This protocol aims to evaluate the anti-tumor efficacy of ruscogenin in nasopharyngeal carcinoma cells. It details comprehensive methodologies—including viability, migration, apoptosis, and ferroptosis assays—to systematically elucidate how this compound induces dual cell death by targeting and downregulating KDM1A.
Research Article
Ruscogenin Suppresses Nasopharyngeal Carcinoma Progression by Downregulating KDM1A to Co-induce Ferroptosis and Apoptosis
In This Article
Summary
Abstract
Nasopharyngeal carcinoma (NPC) is a highly aggressive malignancy. The natural compound ruscogenin (Rus) shows anti-cancer potential. This study elucidates the specific mechanism of action in nasopharyngeal carcinoma (NPC) by investigating its effects on NPC cells and the underlying molecular pathway. The human nasopharyngeal carcinoma (NPC) cell line, C666-1, was treated with various concentrations of Rus. A series of in vitro assays was performed to evaluate cell viability, proliferation, migration, invasion, apoptosis, and ferroptosis. The role of lysine-specific demethylase 1A (KDM1A) was investigated using Western blot and molecular docking, and its mediating role was validated through overexpression rescue experiments. Ruscogenin dose-dependently diminished the proliferative, clonogenic, migratory, and invasive capacities of C666-1 cells. Mechanistically, Rus treatment induced both apoptosis, evidenced by the activation of executioner caspase-3 and a shift towards pro-apoptotic signaling via the Bax/Bcl-2 balance, and ferroptosis, marked by elevated lipid peroxidation and intracellular iron. These anti-tumor effects were strongly correlated with the downregulation of KDM1A. Crucially, the forced overexpression of KDM1A significantly attenuated Rus-induced apoptosis and ferroptosis, and reversed the suppression of malignant phenotypes, confirming KDM1A's critical mediating role. Ruscogenin inhibits the malignant progression of nasopharyngeal carcinoma by downregulating KDM1A, thereby promoting apoptosis and ferroptosis. These findings identify the Rus-KDM1A axis as a promising therapeutic target for NPC treatment.
Introduction
Nasopharyngeal carcinoma (NPC) is a kind of cancer caused by the cooperation of genetic and environmental factors1. Although radiotherapy has made remarkable advances in treatment, the overall survival of NPC patients remains unsatisfactory. This is probably because NPC cells can proliferate rapidly and are highly prone to metastasize to regional lymph nodes and distant organs2. Therefore, it is essential to explore the molecular mechanisms promoting the progression of NPC. Recently, ferroptosis, a new modality of cell death, regulated by Fe2⁺ and lipid peroxides in an iron-dependent manner, has been found to be critically involved in NPC3. The present evidence has demonstrated that ferroptosis plays an important role in NPC, and its induction can enhance cancer radiotherapy4,5.
Owing to the minimal toxicity and fewer side effects, Chinese herbal formulas have been widely used to treat cancer for thousands of years6,7. As a kind of multi-component, multi-target, and synergistic effect medicine, traditional Chinese medicine (TCM) prescriptions have been used as an effective complementary therapy in preventing and treating cancer, including NPC8,9,10. Radix ophiopogonis (RO), the root of the perennial herbaceous plant Ophiopogon japonicus, has been reported to effectively alleviate NPC as an adjuvant therapy11,12. Ruscogenin (Rus), the major component in RO, has been reported to possess potent anti-cancer effects13. In mouse models of colorectal cancer, Rus can improve tight junction injury and inhibit tumor migration14. Importantly, Rus can also decrease the viability of pancreatic cancer cells via inducing ferroptosis. However, the effect of Rus on NPC is still unclear.
Epigenetics is the main field of research for cancer pathogenesis and provides multiple effective treatments15. As one of the well-studied epigenetic modifications, histone methylation is involved in the development of cancer and is also an important target of drug design16. Dynamic modulation of histone methylation is mediated by methyltransferases and demethylases17. KDM1A is a histone lysine demethylase that has been found to be critically involved in carcinogenesis due to its involvement in epigenetic modulation18. In addition, KDM1A is found to be an oncoprotein with high expression in multiple cancers, such as lung and pancreatic cancer19. Guo et al. reported that KDM1A elevation could suppress phosphatase and tensin homolog (PTEN) expression and enhance NPC cell proliferation and migration while inhibiting apoptosis20. Meanwhile, it was also found that upregulation of KDM1A could repress ferroptosis in lung cancer21. Super-PRED database analysis indicated that KDM1A is a potential target of Rus.
Despite the recognized anti-tumor potential of Rus in other cancers, its specific role and molecular mechanisms in nasopharyngeal carcinoma remain completely unestablished, representing a significant research gap. Furthermore, while previous reports have separately linked KDM1A to tumor proliferation and ferroptosis resistance, no study has yet explored whether targeting KDM1A can simultaneously trigger multiple cell death pathways in NPC. Therefore, this study provides a strong rationale to hypothesize that Rus might suppress the malignant progression of NPC cells by directly downregulating KDM1A.
