Ferroptosis Induction in Glioma by Calceolarioside A via Modulation of the PI3K/Akt/Nrf2 Pathway

Summary

This study explores the therapeutic potential of Calceolarioside A in glioma, demonstrating its ability to inhibit PI3K/Akt signaling and promote Nrf2 degradation, thereby inducing ferroptosis and suppressing tumor progression both in vitro and in vivo.

Abstract

This protocol demonstrates the experimental workflow used to investigate the effects of Calceolarioside A (CaA) on ferroptosis induction and modulation of the Phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt)/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in glioma models. The procedures include a series of cell-based assays in U251 and U87 human glioma lines to evaluate cell viability, proliferation, and chemosensitivity following CaA treatment. Ferroptosis-associated changes are assessed by measuring reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and labile iron levels, along with expression of ferroptosis-related proteins such as glutathione peroxidase 4 (GPX4), cysteine/glutamate antiporter subunit (xCT), and ferritin via western blot. To assess pathway involvement, the protocol details qRT-PCR, western blot, and immunoprecipitation-based assays for Nrf2 expression and ubiquitination. Nrf2 overexpression experiments are included to confirm its role in ferroptosis regulation. The protocol further demonstrates the use of a mouse xenograft model, where U87 cells are implanted subcutaneously, followed by intraperitoneal CaA administration to evaluate in vivo tumor growth and toxicity. Histological analysis of major organs is performed to assess systemic safety. Additionally, molecular docking is used to predict direct binding between CaA and the PI3K p110α subunit (PIK3CA). Together, these procedures provide a reproducible framework to examine ferroptosis mechanisms and the therapeutic efficacy of natural compounds in glioma research.

Introduction

Gliomas, particularly high-grade glioblastomas, represent one of the most lethal forms of brain tumors, characterized by rapid proliferation, treatment resistance, and poor prognosis¹. In recent years, various anti-glioma agents have been explored, including traditional chemotherapeutics like temozolomide and a range of natural compounds such as curcumin², resveratrol³, and epigallocatechin gallate⁴. These agents exert their effects through multiple mechanisms, including induction of apoptosis, inhibition of angiogenesis, and modulation of oxidative stress pathways. However, many of these compounds face limitations such as poor bioavailability or incomplete mechanistic characterization. This highlights the critical need for comprehensive methodologies that not only confirm anti-tumor activity but also systematically dissect the underlying pharmacological mechanisms. Establishing such mechanistic workflows is especially important for accelerating the development of novel anti-glioma compounds from discovery to preclinical validation.

To support this goal, an increasing number of studies now integrate advanced computational and experimental tools to enhance mechanistic resolution^5,6. Structure-based drug design and artificial intelligence (AI) models -- such as AlphaFold3 and molecular docking -- are facilitating the identification of promising drug-target interactions with unprecedented speed and accuracy^7,8. These tools complement experimental methods by predicting binding affinities and identifying potential molecular targets before laboratory validation. At the same time, the emergence of pathway-based functional assays, live-cell imaging, and high-resolution proteomic techniques allow researchers to evaluate the cellular impact of new compounds across multiple regulatory axes, including oxidative stress responses, signal transduction cascades, and cell death modalities such as ferroptosis^9,10,11.

Among the signaling pathways involved in glioma biology, the Phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt) and Nuclear factor erythroid 2-related factor 2 (Nrf2) pathways are of particular interest due to their roles in tumor growth, redox homeostasis, and therapy resistance^12,13. PI3K/Akt signaling promotes survival, proliferation, and metabolic adaptation in glioma cells, while Nrf2 functions as a master regulator of the antioxidant response, suppressing ferroptotic cell death¹⁴. Persistent activation of these pathways contributes to treatment evasion, making them attractive targets for pharmacological intervention¹⁵. Conventional methods for pathway interrogation include western blotting for total and phosphorylated proteins, as well as qRT-PCR for gene expression analysis. However, in-depth analysis of Nrf2 signaling requires additional assays that assess protein stability, nuclear translocation, and post-translational modifications such as ubiquitination. These multifaceted detection strategies are essential for understanding how specific compounds disrupt or reinforce redox regulation and ferroptotic control.

