Regulation of the AKT/Wnt/β-catenin Pathway and Induction of Cuproptosis by Curcumin in Glioblastoma

Summary

Curcumin (CUM) inhibits glioblastoma (GBM) progression by inducing cuproptosis through suppression of AKT/Wnt/β-catenin axis. This protocol investigates the molecular mechanism by which CUM impedes malignant progression in GBM and describes methods to evaluate how CUM induces cuproptosis by modulating this pathway.

Abstract

Curcumin (CUM) can regulate the malignant behavior of glioblastoma (GBM). This study investigates whether CUM suppresses GBM progression by inducing cuproptosis via the AKT/Wnt/β-catenin signaling pathway. Optimal curcumin concentrations were determined using the MTT assay. A172 and U251 cells were treated with CUM, AKT inhibitor MK-2206, Wnt inhibitor LGK974, and Elsm-Cu (Elesclomol + CuCl₂). Cell proliferation, migration, invasion, and apoptosis were assessed using carboxyfluorescein succinimidyl amino ester staining, scratch assays, Transwell assays, and flow cytometry, respectively. MitoSOX fluorescence, Seahorse metabolic analysis, immunofluorescence, Cu²⁺ detection, and Western blotting were used to evaluate mitochondrial oxidative stress and cuproptosis. The AKT/Wnt/β-catenin axis was analyzed using a TCF/LEF1 reporter kit and western blotting. A GBM xenograft model was established, and CUM was administered by gavage for five weeks. CUM effects on tumor growth, cuproptosis, and AKT/Wnt/β-catenin pathway protein expression were evaluated. Cells were treated with 10 µM and 20 µM CUM in vitro. CUM treatment reduced proliferation, migration, and invasion, while promoting oxidative stress and cuproptosis. CUM also suppressed Wnt/β-catenin signaling activity. Pathway inhibition increased reactive oxygen species (3.7-fold) and Cu²⁺ (3.1-fold) levels, and decreased dihydrolipoamide acetyltransferase expression, thereby restraining malignant behavior. In nude mice, CUM significantly reduced tumor growth, promoted cuproptosis, and inhibited AKT/Wnt/β-catenin axis activation. Our results indicate that CUM suppresses AKT/Wnt/β-catenin signaling, promotes cuproptosis, and interferes with GBM progression.

Introduction

Glioma is the most prevalent primary intracranial malignant tumor in the central nervous system (CNS)¹. Grade 4 glioma usually refers to glioblastoma (GBM), with poor prognosis². GBM is a highly invasive brain tumor, accounting for nearly 50% of CNS malignancies, with a median survival of less than 1 year³. It is known for its high malignancy and high recurrence rate^4,5. Presently, GBM treatment strategies primarily relies on surgery, supplemented by radiotherapy, chemotherapy, and other comprehensive treatment methods⁶

Protocol

All animal procedures were performed in compliance with established institutional standards for the humane care and use of laboratory animals and were formally approved by the institutional Animal Ethics Committee. Measures were taken to reduce discomfort and distress to the animals whenever possible.

Work involving chemical compounds, dyes, and biological samples followed established biosafety practices to mitigate potential risks. Potentially hazardous substances-including paraformaldehyde, copper-based reagents, and DHE-were handled with appropriate personal protective equipment, including gloves, face masks, and safety glasses. All wast....

Results

Inhibition of GBM cell viability and induced cell death
First, there was no significant change in NHA cell viability after 2 h, 48 h, and 72 h of CUM intervention (Figure 2A), indicating that CUM concentrations used had no toxic effect on normal cells. In contrast, the viability of GBM cell lines (A172, U251, LN229, and SHG-44) was significantly reduced at CUM concentrations ≥ 1 µM (P < 0.05-0.001) (Figure 2B-E