This study aimed to address this gap by systematically evaluating the effect of Rus on the proliferation, migration, and invasion of NPC cells. The primary novelty of this research lies in uncovering a dual-mechanism strategy. The focus was placed on the capacity of Rus to concurrently induce both classical apoptosis and iron-dependent ferroptosis. By validating the novel Rus-KDM1A signaling axis, this study significantly advances current knowledge by demonstrating how a single natural compound can overcome tumor resilience through simultaneous multi-pathway blockade, offering a promising preclinical rationale for targeted NPC therapy.
Protocol
Cell culture, treatment, and transfection
The human nasopharyngeal carcinoma (NPC) cell line, C666-1 (RRID: CVCL_M597), and the non-malignant human nasopharyngeal epithelial cell line, NP69 (RRID: CVCL_F755), were purchased from a commercial supplier. Both cell lines were authenticated by Short Tandem Repeat (STR) profiling and were confirmed to be free of mycoplasma contamination before use. For routine culture, C666-1 cells were propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. NP69 cells were cultured in keratinocyte serum-free medium supplemented with 10% FBS, 10 ng/mL recombinant epidermal growth factor, and 1% penicillin-streptomycin. Ruscogenin (Rus; purity ≥ 98%) was purchased from a commercial supplier. Both cell lines were maintained in a humidified incubator at 37 °C under 5% CO₂. For treatments, cells were exposed to Ruscogenin (Rus) at varying concentrations (10, 20, 50 µM) for 24 h22. For transfection, plasmids encoding lysine-specific demethylase 1A for overexpression (oe-KDM1A) and a corresponding negative control empty vector (oe-NC), procured from a commercial supplier, were transfected into C666-1 cells when the cell confluency reached 70–80%. Specifically, 2.5 µg of plasmid DNA and 5 µL of a lipid-based transfection reagent were each diluted in 150 µL of a reduced serum medium, gently mixed, incubated for 20 min at room temperature, and then added to each well of a 6-well plate according to the manufacturer's protocol. Cells were incubated for 48 h at 37 °C, and transfection efficacy was subsequently confirmed.
Cell counting kit-8 (CCK-8) assay
Cell viability following Rus treatment was quantified using the CCK-8 assay. For this procedure, cells were first seeded into 96-well plates and cultured overnight. The assay was initiated by adding 10 µL of CCK-8 solution, and after 2 h of incubation, the resulting colorimetric change was measured as the optical density (OD) at 450 nm using a plate reader.
Colony formation assay
To assess clonogenic survival, C666-1 cells were seeded into 6-well plates and cultured for 14 days. Following the incubation period, the resulting colonies were fixed with 100% methanol for 10 min, then visualized with 0.5% crystal violet for 30 min. The number of stained colonies was then quantified by microscopic imaging and counting.
Western blot
For Western blot analysis, total protein was first extracted from cells with RIPA buffer, and the resulting lysates were centrifuged (12,000 × g) to collect the supernatant. A BCA assay was used to quantify protein levels. Equal amounts of total protein (30 µg per lane) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, proteins were transferred to polyvinylidene fluoride (PVDF) membranes at a constant voltage of 100 V for 90 min on ice. The membranes were then blocked in 5% bovine serum albumin (BSA) prepared in tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature. The membranes were subsequently incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer (all at 1:1000): Ki67, PCNA, MMP2, MMP9, Bcl-2, Bax, KDM1A, SLC7A11, GPX4, ACSL4, TFR1, and β-actin. Finally, after a 2-h incubation with HRP-conjugated secondary antibodies, the immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection reagent, and densitometric analysis was performed with Image analysis software.
Wound scratch assay
For the wound-healing assay, C666-1 cells were cultured to 80% confluency in 6-well plates. A uniform wound was introduced into the monolayer by scraping with a 200 µL pipette tip. Detached cells were removed with a PBS wash, after which the culture medium was replaced with serum-free medium for a 24-h incubation. Images of the wound area were captured at 0 h and 24 h using a light microscope to assess cell migration.
Transwell assay
Cell invasion was evaluated using transwell chambers (8-µm pore size) pre-coated with 50 µL of basement membrane matrix. The invasive capacity of C666-1 cells was assessed using Transwell chambers. The upper inserts were seeded with cells suspended in serum-starved medium, while the bottom wells contained DMEM with 10% FBS. Following a 24-h incubation, cells remaining on the apical side of the membrane were carefully removed. For quantification, cells on the basolateral surface were fixed in methanol, stained with 0.1% crystal violet, and enumerated from images captured by light microscopy.
