.0001 vs. control).TMZ + capsaicin also triggered a 29.3% increase in intracellular iron, a 51.4% increase in TOS, and a 38.5% increase in MDA levels (P < 0.0001 vs. control).

Capsaicin Treatment Regulated Wound Healing in U87-R and U251-R Cells.
As shown in Figure 3, capsaicin clearly suppressed the migration ability of U87-R and U251-R cells according to the wound healing assay.Initially, in the untreated cells, a substantial portion of the initial wound area was observed to close within a span of 48 hours.Tis closure amounted to 84.26% in the case of U87-R cells and 77.52% in U251-R cells, indicating a signifcant wound-healing process (P < 0.0001 compared to control cells).However, with capsaicin treatment, a remarkable deceleration in the rate of wound healing was evident in both U87-R and U251-R cells.In particular, following 48 hours of treatment with TMZ + capsaicin, U87-R cells exhibited a 25.83% expansion in the wounded region (P < 0.0001 compared to control cells; Figure 3(a)).Similarly, U251-R cells experienced an expansion of 20.74% (P < 0.0001 compared to control cells; Figure 3(a)) after the same treatment duration.Tese outcomes strongly underscore the inhibitory efect of capsaicin treatment on the migratory capacity of both U87-R and U251-R cells.

Capsaicin-Regulated FHOD1 and IRF2 Expressions and
Protein Levels in U87-R and U251-R Cells.Te observations presented in Figures 4 and 5 reveal substantial changes in the expression and protein levels of FHOD1 and IRF2 subsequent to capsaicin treatment in both U87-R and U251-R cells.Notably, TMZ treatment in U87-R and U251-R cells did not elicit statistically signifcant alterations in the mRNA levels of FHOD1 and IRF2.However, as depicted in Figures 4(a) and 4(b), FHOD1 mRNA levels in U87-R and U251-R cells experienced reductions of 34.7% and 41.3%, respectively, under TMZ + capsaicin treatment (P < 0.0001).Furthermore, IRF2 mRNA levels in U87-R and U251-R cells treated with TMZ + capsaicin exhibited reductions of 25.8% and 37.2%, respectively (P < 0.0001; Figures 4(c) and 4(d)).
In alignment with the mRNA outcomes, Western blot analysis indicated that capsaicin treatment led to diminished levels of FHOD1 and IRF2 proteins in both U87-R and U251-R cells.Notably, a more pronounced suppressive efect of capsaicin on these proteins was evident in U251-R cells when compared to capsaicin-treated U87-R cells.

Discussion
Drug resistance plays a pivotal role in clinical outcomes, infuencing tumor relapse rates and patient survival.In the context of GBM, TMZ resistance is a key contributor to treatment failure.Researchers have vigorously examined the molecular underpinnings of this chemoresistance in GBM, particularly focusing on genes involved in DNA repair mechanisms [19].However, despite extensive research, the mechanism of resistance developed by GBM cells against TMZ is not clearly understood.Te primary objective of this study was to elucidate the roles played by FHOD1 and IRF2 in the regulation of ferroptosis in TMZ-resistant GBM cells, particularly through the application of capsaicin treatment.Notably, previous research has observed signifcantly elevated FHOD1 and IRF2 levels in both glioma tissues and cell lines [20].Consequently, we uncovered that the downregulation of FHOD1 and IRF2 through capsaicin treatment in TMZ-resistant GBM cells led to a noteworthy increase in both drug sensitivity and susceptibility to ferroptosis.Tese fndings collectively contribute to elucidating potential avenues for enhancing therapeutic strategies in TMZ-resistant GBM cases.% cell viability of each cell cycle phase (G0/G1, S, and G2/M).