Calceolarioside A (CaA), a hydroxycinnamic acid derivative, offers a dual mechanism by simultaneously inhibiting PI3K/Akt signaling and promoting proteasomal degradation of Nrf2, thus enhancing ferroptotic sensitivity. This dual-targeting strategy may improve reproducibility and mechanistic depth over existing agents like erastin or RSL3, which act downstream of antioxidant defenses. This protocol presents an integrated experimental approach to investigate the pharmacological mechanism of CaA, which includes molecular docking to identify PI3K binding sites, immunoblotting and immunoprecipitation to detect Nrf2 expression and ubiquitination, and ferroptosis markers such as reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA). Furthermore, a xenograft mouse model is used to assess CaA's therapeutic efficacy and systemic safety. This comprehensive workflow provides a reproducible and informative platform for studying the mechanism of action of anti-cancer agents, with emphasis on integrating signal pathway modulation and cell death regulation.

Protocol

Cell culture and treatment
Murine HT22 neuronal cells and human glioma U87 and U251 cell lines were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 4 mM L-glutamine, under humidified conditions at 37 °C with 5% CO₂. To streamline experimental consistency, all cell treatments were standardized and described here. This section outlines the specific conditions used in each treatment group and the corresponding downstream assays.

CaA treatment:
HT22 cells were treated with increasing concentrations of CaA (0, 0.1, 0.5, 1, 10, 50, 100, 250, and 500 µM) for 72 h. U87 and U251 glioma cells were treated with CaA at concentrations of 5, 20, or 50 µM for 24 h for most experiments, or with 50 µM for 0, 6, 12, or 18 h for time-course studies. These treatments were used in the following analyses: western blotting for cysteine/glutamate antiporter subunit (xCT), glutathione peroxidase 4 (GPX4), ferritin (FTH1), glutaminase, and PI3K/Akt/mammalian target of Rapamycin (mTOR)/ eukaryotic Initiation Factor 4B (EIF4B) (total and phosphorylated levels); real-time quantitative polymerase chain reaction (RT-qPCR) for Nrf2 and Kelch-like ECH-associated protein 1 (Keap1); ROS measurement using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA); GSH and MDA quantification; FerroOrange-based Fe²⁺ detection; 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay; Colony formation assay; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.

Liproxstatin-1 (Lip1) co-treatment:
Cells were pretreated with 50 nM Lip1 for 2 h before the addition of 50 µM CaA. These conditions were used in MTT assay (0, 24, 48, 72 h); colony formation assay; EdU assay; Annexin V/PI apoptosis assay (with/without 20 nM cisplatin).

MG132 co-treatment:
Cells were treated with 10 µM MG132 alone or combined with 50 µM CaA for 24 h. This was applied to western blotting for Nrf2 protein stabilization (total and nuclear); Immunoprecipitation (IP) assays for ubiquitinated Nrf2 and Keap1.

Cisplatin co-treatment:
Cells were treated with 20 nM cisplatin alone or in combination with 20 µM CaA for 24 h. These treatments were used in Annexin V/propidium iodide (PI) apoptosis assay.

Nrf2 overexpression:
Cells were transfected with 2.5 µg Nrf2 overexpression plasmid or empty vector using transfection agent for 18 h, then treated with 50 µM CaA for 24 h. This condition was used in western blotting for xCT, GPX4, FTH1, and glutaminase; ROS, MDA, GSH assays; FerroOrange staining for intracellular iron.

A workflow diagram (Supplemental Figure S1) summarizes the above experimental conditions and corresponding analyses. This figure visually links each treatment protocol to its specific downstream assays for improved clarity and reproducibility.

