# Erk1/2 Orchestrates SSPH I‐Induced Oxidative Stress, Mitochondrial Dysfunction and Ferroptosis in Hepatocellular Carcinoma

## TL;DR

This study shows that Erk1/2 plays a central role in how the compound SSPH I causes oxidative stress and cell death in liver cancer.

## Contribution

The study identifies Erk1/2 as the upstream regulator of SSPH I-induced ferroptosis and oxidative stress in hepatocellular carcinoma.

## Key findings

- SSPH I induces ferroptosis and oxidative stress through Erk1/2 activation in HCC cells.
- Erk1/2 inhibition blocks SSPH I's effects on Nrf1/2-HO-1 axis and ferroptosis-related proteins.
- Combining SSPH I with Erk1/2 inhibitors leads to antagonistic anti-tumor effects in xenograft models.

## Abstract

Although Erk1/2 has been linked to oxidative stress regulation in hepatocellular carcinoma (HCC), the interplay among Erk1/2, reactive oxygen species (ROS), and iron metabolism remains poorly characterised. The steroidal saponin SSPH I, a recognised ferroptosis inducer, exerts dual pharmacological effects via Erk1/2 and ROS‐dependent pathways. This study aimed to investigate the regulatory mechanisms of Erk1/2 in ferroptosis and oxidative stress and analyse their feedback regulatory effects on Erk1/2 in HCC using SSPH I as a pharmacological probe, and further elucidate the anti‐HCC effects and mechanisms of SSPH I in vitro and in vivo. Mechanistic studies utilised three inhibitors: U0126 (Erk1/2 phosphorylation inhibitor), Ferrostatin‐1 (ferroptosis inhibitor), and N‐acetyl cysteine (ROS scavenger), combined with SSPH I to delineate its effects on cell viability, mitochondrial dynamics, ferroptosis induction and oxidative stress. Mechanistically, SSPH I disrupted mitochondrial function and suppressed HCC cell survival through iron accumulation and ROS generation, while concurrently activating Erk1/2 signalling. Pharmacological inhibition of ROS or iron pathways partially attenuated SSPH I‐induced ferroptosis and ROS generation, but failed to abrogate these effects. Erk1/2 inhibition completely abolished SSPH I‐mediated regulation of the Nrf1/2‐HO‐1 axis and ferroptosis‐related protein expression in cellular and animal models, identifying Erk1/2 as the upstream regulatory node. Notably, while both SSPH I and U0126 monotherapies inhibited xenograft growth, their combined use resulted in antagonistic effects. These findings establish Erk1/2 activation as the central molecular mechanism orchestrating SSPH I‐driven oxidative stress amplification, mitochondrial dysfunction and ferroptosis execution in HCC.