Flow cytometry
Apoptosis was quantified using flow cytometry. To prepare the samples, C666-1 cells were collected, rinsed with ice-cold PBS, and then resuspended in 100 µL of binding buffer. The cells were then co-stained with Annexin-V-FITC (20 µg/mL) and propidium iodide (PI). This staining was performed for 15 min on ice, protected from light. Data were immediately acquired on a flow cytometer to determine the percentage of apoptotic cells.
Detection of caspase-3 activity
Caspase-3 activity was measured using a commercial assay kit according to the manufacturer's instructions. Briefly, C666-1 cells were lysed, and the supernatants were collected after centrifugation at 14,000 × g. Following a 2-h incubation with the provided reagent, the absorbance of each supernatant was quantified at 405 nm.
RT-qPCR
Total RNA was purified from C666-1 cells using a commercial RNA extraction reagent. The resulting RNA was reverse-transcribed into first-strand cDNA using a commercial kit according to the manufacturer's protocol. Gene expression analysis was subsequently performed by quantitative PCR (qPCR) on a real-time PCR system with a SYBR Green-based master mix. The comparative Ct (2⁻△△Ct) method was employed to determine relative transcript levels. The primer sequences used for amplification were: β-actin (Forward: 5'-CATGTACGTTGCTATCCAGGC-3'; Reverse: 5'-CTCCTTAATGTCACGCACGAT-3') and KDM1A (Forward: 5'-TGACCGGATGACTTCTCAAGA-3'; Reverse: 5'-GTTGGAGAGTAGCCTC AAATGTC-3').
Thiobarbituric acid reactive substances (TBARS) assay
To measure lipid peroxidation, supernatants were first prepared from C666-1 cells by precipitation (15% TCA, 500 mM BHA) and centrifugation for 10 min at 10,000 × g. These supernatants were then reacted with 0.375% thiobarbituric acid, heated at 100 °C for 10 min to generate the MDA-TBA adduct. The level of this adduct, indicative of lipid peroxidation, was determined by measuring the absorbance at 532 nm on a microplate reader23.
Detection of lipid reactive oxygen species (ROS)
Lipid peroxidation was detected using a lipid peroxidation fluorescent probe. C666-1 cells were seeded in 6-well plates, cultured overnight, and then exposed to the fluorescent probe (2 µM). This probe exhibits a fluorescence shift from red to green upon oxidation. The green/red fluorescence ratio was analyzed using a laser scanning confocal microscope and Image analysis software. The plotted green/red fluorescence ratio was clearly defined and utilized as the quantitative indicator of lipid peroxidation levels24.
Detection of Fe2+
The intracellular ferrous iron (Fe2⁺) level was measured using an intracellular iron fluorescent probe. C666-1 cells were incubated with 10 µM fluorescent probe for 10 min. After washing with PBS to remove excess probe, the cells were analyzed by fluorescence microscopy.
Molecular docking analysis
Molecular docking is a pivotal computational method that facilitates novel drug design by predicting the binding affinity and interaction between a ligand and its target protein through the evaluation of their binding modes25. From the RCSB PDB webpage (https://www.rcsb.org/), the 3D structure of KDM1A (PDB ID: 6NQU) was obtained. For the preparation of the KDM1A protein structure, the water was removed, and irrelevant small ligands were removed. Molecular docking simulation was carried out via molecular docking software using the Lamarckian Genetic Algorithm, and the visualization was conducted utilizing molecular visualization software26.
Statistical analysis
All experiments were performed using at least three independent biological replicates, and the results are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using statistical analysis software. Differences among multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test. A P-value of less than 0.05 was considered to indicate a statistically significant difference.
Results
Ruscogenin selectively inhibits the viability and proliferation of nasopharyngeal carcinoma cells
The study first investigated the effect of ruscogenin (Rus) on the viability of both normal nasopharyngeal epithelial cells (NP69) and nasopharyngeal carcinoma (NPC) cells (C666-1). As determined by the CCK-8 assay, Rus treatment at concentrations of 10, 20, and 50 µM showed no significant toxicity towards the normal NP69 cells. Conversely, Ruscogenin (Rus) significantly impaired the survival of C666-1 cancer cells in a manner dependent on both concentration and incubation time. The most prominent cytotoxic activity was observed at 50 µM (p < 0.001) (Figure 1B-C). Next, the colony formation assay was performed to evaluate the clonogenic survival of C666-1 cells after Rus treatment, and the results showed that Rus significantly inhibited the clonogenic survival of C666-1 cells in a highly significant dose-dependent manner (Figure 1D). To validate these results at the molecular level, the expression of proliferation-related proteins Ki67 and PCNA was analyzed by Western blot (Figure 1E). These results demonstrate that Ruscogenin exhibits an anti-proliferative effect that is selective for NPC cells when compared with normal nasopharyngeal epithelial cells. This selectivity was further confirmed by the significant, dose-dependent reduction in the protein levels of Ki67 and PCNA in C666-1 cells (p < 0.001 at 50 µM) (Figure 1E).
