Ruscogenin Suppresses Nasopharyngeal Carcinoma Progression by Downregulating KDM1A to Co-induce Ferroptosis and Apoptosis

Summary

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.

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 radiotherapy^4,5.

Owing to the minimal toxicity and fewer side effects, Chinese herbal formulas have been widely used to treat cancer for thousands of years^6,7. 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 NPC^8,9,10. Radix ophiopogonis (RO), the root of the perennial herbaceous plant Ophiopogon japonicus, has been reported to effectively alleviate NPC as an adjuvant therapy^11,12. 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 ...

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...

Materials

List of materials used in this article

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