Assessing cell viability
Cell viability was assessed using the conventional MTT colorimetric method as previously described¹⁶. HT22, U251 and U87 cells were plated into 96-well culture plates and incubated overnight to allow adherence. After completing the treatment period, 15 µL of MTT solution was introduced into each well and the plates were incubated for 4 h at 37 °C. The medium was then discarded, and 150 µL of dimethyl sulfoxide (DMSO) was added to solubilize the formed formazan crystals. Absorbance was subsequently recorded at 490 nm using a microplate reader. Higher absorbance values indicate greater cell viability, whereas a reduction in absorbance suggests cytotoxic effects.

EdU incorporation assay
For assessing DNA synthesis and cell proliferation, EdU incorporation assays were performed using an EdU Imaging Kit as previously described¹⁷. Briefly, U251 and U87 glioma cells were seeded in 24-well plates. After treatment, cells were incubated with EdU (10 µM final concentration) and cultured for an additional 24 h. Following the incubation step, the cells were fixed using 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100., and stained according to the kit instructions. The percentage of EdU-positive cells (proliferating cells) was quantified by fluorescence microscopy and normalized to total 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei.

Colony formation assay
For the colony formation assay, U251 and U87 glioma cells were plated in 3.5 cm dishes at low density (500 cells per dish). Following treatment, the culture medium was exchanged for fresh, drug-free medium, and the cells were maintained for another 14 days to permit colony formation, with medium changes performed every three days. The resulting colonies were subsequently fixed using 4% paraformaldehyde, stained with 0.1% crystal violet., and counted manually under a microscope. Clusters exceeding 75 µm in diameter were scored as colonies. Each experiment was performed in triplicate and repeated three times. This assay followed commonly accepted protocols for evaluating long-term proliferative capacity¹⁸. Colonies should appear as dense, circular clusters; fewer and smaller colonies suggest reduced proliferative capacity.

Assessing cell apoptosis
Apoptotic cell death was assessed using the FITC-Annexin V apoptosis detection kit as previously described¹⁶. U87 and U251 glioma cells were treated with 20 nM cisplatin in the presence or absence of 20 µM CaA for 24 h prior to staining. For experiments involving ferroptosis inhibition, cells were pretreated with 50 nM Lip1 for 2 h, followed by co-treatment with 20 µM CaA and 20 nM cisplatin for an additional 24 h. After the indicated treatments, cells were harvested (300 × g, 5 min) and stained with Annexin V-FITC and PI according to the manufacturer's instructions. After labeling, samples were examined on a flow cytometer. Data acquisition was performed using FL1 (FITC) and FL2 (PI) channels, with compensation settings applied to minimize spectral overlap. The proportion of Annexin V-positive cells was subsequently calculated to determine the degree of apoptosis.

Western blot analysis
Western blotting was conducted according to established procedures^19,20. Briefly, after the indicated treatments, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and the protein levels were determined using the Bradford assay. For experiments assessing Nrf2 subcellular distribution, nuclear and cytoplasmic proteins were extracted using a commercial nuclear extraction kit according to the manufacturer's instructions.

A total of 40 µg of protein from each sample was resolved on a 10% SDS-PAGE gel and subsequently transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk prepared in PBST (PBS with 0.1% Tween-20) for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. After washing, HRP-conjugated secondary antibodies (1:5,000) were applied for 1 h at 37 °C, and the signals were detected using an enhanced chemiluminescence (ECL) system.

The expression levels of xCT, glutaminase, GPX4, and FTH1 were measured, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. Phosphorylated proteins (p-PI3K, p-Akt, p-mTOR, and p-EIF4B) were quantified relative to their respective total protein levels, while the total proteins were normalized against GAPDH. Nuclear Nrf2 was normalized to Lamin B. Expected outcomes include a dose- and time-dependent reduction in Nrf2 and p-PI3K/Akt/mTOR/EIF4B, and downregulation of ferroptosis-related markers in CaA-treated cells.

For ubiquitination assays, cells were treated with MG132 (10 µM) with or without CaA (50 µM) for 24 h. After lysis, protein complexes were immunoprecipitated using anti-Nrf2 or anti-Keap1 antibodies; normal rabbit IgG was used as a negative control. The immunoprecipitates were then probed with anti-ubiquitin antibodies to detect ubiquitinated forms, while total lysates were also probed for Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) levels to confirm input.