Ruscogenin inhibits the migration and invasion of C666-1 cells
Given that metastasis is a key feature of malignant tumors, the study next explored the impact of Rus on the migratory and invasive capabilities of C666-1 cells. The wound scratch assay revealed that Rus treatment significantly inhibited the wound healing ability of C666-1 cells in a dose-dependent manner (p < 0.001 for 50 µM), suggesting a reduction in cell migration (Figure 2A). Similarly, the transwell assay showed that the number of invasive cells was substantially decreased following Rus treatment, with the effect being highly significant at higher concentrations (p < 0.001 for 50 µM) (Figure 2B). This inhibitory effect was further supported by western blot analysis of Matrix Metalloproteinases (MMPs). Ruscogenin dose-dependently downregulated MMP2 and MMP9 expression in C666-1 cells, reaching high statistical significance at 50 µM (p < 0.001) (Figure 2C). These results demonstrate that Rus effectively suppresses the migration and invasion of NPC cells with high statistical significance.
Ruscogenin induces apoptosis in C666-1 cells
To determine if the observed reduction in cell viability was due to programmed cell death, the study assessed the level of apoptosis. Flow cytometry confirmed that ruscogenin potently promoted apoptosis in C666-1 cells in a dose-dependent manner, an effect that was highly significant at 50 µM (p < 0.001) (Figure 3A). This was accompanied by a corresponding and statistically significant increase in the activity of caspase-3, a key executioner enzyme in the apoptotic cascade (p < 0.001 for 50 µM) (Figure 3B). To further investigate the apoptotic mechanism, key regulatory proteins were assessed. Western blotting demonstrated that Ruscogenin treatment prompted a significant, dose-dependent shift towards a pro-apoptotic state by significantly decreasing the expression of the anti-apoptotic protein Bcl-2 and increasing the expression of the pro-apoptotic protein Bax (p < 0.001 at 50 µM) (Figure 3C). These significant changes in protein expression further confirm that Rus induces apoptosis in C666-1 cells.
Ruscogenin promotes ferroptosis in C666-1 cells by downregulating KDM1A
The study then investigated ferroptosis and its connection to the protein KDM1A. It was first observed that Rus treatment dose-dependently and significantly decreased the expression of KDM1A in C666-1 cells (p < 0.001 for 50 µM) (Figure 4A), and molecular docking analysis predicted a direct interaction between Rus and the KDM1A protein (Figure 4B). To establish a causal link, C666-1 cells were transfected to significantly overexpress KDM1A (p < 0.001) (Figure 4C). The results showed that Rus treatment alone significantly increased the markers of ferroptosis, including TBARS production (p < 0.001), lipid ROS accumulation (p < 0.001), and the level of intracellular ferrous iron (Fe2⁺). Crucially, the overexpression of KDM1A significantly reversed these effects, reducing the levels of these ferroptosis markers in Rus-treated cells (p < 0.001 for all comparisons) (Figure 4D–F).
Furthermore, analysis of key ferroptosis-related proteins revealed that Rus treatment significantly decreased SLC7A11 and GPX4 levels while increasing ACSL4 and TFR1 levels (p < 0.001 for all). Overexpression of KDM1A significantly counteracted these changes (p < 0.001 for all), restoring the expression of these proteins towards their baseline levels (Figure 4G). These data strongly suggest that Rus promotes ferroptosis in NPC cells by downregulating KDM1A.
KDM1A overexpression reverses the inhibitory effects of Ruscogenin on NPC cell proliferation, migration, and invasion
To confirm that the anti-cancer effects of Rus are mediated through KDM1A, rescue experiments were performed. Overexpression of KDM1A was found to significantly counteract the anti-proliferative effects of Ruscogenin in C666-1 cells, as confirmed by both cell viability and colony formation assays (Figure 5A–B). Similarly, the significant Rus-induced reduction in Ki67 and PCNA protein levels was also partially but significantly restored by KDM1A overexpression (p < 0.001) (Figure 5C). Similarly, the potent anti-migratory and anti-invasive activity of Ruscogenin (p < 0.001) was negated by the overexpression of KDM1A. Both the wound scratch and transwell assays showed a significant recovery in migratory and invasive capabilities in cells overexpressing KDM1A (p < 0.001) (Figure 5D). This was accompanied by a significant restoration of MMP2 and MMP9 protein expression (p < 0.001) (Figure 5E).
