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

doi:10.21203/rs.3.rs-4827626/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-4827626/v1

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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

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.

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 1×10⁵ to 6×10⁵ 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.

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. 1A). The calculated IC50 for celastrol was 3.420 µM (Fig. 1A). 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. 1B). The IC50 for Curcumin was 56.69 µM (Fig. 1B).

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. 1C). 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. 1D). similarly, mitochondrial membrane potential decreased significantly with 12.5 µM, 25 µM, and 35 µM curcumin treatment (Fig. 1E), accompanied by elevated ROS levels in these groups (Fig. 1F). 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. 2A). 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. 2A). The IC50 for curcumin was reduced to 48.49 µM in the presence of 0.7 µM celastrol (Fig. 2B). 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. 3A, B). Additionally, the protein expression ratio of cleaved caspase-3/caspase-3 also remained unchanged across all treatments (Fig. 3C, 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. 4A, B). However, LC3-II levels significantly increased with both curcumin (10 µM, 25 µM, and 35 µM) alone and combined treatment (Fig. 4A, 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 Fe²⁺ 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. 5A, 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. 5C,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. 5E,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. 5E,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. 6A, 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. 6A, C). These data indicated that Drp1 phosphorylation-mediated mitochondrial fission may contribute to combined celastrol and curcumin-induced cell death.

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. 3B). 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. 3A, 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. 4A, B) and increasing ROS levels (Fig. 1F). 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. 4A, B), phosphorylation of Drp1^Ser616, which promotes mitochondrial fission, was only increased in the combined treatment and not with curcumin alone (Fig. 6A, 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. 5C-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. 5A, 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. 5E, 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.

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.

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

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

¹School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China. ²Graduate 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. ⁴Modern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, Guangxi, 533000, China. ⁵Department of Physiology, School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, Guangxi,533000, China.

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

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Cell Lines and Culture

Cell viability by CCK8 assay

CNE1 mitochondrial ROS levels

CNE1 mitochondrial membrane potential levels

Western blot analysis

Statistical analysis

Results

Celastrol or curcumin effectively inhibited the proliferation of CNE1 cells and induced mitochondrial dysfunction

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

Ferroptosis plays a critical role in low-dose celastrol plus curcumin-induces cell death

Mitochondrial dynamics may be a key player in combined treatment-induced cell death

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1