Celastrol combined with curcumin inhibits proliferation and causes cell death in nasopharyngeal carcinoma CNE1 cell line by inducing ferroptosis

Celastrol combined with curcumin inhibits proliferation and causes cell death in nasopharyngeal carcinoma CNE1 cell line by inducing ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Celastrol combined with curcumin inhibits proliferation and causes cell death in nasopharyngeal carcinoma CNE1 cell line by inducing ferroptosis Tao Feng, Yinjun Luo, Xin Zhang, Ziyang Fang, Ying Li, Shijing Ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4827626/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nasopharyngeal carcinoma (NPC) is a highly invasive form of head and neck cancer that arises from nasopharyngeal epithelial cells. The treatment of advanced NPC with radiotherapy presents significant challenges due to cellular resistance, which has spurred interest in natural small molecule drugs. Celastrol and curcumin, both derived from plants, have exhibited anti-tumor properties. However, the clinical development of celastrol is hindered by its low bioavailability and associated toxic side effects, while curcumin, although non-toxic, also suffers from limited bioavailability. The combination of drugs is a fundamental principle of traditional Chinese medicine, as it enhances therapeutic efficacy while reducing toxicity, suggesting a potential synergistic use of celastrol and curcumin. Furthermore, ferroptosis is crucial for tumor cell death. Consequently, our study aims to investigate whether the combination of celastrol and curcumin can induce ferroptosis in NPC cells and assess its antiproliferative effects. Methods Human nasopharyngeal carcinoma cell lines were used for in vitro cell analysis. CCK8 was used to evaluate the effect of treatment with different concentrations of Celastrol and curmin on cell viability in a human nasopharyngeal carcinoma CNE1 cell line. Mitochondrial reactive oxygen species and mitochondrial membrane potential were detected to determine mitochondrial oxidative stress and function. Western blot was used to detect apoptosis, autophagy and ferritin-related proteins expression. Results The combination of celastrol and curcumin exhibited a more pronounced antiproliferative effect on CNE1 cells. Following treatment with these compounds, mitochondria generated substantial amounts of reactive oxygen species, resulting in impaired mitochondrial function. Moreover, the cell death induced by the combination of celastrol and curcumin was found to be independent of apoptosis, instead, it was correlated with increased cellular autophagy, enhanced mitochondrial fission, and the induction of ferroptosis. Conclusion Low doses of celastrol combined with curcumin exhibited a greater inhibition of CNE1 cell growth compared to curcumin alone. This enhanced efficacy of the combination therapy is likely attributable to its effects on mitochondrial fission and the induction of ferroptosis. Nasopharyngeal Carcinoma celastrol curcumin combined treatment ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nasopharyngeal Carcinoma (NPC) is a malignant tumor originating from the epithelial cells of the nasopharynx. Regions with a high incidence of NPC include Southeast Asia, southern China, North Africa, and Greenland[ 1 ]. The development of NPC is influenced by factors such as Epstein-Barr virus infection, genetic predisposition, environmental influences, and lifestyle choices[ 2 ]. Chemotherapy and radiotherapy are commonly used as initial treatments for NPC, showing promising results in early-stage patients. However, these treatments can lead to severe side effects and the development of resistance. Despite progress in treatment, advanced NPC patients still face challenges such as distant relapse and metastasis, resulting in low survival rates[ 3 ]. Hence, there is a critical need to devise more effective strategies to combat NPC. Natural products and their derivatives offer a valuable and sustainable source of scaffolds for contemporary drug development, constituting over 60% of FDA-approved anti-cancer medications[ 4 , 5 ]. Celastrol, a triterpenoid compound extracted from Trypterygium wilfordii Hook F (commonly referred to as the Thunder of God Vine), has long been used in traditional Chinese medicine for the treatment of inflammatory diseases[ 6 ]. Recent studies, using in vitro and/or in vivo models, suggested that Celastrol is a promising anti-cancer reagent against breast cancer, leukemia, hepatocyte carcinoma, melanoma, myeloma, and prostate cancer[ 7 – 11 ]. However, toxic side effects, low water solubility, and poor oral bioavailable have limited further clinical application of celastrol[ 12 ]. While structural modifications of celastrol have been explored to enhance solubility or mitigate toxicity, these alterations have often reduced the derivatives' anticancer efficacy[ 13 ]. There is a pressing requirement for innovative strategies to mitigate the toxicity of celastrol without compromising its anti-cancer efficacy. Curcumin, a polyphenolic substance in turmeric (Curcuma longa), has been historically used as a food additive and dye colorant in Southeast Asia[ 14 ]. In addition to its well-known antioxidant, anti-inflammatory, antimicrobial, and analgesic properties, curcumin has displayed potential in treating various cancers, including liver, lung, breast, and gastric cancers[ 15 , 16 ]. Although curcumin is recognized as a safe compound even at high doses, its low bioavailability poses a therapeutic challenge for clinical application. Interestingly, the bioavailability of curcumin can be increased by 2000% when combined with piperine[ 17 ]. This suggests that combination therapy may be an effective strategy to enhance the bioavailability of curcumin and improve its anti-cancer effects. The compatibility theory of Chinese medicine is essential in traditional Chinese medicine prescriptions, intending to improve effectiveness and minimize toxicity. However, there is a gap in research regarding the combined utilization of celastrol and curcumin for treating nasopharyngeal carcinoma. This study aims to investigate the potential anti-cancer properties of combining celastrol and curcumin. We hypothesize that the combination increases the anti-cancer effects while reducing the dosage requirement, optimizing toxicity levels, and reducing the risk of drug resistance development. Materials and Methods Cell Lines and Culture Human NPC cell lines CNE1 was obtained from the Shanghai Aolu Biological Technology Co., Ltd (Shanghai, China). CNE1 cell was cultured in RPMI 1640 medium(Gibco, America), containing 10% fetal bovine serum (Procell, China) and 1% penicillin/streptomycin (Solarbio, China) at 37°C in a 5% CO2 incubator. Cell viability by CCK8 assay Cell viability was assessed using the CCK8 (Dojindo, Japan) method. CNE-1 cells in logarithmic growth phase were trypsinized, seeded in 96-well plates at a density of 8,000 cells per well, and incubated at 37℃ in a 5% CO2 environment. Following cell attachment, the culture medium was replaced with fresh medium containing varying concentrations of curcumin, nansenin, or a combination of both. After 24 hours of incubation, cellular proliferation was measured using the CCK-8 assay. Cell viability was determined based on the absorbance at 450 nm (OD450) to evaluate the effects of celastrol (Sigma, America), curcumin (Sigma, America), and their combination on CNE1 cells. All compounds were DMSO (Solarbio, China) for solubilization. CNE1 mitochondrial ROS levels The levels of mitochondrial reactive oxygen species (ROS) in CNE1 cells were evaluated using the Mitochondrial ROS Activity Assay Kit (Beyotime, China), following the manufacturer's instructions. A dilution of dichlorofluorescin diacetate at a ratio of 1:1000 was prepared with serum-free culture medium. Cells from each group were collected and resuspended in the diluted DCFH-DA, achieving a cell density of 1×10^6 cells/mL. They were subsequently incubated at 37ºC in a cell culture incubator for 20 minutes. Throughout this period, the mixture was gently inverted every 3–5 minutes to facilitate adequate interaction between the DCFH-DA and the cells. After incubation, cells were washed three times with serum-free cell culture medium to remove any residual DCFH-DA that had not penetrated the cells. Rosup was added to the positive control. The incubated cells were then evenly distributed into 96-well plates with 6 wells per group, and analyzed using a fully automated enzyme labeling instrument with the excitation and emission wavelengths set at 488 nm and 525 nm, respectively. Fluorescence intensity was measured before and after stimulation in real-time or at specified time points. All procedures were conducted in a dark environment. CNE1 mitochondrial membrane potential levels Mitochondrial membrane potential was assessed using the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, China). Cells were harvested in fresh cell culture medium at a concentration ranging from 1×10 5 to 6×10 5 cells per group. Subsequently, 0.5 mL of JC-1 working solution was added to the cells, mixed thoroughly, and then incubated for 20 minutes at 37ºC in a cell culture incubator. Following the incubation period, the cells underwent centrifugation at 4ºC, 600 g for 3 minutes. The supernatant was removed, and the cells were washed twice with 1 mL of JC-1 buffer. This washing procedure was repeated twice. Each group of cells was resuspended in an appropriate volume of JC-1 staining buffer and transferred to a 96-well plate for analysis using a fully automated enzyme labeling instrument. For detection of JC-1 monomers, the excitation light wavelength was set to 490 nm and the emission light wavelength to 530 nm. In the case of JC-1 polymers, the excitation light wavelength was set to 525 nm and the emission light wavelength to 590 nm. Western blot analysis Cell were ground in PMSF (Solarbio, China) with a high dose of RIPA buffer (ABExBIO, USA) and protein phosphatase inhibitors (Solarbio, China). The concentration of protein was measured through a bicinchoninic acid assay (Beyotime, China). Proteins were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto a PVDF membrane (Innobilon, USA). Membranes were blocked for 2 hours by adding 5% skimmed milk or 5% BSA to three-phase buffered saline (pH 7.4) containing 0.1% Tween 20 (Solarbio, China). Then incubated with Mfn2 (1:500 dilution, Santa Cruz), Drp1 (1:1000 dilution, Abcam), p-Drp1 Ser616 (1:1000 dilution, Abcam), TFRC (1:1000 dilution, Boster), SLC7A11 (1:1000 dilution, Thermo Fisher), GPX4 (1:1000 dilution, Proteintech), ACSL4 (1:1000 dilution, Santa Cruz), Bax (1:1000 dilution, Boster), Caspase-3 (1:1000 dilution, Cell signaling), Cleaved caspase-3 (1:1000 dilution, Cell signaling), LC3 I/II(1:1000 dilution, Cell signaling) primary antibodies at 4°C overnight, then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature and developed with ECL blotting substrate (ABclonal, China). Western blot images were analyzed using Image J software. Statistical analysis Data from every experiment were represented as mean ± SEM and analyzed utilizing GraphPad Prism (GraphPad Software, Inc., USA). Comparisons between groups were evaluated with an unpaired t-test. A p-value less than 0.05 (p < 0.05) was deemed statistically significant. Results Celastrol or curcumin effectively inhibited the proliferation of CNE1 cells and induced mitochondrial dysfunction CNE cell viability was measured after administering different concentrations of celastrol or curcumin. Our results showed that celastrol at low dose (0.3 µM and 0.625 µM) did not significantly affect cell viability compared to the control group, while higher concentrations (1.25 µM, 2.5 µM, 5 µM, and 10 µM) led to a concentration-dependent decrease in cell viability ( Fig. 1 A ) . The calculated IC50 for celastrol was 3.420 µM ( Fig. 1 A ) . Similarly, curcumin at low dose (3.125 µM and 6.25 µM) showed no significant changes in cell viability compared to the control group, but at higher concentrations (12.5 µM, 25 µM, 50 µM, and 100 µM), there was a concentration-dependent decrease ( Fig. 1 B ) . The IC50 for Curcumin was 56.69 µM ( Fig. 1 B ) . Next, mitochondrial function in CNE1 cells was assessed in response to therapeutic doses of celastrol or curcumin. Our results showed a significant decrease in mitochondrial membrane potential following treatment with 1.25 µM, 2.5 µM, and 3.5 µM of celastrol compared to the control group (Fig. 1 C). Mitochondrial ROS levels remained stable at 1.25 µM and 2.5 µM of celastrol, whilst 3.5 µM celastrol notably increase mitochondrial ROS levels compared to the control group (Fig. 1 D). similarly, mitochondrial membrane potential decreased significantly with 12.5 µM, 25 µM, and 35 µM curcumin treatment (Fig. 1 E), accompanied by elevated ROS levels in these groups (Fig. 1 F). These data suggested that either therapeutic dose of celastrol or curcumin effectively inhibited the proliferation and induced mitochondrial dysfunction of CNE1 cells. Low-dose celastrol enhances curcumin-induced cytotoxic effects through non-apoptotic cell death One issue with the clinical application of celastrol is its limited therapeutic range, which is affected by dosage constraints and possible adverse effects. Next, we investigated whether curcumin could sensitize CNE1 cells to low-dose celastrol. In our study, CNE1 cells were treated with a low dose of celastrol (0.7 µM) in combination with varying concentrations of curcumin, and cell viability was measured. Our results showed that 0.7 µM celastrol alone did not significantly reduce cell viability compared to the control group ( Fig. 2 A ) . When combined with 0.7 µM celastrol, curcumin notably enhanced the inhibitory effects on CNE1 cells, surpassing the effects of the same concentration of curcumin without celastrol ( Fig. 2 A ) . The IC50 for curcumin was reduced to 48.49 µM in the presence of 0.7 µM celastrol ( Fig. 2 B ) . The results suggested that the combination exhibited remarkable effects when compared to either low-dose celastrol or curcumin used separately in CNE1 cells. Next, we examined the type of cell death involved in treating CNE1 cells with combined curcumin and low-dose celastrol. Initially, we assessed the levels of apoptosis-related proteins BCL2-associated X (Bax), cleaved cysteine-aspartic acid protease 3 (cleaved caspase-3), and cysteine-aspartic acid protease 3 (caspase-3) in CNE1 cells. Our results showed that there was no significant alteration in the protein expression level of Bax in either the curcumin alone or the celastrol combined with the curcumin treatment when compared to the control group (Fig. 3 A, B ) . Additionally, the protein expression ratio of cleaved caspase-3/caspase-3 also remained unchanged across all treatments (Fig. 3 C, D). These results indicate that the low-dose celastrol enhances curcumin-induced cytotoxic effects through non-apoptotic cell death. Combined treatment with curcumin and low-dose celastrol induced autophagy in the CNE1 cell line Autophagy, a highly conserved degradation pathway, plays a crucial role in various cellular processes. Research has shown that in cancer cells, autophagy can act as an alternative to apoptosis, particularly in cells resistant to apoptosis[ 18 ]. As the increase of microtubule-associated protein 1A/1B-light chain 3, lipidated form (LC3-II) can indicate an enhanced autophagy induction, we examined the levels of autophagy-related proteins LC3 II in CNE1 cells after drug treatments. Our results showed that treatment with 0.7µM celastrol for 24 hours did not affect the LC3 II level in CNE1 cells compared to the control group (Fig. 4 A, B). However, LC3-II levels significantly increased with both curcumin (10 µM, 25 µM, and 35 µM) alone and combined treatment (Fig. 4 A, B). In addition, the increased LC3-II levels were comparable between curcumin alone and the combined treatment. These findings indicate that either curcumin alone or combined treatment induced autophagy in CNE1 cells. Ferroptosis plays a critical role in low-dose celastrol plus curcumin-induces cell death Ferroptosis pathways were determined to investigate the potential of low-dose celastrol in enhancing the anti-cancer effects of curcumin. During ferroptosis, the transferrin receptor (TFRC) aids in the uptake of transferrin-bound iron into cells, where it is converted to Fe 2+ through reduction, triggering a strong Fenton reaction and subsequent surge in ROS. Glutathione peroxidase 4 (GPX4) and Solute carrier family 7 member 11 (SLC7A11) are essential for counteracting lipid peroxidation by utilizing glutathione (GSH). Conversely, Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key player in initiating lipid peroxidation and a contributor to ferroptosis, catalyzes the esterification of free polyunsaturated fatty acids (PUFAs) to generate PUFA-CoA. Our results showed that while TFRC protein levels did not exhibit statistical significance across all treatments compared to the control group, there was a trend towards an increase in the 0.7 µM celastrol combined with 35 µM curcumin treatment (p-value = 0.067) ( Fig. 5 A, B ) . SLC7A11 levels were significantly lower in the 25 µM and 35 µM curcumin groups compared to the control, with a further reduction observed in the 0.7 µM celastrol combined with 35 µM curcumin group ( Fig. 5 C,D ) . Additionally, GPX4 levels were significantly increased in both the curcumin and the curcumin groups when compared to the control, with a more significant increase observed in cells treated with 0.7 µM celastrol combined with 35 µM curcumin ( Fig. 5 E,F ) . Only the combination of 0.7 µM celastrol and 35 µM curcumin led to a significant increase in ACSL4 levels, while other treatment regimens did not affect ACSL4 levels compared to the control group ( Fig. 5 E,G ) . These findings suggest that the enhanced inhibitory effects on CNE1 cells by the combination therapy may be primarily attributed to ferroptosis. Mitochondrial dynamics may be a key player in combined treatment-induced cell death There is a growing body of literature that connects mitochondrial fusion/fission to cancer cell metabolism and death[ 19 , 20 ]. We therefore considered whether mitochondrion dynamics could play a role in cell death induced by the combined treatment. The expression of mitochondrial dynamin-related protein 1 (Drp1) and mitochondrial mitofusin 2 protein (Mfn2) in CNE1 cells was evaluated following treatment with low-dose celastrol, curcumin alone, and their combination. Our results showed that 0.7 µM celastrol and 10 µM or 25 µM curcumin alone did not affect the p-Drp1/Drp1 ratio compared to the control group (Fig. 6 A, B). Only a high dose of curcumin (35 µM) led to a significant increase in the p-Drp1/Drp1 ratio. However, combining 25 µM or 35 µM curcumin with 0.7 µM celastrol resulted in a notable increase in the p-Drp1/Drp1 ratio compared to the control group. Furthermore, when 35 µM curcumin was combined with 0.7 µM celastrol, there was a significant increase in the p-Drp1/Drp1 ratio, surpassing the effects of curcumin alone. Mfn2 expression, a mitochondrial fusion-related protein, showed no significant difference from the control group in any treatment group (Fig. 6 A, C). These data indicated that Drp1 phosphorylation-mediated mitochondrial fission may contribute to combined celastrol and curcumin-induced cell death. Discussion Natural substances provide most small molecule drug candidates for potential use as anticancer agents[ 21 ]. Compounds derived from plants offer unique and innovative structures that serve as valuable tools for exploring protein function and mechanisms of cell death[ 22 ]. High doses of celastrol and curcumin have demonstrated significant promise as effective antitumor agents, exhibiting notable antiproliferative effects[ 23 , 24 ]. However, the severe side effects associated with high-dose celastrol restrict its clinical application. While the FDA has classified curcumin as safe based on prior clinical trials [ 25 , 26 ], it is crucial to emphasize that its low bioavailability presents a therapeutic challenge for clinical use[ 27 ]. This study investigates the potential of combination therapy utilizing low-dose celastrol and curcumin to enhance anticancer effects while minimizing unwanted cytotoxicity. In this study, we found that celastrol or curcumin effectively inhibited CNE1 cell proliferation and induced mitochondrial dysfunction. We hypothesized that curcumin may enhance the sensitivity of CNE1 cells to low-dose celastrol. Our study used a very low dose of celastrol (0.7 µM), which may not induce inhibition of cell proliferation or cell death on its own. However, the combination of 0.7 µM celastrol with curcumin demonstrates enhanced inhibitory effects on CNE1 cells compared to curcumin alone at the same concentration. In addition, we provide novel evidence that the combination therapy's enhanced inhibitory effects on CNE1 cells may be primarily attributed to ferroptosis but not apoptosis. Cell death pathways were assessed in this study to discuss the underlying mechanism of the combined treatment. Previous studies have demonstrated that celastrol and curcumin possess strong anti-tumour activity against various cancer cell lines, such as lung, breast, liver, and gastric cancers[ 7 , 28 – 34 ], by triggering cell apoptosis and related pathways. However, the impact of curcumin on apoptotic cell death remains a topic of debate. Our findings align with the previous studies[ 35 , 36 ], showing that curcumin-induced cell death occurs independently of caspase-3 activation (Fig. 3 B). We speculated that the effects of curcumin on cell death may vary based on concentration and cell type specificity. Unexpectedly, the combination of curcumin and low-dose celastrol did not result in significant changes in the expression of apoptosis-related proteins, such as Bax and the cleaved caspase-3/caspase-3 ratio (Fig. 3 A, B). This suggests that an alternative mechanism of cell death, other than apoptosis, may be involved in the combined treatment. Autophagy-dependent cell death is a complex process that shows varied associations with different types of regulated cell death[ 37 ]. Depending on the specific cellular environment and drug-cell interactions, autophagy can have a dual function, providing protective effects or inducing autophagic cell death. Cancer cells, including those of nasopharyngeal carcinoma, experience constant cellular stress due to heightened metabolic activity and increased nutrient requirements. The proper functioning of autophagic pathways is essential to maintain the mitochondrial metabolites needed for tumor cell growth[ 38 , 39 ]. However, excessive activation of autophagy can lead to pro-death signaling, ultimately resulting in the elimination of cancer cells[ 40 , 41 ]. Research has indicated that autophagy can serve as an alternative to apoptosis, particularly in cancer cells resistant to apoptosis[ 18 ]. Because of its dual nature, there have been successful reports of tumor cell suppression through both the inhibition and induction of autophagy. While autophagy can have both pro-death and pro-survival effects, most studies reported that curcumin-induced autophagy acts as a pro-death signal in various types of tumors, such as glioma [ 42 ]and gastric cancer cells[ 34 ], by inhibiting PI3K and Akt signaling pathways. In addition, curcumin has been demonstrated to increase ROS production, promoting apoptosis in colorectal cancer[ 43 ]. However, it is widely recognized that ROS can also trigger and maintain autophagy in tumor cells[ 44 ]. Consistent with these findings, our data showed that curcumin at concentrations of 10, 25, and 35µM induced autophagy by increasing LC3 levels (Fig. 4 A, B) and increasing ROS levels (Fig. 1 F). Our results suggest that ROS induced by curcumin play a role in the conversion of LC3-I to LC3-II, ultimately activating autophagy. While the combined treatment resulted in a similar level of autophagy in CNE1 cells compared to curcumin treatment alone (Fig. 4 A, B), phosphorylation of Drp1 Ser616 , which promotes mitochondrial fission, was only increased in the combined treatment and not with curcumin alone (Fig. 6 A, B). These findings indicated that mitochondrial fission may