KDM1A overexpression attenuates Ruscogenin-induced apoptosis in C666-1 cells
Finally, the study examined whether KDM1A also plays a role in Rus-induced apoptosis. Flow cytometry and caspase-3 activity assays demonstrated that KDM1A treatment significantly attenuated the pro-apoptotic effect of Rus (p < 0.001), leading to a lower apoptotic rate and reduced caspase-3 activity compared to cells treated with Rus alone (Figure 6A–B). This was also confirmed by Western blot, in which Rus overexpression significantly changed the expression of Bcl-2 and Bax in NPC cells, and KDM1A overexpression markedly reversed Rus-induced alterations in Bcl-2 and Bax expression (p < 0.001) (Figure 6C), and the balance was reversed from cell survival to cell death (Figure 6C). Therefore, the rescue experiments demonstrated that ruscogenin suppresses proliferation and invasion and induces apoptosis and ferroptosis in nasopharyngeal carcinoma cells, and Ruscogenin-mediated effects were significantly mediated through downregulation of KDM1A.
DATA AVAILABILITY:
The raw data supporting the conclusions of this article are openly available in the Zenodo repository at https://doi.org/10.5281/zenodo.21852421.
Figure 1: Ruscogenin selectively inhibits the viability and proliferation of nasopharyngeal carcinoma cells (n = 3). (A) The chemical structure of Ruscogenin (Rus). (B) A Cell Counting Kit-8 (CCK-8) assay was used to measure the viability of normal NP69 nasopharyngeal epithelial cells following a 24-h treatment with various concentrations of Rus. (C) C666-1 NPC cells were treated with the indicated concentrations of Rus for 24, 48, or 72 h, and their viability was subsequently determined by a CCK-8 assay. (D) Clonogenic survival of C666-1 cells after a 14-day exposure to Rus. The panel shows representative images and the corresponding quantification. (E) The expression levels of proliferation-associated proteins Ki67 and PCNA in C666-1 cells were analyzed by Western blot after a 24-h Rus treatment, with quantification shown below. Values represent the mean ± standard deviation (SD) from three separate experiments. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. Please click here to view a larger version of this figure.
Figure 2: Ruscogenin inhibits the migration and invasion of C666-1 cells (n = 3). (A) A wound healing assay was performed to evaluate the migratory ability of C666-1 cells. Representative images were captured at 0 and 24 h after treatment with various concentrations of Rus. The graph shows the quantification of the cell migration rate. (B) A Transwell assay was conducted to assess the invasive ability of C666-1 cells treated with Rus. Representative images show the stained cells that invaded through the Matrigel-coated membrane. The graph shows the quantification of invaded cells. (C) Western blot analysis and quantification of the migration- and invasion-related proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in C666-1 cells. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. Please click here to view a larger version of this figure.
Figure 3: Ruscogenin induces apoptosis in C666-1 cells (n = 3). (A) Apoptosis in C666-1 cells treated with various concentrations of Rus was analyzed by flow cytometry using Annexin-V-fluorescein isothiocyanate/propidium iodide (Annexin-V-FITC/PI) staining. The graph shows the quantification of the apoptotic cell percentage (total apoptosis was calculated as the sum of early- and late-apoptotic populations, Q3 + Q2). (B) The activity of caspase-3, an executioner caspase of apoptosis, was measured in Rus-treated C666-1 cells. (C) Western blot analysis and quantification of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax. The contrasting changes in Bax and Bcl-2 expression levels indicate a pro-apoptotic effect. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. Please click here to view a larger version of this figure.
Figure 4: Ruscogenin promotes ferroptosis in C666-1 cells by downregulating lysine-specific demethylase 1A (KDM1A) (n = 3). (A) Western blot showing the dose-dependent downregulation of KDM1A protein by Rus. (B) Molecular docking simulation showing the predicted binding interaction between Ruscogenin and KDM1A. (C) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses validating the transfection efficacy of the KDM1A overexpression plasmid (oe-KDM1A) at the mRNA and protein levels, respectively. (D–G) C666-1 cells were treated with Rus (50 µM) with or without KDM1A overexpression. (D) Quantification of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) assay. (E) Detection of lipid peroxidation using the BODIPY 581/591 C11 probe. The plotted green/red fluorescence ratio indicates the level of lipid peroxidation. (F) Detection of intracellular Fe2⁺ using the Phen Green SK probe. (G) Western blot analysis of key ferroptosis-regulatory proteins (SLC7A11, GPX4, ACSL4, and TFR1). Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups.
Figure 5: KDM1A overexpression reverses the inhibitory effects of Ruscogenin on nasopharyngeal carcinoma (NPC) cell proliferation, migration, and invasion (n = 3). C666-1 cells were divided into four groups: Control, Rus (50 µM), Rus + oe-NC (empty vector), and Rus + oe-KDM1A. (A) Cell viability was assessed by CCK-8 assay. (B) Proliferative capacity was evaluated by colony formation assay. (C) Western blot analysis of proliferation markers, such as Ki-67 (Ki67) and proliferating cell nuclear antigen (PCNA). (D) Cell migration and invasion were assessed by wound healing and transwell assays, respectively. (E) Western blot analysis of migration-related proteins MMP2 and MMP9. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. Please click here to view a larger version of this figure.