RNA extraction and quantitative real-time PCR (qRT-PCR)
RNA extraction and qRT-PCR were performed following standard protocols²¹. Briefly, U87 and U251 glioma cells were treated with or without 50 µM CaA for different durations (6 and 18 h). Total RNA was isolated and converted into cDNA through reverse transcription. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Green qPCR Master Mix on a real-time PCR platform. Relative gene expression was determined using the 2-ΔΔCt calculation. The primer sequences used for Nrf2, Keap1, and the housekeeping gene GAPDH were as follows:

Nrf2 forward, 5′-CACATCCAGTCAGAAACCAGTGG-3′;
Nrf2 reverse, 5′-GGAATGTCTGCGCCAAAAGCTG-3′

Keap1 forward, 5′-CAACTTCGCTGAGCAGATTGGC-3′;
Keap1 reverse, 5′-TGATGAGGGTCACCAGTTGGCA-3′

GAPDH forward, 5′-CGGAGTCAACGGATTTGGTCG-3′;
GAPDH reverse, 5′-AGCCTTCTACATGGTGGTGAAGAC-3′

Nrf2 transduction
U251 and U87 glioma cells were transiently transfected with either an Nrf2 overexpression plasmid or a corresponding empty control vector using the lipid-based transfection reagent; the procedure was based on conventional transient transfection methods²². Briefly, cells were seeded in 6-well plates at a density of 3 × 10⁵ cells per well and cultured overnight to achieve approximately 70-80% confluence at the time of transfection. For each well, 2.5 µg of plasmid DNA was diluted in 125 µL of Reduced Serum Medium and mixed with 5 µL of the transfection reagent. Separately, 3.75 µL of the transfection reagent was diluted in another 125 µL of the reduced serum medium. The diluted DNA and transfection reagent mixtures were then combined, gently mixed, and incubated for 10-15 min at room temperature to allow complex formation. The resulting transfection complexes (total volume 250 µL) were added dropwise to each well containing cells in fresh complete medium.

After 18 h of incubation at 37 °C in a CO₂ incubator, the transfection medium was replaced with fresh culture medium. Cells were then subsequent assays, including western blotting, ROS analysis, MDA/GSH quantification, and iron accumulation studies. This protocol follows widely accepted transient gene expression procedures and allows for robust Nrf2 expression within 24-48 h post transfection.

Measurement of intracellular iron
Intracellular Fe²⁺ levels were measured using the fluorescent indicator FerroOrange. Brief, U251 and U87 glioma cells were seeded in 24-well plates and treated with varying concentrations of CaA (5, 20, and 50 µM). To assess the effect of Nrf2 overexpression on ferroptosis, U87 and U251 cells were transfected with an Nrf2 expression plasmid or empty vector. After 18 h, medium was replaced, and cells were treated with 50 µM CaA for 24 h. Subsequently, cells were exposed to 1 µM FerroOrange in serum-free medium and incubated at 37 °C for 30 min before analysis. After incubation, cells were rinsed and observed under a fluorescence microscope, using a TRITC filter set (excitation ~543 nm, emission 560-600 nm), where increased orange fluorescence intensity indicates higher levels of labile Fe²⁺, typically localized in the cytoplasm. This method was adapted from established fluorescent probe-based iron assays²³.

Measurement of GSH and MDA
Intracellular GSH and MDA levels were determined following standardized evaluation protocols to evaluate oxidative stress²⁴. Brief, U251 and U87 glioma cells were seeded in 24-well plates and treated with varying concentrations of CaA (5, 20, and 50 µM). To assess the effect of Nrf2 overexpression on ferroptosis, U87 and U251 cells were transfected with an Nrf2 expression plasmid or empty vector. After 18 h, medium was replaced and cells were treated with 50 µM CaA for 24 h. Subsequently, cells were lysed in RIPA buffer, and total protein concentrations were measured using a BCA protein quantification kit. GSH and MDA contents were subsequently analyzed using commercial detection kits in accordance with the manufacturers' protocols.