Discussion

The recurrence rate of GBM is associated with its malignant biological characteristics³⁶. CUM exerts anti-GBM effects by regulating oxidative stress, apoptosis, autophagy, metastasis, invasion, and key molecular targets³⁷. In this study, cellular-level experiments demonstrated that CUM significantly increased GBM cell mortality and inhibited proliferation, colony formation, migration, and invasion -- findings consistent with previous reports²⁰.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% crystal violet staining solution	Servicebio	G1014	Labeling cells
4% paraformaldehyde	Beyotime	P0099	Fix cells or tissues
AKT inhibitor (MK-2206)	Selleck.cn, Shanghai, China	S1078	Specific AKT inhibitor
Antibodies (primary and secondary antibodies)	ABclonal Technology, Wuhan, China / abcam, Cambridge, UK / Servicebio, Wuhan, China / Beyotime, Shanghai, China / Cell Signaling, Danvers, MA, USA	A8814, ab32572, GB25303, ab15580, ab108257, ab246917, A8814, AF6708, AA326, 4060, ab219412, 280376, 9322, ab97051, ab229025	Detection of protein expression levels
Cell apoptosis detection kit	Servicebio, Wuhan, China	G1021	Detection of apoptosis
Cell Trace CFSE dye	Invitrogen,Carlsbad, CA, USA	C34570	Cell proliferation detection
Chemiluminescence imaging system	Bio-Rad, Hercules, CA, USA	Chemi Doc XRS+	Ultra-high sensitivity detection of protein
Cu2+ colorimetric assay kit	Elabscience, Wuhan, China	E-BC-K300-M	Detection of Cu2+ content in tumor tissues and GBM cells
CuCl2	Sigma-Aldrich, Darmstadt, Germany	751944	Cuproptosis inducer
Curcumin (CUM)	Shanghai source leaf, Shanghai, China	S19245	Polyphenolic compound
DHE detection kit	Beyotime, Shanghai, China	S0063	Detection of ROS levels
Dimethyl sulfoxide (DMSO)	Servicebio	G4101-2	Solvent for MTT assay
ECL developer	Servicebio	G2161	Used for Western blot
Elesclomol	Selleck.cn	S1052	Cuproptosis inducer
Flow cytometry	BD Biosciences, New Jersey, USA	BD FACSAria III	Used for cell death and CFSE assays
Fluorescence microscope	OLYMPUS, Tokyo, Japan	IX73	Used for Immunofluorescence, ROS, TUNEL, DHE and MMP assays
Glioblastoma cell lines A172 (RRID: CVCL_0131), U251 (RRID: CVCL_0021), LN229 (RRID: CVCL_0393) and SHG-44 (RRID: CVCL_6902)	Punosai Biotechnology Co., Ltd., Wuhan, China	CL-0012, CL-0237, CL-0578, CL-0207	Human glioblastoma cell lines
GraphPad Prism 9.0 software	GraphPad Software, LLC	Version 9.0	Statistical analysis
ImageJ 1.53k software	National Institute of Mental Health	Version 1.53k	statistical calculation
JC-1 working solution	Solarbio, Beijing, China	M8650	Detection of MMP
Male BALB/c nude mice	Slack Kingda Laboratory Animal Co., Ltd., Hunan, China		Mouse experiment
Microplate reader	Bio-Tek, Vermont, USA	ELX-800	For OD450 nm value measurement
Microscope	OLYMPUS	BX53	Used for cell scratch test, Transwell experiment, and immunohistochemistry assays
MitoSOX probe	Thermo Fisher Scientific, Waltham, MA, USA	M36008	Used for ROS detection
Mitotracker Red CMXRos	Thermo Fisher Scientific	M7512	Specific labeling of mitochondria in living cells
MTT detection kit	Servicebio	G4101-1	Detection of cell viability
Normal human brain astrocyte NHA cells (RRID:CVCL_B0DK )	Huatuo Biotechnology Co., Ltd., Shenzhen, China	HTX2408	Cell line for control experiments
Nuclear protein extraction kit	Beyotime	P0027	Nuclear protein extraction
Seahorse cell energy metabolism analyzer	Agilent,  Beijing, China	Seahorse XF	Used for OCR detection
TCF/LEF1 luciferase reporter kit	Biolab Technology Co., Ltd, Beijing, China		Detection of the activity of Wnt/β-catenin signaling pathway
TUNEL detection kit	Beyotime	C1088	Detection of apoptosis in tumor tissues
Wnt inhibitor (LGK974)	Selleck.cn	S7143	Specific Wnt inhibitor