play a critical role in combined treatment-induced cell death. Mitochondria create a dynamic network that can undergo fusion and fission events, collectively referred to as mitochondrial dynamics. The regulation of these events is controlled by Mfn 1 and 2, OPA-1, Drp1, and mitochondrial Fis1 proteins. Changes in the phosphorylation or expression levels of Drp1 are closely associated with the imbalance of mitochondrial fission and fusion. Disruption in mitochondrial dynamics, characterized by increased fission resulting in fragmented mitochondria, has been implicated in cancer progression[ 45 , 46 ] and metastasis[ 47 ]. However, several studies have demonstrated that excessive mitochondrial fission significantly enhances cancer cell death[ 48 ]. On a molecular scale, mitochondrial fission generates a substantial number of mitochondrial fragments that contain damaged DNA and contribute to an overload of ROS, mediating oxidative stress in cancer. Furthermore, mitochondrial division facilitates the opening of the mitochondrial permeability transition pore (mPTP), a key indicator of mitochondrial death. Additionally, fragmented mitochondria are unable to produce sufficient ATP to sustain cancer metabolism. Based on these mechanisms, mitochondrial division has been recognized as a promising target for enhancing cancer cell death[ 49 ]. Among various forms of regulated cell death, ferroptosis is a unique form of cell death, distinguished by cell volume shrinkage and heightened mitochondrial membrane density, without the usual signs of apoptosis or necrosis[ 50 ]. A previous study found that curcumin triggers ferroptosis in breast cancer cells by promoting iron accumulation and downregulating GPX4[ 51 ]. However, Zhou’s team found that nasopharyngeal carcinoma cells show a reduced number of active reactions to ferroptosis inducers compared to both tongue squamous cell carcinoma cells and laryngeal squamous cell carcinoma cells[ 52 ]. Furthermore, ferroptosis has been reported to correlate with chemoresistance in nasopharyngeal carcinoma[ 53 ]. Therefore, ferroptosis has become a promising target for innovative drug development in treating nasopharyngeal carcinoma. In our current research, curcumin was found to effectively suppress the expression of GPX4 and SLC7A11 (Fig. 5 C-F). SLC7A11, a transmembrane protein involved in cystine-glutamate transport, is critical in maintaining cellular redox balance by facilitating glutathione synthesis[ 54 ]. However, TFRC, essential for regulating intracellular iron levels[ 55 ], did not show any change after curcumin treatment (Fig. 5 A, B). Interestingly, the combination treatment resulted in a further decrease in GPX4 and SLC7A11 compared to curcumin treatment alone. Furthermore, ACSL4, a contributor to ferroptosis by enhancing lipid peroxidation, only increased with the combined treatment and not with curcumin alone (Fig. 5 E, G). This suggests that curcumin enhances the sensitivity of CNE1 cells to ferroptosis, although it is insufficient to induce ferroptosis independently at a concentration of 35 µM. Interestingly, the addition of low-dose celastrol further enhanced curcumin-induced ferroptosis, highlighting the critical role of ferroptosis in cell death induced by the combined treatment. In addition, the combined treatment led to ferroptosis accompanied by increased phosphorylation of Drp1. These results align with previous studies demonstrating the association between ferroptosis and heightened mitochondrial fragmentation[ 20 , 56 ]. We speculated that mitochondrial fission may be a crucial factor in the combined treatment-induced ferroptotic cell death; however, the exact relationship remains to be clarified. The compatibility of medicines is fundamental to traditional Chinese medicine prescriptions, which can serve as a safe and cost-effective approach for the co-treatment of various cancer types, including nasopharyngeal carcinoma. However, it is important to note that plant compounds should not be viewed as standalone cures for cancer. Instead, their significance lies in their role as a continuous prophylactic or complementary supplement to cytostatic therapy. Conclusion Low doses of celastrol combined with curcumin exhibited greater inhibition of CNE1 cell growth compared to curcumin alone. This enhanced efficacy of the combination therapy is likely attributable to effects on mitochondrial fission and the induction of ferroptosis. These findings not only demonstrate a potential antitumor effect of combining celastrol with curcumin but also open avenues for future investigations into the possible co-therapeutic properties of these phytotherapeutic agents in conjunction with chemoradiotherapy, thereby facilitating the development of a safe and effective strategy for cancer treatment. Abbreviations NPC Nasopharyngeal carcinoma ROS Reactive oxygen species mPTP Mitochondrial permeability transition pore Bax BCL2-associated X Cleaved caspase-3 Cleaved cysteine-aspartic acid protease 3 Caspase-3 Cysteine-aspartic acid protease 3 LC3 II Microtubule-associated protein 1A/1B-light chain 3, lipidated form TFRC Transferrin receptor GPX4 Glutathione peroxidase 4 SLC7A11 Solute carrier family 7 member 11 ACSL4 Acyl-CoA synthetase long-chain family member 4 Drp1 Dynamin-Related Protein 1 Mfn2 Mitofusin 2 protein GSH Glutathione PUFAs Polyunsaturated fatty acids Declarations Acknowledgements Not applicable. Author contributions SL: Conceptualization, funding acquisition, and resources; TF, YL, XZ, ZF, XF, BL, YL, JW, SM: Performed experiments; TF, SL: Formal analysis; SL, TF: Writing-original draft; SL, JW: Writing-review and editing. All authors reviewed the manuscript. Corresponding authors Correspondence to Suchan Liao. Funding This study was supported by Baise scientific research and technology development plan of regionally frequently occurring diseases, China (NO.20224129 to SL); The basic scientific research ability improvement project of young and middle-aged university teachers in Guangxi, China (NO.2019KY0568 to SL); Natural Science Foundation of Youjiang Medical University for Nationalities (NO. yy2018ky001 to SL). Availability of data materials The data used in this study are available from the corresponding authors on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China. 2 Graduate School, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. 3 Laboratory Center of Medical Science Morphology, School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. 4 Modern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. 5 Department of Physiology, School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China. References Chen Y-P, Chan AT, Le Q-T, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. The Lancet. 2019;394(10192):64-80. Tao Q, Chan AT. Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments. Expert reviews in molecular medicine. 2007;9(12):1-24. Salvador-Coloma C, Cohen EJJoop. Multidisciplinary care of laryngeal cancer. Journal of oncology practice 2016;12(8):717-24. Newman DJ, Cragg GM. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J Nat Prod. 2020;83(3):770-803. Tsang NY, Li WF, Varhegyi E, Rong L, Zhang HJ. Ebola Entry Inhibitors Discovered from Maesa perlarius. Int J Mol Sci. 2022;23(5). Duan J, Zhan JC, Wang GZ, Zhao XC, Huang WD, Zhou GB. The red wine component ellagic acid induces autophagy and exhibits anti-lung cancer activity in vitro and in vivo. J Cell Mol Med. 2019;23(1):143-54. Chen P, Wang B, Li M, Cui C, Liu F, Gao Y. Celastrol inhibits the proliferation and migration of MCF-7 cells through the leptin-triggered PI3K/AKT pathway. Computational and Structural Biotechnology Journal. 2022;20:3173-81. Chen M, Yang J, Li L, Lu X, Sun R, Wang Y, Zhang X. Metabolomics Reveals Cysteine Metabolism Plays a Role in Celastrol-Induced Mitochondrial Apoptosis of Acute Promyelocytic Leukemia Cells. Available at SSRN 3335016. 2019. Du S, Song X, Li Y, Cao Y, Chu F, Durojaye OA, et al. Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells. Scientific Reports. 2020;10(1):11273. Kannaiyan R, Hay HS, Rajendran P, Li F, Shanmugam MK, Vali S, et al. Celastrol inhibits proliferation and induces chemosensitization through down‐regulation of NF‐κB and STAT3 regulated gene products in multiple myeloma cells. British journal of pharmacology. 2011;164(5):1506-21. Arisan ED, Rencuzogullari O, Coban M, Sevgin B, Obakan-Yerlikaya P, Çoker-Gürkan A, Palavan-Unsal N. The role of the PI3K/AKT/mTOR signaling axis in the decision of the celastrol-induced cell death mechanism related to the lipid regulatory pathway in prostate cancer cells. Phytochemistry Letters. 2020;39:73-83. Yu X, Meng X, Xu M, Zhang X, Zhang Y, Ding G, et al. Celastrol ameliorates cisplatin nephrotoxicity by inhibiting NF-κB and improving mitochondrial function. EBioMedicine. 2018;36:266-80. Hou W, Liu B, Xu H. Celastrol: Progresses in structure-modifications, structure-activity relationships, pharmacology and toxicology. Eur J Med Chem. 2020;189:112081. Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod. 2007;70(3):461-77. Priyadarsini KI, Maity DK, Naik G, Kumar MS, Unnikrishnan M, Satav J, Mohan H. Role of phenolic OH and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radical Biology and Medicine. 2003;35(5):475-84. Barati N, Momtazi‐Borojeni AA, Majeed M, Sahebkar A. Potential therapeutic effects of curcumin in gastric cancer. Journal of cellular physiology. 2019;234(3):2317-28. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med. 1998;64(4):353-6. Brockmueller A, Ruiz de Porras V, Shakibaei M. Curcumin and its anti-colorectal cancer potential: From mechanisms of action to autophagy. Phytother Res. 2024;38(7):3525-51. Vyas S, Zaganjor E, Haigis MC. Mitochondria and Cancer. Cell. 2016;166(3):555-66. Li C, Zhang Y, Liu J, Kang R, Klionsky DJ, Tang D. Mitochondrial DNA stress triggers autophagy-dependent ferroptotic death. Autophagy. 2021;17(4):948-60. Shanmugam MK, Lee JH, Chai EZP, Kanchi MM, Kar S, Arfuso F, et al., editors. Cancer prevention and therapy through the modulation of transcription factors by bioactive natural compounds. Seminars in cancer biology; 2016: Elsevier. Dhankhar S, Ruhil S, Balhara M, Dhankhar S, Chhillar A. Aegle marmelos (Linn.) Correa: A potential source of Phytomedicine. J Med Plant Res. 2011;5(9):1497-507. Guneydas G, Topcul MR. Antiproliferative effects of curcumin different types of breast cancer. Asian Pacific Journal of Cancer Prevention: APJCP. 2022;23(3):911. Zhao Z, Wang Y, Gong Y, Wang X, Zhang L, Zhao H, et al. Celastrol elicits antitumor effects by inhibiting the STAT3 pathway through ROS accumulation in non-small cell lung cancer. J Transl Med. 2022;20(1):525. Cox KH, Pipingas A, Scholey AB. Investigation of the effects of solid lipid curcumin on cognition and mood in a healthy older population. J Psychopharmacol. 2015;29(5):642-51. Dhillon N, Aggarwal BB, Newman RA, Wolff RA, Kunnumakkara AB, Abbruzzese JL, et al. Phase II trial of curcumin in patients with advanced pancreatic cancer. Clin Cancer Res. 2008;14(14):4491-9. Koronyo Y, Biggs D, Barron E, Boyer DS, Pearlman JA, Au WJ, et al. Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease. JCI Insight. 2017;2(16). Ashrafizadeh M, Najafi M, Makvandi P, Zarrabi A, Farkhondeh T, Samarghandian S. Versatile role of curcumin and its derivatives in lung cancer therapy. Journal of cellular physiology. 2020;235(12):9241-68. Zhao Z, Wang Y, Gong Y, Wang X, Zhang L, Zhao H, et al. Celastrol elicits antitumor effects by inhibiting the STAT3 pathway through ROS accumulation in non-small cell lung cancer. Journal of Translational Medicine. 2022;20(1):525. Farghadani R, Naidu R. Curcumin as an enhancer of therapeutic efficiency of chemotherapy drugs in breast cancer. International Journal of Molecular Sciences. 2022;23(4):2144. Ren B, Luo S, Tian X, Jiang Z, Zou G, Xu F, et al. Curcumin inhibits liver cancer by inhibiting DAMP molecule HSP70 and TLR4 signaling. Oncology reports. 2018;40(2):895-901. Zhou M, Liao J, Lai W, Xu R, Liu W, Xie D, et al. A celastrol-based nanodrug with reduced hepatotoxicity for primary and metastatic cancer treatment. EBioMedicine. 