Real-Time Monitoring of APE1 during Cancer Cell Pyroptosis Induced by Electrical Stimulation with DNA Nanoprobes
Abstract
Apurinic/apyrimidinic endonuclease 1 (APE1) is a pivotal biomarker frequently overexpressed in cancer cells, hence in situ monitoring of its dynamic alterations during cell death is crucial for therapeutic evaluation. Herein, we designed highly sensitive and selective fluorescent nanoprobes based on DNA-functionalized gold nanoparticles (AuNPs) for real-time imaging and monitoring of APE1 activity in living cells. The nanoprobes, composed of APE1-responsive double-stranded DNA containing apurinic/apyrimidinic (AP) sites and conjugated with black hole quencher 2 (BHQ2), are immobilized on the surface of AuNPs. Our results demonstrate that electrical stimulation (ES) selectively induces pyroptosis of HeLa cells, and nearly complete suppression of APE1 activity as ES-induced pyroptosis progressed was revealed via confocal microscopy analyses. The ES treatment leads to a marked accumulation of intracellular reactive oxygen species (ROS) and the resulting oxidative stress causes a pronounced up-regulation of γ-H2AX, indicating severe DNA double-strand breaks. We propose that the ROS-mediated down-regulation of APE1 contributes to impaired base excision repair (BER) and exacerbates genomic instability during ES-induced pyroptosis. Notably, this phenomenon did not occur in normal H8 cells. This study establishes a nanoprobe sensing platform for real-time monitoring of APE1 dynamics and identifies the down-regulation of APE1 as an indicator of effective ES-induced pyroptosis in…
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