Journal of Food Biochemistry
Te phenomenon of continuous cell proliferation is a hallmark associated with resistance to TMZ and is a key factor contributing to tumor recurrence [21].In this study, we observed that U87 and U251 cells, which did not possess TMZ resistance, exhibited a higher susceptibility to TMZ treatment than the TMZ-resistant U87-R and U251-R cells.Importantly, even after TMZ treatment, the resistant U87-R and U251-R cells displayed rapid regrowth in the cell migration assay.Furthermore, the viability and proliferation of TMZ-resistant U87-R and U251-R cells were found to be sustained even in the presence of higher concentrations of TMZ, contrasting with the response of U87 and U251 cells.Tis suggests that the resistant cells have developed mechanisms to withstand the cytotoxic efects of TMZ.Tis resistance to the efects of TMZ implies a signifcant potential for tumor recurrence and dissemination of these resistant cells.
Ferroptosis is a form of programmed cell death characterized by the accumulation of cellular iron and reactive oxygen species (ROS) [10].In a previous study, it was observed that the suppression of FHOD1 signifcantly increased elastin-induced ferroptosis in T98G and U251 GBM cells [9].Tese outcomes strongly suggest that FHOD1linked signaling pathways potentially hold a critical role in governing resistance to ferroptosis.Furthermore, by employing a knockdown strategy, this study ofered pioneering evidence of the indispensability of FHOD1 expression for the efcient migration of U87, U138, T86, and UTGB7 GBM cells [22].Tis aligns with fndings at the cellular level, where FHOD1 knockdown in basal-like breast cancer cell lines resulted in larger cell areas and compromised abilities to migrate, invade, and proliferate [8].Besides, FHOD1 expression and functional relevance have been observed in other cancer types such as oral squamous cell carcinoma and melanoma [7,23].Tese studies have indicated that FHOD1 contributes to cellular migration and invasion in vitro, suggesting a broader role for FHOD1 in promoting these processes across multiple cancer types.Tese fndings harmonize with our data that identifed FHOD1 as a pivotal contributor to facilitating cellular migration.Furthermore, our results showed that when capsaicin was administered to TMZ-resistant U87-R and U251-R cells, it led to the reduction of FHOD1, GSH, and GPX4 levels.Tis reduction, in turn, triggered an accumulation of intracellular iron within these cells.Consequently, this increase in intracellular iron, in combination with the reduction of FHOD1, GSH, and GPX4 levels, led to the heightened oxidative stress and a surge in lipid peroxidation.Members of the IRF family have emerged as important regulators of infammatory and immune microenvironment signaling pathways, exerting crucial roles in the development and progression of cancer [24].Dysregulation of the IRF family members has been documented in diverse malignancies [25].However, the comprehensive role of IRFs in glioma remains relatively understudied.In this study, we initiated an investigation into the expression profle, prognostic implications, and biological functions of individual IRF2 within the context of TMZ-resistant U87-R and U251-R cells.As previously mentioned, the levels of IRF family members were noted to be elevated in GBM cells compared to normal cells [20].Furthermore, the expression levels of diferent IRFs elevate with tumor progression, yet patients exhibiting lower IRF expression experience signifcantly prolonged survival [12].For instance, Liang et al. demonstrated that IRF1 knockdown in a glioma animal model led to an increased apoptosis and enhanced the efectiveness of anti-VEGF treatment, highlighting elevated IRF1 expression in glioma cell lines [26].Similarly, Jin et al. reported overexpression of IRF7 in glioma cells and linked these elevated levels to decreased patient survival [27].Moreover, patients with high IRF2 expression in gliomas exhibited poor overall survival [20].
Tis research efectively employed extensive clinical data to identify heightened IRF2 expression as a noteworthy trait in gliomas, indicating IRF2's potential as a biomarker for glioma.Numerous studies have uncovered IRF2's connections with tumorigenesis and tumor progression [28].In liver cancer cells, reducing IRF2 has been associated with decreased invasiveness [29].Conversely, in pancreatic cancer, IRF2 has been linked to the depth of tumor invasion and the promotion of tumor cell growth [30].It is crucial to acknowledge, however, that the role of IRF2 may vary in diferent tumor contexts.
In summary, our study provides strong evidence underlining the critical roles of FHOD1 and IRF2 in conferring resistance to TMZ in glioma cells.Our fndings Journal of Food Biochemistry demonstrate that capsaicin treatment sensitizes glioma cells to TMZ and ferroptosis, leading to a concurrent reduction in FHOD1 and IRF2 levels.However, an important limitation of our study is that silecing and overexpressing experiments of the targeted FHOD1 and IRF2 genes could not be performed.Tese results highlight FHOD1 and IRF2 as promising targets for the development of chemotherapeutic drugs and suggest their potential as novel diagnostic and predictive biomarkers.
Our study underscores the importance of unraveling the mechanisms behind TMZ resistance, given its substantial impact on tumor behavior and the efectiveness of treatment strategies.

Figure 3 :
Figure 3: Capsaicin treatment inhibited cell migration rate in U87-R and U251-R cells: (a) microscope images of cell migration rates of capsaicin treatment for 48 hours in U87-R cells and (b) microscope images of cell migration rates of capsaicin treatment for 48 hours in U251-R cells.* * * P < 0.0001 vs. control groups.