2023;94. Chen X, Zhao Y, Luo W, Chen S, Lin F, Zhang X, et al. Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells. Theranostics. 2020;10(22):10290. Fu H, Wang C, Yang D, Wei Z, Xu J, Hu Z, et al. Curcumin regulates proliferation, autophagy, and apoptosis in gastric cancer cells by affecting PI3K and P53 signaling. Journal of cellular physiology. 2018;233(6):4634-42. O'Sullivan-Coyne G, O'Sullivan GC, O'Donovan TR, Piwocka K, McKenna SL. Curcumin induces apoptosis-independent death in oesophageal cancer cells. Br J Cancer. 2009;101(9):1585-95. Seo SU, Woo SM, Lee HS, Kim SH, Min KJ, Kwon TK. mTORC1/2 inhibitor and curcumin induce apoptosis through lysosomal membrane permeabilization-mediated autophagy. Oncogene. 2018;37(38):5205-20. Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. Cell Res. 2019;29(5):347-64. Amaravadi R, Kimmelman AC, White E. Recent insights into the function of autophagy in cancer. Genes Dev. 2016;30(17):1913-30. Amaravadi RK, Kimmelman AC, Debnath J. Targeting Autophagy in Cancer: Recent Advances and Future Directions. Cancer Discov. 2019;9(9):1167-81. Kim KW, Hwang M, Moretti L, Jaboin JJ, Cha YI, Lu B. Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer. Autophagy. 2008;4(5):659-68. Nazim UM, Yin H, Park SY. Downregulation of c‑FLIP and upregulation of DR‑5 by cantharidin sensitizes TRAIL‑mediated apoptosis in prostate cancer cells via autophagy flux. Int J Mol Med. 2020;46(1):280-8. Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol Pharmacol. 2007;72(1):29-39. Liu C, Rokavec M, Huang Z, Hermeking H. Curcumin activates a ROS/KEAP1/NRF2/miR-34a/b/c cascade to suppress colorectal cancer metastasis. Cell Death Differ. 2023;30(7):1771-85. Tang ZH, Cao WX, Wang ZY, Lu JH, Liu B, Chen X, Lu JJ. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells. Redox Biol. 2017;12:367-76. Tran Q, Lee H, Jung JH, Chang SH, Shrestha R, Kong G, et al. Emerging role of LETM1/GRP78 axis in lung cancer. Cell Death Dis. 2022;13(6):543. Li T, Han J, Jia L, Hu X, Chen L, Wang Y. PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation. Protein Cell. 2019;10(8):583-94. Cheng CT, Kuo CY, Ouyang C, Li CF, Chung Y, Chan DC, et al. Metabolic Stress-Induced Phosphorylation of KAP1 Ser473 Blocks Mitochondrial Fusion in Breast Cancer Cells. Cancer Res. 2016;76(17):5006-18. Tang Q, Liu W, Zhang Q, Huang J, Hu C, Liu Y, et al. Dynamin-related protein 1-mediated mitochondrial fission contributes to IR-783-induced apoptosis in human breast cancer cells. J Cell Mol Med. 2018;22(9):4474-85. You Y, He Q, Lu H, Zhou X, Chen L, Liu H, et al. Silibinin Induces G2/M Cell Cycle Arrest by Activating Drp1-Dependent Mitochondrial Fission in Cervical Cancer. Front Pharmacol. 2020;11:271. Zhang Z, Yao Z, Wang L, Ding H, Shao J, Chen A, et al. Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells. Autophagy. 2018;14(12):2083-103. Li R, Zhang J, Zhou Y, Gao Q, Wang R, Fu Y, et al. Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. Oxid Med Cell Longev. 2020;2020:3469840. Liu S, Yan S, Zhu J, Lu R, Kang C, Tang K, et al. Combination RSL3 Treatment Sensitizes Ferroptosis- and EGFR-Inhibition-Resistant HNSCCs to Cetuximab. Int J Mol Sci. 2022;23(16). Xu Y, Du Y, Zheng Q, Zhou T, Ye B, Wu Y, et al. Identification of Ferroptosis-Related Prognostic Signature and Subtypes Related to the Immune Microenvironment for Breast Cancer Patients Receiving Neoadjuvant Chemotherapy. Front Immunol. 2022;13:895110. de la Vega MR, Chapman E, Zhang DD. NRF2 and the Hallmarks of Cancer. Cancer cell. 2018;34(1):21-43. Zhu G, Murshed A, Li H, Ma J, Zhen N, Ding M, et al. O-GlcNAcylation enhances sensitivity to RSL3-induced ferroptosis via the YAP/TFRC pathway in liver cancer. Cell Death Discovery. 2021;7(1):83. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-72. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4827626","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345357440,"identity":"84942359-9638-4e6c-940f-96581d7446ca","order_by":0,"name":"Tao Feng","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Feng","suffix":""},{"id":345357441,"identity":"ba79f181-aa36-418a-a32b-39169e0cf43a","order_by":1,"name":"Yinjun Luo","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Yinjun","middleName":"","lastName":"Luo","suffix":""},{"id":345357443,"identity":"58db16f1-4020-4507-bbb2-49b7076e5135","order_by":2,"name":"Xin Zhang","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":345357448,"identity":"dfd0dd78-34d5-498b-9088-b0ee2045bd42","order_by":3,"name":"Ziyang Fang","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Ziyang","middleName":"","lastName":"Fang","suffix":""},{"id":345357449,"identity":"1eb4eb74-4b47-4216-ac1d-b940b481bb90","order_by":4,"name":"Ying Li","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Li","suffix":""},{"id":345357451,"identity":"68f423fa-46a8-44af-aa0f-5cde896b71f0","order_by":5,"name":"Shijing Ma","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Shijing","middleName":"","lastName":"Ma","suffix":""},{"id":345357453,"identity":"17133d48-316f-42c1-b593-c8a8cb3bff97","order_by":6,"name":"Jingting Wei","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Jingting","middleName":"","lastName":"Wei","suffix":""},{"id":345357454,"identity":"09d620a3-6ea8-4b9f-89e8-793e3d2e7756","order_by":7,"name":"Xiaoyan Fang","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Fang","suffix":""},{"id":345357455,"identity":"17d2913a-d572-4a69-91db-23a009bd6a9b","order_by":8,"name":"Biao Li","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Li","suffix":""},{"id":345357456,"identity":"ef6e64ef-bd57-4bd3-a089-840ee0c778ba","order_by":9,"name":"Jinhua Wang","email":"","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Wang","suffix":""},{"id":345357457,"identity":"38568da1-5e59-49ec-8b0c-7a251310d9b4","order_by":10,"name":"Suchan Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACPmYIncDAwHyAAc7GB9gQWthgSglpQSjjMSBSCzvzw4c/2+zy+Gf3fJO62XaHgZ89x4Dh5w58DmMzNuZtSy6WuHN2m3Ru2zMGyZ43Boy9Z/D6xUyase1AYsONXJCWwwwGN3IMmBnb8Glh/yb5E6hl/o2cZ2At9oS18JhJ8AK1bLiRwwaxRYKwlmJjnnPJiRtvpBlb55w7zCNx5lnBwV48Wvj5j298+KPMLnHejeSHt3PKDsvxtydvfPATjxZkwCIBJHhArAPEaQCmmA/EqhwFo2AUjIKRBQAXH0z6klHeywAAAABJRU5ErkJggg==","orcid":"","institution":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities","correspondingAuthor":true,"prefix":"","firstName":"Suchan","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2024-07-30 09:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4827626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4827626/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63398084,"identity":"cdaedf0d-bca7-4c74-bfb4-b577ee7c0b5a","added_by":"auto","created_at":"2024-08-27 17:48:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":204768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCelastrol or curcumin effectively inhibited the proliferation of CNE1 cells and induced mitochondrial dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Percentage of cell viability after treatment of CNE1 cells with 0.3 μM, 0.625 μM, 1.25 μM,2.5 μM,5 μM, and 10 μM of celastrol for 24 h, respectively. \u0026nbsp;And IC50 values of CNE1 after 24h of celastrol treatment. \u0026nbsp;(B) Percentage of cell viability after treatment of CNE1 cells with 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM of curcumin for 24 h, respectively.\u0026nbsp; And IC50 value of CNE1 after 24h of curcumin treatment.\u0026nbsp; (C) Mitochondrial membrane potential levels after treatment of CNE1 cells with 1.25 μM, 2.5 μM and 3.5 μM celastrol, respectively.\u0026nbsp; Data are presented as mean ± SEM.\u0026nbsp; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u0026nbsp; (D) ROS levels after treatment of CNE1 cells with 1.25 μM, 2.5 μM and 3.5 μM Celastrol, respectively.\u0026nbsp; Data are presented as mean ± SEM.\u0026nbsp; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u0026nbsp; (E) Mitochondrial membrane potential levels after treatment of CNE1 cells with 12.5 μM, 25 μM and 35 μM curcumin, respectively. \u0026nbsp;Data are presented as mean ± SEM.\u0026nbsp; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u0026nbsp; (F) ROS levels after treatment of CNE1 cells with 12.5 μM, 25 μM and 35 μM curcumin, respectively.\u0026nbsp; Data are presented as mean ± SEM.\u0026nbsp; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/d80abc6975f2fc239f5e3f3e.png"},{"id":63398081,"identity":"c4cf6cd6-2431-4394-a8f0-26b4754ec4c3","added_by":"auto","created_at":"2024-08-27 17:48:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCelastrol combined with curcumin more significantly inhibited CNE1 cell proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell viability of CNE1 cell lines using 0.7 μM Celastrol combined with 5 μM, 10 μM,15 μM,20 μM, 25 μM, 30 μM, 40 μM, 45 μM and 50 μM curcumin and 5 μM, 10 μM,15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 45 μM and 50 μM alone. \u0026nbsp;Curcumin for cell viability after 24 h of treatment of CNE1 cell line.\u0026nbsp; Data are presented as mean ± SEM.\u0026nbsp; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control. \u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with curcumin at the same concentration.\u0026nbsp; (B) IC50 values for curcumin when 0.7 μM celastrol is combined with 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 45 μM, and 50 μM curcumin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/cdf0dd7bb0462e90cca9d3d2.png"},{"id":63398086,"identity":"f593bea2-cd10-46d0-b067-b11428abab37","added_by":"auto","created_at":"2024-08-27 17:48:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeath of CNE1 cells is not dependent on apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot detecting Bax protein levels. \u0026nbsp;(B) Quantitative data of (A) showed the Bax protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;(C) Western blot detecting cleaved caspase-3 and caspase-3 protein levels. \u0026nbsp;(D) Quantitative data of (C) showed the ratio of cleaved caspase-3/caspase-3 protein expression level. \u0026nbsp;Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/d5ac117bda569d64635892ce.png"},{"id":63398329,"identity":"2bd64941-a33e-429a-bb11-6a1f11d7cbfe","added_by":"auto","created_at":"2024-08-27 17:56:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCelastrol combined with curcumin induces autophagy in the CNE1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot detecting LC3 II protein levels. \u0026nbsp;(B) Quantitative data of (A) showed the ratio of LC3 II protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/7bce446bf74df25571b44b71.png"},{"id":63398328,"identity":"8c271459-4218-4020-8fdf-2fd9588c6264","added_by":"auto","created_at":"2024-08-27 17:56:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":293495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCelastrol combined with curcumin induces\u003c/strong\u003e \u003cstrong\u003eferroptosis in CNE1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot detecting TFRC protein levels. \u0026nbsp;(B) Quantitative data of (A) showed the TFRC protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u0026nbsp; (C) Western blot detecting SLC7A11 protein levels. \u0026nbsp;(D) Quantitative data of (C) showed the TFRC protein expression protein level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with curcumin at the same concentration.\u0026nbsp; (E) Western blot detecting GPX4 and ACSL4 protein levels. \u0026nbsp;(F) Quantitative data of (E) showed the GPX4 protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with the control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with curcumin at the same concentration.\u0026nbsp; (G) Quantitative data of (E) showed the ACSL4 protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/f696d202e162395943eaa35e.png"},{"id":63398082,"identity":"24003425-9e00-44c8-b157-7f58c86bb28b","added_by":"auto","created_at":"2024-08-27 17:48:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":197333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced mitochondrial fission by the combination of celastrol and curcumin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot detecting Drp1, p-Drp1\u003csup\u003eser616\u003c/sup\u003e protein levels. \u0026nbsp;(B) Quantitative data of (D) showed the ratio of p-Drp1\u003csup\u003eser616\u003c/sup\u003e/Drp1 protein expression level. \u0026nbsp;Data are presented as mean ± SEM. \u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with the control, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e＜0.05 with t test compared with curcumin at the same concentration.