Figure 6: KDM1A overexpression attenuates Ruscogenin-induced apoptosis in C666-1 cells (n = 3). Cells were treated as described in Figure 5. (A) Apoptotic cells were quantified by flow cytometry after Annexin-V/PI staining. The total apoptotic cell percentage was calculated as the sum of early- and late-apoptotic populations (Q3 + Q2). (B) Caspase-3 activity was measured to assess the level of apoptosis execution. (C) Western blot analysis was performed to determine the expression levels of the apoptosis-related proteins Bcl-2 and Bax. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. Please click here to view a larger version of this figure.
Discussion
Nasopharyngeal carcinoma (NPC) is a severe malignancy that carries a high prevalence of metastasis, which explains why better interventions are urgently required27. Here, it is shown that Ruscogenin (Rus), a natural product obtained as a derivative of Radix ophiopogonis, has strong anti-tumor effects on NPC cells. It is also concluded that Rus prevents the NPC progression by suppressing the histone demethylase KDM1A, which subsequently promotes two different types of programmed cell death, namely, apoptosis and ferroptosis. This paper expounds on a previously unknown Rus-KDM1A signaling axis in NPC, including a dual mechanism of action that can improve its therapeutic potential.
The invader and migratory abilities of the cancer cells are the determinants of tumor metastasis. Cancer cell migration and invasion capacity are two of the major causes of metastasis, and they are closely linked to adverse clinical outcomes. This is, in most cases, facilitated by proteolytic enzymes like the Matrix Metalloproteinase 2 (MMP 2) and MMP 9, which are highly expressed in this situation 28. These findings strongly support and expand upon prior studies regarding the anti-tumor properties of Ruscogenin. Consistent with previous reports demonstrating its anti-metastatic effects in hepatocellular carcinoma22 and its capacity to induce ferroptosis in pancreatic cancer14, these results confirm the broad-spectrum anti-cancer potential of this natural compound. However, this study significantly advances current knowledge by uncovering a unique dual-death mechanism in NPC. Traditional chemotherapies often encounter clinical bottlenecks due to acquired resistance to apoptosis in tumor cells. By simultaneously triggering both classical apoptosis and iron-dependent ferroptosis—two distinct and non-overlapping cell death modalities—Ruscogenin effectively bypasses this resistance. This dual-pronged attack creates a severe synthetic vulnerability in NPC cells, providing a compelling translational rationale for its development as a therapeutic agent for refractory or chemoresistant tumors. With the conduct of wound scratch assay, transwell assay, and western blot, the data showed that Rus treatment exerted suppressive effects on C666-1 cell migratory and invasive capabilities, coupled with reduced MMP2 and MMP9 levels. A disruption in the delicate balance between cell proliferation and apoptosis is a key driver of tumorigenesis29. Consequently, inducing apoptosis while inhibiting proliferation represents a cornerstone therapeutic strategy to impede tumor progression 30. It is known that the Bcl-2 protein suppresses cell apoptosis via interrupting the early linkage to programmed cell death, while the Bax protein accelerates cell apoptosis by forming a dimer with the Bcl-2 protein to inactivate the Bcl-231. Ki67 and PCNA are extensively adopted markers in cancer cell proliferation32. It was demonstrated that Rus effectively halts the cell cycle and induces apoptosis, as evidenced by the downregulation of proliferation markers Ki67 and PCNA, a decrease in the anti-apoptotic protein Bcl-2, and an increase in the pro-apoptotic protein Bax. This shift in the Bax/Bcl-2 ratio, coupled with elevated caspase-3 activity, confirms that Rus redirects the cellular fate of NPC cells towards apoptotic demise, a cornerstone of effective cancer therapy.
The second element of this research that is newer is the contribution of Rus towards the induction of ferroptosis. The common regulated cell death, including ferroptosis, which is a special type of iron and lipid peroxide-dependent accumulation, is now a significant therapeutic weakness to be used to prevent the progression of NPCs33. This disease can therefore be treated by induction of ferroptosis as it is now deemed an efficient treatment mode34. A variety of core enzymes strictly take care of this process. It is important to note that GPX4 is a central antioxidant inhibitor; it inhibits the development of toxic lipid peroxides in an iron-dependent fashion. Also, the activity of GPX4 depends on a cystine/glutamate antiporter System Xc⁻, with SLC7A11 being one of the major subunits. SLC7A11 aids in the absorption of cystine to synthesize glutathione, which lowers lipid oxidation and inhibits ferroptosis35. The data have indicated that Rus treatment caused the cardinal symptoms of ferroptosis, which included lipid ROS accumulation, elevated intracellular levels of Fe2+, and increased production of TBARS. Intuitively, Rus did this in a remodeling of the expression of major ferroptotic regulators. It repressed the system Xc⁻ antiporter subunit SLC7A11 and glutathione peroxidase GPX4, which constitute primary defenses against lipid peroxidation. In parallel, Rus increased the expression of ACSL4 and TFR1, which are essential proteins of lipid metabolism and iron absorption, respectively, and thus, cells are predisposed to ferroptotic death. This once again indicates that Rus is capable of using the ferroptosis pathway in an efficient manner, and it presents a good approach towards dealing with resistance to traditional apoptosis-based medicines.