ROS assay
Intracellular ROS generation was assessed using the fluorescent probe DCFH-DA in accordance with established protocols²⁵. Brief, U251 and U87 glioma cells were seeded in 24-well plates and treated with varying concentrations of CaA (5, 20, and 50 µM). To assess the effect of Nrf2 overexpression on ferroptosis, U87 and U251 cells were transfected with an Nrf2 expression plasmid or empty vector. After 18 h, medium was replaced and cells were treated with 50 µM CaA for 24 h. Subsequently, cells were treated with 5 µM DCFH-DA at 37 °C for 1 h, followed by PBS washes to remove excess dye. Fluorescence signals were visualized using an inverted fluorescence microscope, equipped with a FITC filter (excitation 488 nm, emission 525 nm), and images were captured for analysis. Increased green fluorescence intensity corresponds to elevated intracellular ROS levels.

Animal experiments
All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Weifang Peoples Hospital under approval number [2025SDL579]. To generate the glioma xenograft model, 2 × 10⁶ U87 cells were resuspended in 0.2 mL of PBS and subcutaneously inoculated into the left flank of 4-week-old male BALB/c nude mice. After allowing tumors to develop for 7 days, the mice were randomly assigned to three groups (n = 8 per group): untreated control, CaA-treated (2.5 mg/kg), and CaA (2.5 mg/kg) combined with Lip1 (10 mg/kg). Treatments were administered via daily intraperitoneal injection for 14 consecutive days. Tumor dimensions were measured every 3 days using calipers, and volumes were calculated using the formula: volume = (length × width²) / 2. On day 21, all mice were euthanized, and tumors were excised and photographed for further analysis.

RNA sequencing and KEGG pathway analysis
RNA sequencing was performed to profile transcriptional changes following CaA exposure. RNA integrity was confirmed by using a microvolume UV-VIS spectrophotometer and capillary-based electrophoresis system prior to sequencing on the Illumina platform. Clean reads were mapped to the human reference genome (GRCh38) with HISAT2, and gene expression was quantified as FPKM. Differentially expressed genes were identified using DESeq2 (FDR < 0.05, |log₂FC| ≥ 1), and functional enrichment was assessed by KEGG pathway analysis (adjusted P < 0.05).

Molecular docking analysis
Molecular docking was employed to predict the interaction between CaA and the PI3K/Akt pathway. The crystal structure of the human PI3K catalytic subunit p110α (PIK3CA) was obtained from the Protein Data Bank (PDB ID: 8BFU). The structure of CaA was retrieved from PubChem and energy-minimized using molecular modeling software. Using molecular docking preparation software, all water molecules and non-essential ligands were removed from the PIK3CA structure, and hydrogen atoms were added. A docking grid was defined around the active site of PIK3CA (based on known binding site coordinates), and a docking algorithm was used to perform the docking. The top-scoring binding poses of CaA were selected according to binding affinity (kcal/mol) and examined for key interactions. Protein-ligand interactions, such as hydrogen bonds and hydrophobic contacts, were visualized with molecular visualization software and a 2D interaction mapping tool.

Safety and waste disposal
All experiments involving chemical reagents such as DMSO, fluorescent dyes (DCFH-DA, FerroOrange), and fixatives were conducted in a fume hood with appropriate personal protective equipment (PPE), including gloves and safety goggles. DMSO and fluorescent probes were handled with caution due to their ability to penetrate skin and potential cytotoxicity. Contaminated materials, including pipette tips, culture plates, and gloves, were disposed of in designated hazardous waste containers. Animal tissues and carcasses were treated as biohazardous waste and were incinerated or autoclaved according to institutional biosafety regulations and local environmental guidelines.

Statistical analysis
All results are presented as the mean ± standard deviation (SD). Differences between two groups were evaluated using a two-tailed Student's t-test. For analyses involving multiple groups, one-way ANOVA followed by suitable post hoc comparisons (such as Tukey's test) was applied. A p-value below 0.05 was regarded as indicative of statistical significance.