\u0026nbsp; (C) Western blot detecting Mfn2 protein levels. \u0026nbsp;(D) Quantitative data of (C) showed the Mfn2 protein expression level. \u0026nbsp;Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/db9a945fb35ac76953ad9fce.png"},{"id":64228430,"identity":"5dd3bf67-e06e-4944-954a-a92ece6d01dc","added_by":"auto","created_at":"2024-09-10 14:22:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1834168,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4827626/v1/629ae35f-a27f-45f2-8f40-396eb71f58da.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Celastrol combined with curcumin inhibits proliferation and causes cell death in nasopharyngeal carcinoma CNE1 cell line by inducing ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNasopharyngeal Carcinoma (NPC) is a malignant tumor originating from the epithelial cells of the nasopharynx. Regions with a high incidence of NPC include Southeast Asia, southern China, North Africa, and Greenland[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The development of NPC is influenced by factors such as Epstein-Barr virus infection, genetic predisposition, environmental influences, and lifestyle choices[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chemotherapy and radiotherapy are commonly used as initial treatments for NPC, showing promising results in early-stage patients. However, these treatments can lead to severe side effects and the development of resistance. Despite progress in treatment, advanced NPC patients still face challenges such as distant relapse and metastasis, resulting in low survival rates[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, there is a critical need to devise more effective strategies to combat NPC.\u003c/p\u003e \u003cp\u003eNatural products and their derivatives offer a valuable and sustainable source of scaffolds for contemporary drug development, constituting over 60% of FDA-approved anti-cancer medications[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Celastrol, a triterpenoid compound extracted from \u003cem\u003eTrypterygium wilfordii\u003c/em\u003e Hook F (commonly referred to as the Thunder of God Vine), has long been used in traditional Chinese medicine for the treatment of inflammatory diseases[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies, using \u003cem\u003ein vitro\u003c/em\u003e and/or \u003cem\u003ein vivo\u003c/em\u003e models, suggested that Celastrol is a promising anti-cancer reagent against breast cancer, leukemia, hepatocyte carcinoma, melanoma, myeloma, and prostate cancer[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, toxic side effects, low water solubility, and poor oral bioavailable have limited further clinical application of celastrol[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While structural modifications of celastrol have been explored to enhance solubility or mitigate toxicity, these alterations have often reduced the derivatives' anticancer efficacy[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There is a pressing requirement for innovative strategies to mitigate the toxicity of celastrol without compromising its anti-cancer efficacy.\u003c/p\u003e \u003cp\u003eCurcumin, a polyphenolic substance in turmeric (Curcuma longa), has been historically used as a food additive and dye colorant in Southeast Asia[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition to its well-known antioxidant, anti-inflammatory, antimicrobial, and analgesic properties, curcumin has displayed potential in treating various cancers, including liver, lung, breast, and gastric cancers[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although curcumin is recognized as a safe compound even at high doses, its low bioavailability poses a therapeutic challenge for clinical application. Interestingly, the bioavailability of curcumin can be increased by 2000% when combined with piperine[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This suggests that combination therapy may be an effective strategy to enhance the bioavailability of curcumin and improve its anti-cancer effects.\u003c/p\u003e \u003cp\u003eThe compatibility theory of Chinese medicine is essential in traditional Chinese medicine prescriptions, intending to improve effectiveness and minimize toxicity. However, there is a gap in research regarding the combined utilization of celastrol and curcumin for treating nasopharyngeal carcinoma. This study aims to investigate the potential anti-cancer properties of combining celastrol and curcumin. We hypothesize that the combination increases the anti-cancer effects while reducing the dosage requirement, optimizing toxicity levels, and reducing the risk of drug resistance development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines and Culture\u003c/h2\u003e \u003cp\u003eHuman NPC cell lines CNE1 was obtained from the Shanghai Aolu Biological Technology Co., Ltd (Shanghai, China). CNE1 cell was cultured in RPMI 1640 medium(Gibco, America), containing 10% fetal bovine serum (Procell, China) and 1% penicillin/streptomycin (Solarbio, China) at 37\u0026deg;C in a 5% CO2 incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell viability by CCK8 assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the CCK8 (Dojindo, Japan) method. CNE-1 cells in logarithmic growth phase were trypsinized, seeded in 96-well plates at a density of 8,000 cells per well, and incubated at 37℃ in a 5% CO2 environment. Following cell attachment, the culture medium was replaced with fresh medium containing varying concentrations of curcumin, nansenin, or a combination of both. After 24 hours of incubation, cellular proliferation was measured using the CCK-8 assay. Cell viability was determined based on the absorbance at 450 nm (OD450) to evaluate the effects of celastrol (Sigma, America), curcumin (Sigma, America), and their combination on CNE1 cells. All compounds were DMSO (Solarbio, China) for solubilization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCNE1 mitochondrial ROS levels\u003c/h2\u003e \u003cp\u003eThe levels of mitochondrial reactive oxygen species (ROS) in CNE1 cells were evaluated using the Mitochondrial ROS Activity Assay Kit (Beyotime, China), following the manufacturer's instructions. A dilution of dichlorofluorescin diacetate at a ratio of 1:1000 was prepared with serum-free culture medium. Cells from each group were collected and resuspended in the diluted DCFH-DA, achieving a cell density of 1\u0026times;10^6 cells/mL. They were subsequently incubated at 37\u0026ordm;C in a cell culture incubator for 20 minutes. Throughout this period, the mixture was gently inverted every 3\u0026ndash;5 minutes to facilitate adequate interaction between the DCFH-DA and the cells. After incubation, cells were washed three times with serum-free cell culture medium to remove any residual DCFH-DA that had not penetrated the cells. Rosup was added to the positive control. The incubated cells were then evenly distributed into 96-well plates with 6 wells per group, and analyzed using a fully automated enzyme labeling instrument with the excitation and emission wavelengths set at 488 nm and 525 nm, respectively. Fluorescence intensity was measured before and after stimulation in real-time or at specified time points. All procedures were conducted in a dark environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCNE1 mitochondrial membrane potential levels\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential was assessed using the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, China). Cells were harvested in fresh cell culture medium at a concentration ranging from 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e to 6\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per group. Subsequently, 0.5 mL of JC-1 working solution was added to the cells, mixed thoroughly, and then incubated for 20 minutes at 37\u0026ordm;C in a cell culture incubator. Following the incubation period, the cells underwent centrifugation at 4\u0026ordm;C, 600 g for 3 minutes. The supernatant was removed, and the cells were washed twice with 1 mL of JC-1 buffer. This washing procedure was repeated twice. Each group of cells was resuspended in an appropriate volume of JC-1 staining buffer and transferred to a 96-well plate for analysis using a fully automated enzyme labeling instrument. For detection of JC-1 monomers, the excitation light wavelength was set to 490 nm and the emission light wavelength to 530 nm. In the case of JC-1 polymers, the excitation light wavelength was set to 525 nm and the emission light wavelength to 590 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCell were ground in PMSF (Solarbio, China) with a high dose of RIPA buffer (ABExBIO, USA) and protein phosphatase inhibitors (Solarbio, China). The concentration of protein was measured through a bicinchoninic acid assay (Beyotime, China). Proteins were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto a PVDF membrane (Innobilon, USA). Membranes were blocked for 2 hours by adding 5% skimmed milk or 5% BSA to three-phase buffered saline (pH 7.4) containing 0.1% Tween 20 (Solarbio, China). Then incubated with Mfn2 (1:500 dilution, Santa Cruz), Drp1 (1:1000 dilution, Abcam), p-Drp1\u003csup\u003eSer616\u003c/sup\u003e (1:1000 dilution, Abcam), TFRC (1:1000 dilution, Boster), SLC7A11 (1:1000 dilution, Thermo Fisher), GPX4 (1:1000 dilution, Proteintech), ACSL4 (1:1000 dilution, Santa Cruz), Bax (1:1000 dilution, Boster), Caspase-3 (1:1000 dilution, Cell signaling), Cleaved caspase-3 (1:1000 dilution, Cell signaling), LC3 I/II(1:1000 dilution, Cell signaling) primary antibodies at 4\u0026deg;C overnight, then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature and developed with ECL blotting substrate (ABclonal, China). Western blot images were analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData from every experiment were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analyzed utilizing GraphPad Prism (GraphPad Software, Inc., USA). Comparisons between groups were evaluated with an unpaired t-test. A p-value less than 0.05 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCelastrol or curcumin effectively inhibited the proliferation of CNE1 cells and induced mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003eCNE cell viability was measured after administering different concentrations of celastrol or curcumin. Our results showed that celastrol at low dose (0.3 \u0026micro;M and 0.625 \u0026micro;M) did not significantly affect cell viability compared to the control group, while higher concentrations (1.25 \u0026micro;M, 2.5 \u0026micro;M, 5 \u0026micro;M, and 10 \u0026micro;M) led to a concentration-dependent decrease in cell viability \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The calculated IC50 for celastrol was 3.420 \u0026micro;M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Similarly, curcumin at low dose (3.125 \u0026micro;M and 6.25 \u0026micro;M) showed no significant changes in cell viability compared to the control group, but at higher concentrations (12.5 \u0026micro;M, 25 \u0026micro;M, 50 \u0026micro;M, and 100 \u0026micro;M), there was a concentration-dependent decrease \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The IC50 for Curcumin was 56.69 \u0026micro;M \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, mitochondrial function in CNE1 cells was assessed in response to therapeutic doses of celastrol or curcumin. Our results showed a significant decrease in mitochondrial membrane potential following treatment with 1.25 \u0026micro;M, 2.5 \u0026micro;M, and 3.5 \u0026micro;M of celastrol compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Mitochondrial ROS levels remained stable at 1.25 \u0026micro;M and 2.5 \u0026micro;M of celastrol, whilst 3.5 \u0026micro;M celastrol notably increase mitochondrial ROS levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). similarly, mitochondrial membrane potential decreased significantly with 12.5 \u0026micro;M, 25 \u0026micro;M, and 35 \u0026micro;M curcumin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), accompanied by elevated ROS levels in these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These data suggested that either therapeutic dose of celastrol or curcumin effectively inhibited the proliferation and induced mitochondrial dysfunction of CNE1 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLow-dose celastrol enhances curcumin-induced cytotoxic effects through non-apoptotic cell death\u003c/h2\u003e \u003cp\u003eOne issue with the clinical application of celastrol is its limited therapeutic range, which is affected by dosage constraints and possible adverse effects. Next, we investigated whether curcumin could sensitize CNE1 cells to low-dose celastrol. In our study, CNE1 cells were treated with a low dose of celastrol (0.7 \u0026micro;M) in combination with varying concentrations of curcumin, and cell viability was measured. Our results showed that 0.7 \u0026micro;M celastrol alone did not significantly reduce cell viability compared to the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. When combined with 0.7 \u0026micro;M celastrol, curcumin notably enhanced the inhibitory effects on CNE1 cells, surpassing the effects of the same concentration of curcumin without celastrol \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The IC50 for curcumin was reduced to 48.49 \u0026micro;M in the presence of 0.7 \u0026micro;M celastrol \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The results suggested that the combination exhibited remarkable effects when compared to either low-dose celastrol or curcumin used separately in CNE1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined the type of cell death involved in treating CNE1 cells with combined curcumin and low-dose celastrol. Initially, we assessed the levels of apoptosis-related proteins BCL2-associated X (Bax), cleaved cysteine-aspartic acid protease 3 (cleaved caspase-3), and cysteine-aspartic acid protease 3 (caspase-3) in CNE1 cells. Our results showed that there was no significant alteration in the protein expression level of Bax in either the curcumin alone or the celastrol combined with the curcumin treatment when compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Additionally, the protein expression ratio of cleaved caspase-3/caspase-3 also remained unchanged across all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These results indicate that the low-dose celastrol enhances curcumin-induced cytotoxic effects through non-apoptotic cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCombined treatment with curcumin and low-dose celastrol induced autophagy in the CNE1 cell line\u003c/h2\u003e \u003cp\u003eAutophagy, a highly conserved degradation pathway, plays a crucial role in various cellular processes. Research has shown that in cancer cells, autophagy can act as an alternative to apoptosis, particularly in cells resistant to apoptosis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As the increase of microtubule-associated protein 1A/1B-light chain 3, lipidated form (LC3-II) can indicate an enhanced autophagy induction, we examined the levels of autophagy-related proteins LC3 II in CNE1 cells after drug treatments. Our results showed that treatment with 0.7\u0026micro;M celastrol for 24 hours did not affect the LC3 II level in CNE1 cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). However, LC3-II levels significantly increased with both curcumin (10 \u0026micro;M, 25 \u0026micro;M, and 35 \u0026micro;M) alone and combined treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). In addition, the increased LC3-II levels were comparable between curcumin alone and the combined treatment. These findings indicate that either curcumin alone or combined treatment induced autophagy in CNE1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFerroptosis plays a critical role in low-dose celastrol plus curcumin-induces cell death\u003c/h2\u003e \u003cp\u003eFerroptosis pathways were determined to investigate the potential of low-dose celastrol in enhancing the anti-cancer effects of curcumin. During ferroptosis, the transferrin receptor (TFRC) aids in the uptake of transferrin-bound iron into cells, where it is converted to Fe\u003csup\u003e2+\u003c/sup\u003e through reduction, triggering a strong Fenton reaction and subsequent surge in ROS. Glutathione peroxidase 4 (GPX4) and Solute carrier family 7 member 11 (SLC7A11) are essential for counteracting lipid peroxidation by utilizing glutathione (GSH). Conversely, Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key player in initiating lipid peroxidation and a contributor to ferroptosis, catalyzes the esterification of free polyunsaturated fatty acids (PUFAs) to generate PUFA-CoA. Our results showed that while TFRC protein levels did not exhibit statistical significance across all treatments compared to the control group, there was a trend towards an increase in the 0.7 \u0026micro;M celastrol combined with 35 \u0026micro;M curcumin treatment (p-value\u0026thinsp;=\u0026thinsp;0.067) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. SLC7A11 levels were significantly lower in the 25 \u0026micro;M and 35 \u0026micro;M curcumin groups compared to the control, with a further reduction observed in the 0.7 \u0026micro;M celastrol combined with 35 \u0026micro;M curcumin group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D\u003cb\u003e)\u003c/b\u003e. Additionally, GPX4 levels were significantly increased in both the curcumin and the curcumin groups when compared to the control, with a more significant increase observed in cells treated with 0.7 \u0026micro;M celastrol combined with 35 \u0026micro;M curcumin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,F\u003cb\u003e)\u003c/b\u003e. Only the combination of 0.7 \u0026micro;M celastrol and 35 \u0026micro;M curcumin led to a significant increase in ACSL4 levels, while other treatment regimens did not affect ACSL4 levels compared to the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,G\u003cb\u003e)\u003c/b\u003e. These findings suggest that the enhanced inhibitory effects on CNE1 cells by the combination therapy may be primarily attributed to ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial dynamics may be a key player in combined treatment-induced cell death\u003c/h2\u003e \u003cp\u003eThere is a growing body of literature that connects mitochondrial fusion/fission to cancer cell metabolism and death[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We therefore considered whether mitochondrion dynamics could play a role in cell death induced by the combined treatment. The expression of mitochondrial dynamin-related protein 1 (Drp1) and mitochondrial mitofusin 2 protein (Mfn2) in CNE1 cells was evaluated following treatment with low-dose celastrol, curcumin alone, and their combination. Our results showed that 0.7 \u0026micro;M celastrol and 10 \u0026micro;M or 25 \u0026micro;M curcumin alone did not affect the p-Drp1/Drp1 ratio compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Only a high dose of curcumin (35 \u0026micro;M) led to a significant increase in the p-Drp1/Drp1 ratio. However, combining 25 \u0026micro;M or 35 \u0026micro;M curcumin with 0.7 \u0026micro;M celastrol resulted in a notable increase in the p-Drp1/Drp1 ratio compared to the control group. Furthermore, when 35 \u0026micro;M curcumin was combined with 0.7 \u0026micro;M celastrol, there was a significant increase in the p-Drp1/Drp1 ratio, surpassing the effects of curcumin alone. Mfn2 expression, a mitochondrial fusion-related protein, showed no significant difference from the control group in any treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C). These data indicated that Drp1 phosphorylation-mediated mitochondrial fission may contribute to combined celastrol and curcumin-induced cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNatural substances provide most small molecule drug candidates for potential use as anticancer agents[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Compounds derived from plants offer unique and innovative structures that serve as valuable tools for exploring protein function and mechanisms of cell death[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. High doses of celastrol and curcumin have demonstrated significant promise as effective antitumor agents, exhibiting notable antiproliferative effects[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the severe side effects associated with high-dose celastrol restrict its clinical application. While the FDA has classified curcumin as safe based on prior clinical trials [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], it is crucial to emphasize that its low bioavailability presents a therapeutic challenge for clinical use[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This study investigates the potential of combination therapy utilizing low-dose celastrol and curcumin to enhance anticancer effects while minimizing unwanted cytotoxicity. In this study, we found that celastrol or curcumin effectively inhibited CNE1 cell proliferation and induced mitochondrial dysfunction. We hypothesized that curcumin may enhance the sensitivity of CNE1 cells to low-dose celastrol. Our study used a very low dose of celastrol (0.7 \u0026micro;M), which may not induce inhibition of cell proliferation or cell death on its own. However, the combination of 0.7 \u0026micro;M celastrol with curcumin demonstrates enhanced inhibitory effects on CNE1 cells compared to curcumin alone at the same concentration. In addition, we provide novel evidence that the combination therapy's enhanced inhibitory effects on CNE1 cells may be primarily attributed to ferroptosis but not apoptosis.\u003c/p\u003e \u003cp\u003eCell death pathways were assessed in this study to discuss the underlying mechanism of the combined treatment. Previous studies have demonstrated that celastrol and curcumin possess strong anti-tumour activity against various cancer cell lines, such as lung, breast, liver, and gastric cancers[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], by triggering cell apoptosis and related pathways. However, the impact of curcumin on apoptotic cell death remains a topic of debate. Our findings align with the previous studies[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], showing that curcumin-induced cell death occurs independently of caspase-3 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We speculated that the effects of curcumin on cell death may vary based on concentration and cell type specificity. Unexpectedly, the combination of curcumin and low-dose celastrol did not result in significant changes in the expression of apoptosis-related proteins, such as Bax and the cleaved caspase-3/caspase-3 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). This suggests that an alternative mechanism of cell death, other than apoptosis, may be involved in the combined treatment.