In this research, KDM1A, a histone demethylase, serves as the mechanistic linchpin of Rus's action. KDM1A, known as an oncoprotein in NPC and other cancers, promotes cell proliferation and survival. There is a general involvement of aberrant KDM1A expression in the development of various malignancies, including glioma, endometrial adenocarcinoma, and NPC36. What is more, KDM1A has been implicated in various biological processes that contribute to cancer progression, including multidrug resistance, proliferation, and EMT37. Notably, recent studies have reported that KDM1A overexpression suppresses ferroptosis in lung cancer21 and promotes cell cycle progression in pancreatic cancer. The findings in NPC align perfectly with these existing studies, demonstrating that the targeted downregulation of KDM1A is sufficient to lift the blockade on ferroptosis and halt proliferation. More importantly, novel biological evidence is provided showing that pharmacologically targeting a single epigenetic regulator (KDM1A) with Ruscogenin can concurrently dismantle both apoptosis evasion and ferroptosis resistance. This highlights the profound biological significance of epigenetic intervention, positioning the KDM1A axis as a master switch for dictating cancer cell fate. The molecular docking analysis showed a potential direct interaction between Rus and KDM1A, which was subsequently supported by experiments in which Rus treatment significantly downregulated KDM1A production. The rescue experiments showed beyond any doubt the essentiality of KDM1A. KDM1A overexpression also significantly reversed proliferation, migration, and invasion, and neutralized the pro-ferroptotic and pro-apoptotic effects of Rus. This is a strong indication that KDM1A is a major downstream inflammatory effector in the heart of Rus's anti-tumor effect, and thus the Rus-KDM1A axis is a dominant therapeutic focus.
Despite these promising findings, several limitations of the current study outline important future directions. First, the current evidence is primarily based on a single NPC cell line (C666-1) and entirely in vitro experiments. While this provides a strong foundational mechanistic proof-of-concept, the biological heterogeneity of NPC requires that future studies validate the Rus-KDM1A axis across multiple NPC cell lines and patient-derived organoid models. Second, to fully translate these preliminary therapeutic implications into clinical potential, rigorous in vivo studies using murine xenograft models are urgently needed. Such studies will be critical to evaluate the systemic toxicity, pharmacokinetics, and the true anti-tumor efficacy of Ruscogenin within a complex, living tumor microenvironment. Lastly, in spite of the fact that KDM1A was identified as a major mediator, Ruscogenin, being a natural compound, might also have other types of molecular targets, which lead to its comprehensive anti-cancer activity. Prospective research using proteomic or transcriptomic methods can be used to demarcate a more extensive mechanism of action.
Conclusively, this research shows that Ruscogenin demonstrates significant efficacy in preventing the malignancy of nasopharyngeal carcinoma. It achieves this by suppressing KDM1A that induces a dual pronged assault on the cancer cells by comitantly inducing apoptosis and ferroptosis. The results not only provide a compelling justification for developing Ruscogenin as a therapeutic agent in NPC but also support the idea that KDM1A is an important target and that this effect can be achieved with appropriate drugs.
The present study provides evidence that Ruscogenin is a potential therapeutic candidate for inhibiting the malignant transformation of nasopharyngeal carcinoma by suppressing cell proliferation, migration, and invasion. The main process through which this anti-tumor action is realized is the specific inhibition of the histone demethylase KDM1A. This KDM1A inhibition by Ruscogenin triggers a two-way pro-death signaling pathway that results in the simultaneous elevation of classical apoptosis, which involves the activation of caspase-3, and ferroptosis, which involves iron-dependent lipid peroxidation. Rescue experiments clearly showed that the Rus-KDM1A axis plays an essential role, as overexpression of KDM1A reversed the anti-tumor effects of Ruscogenin. The results of this study are also valuable because, in addition to explaining a novel pathway of Ruscogenin, they point to a possible approach to overcoming therapeutic resistance by activating two distinct cell death pathways simultaneously. Hence, these results are a solid indicator of the continued evolution of Ruscogenin and confirm the KDM1A pathway as a new therapeutic target in nasopharyngeal carcinoma.
Disclosures
The authors declare no competing financial interests. The authors declare that no generative AI tools were used in the study's conception, data collection, data analysis, or figure generation. AI-assisted technologies were used solely for minor language editing and grammar correction during the drafting of this manuscript. The authors take full responsibility for the content and integrity of the published work.
