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This research aims to understand how Aloperine affects cell death mechanisms in colorectal cancer cells through the Nrf2 pathway.

March 22, 2026Open Access

Aloperine Induces Ferroptosis of Colorectal Cancer Cells via the Nrf2 Pathway

Key Points

This research aims to understand how Aloperine affects cell death mechanisms in colorectal cancer cells through the Nrf2 pathway.
Colorectal cancer cell lines HCT116 and SW480 were treated with varying concentrations of Aloperine (ALO).
Cell proliferation was assessed using the CCK-8 assay and apoptosis via flow cytometry.
Lipid peroxidation and GSH levels were measured through oxidation assays and the DTNB method, respectively.
Molecular docking analyzed interactions between ALO and Nrf2, while Western blot quantified apoptosis and ferroptosis-related proteins.
The effects of Nrf2 overexpression on ALO’s impact on cells were evaluated.
ALO treatment reduced cell viability and increased both apoptosis and ferroptosis in a dose-dependent manner.
It elevated levels of lipid peroxidation and intracellular iron while lowering GSH content.
Ferroptosis inhibitors alleviated cell death and apoptosis caused by ALO.
ALO led to increased reactive oxygen species (ROS) and altered apoptosis-related proteins.
Molecular docking showed ALO binds to Nrf2, compromising its expression and downstream protective proteins.

Abstract

Objective: This study investigated the effects of ALO on apoptosis and ferroptosis in colorectal cancer HCT116 and SW480 cells and the underlying mechanisms. Methods: Cells were treated with varying ALO concentrations. CCK-8 assay assessed proliferation. Flow cytometry detected apoptosis. Lipid peroxidation was measured by BODIPY 581/591 C11 dye oxidation and TBA method. GSH content was determined by DTNB method. ROS and intracellular iron levels were assessed using fluorescent probes and iron assays. Molecular docking analyzed ALO-Nrf2 binding. Immunofluorescence detected Nrf2 expression. Western blot quantified apoptosis-related proteins (Bax, Bcl-2) and ferroptosis-related proteins (Nrf2, GPX4, xCT, DMT1). Effects of Nrf2 overexpression on ALO-treated cells were observed. Results: ALO inhibited cell viability and increased apoptosis dose-dependently. It elevated lipid peroxidation and intracellular iron while reducing GSH. Ferroptosis inhibitors DFO and Fer-1 reversed cell death and reduced apoptosis. ALO induced ROS production, upregulated Bax/Caspase3, and downregulated Bcl-2. Molecular docking suggested ALO binds to Nrf2 via hydrogen bonding. Immunofluorescence and Western blot showed ALO suppressed Nrf2, GPX4, xCT, and DMT1 expression concentration-dependently. Nrf2 overexpression significantly attenuated ALO’s inhibitory effects on proliferation and its induction of ferroptosis and apoptosis. Conclusion: ALO suppresses colorectal cancer cell proliferation by inducing apoptosis and ferroptosis via inhibition of the Nrf2 signaling pathway. Plain Language Summary: Colorectal cancer is a leading global health problem, and new therapeutic strategies are urgently needed. This study investigated the inhibitory effects of ALO on colorectal cancer cells and the underlying molecular mechanisms. We treated two colorectal cancer cell lines (HCT116 and SW480) with different concentrations of ALO. ALO inhibited cancer cell proliferation in a dose-dependent manner by inducing two forms of cell death: apoptosis (programmed cell death) and ferroptosis (iron-dependent cell death). ALO increased reactive oxygen species (ROS) and lipid peroxidation, reduced the antioxidant GSH level, and elevated intracellular iron content. At the molecular level, ALO binds to and inhibits the key protein Nrf2, which is critical for cellular stress defense. This inhibition downregulated proteins that protect against ferroptosis. Overexpression of Nrf2 significantly reversed the effects of ALO, confirming its key role in the mechanism. Our findings demonstrate that ALO induces apoptosis and ferroptosis in colorectal cancer cells by suppressing the Nrf2 pathway, suggesting its potential as a promising candidate for colorectal cancer treatment. This study provides a novel strategy for developing more effective anti-cancer therapies. Keywords: colorectal cancer, ALO, ferroptosis, Nrf2 pathway, molecular docking

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An et al. (Sun,) studied this question.

synapsesocial.com/papers/69bf8641f665edcd009e8d61 https://doi.org/https://doi.org/10.2147/ott.s575500

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