\u003c/p\u003e \u003cp\u003eAutophagy-dependent cell death is a complex process that shows varied associations with different types of regulated cell death[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Depending on the specific cellular environment and drug-cell interactions, autophagy can have a dual function, providing protective effects or inducing autophagic cell death. Cancer cells, including those of nasopharyngeal carcinoma, experience constant cellular stress due to heightened metabolic activity and increased nutrient requirements. The proper functioning of autophagic pathways is essential to maintain the mitochondrial metabolites needed for tumor cell growth[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, excessive activation of autophagy can lead to pro-death signaling, ultimately resulting in the elimination of cancer cells[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Research has indicated that autophagy can serve as an alternative to apoptosis, particularly in cancer cells resistant to apoptosis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Because of its dual nature, there have been successful reports of tumor cell suppression through both the inhibition and induction of autophagy. While autophagy can have both pro-death and pro-survival effects, most studies reported that curcumin-induced autophagy acts as a pro-death signal in various types of tumors, such as glioma [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]and gastric cancer cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], by inhibiting PI3K and Akt signaling pathways. In addition, curcumin has been demonstrated to increase ROS production, promoting apoptosis in colorectal cancer[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, it is widely recognized that ROS can also trigger and maintain autophagy in tumor cells[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Consistent with these findings, our data showed that curcumin at concentrations of 10, 25, and 35\u0026micro;M induced autophagy by increasing LC3 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) and increasing ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Our results suggest that ROS induced by curcumin play a role in the conversion of LC3-I to LC3-II, ultimately activating autophagy. While the combined treatment resulted in a similar level of autophagy in CNE1 cells compared to curcumin treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), phosphorylation of Drp1\u003csup\u003eSer616\u003c/sup\u003e, which promotes mitochondrial fission, was only increased in the combined treatment and not with curcumin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). These findings indicated that mitochondrial fission may play a critical role in combined treatment-induced cell death.\u003c/p\u003e \u003cp\u003eMitochondria create a dynamic network that can undergo fusion and fission events, collectively referred to as mitochondrial dynamics. The regulation of these events is controlled by Mfn 1 and 2, OPA-1, Drp1, and mitochondrial Fis1 proteins. Changes in the phosphorylation or expression levels of Drp1 are closely associated with the imbalance of mitochondrial fission and fusion. Disruption in mitochondrial dynamics, characterized by increased fission resulting in fragmented mitochondria, has been implicated in cancer progression[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and metastasis[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, several studies have demonstrated that excessive mitochondrial fission significantly enhances cancer cell death[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. On a molecular scale, mitochondrial fission generates a substantial number of mitochondrial fragments that contain damaged DNA and contribute to an overload of ROS, mediating oxidative stress in cancer. Furthermore, mitochondrial division facilitates the opening of the mitochondrial permeability transition pore (mPTP), a key indicator of mitochondrial death. Additionally, fragmented mitochondria are unable to produce sufficient ATP to sustain cancer metabolism. Based on these mechanisms, mitochondrial division has been recognized as a promising target for enhancing cancer cell death[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong various forms of regulated cell death, ferroptosis is a unique form of cell death, distinguished by cell volume shrinkage and heightened mitochondrial membrane density, without the usual signs of apoptosis or necrosis[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. A previous study found that curcumin triggers ferroptosis in breast cancer cells by promoting iron accumulation and downregulating GPX4[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, Zhou\u0026rsquo;s team found that nasopharyngeal carcinoma cells show a reduced number of active reactions to ferroptosis inducers compared to both tongue squamous cell carcinoma cells and laryngeal squamous cell carcinoma cells[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, ferroptosis has been reported to correlate with chemoresistance in nasopharyngeal carcinoma[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, ferroptosis has become a promising target for innovative drug development in treating nasopharyngeal carcinoma. In our current research, curcumin was found to effectively suppress the expression of GPX4 and SLC7A11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F). SLC7A11, a transmembrane protein involved in cystine-glutamate transport, is critical in maintaining cellular redox balance by facilitating glutathione synthesis[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, TFRC, essential for regulating intracellular iron levels[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], did not show any change after curcumin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Interestingly, the combination treatment resulted in a further decrease in GPX4 and SLC7A11 compared to curcumin treatment alone. Furthermore, ACSL4, a contributor to ferroptosis by enhancing lipid peroxidation, only increased with the combined treatment and not with curcumin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, G). This suggests that curcumin enhances the sensitivity of CNE1 cells to ferroptosis, although it is insufficient to induce ferroptosis independently at a concentration of 35 \u0026micro;M. Interestingly, the addition of low-dose celastrol further enhanced curcumin-induced ferroptosis, highlighting the critical role of ferroptosis in cell death induced by the combined treatment. In addition, the combined treatment led to ferroptosis accompanied by increased phosphorylation of Drp1. These results align with previous studies demonstrating the association between ferroptosis and heightened mitochondrial fragmentation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. We speculated that mitochondrial fission may be a crucial factor in the combined treatment-induced ferroptotic cell death; however, the exact relationship remains to be clarified.\u003c/p\u003e \u003cp\u003eThe compatibility of medicines is fundamental to traditional Chinese medicine prescriptions, which can serve as a safe and cost-effective approach for the co-treatment of various cancer types, including nasopharyngeal carcinoma. However, it is important to note that plant compounds should not be viewed as standalone cures for cancer. Instead, their significance lies in their role as a continuous prophylactic or complementary supplement to cytostatic therapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLow doses of celastrol combined with curcumin exhibited greater inhibition of CNE1 cell growth compared to curcumin alone. This enhanced efficacy of the combination therapy is likely attributable to effects on mitochondrial fission and the induction of ferroptosis. These findings not only demonstrate a potential antitumor effect of combining celastrol with curcumin but also open avenues for future investigations into the possible co-therapeutic properties of these phytotherapeutic agents in conjunction with chemoradiotherapy, thereby facilitating the development of a safe and effective strategy for cancer treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNPC \u0026nbsp;Nasopharyngeal carcinoma\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003emPTP \u0026nbsp;Mitochondrial permeability transition pore\u003c/p\u003e\n\u003cp\u003eBax \u0026nbsp;BCL2-associated X\u003c/p\u003e\n\u003cp\u003eCleaved caspase-3 \u0026nbsp; Cleaved cysteine-aspartic acid protease 3\u003c/p\u003e\n\u003cp\u003eCaspase-3 \u0026nbsp;Cysteine-aspartic acid protease 3\u003c/p\u003e\n\u003cp\u003eLC3 II \u0026nbsp;Microtubule-associated protein 1A/1B-light chain 3, lipidated form\u003c/p\u003e\n\u003cp\u003eTFRC \u0026nbsp;Transferrin receptor\u003c/p\u003e\n\u003cp\u003eGPX4 \u0026nbsp;Glutathione peroxidase 4\u003c/p\u003e\n\u003cp\u003eSLC7A11 \u0026nbsp;Solute carrier family 7 member 11\u003c/p\u003e\n\u003cp\u003eACSL4 \u0026nbsp; Acyl-CoA synthetase long-chain family member 4\u003c/p\u003e\n\u003cp\u003eDrp1 \u0026nbsp;Dynamin-Related Protein 1\u003c/p\u003e\n\u003cp\u003eMfn2 \u0026nbsp;Mitofusin 2 protein\u003c/p\u003e\n\u003cp\u003eGSH \u0026nbsp;Glutathione\u003c/p\u003e\n\u003cp\u003ePUFAs \u0026nbsp;Polyunsaturated fatty acids\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSL: Conceptualization, funding acquisition, and resources; TF, YL, XZ, ZF, XF, BL, YL, JW, SM: Performed experiments; TF, SL: Formal analysis; SL, TF: Writing-original draft; SL, JW: Writing-review and editing. \u0026nbsp;All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Suchan Liao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Baise scientific research and technology development plan of regionally frequently occurring diseases, China (NO.20224129 to SL); The basic scientific research ability improvement project of young and middle-aged university teachers in Guangxi, China (NO.2019KY0568 to SL); Natural Science Foundation of Youjiang Medical University for Nationalities (NO. yy2018ky001 to SL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eSchool of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China. \u0026nbsp;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eGraduate School, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. \u0026nbsp;\u003csup\u003e3\u003c/sup\u003e Laboratory Center of Medical Science Morphology, School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. \u0026nbsp;\u003csup\u003e4\u003c/sup\u003eModern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. \u0026nbsp;\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eDepartment of Physiology, School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen Y-P, Chan AT, Le Q-T, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. The Lancet. 2019;394(10192):64-80.\u003c/li\u003e\n\u003cli\u003eTao Q, Chan AT. Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments. Expert reviews in molecular medicine. 2007;9(12):1-24.\u003c/li\u003e\n\u003cli\u003eSalvador-Coloma C, Cohen EJJoop. Multidisciplinary care of laryngeal cancer. Journal of oncology practice 2016;12(8):717-24.\u003c/li\u003e\n\u003cli\u003eNewman DJ, Cragg GM. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J Nat Prod. 2020;83(3):770-803.\u003c/li\u003e\n\u003cli\u003eTsang NY, Li WF, Varhegyi E, Rong L, Zhang HJ. Ebola Entry Inhibitors Discovered from Maesa perlarius. Int J Mol Sci. 2022;23(5).\u003c/li\u003e\n\u003cli\u003eDuan J, Zhan JC, Wang GZ, Zhao XC, Huang WD, Zhou GB. The red wine component ellagic acid induces autophagy and exhibits anti-lung cancer activity in vitro and in vivo. J Cell Mol Med. 2019;23(1):143-54.\u003c/li\u003e\n\u003cli\u003eChen P, Wang B, Li M, Cui C, Liu F, Gao Y. Celastrol inhibits the proliferation and migration of MCF-7 cells through the leptin-triggered PI3K/AKT pathway. Computational and Structural Biotechnology Journal. 2022;20:3173-81.\u003c/li\u003e\n\u003cli\u003eChen M, Yang J, Li L, Lu X, Sun R, Wang Y, Zhang X. Metabolomics Reveals Cysteine Metabolism Plays a Role in Celastrol-Induced Mitochondrial Apoptosis of Acute Promyelocytic Leukemia Cells. Available at SSRN 3335016. 2019.\u003c/li\u003e\n\u003cli\u003eDu S, Song X, Li Y, Cao Y, Chu F, Durojaye OA, et al. Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells. Scientific Reports. 2020;10(1):11273.\u003c/li\u003e\n\u003cli\u003eKannaiyan R, Hay HS, Rajendran P, Li F, Shanmugam MK, Vali S, et al. Celastrol inhibits proliferation and induces chemosensitization through down‐regulation of NF‐\u0026kappa;B and STAT3 regulated gene products in multiple myeloma cells. British journal of pharmacology. 2011;164(5):1506-21.\u003c/li\u003e\n\u003cli\u003eArisan ED, Rencuzogullari O, Coban M, Sevgin B, Obakan-Yerlikaya P, \u0026Ccedil;oker-G\u0026uuml;rkan A, Palavan-Unsal N. The role of the PI3K/AKT/mTOR signaling axis in the decision of the celastrol-induced cell death mechanism related to the lipid regulatory pathway in prostate cancer cells. Phytochemistry Letters. 