Acknowledgements
Not applicable.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 7500 Fast Real-Time PCR System | Applied Biosystems | 4351106 | RT-qPCR analysis |
| Annexin-V-FITC/PI Apoptosis kit | BD Biosciences | 556547 | Flow cytometry apoptosis detection |
| Anti-ACSL4 antibody | Abcam | ab155282 | Primary antibody for Western blot |
| Anti-Bax antibody | Cell Signaling Technology (CST) | 5023 | Primary antibody for Western blot |
| Anti-Bcl-2 antibody | Cell Signaling Technology (CST) | 15071 | Primary antibody for Western blot |
| Antibodies | |||
| Anti-GPX4 antibody | Abcam | ab125066 | Primary antibody for Western blot |
| Anti-KDM1A (LSD1) antibody | Cell Signaling Technology (CST) | 2139 | Primary antibody for Western blot |
| Anti-Ki67 antibody | Abcam | ab16667 | Primary antibody for Western blot |
| Anti-MMP2 antibody | Abcam | ab92536 | Primary antibody for Western blot |
| Anti-MMP9 antibody | Abcam | ab76003 | Primary antibody for Western blot |
| Anti-PCNA antibody | Cell Signaling Technology (CST) | 13110 | Primary antibody for Western blot |
| Anti-rabbit IgG, HRP-linked | Cell Signaling Technology (CST) | 7074 | Secondary antibody |
| Anti-SLC7A11 (xCT) antibody | Abcam | ab175186 | Primary antibody for Western blot |
| Anti-TFR1 antibody | Abcam | ab84036 | Primary antibody for Western blot |
| Anti-β-actin antibody | Cell Signaling Technology (CST) | 4970 | Loading control primary antibody |
| Assay Kits | |||
| Autodock 4.2 | Scripps Research Institute | N/A | Molecular docking simulation |
| BCA protein assay kit | Beyotime Biotechnology | P0012 | Protein quantification |
| BODIPY 581/591 C11 probe | Invitrogen (Thermo Fisher Scientific) | D3861 | Lipid ROS detection (2 μM) |
| C666-1 cell line | iCell Bioscience Inc. | iCell-h378 | Human nasopharyngeal carcinoma cell line |
| Caspase-3 activity assay kit | Beyotime Biotechnology | C1115 | Apoptosis evaluation |
| CCK-8 assay kit | Dojindo Molecular Technologies | CK04 | Cell viability measurement |
| Chemicals & Reagents | |||
| CytoFLEX flow cytometer | Beckman Coulter | CytoFLEX | Apoptosis analysis |
| ECL detection reagent | MilliporeSigma | WBKLS0500 | Western blot visualization (Immobilon) |
| Equipment | |||
| Fetal Bovine Serum (FBS) | Gibco (Thermo Fisher Scientific) | 10099141 | Cell culture supplement |
| GraphPad Prism 8.0 | GraphPad Software | N/A | Statistical analysis and graphing |
| ImageJ software | NIH | N/A | Image densitometry analysis |
| Keratinocyte serum-free medium | iCell Bioscience Inc. | iCell-019 | For NP69 cell culture |
| Laser scanning confocal microscope | Carl Zeiss | LSM 880 | BODIPY C11 fluorescence imaging |
| Light / Fluorescence microscope | Olympus | IX73 | Wound scratch, Transwell & Fe2+ imaging |
| LightCycler 480 SYBR Green I Master | Roche Diagnostics | 4887352001 | Quantitative real-time PCR |
| Lipofectamine 2000 reagent | Invitrogen (Thermo Fisher Scientific) | 11668019 | Cell transfection reagent |
| Matrigel | Corning | 356234 | Basement membrane matrix for invasion assay |
| Microplate reader | Molecular Devices | SpectraMax M5 | Absorbance measurement (405, 450, 532 nm) |
| NP69 cell line | iCell Bioscience Inc. | iCell-h431 | Normal human nasopharyngeal epithelial cell line |
| oe-KDM1A & oe-NC plasmids | Shanghai GenePharma Co., Ltd. | Custom Synthesis | Plasmids for KDM1A overexpression |
| Phen Green SK (PGSK) probe | Invitrogen (Thermo Fisher Scientific) | P14312 | Intracellular Fe2+ detection (10 μM) |
| Pymol 3.0 | Schrödinger | N/A | Molecular docking visualization |
| Reverse transcription kit | Roche Diagnostics | 4379012001 | Transcriptor First Strand cDNA Synthesis Kit |
| RIPA lysis buffer | Solarbio (Beijing, China) | R0010 | Total protein extraction |
| RPMI 1640 medium | iCell Bioscience Inc. | iCell-0002 | For C666-1 cell culture |
| Ruscogenin (Rus) | MedChemExpress (MCE) | HY-N0193 | Drug treatment, purity ≥ 98% |
| Software | |||
| TBARS assay kit | Cayman Chemical | 10009055 | Lipid peroxidation measurement |
| TRizol reagent | Invitrogen (Thermo Fisher Scientific)* | 15596026 | Total RNA extraction |
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