Results

Proliferation suppression and enhanced chemosensitivity of U251 and U87 cells in vitro by CaA
Figure 1A illustrates the structure of CaA. We first evaluated the cytotoxic effects of CaA using a MTT assay on normal murine HT22 neuronal cells and human glioma U251 and U87 cells. Compared with untreated cells (0 µM CaA), CaA had minimal impact on HT22 cell viability at concentrations below 100 µM over 72 h (Figure 1B). However, treatme...

Discussion

CaA exhibits a compelling antitumor effect in glioblastoma by uniquely modulating two critical survival axes: the PI3K/Akt/mTOR pathway and the Nrf2-ferroptosis defense mechanism. The PI3K/Akt pathway is frequently hyperactivated in glioblastoma multiforme (GBM, altered in up to ~88-90% of cases) due to PTEN loss or RTK activation, driving proliferation and therapy resistance²⁶. Yet, targeting this pathway alone has yielded limited apoptosis in GBM, as tumors often escape PI3K inhibition via cytos...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agilent 2100 Bioanalyzer			capillary-based electrophoresis system
Annexin V-FITC Apoptosis Kit	Roche Diagnostics	11858777001	N/A
AutoDock Tools 1.5.7 (AutoDock Vina)			molecular docking preparation software (docking algorithm)
Calceolarioside A (CaA)	Sigma-Aldrich	SMB00246	N/A
Chem3D			molecular modeling software
cDNA Synthesis Kit	Vazyme Biotech	R223	N/A
DCFH-DA	Sigma-Aldrich	D6883	N/A
DMEM	Thermo Fisher Scientific	11965092	N/A
DMSO	Merck	D8418	N/A
FerroOrange probe	Dojindo	F374	N/A
Fetal Bovine Serum (FBS)	Sigma-Aldrich	F2442	N/A
Flow cytometer	BD Biosciences	FACSAriaIII Flow Cytometry	Instrument for analyzing Annexin V stained cells
Fluorescence microscope	Olympus	IX73	Instrument for observing FerroOrange and DCFH-DA fluorescence
FTH1 antibody	Abcam	ab75973	AB_1310150
GAPDH antibody	Abcam	ab8245	AB_2107448
GLS antibody	Abcam	ab93434	AB_10563527
GPX4 antibody	Abcam	ab125066	AB_10972289
HT22 murine neuronal cell line	AddexBio	C0011008	CVCL_0321
jetPRIME transfection reagent	Polyplus-transfection	114-07	polymer-based transfection reagent
Keap1 antibody	Abcam	ab139729	AB_2732852
L-Glutamine (200 mM)	Thermo Fisher Scientific	25030081	N/A
LigPlot+			2D interaction mapping tool
Lipofectamine 3000	Thermo Fisher Scientific		lipid-based transfection reagent
Liproxstatin-1	Selleck	S7699	N/A
MG132	Selleck	S2619	N/A
Microplate reader	Thermo Fisher Scientific	H1M	Instrument for measuring absorbance in MTT assay (at 490 nm)
MTT	Sigma-Aldrich	M5655	N/A
NanoDrop			microvolume UV-Vis spectrophotometer
Nrf2 antibody	Abcam	ab62352	AB_945626
Nude BALB/c mice	Vital River, China	N/A	IMSR_CRL:028
OptiMeM reduced serum medium
PyMOL			molecular visualization software
QuantStudio 5 Real-Time PCR System
SYBR Green Master Mix	Vazyme Biotech	Q711	N/A
TRIzol Reagent	Thermo Fisher	15596026	N/A
U251 human glioma cell line	CLS Cell Lines Service	300385	CVCL_0021
U87 human glioma cell line	ATCC	HTB-14	CVCL_0022
Ubiquitin antibody	Abcam	ab7780	AB_306981
xCT/SLC7A11 antibody	Abcam	ab175186	AB_2715513