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....

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....

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