Hypericin photoactivation induces triple-negative breast cancer cells pyroptosis by targeting the ROS/CALR/Caspase-3/GSDME pathway

• Hypericin photoactivation induces pyroptosis of TNBC cells. • Hypericin photoactivation induced pyroptosis is dependent on the caspase-3-mediated cleavage of GSDME. • Endoplasmic reticulum stress is involved in hypericin photoactivation induced pyroptosis in TNBC cells. • CALR is a potential target of hypericin and induces pyroptosis in TNBC cells after photodynamic therapy. • Hypericin photoactivation induces TNBC cells pyroptosis by enhancing ROS production and causing excessive intracellular Ca 2+ elevation. Hypericin (HP), a natural photosensitizer, has demonstrated great efficacy in photodynamic therapy (PDT) for cancer treatment. In addition to the induction of apoptosis and necrosis through reactive oxygen species (ROS) generation, the therapeutic mechanisms and targets of PDT-HP remain unknown. To investigate the direct targets and mechanisms of action of photoactivated hypericin in the inhibition of triple-negative breast cancer (TNBC). Cell pyroptosis was examined via LDH release, SYTOX Green staining, and ELISA. RNA sequencing, network pharmacology, drug affinity target stability (DARTS)-tandem mass spectrometry (MS/MS), and molecular docking were employed to identify drug targets. Furthermore, immunoblotting and flow cytometry were utilized to elucidate the mechanisms of drug action. Our research revealed that PDT-HP can induce pyroptosis in TNBC cells. Further investigation revealed that PDT-HP induces endoplasmic reticulum stress, activating Caspase-3 and gasdermin E (GSDME) to trigger TNBC cell pyroptosis. RNA-seq, network pharmacology, and DARTS-MS/MS proteomic analyses revealed that the endoplasmic reticulum protein calreticulin (CALR) is a potential HP target and that interfering with CALR inhibited PDT-HP-induced pyroptosis. During PDT-HP treatment, the interaction between CALR and SERCA2 inactivates SERCA2, increasing the susceptibility of cells to increased intracellular Ca 2+ levels under oxidative stress. This triggered endoplasmic reticulum stress and activated Caspase3, which further cleaved GSDME, releasing GSDME-N and ultimately leading to pyroptosis in TNBC cells. In this study, we provide insight into the antitumor mechanism by examining the pharmacological mechanism by which PDT-HP regulates TNBC cell pyroptosis via the ROS/CALR/Caspase-3/GSDME signaling axis.