2020;39:73-83.\u003c/li\u003e\n\u003cli\u003eYu X, Meng X, Xu M, Zhang X, Zhang Y, Ding G, et al. Celastrol ameliorates cisplatin nephrotoxicity by inhibiting NF-\u0026kappa;B and improving mitochondrial function. EBioMedicine. 2018;36:266-80.\u003c/li\u003e\n\u003cli\u003eHou W, Liu B, Xu H. Celastrol: Progresses in structure-modifications, structure-activity relationships, pharmacology and toxicology. Eur J Med Chem. 2020;189:112081.\u003c/li\u003e\n\u003cli\u003eNewman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod. 2007;70(3):461-77.\u003c/li\u003e\n\u003cli\u003ePriyadarsini KI, Maity DK, Naik G, Kumar MS, Unnikrishnan M, Satav J, Mohan H. Role of phenolic OH and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radical Biology and Medicine. 2003;35(5):475-84.\u003c/li\u003e\n\u003cli\u003eBarati N, Momtazi‐Borojeni AA, Majeed M, Sahebkar A. Potential therapeutic effects of curcumin in gastric cancer. Journal of cellular physiology. 2019;234(3):2317-28.\u003c/li\u003e\n\u003cli\u003eShoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med. 1998;64(4):353-6.\u003c/li\u003e\n\u003cli\u003eBrockmueller A, Ruiz de Porras V, Shakibaei M. Curcumin and its anti-colorectal cancer potential: From mechanisms of action to autophagy. Phytother Res. 2024;38(7):3525-51.\u003c/li\u003e\n\u003cli\u003eVyas S, Zaganjor E, Haigis MC. Mitochondria and Cancer. Cell. 2016;166(3):555-66.\u003c/li\u003e\n\u003cli\u003eLi C, Zhang Y, Liu J, Kang R, Klionsky DJ, Tang D. Mitochondrial DNA stress triggers autophagy-dependent ferroptotic death. Autophagy. 2021;17(4):948-60.\u003c/li\u003e\n\u003cli\u003eShanmugam MK, Lee JH, Chai EZP, Kanchi MM, Kar S, Arfuso F, et al., editors. Cancer prevention and therapy through the modulation of transcription factors by bioactive natural compounds. Seminars in cancer biology; 2016: Elsevier.\u003c/li\u003e\n\u003cli\u003eDhankhar S, Ruhil S, Balhara M, Dhankhar S, Chhillar A. Aegle marmelos (Linn.) Correa: A potential source of Phytomedicine. J Med Plant Res. 2011;5(9):1497-507.\u003c/li\u003e\n\u003cli\u003eGuneydas G, Topcul MR. Antiproliferative effects of curcumin different types of breast cancer. Asian Pacific Journal of Cancer Prevention: APJCP. 2022;23(3):911.\u003c/li\u003e\n\u003cli\u003eZhao Z, Wang Y, Gong Y, Wang X, Zhang L, Zhao H, et al. Celastrol elicits antitumor effects by inhibiting the STAT3 pathway through ROS accumulation in non-small cell lung cancer. J Transl Med. 2022;20(1):525.\u003c/li\u003e\n\u003cli\u003eCox KH, Pipingas A, Scholey AB. Investigation of the effects of solid lipid curcumin on cognition and mood in a healthy older population. J Psychopharmacol. 2015;29(5):642-51.\u003c/li\u003e\n\u003cli\u003eDhillon N, Aggarwal BB, Newman RA, Wolff RA, Kunnumakkara AB, Abbruzzese JL, et al. Phase II trial of curcumin in patients with advanced pancreatic cancer. Clin Cancer Res. 2008;14(14):4491-9.\u003c/li\u003e\n\u003cli\u003eKoronyo Y, Biggs D, Barron E, Boyer DS, Pearlman JA, Au WJ, et al. Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer\u0026apos;s disease. JCI Insight. 2017;2(16).\u003c/li\u003e\n\u003cli\u003eAshrafizadeh M, Najafi M, Makvandi P, Zarrabi A, Farkhondeh T, Samarghandian S. Versatile role of curcumin and its derivatives in lung cancer therapy. Journal of cellular physiology. 2020;235(12):9241-68.\u003c/li\u003e\n\u003cli\u003eZhao Z, Wang Y, Gong Y, Wang X, Zhang L, Zhao H, et al. Celastrol elicits antitumor effects by inhibiting the STAT3 pathway through ROS accumulation in non-small cell lung cancer. Journal of Translational Medicine. 2022;20(1):525.\u003c/li\u003e\n\u003cli\u003eFarghadani R, Naidu R. Curcumin as an enhancer of therapeutic efficiency of chemotherapy drugs in breast cancer. International Journal of Molecular Sciences. 2022;23(4):2144.\u003c/li\u003e\n\u003cli\u003eRen B, Luo S, Tian X, Jiang Z, Zou G, Xu F, et al. Curcumin inhibits liver cancer by inhibiting DAMP molecule HSP70 and TLR4 signaling. Oncology reports. 2018;40(2):895-901.\u003c/li\u003e\n\u003cli\u003eZhou M, Liao J, Lai W, Xu R, Liu W, Xie D, et al. A celastrol-based nanodrug with reduced hepatotoxicity for primary and metastatic cancer treatment. EBioMedicine. 2023;94.\u003c/li\u003e\n\u003cli\u003eChen X, Zhao Y, Luo W, Chen S, Lin F, Zhang X, et al. Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells. Theranostics. 2020;10(22):10290.\u003c/li\u003e\n\u003cli\u003eFu H, Wang C, Yang D, Wei Z, Xu J, Hu Z, et al. Curcumin regulates proliferation, autophagy, and apoptosis in gastric cancer cells by affecting PI3K and P53 signaling. Journal of cellular physiology. 2018;233(6):4634-42.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Sullivan-Coyne G, O\u0026apos;Sullivan GC, O\u0026apos;Donovan TR, Piwocka K, McKenna SL. Curcumin induces apoptosis-independent death in oesophageal cancer cells. Br J Cancer. 2009;101(9):1585-95.\u003c/li\u003e\n\u003cli\u003eSeo SU, Woo SM, Lee HS, Kim SH, Min KJ, Kwon TK. mTORC1/2 inhibitor and curcumin induce apoptosis through lysosomal membrane permeabilization-mediated autophagy. Oncogene. 2018;37(38):5205-20.\u003c/li\u003e\n\u003cli\u003eTang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. Cell Res. 2019;29(5):347-64.\u003c/li\u003e\n\u003cli\u003eAmaravadi R, Kimmelman AC, White E. Recent insights into the function of autophagy in cancer. Genes Dev. 2016;30(17):1913-30.\u003c/li\u003e\n\u003cli\u003eAmaravadi RK, Kimmelman AC, Debnath J. Targeting Autophagy in Cancer: Recent Advances and Future Directions. Cancer Discov. 2019;9(9):1167-81.\u003c/li\u003e\n\u003cli\u003eKim KW, Hwang M, Moretti L, Jaboin JJ, Cha YI, Lu B. Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer. Autophagy. 2008;4(5):659-68.\u003c/li\u003e\n\u003cli\u003eNazim UM, Yin H, Park SY. Downregulation of c‑FLIP and upregulation of DR‑5 by cantharidin sensitizes TRAIL‑mediated apoptosis in prostate cancer cells via autophagy flux. Int J Mol Med. 2020;46(1):280-8.\u003c/li\u003e\n\u003cli\u003eAoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol Pharmacol. 2007;72(1):29-39.\u003c/li\u003e\n\u003cli\u003eLiu C, Rokavec M, Huang Z, Hermeking H. Curcumin activates a ROS/KEAP1/NRF2/miR-34a/b/c cascade to suppress colorectal cancer metastasis. Cell Death Differ. 2023;30(7):1771-85.\u003c/li\u003e\n\u003cli\u003eTang ZH, Cao WX, Wang ZY, Lu JH, Liu B, Chen X, Lu JJ. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells. Redox Biol. 2017;12:367-76.\u003c/li\u003e\n\u003cli\u003eTran Q, Lee H, Jung JH, Chang SH, Shrestha R, Kong G, et al. Emerging role of LETM1/GRP78 axis in lung cancer. Cell Death Dis. 2022;13(6):543.\u003c/li\u003e\n\u003cli\u003eLi T, Han J, Jia L, Hu X, Chen L, Wang Y. PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation. Protein Cell. 2019;10(8):583-94.\u003c/li\u003e\n\u003cli\u003eCheng CT, Kuo CY, Ouyang C, Li CF, Chung Y, Chan DC, et al. Metabolic Stress-Induced Phosphorylation of KAP1 Ser473 Blocks Mitochondrial Fusion in Breast Cancer Cells. Cancer Res. 2016;76(17):5006-18.\u003c/li\u003e\n\u003cli\u003eTang Q, Liu W, Zhang Q, Huang J, Hu C, Liu Y, et al. Dynamin-related protein 1-mediated mitochondrial fission contributes to IR-783-induced apoptosis in human breast cancer cells. J Cell Mol Med. 2018;22(9):4474-85.\u003c/li\u003e\n\u003cli\u003eYou Y, He Q, Lu H, Zhou X, Chen L, Liu H, et al. Silibinin Induces G2/M Cell Cycle Arrest by Activating Drp1-Dependent Mitochondrial Fission in Cervical Cancer. Front Pharmacol. 2020;11:271.\u003c/li\u003e\n\u003cli\u003eZhang Z, Yao Z, Wang L, Ding H, Shao J, Chen A, et al. Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells. Autophagy. 2018;14(12):2083-103.\u003c/li\u003e\n\u003cli\u003eLi R, Zhang J, Zhou Y, Gao Q, Wang R, Fu Y, et al. Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. Oxid Med Cell Longev. 2020;2020:3469840.\u003c/li\u003e\n\u003cli\u003eLiu S, Yan S, Zhu J, Lu R, Kang C, Tang K, et al. Combination RSL3 Treatment Sensitizes Ferroptosis- and EGFR-Inhibition-Resistant HNSCCs to Cetuximab. Int J Mol Sci. 2022;23(16).\u003c/li\u003e\n\u003cli\u003eXu Y, Du Y, Zheng Q, Zhou T, Ye B, Wu Y, et al. Identification of Ferroptosis-Related Prognostic Signature and Subtypes Related to the Immune Microenvironment for Breast Cancer Patients Receiving Neoadjuvant Chemotherapy. Front Immunol. 2022;13:895110.\u003c/li\u003e\n\u003cli\u003ede la Vega MR, Chapman E, Zhang DD. NRF2 and the Hallmarks of Cancer. Cancer cell. 2018;34(1):21-43.\u003c/li\u003e\n\u003cli\u003eZhu G, Murshed A, Li H, Ma J, Zhen N, Ding M, et al. O-GlcNAcylation enhances sensitivity to RSL3-induced ferroptosis via the YAP/TFRC pathway in liver cancer. Cell Death Discovery. 2021;7(1):83.\u003c/li\u003e\n\u003cli\u003eDixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-72.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nasopharyngeal Carcinoma, celastrol, curcumin, combined treatment, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-4827626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4827626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNasopharyngeal carcinoma (NPC) is a highly invasive form of head and neck cancer that arises from nasopharyngeal epithelial cells. The treatment of advanced NPC with radiotherapy presents significant challenges due to cellular resistance, which has spurred interest in natural small molecule drugs. Celastrol and curcumin, both derived from plants, have exhibited anti-tumor properties. However, the clinical development of celastrol is hindered by its low bioavailability and associated toxic side effects, while curcumin, although non-toxic, also suffers from limited bioavailability. The combination of drugs is a fundamental principle of traditional Chinese medicine, as it enhances therapeutic efficacy while reducing toxicity, suggesting a potential synergistic use of celastrol and curcumin. Furthermore, ferroptosis is crucial for tumor cell death. Consequently, our study aims to investigate whether the combination of celastrol and curcumin can induce ferroptosis in NPC cells and assess its antiproliferative effects.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman nasopharyngeal carcinoma cell lines were used for in vitro cell analysis. CCK8 was used to evaluate the effect of treatment with different concentrations of Celastrol and curmin on cell viability in a human nasopharyngeal carcinoma CNE1 cell line. Mitochondrial reactive oxygen species and mitochondrial membrane potential were detected to determine mitochondrial oxidative stress and function. Western blot was used to detect apoptosis, autophagy and ferritin-related proteins expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe combination of celastrol and curcumin exhibited a more pronounced antiproliferative effect on CNE1 cells. Following treatment with these compounds, mitochondria generated substantial amounts of reactive oxygen species, resulting in impaired mitochondrial function. Moreover, the cell death induced by the combination of celastrol and curcumin was found to be independent of apoptosis, instead, it was correlated with increased cellular autophagy, enhanced mitochondrial fission, and the induction of ferroptosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLow doses of celastrol combined with curcumin exhibited a greater inhibition of CNE1 cell growth compared to curcumin alone. This enhanced efficacy of the combination therapy is likely attributable to its effects on mitochondrial fission and the induction of ferroptosis.\u003c/p\u003e","manuscriptTitle":"Celastrol combined with curcumin inhibits proliferation and causes cell death in nasopharyngeal carcinoma CNE1 cell line by inducing ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 17:48:08","doi":"10.21203/rs.3.rs-4827626/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82f8a17b-ab08-48e2-9ade-b34afba9fbaf","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-10T14:14:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-27 17:48:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4827626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4827626","identity":"rs-4827626","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}