Monitoring the Activation of Caspases-1/3/4 for Describing the Pyroptosis Pathways of Cancer Cells

Pyroptosis is closely related to inhibiting the occurrence and development of tumors. However, the pyroptosis pathways (PPs) impacted by different stimulants are still unknown. To accurately understand the PP in cancer cells, we designed a multicolor fluorescent nanoprobe (Cas-NP) to monitor the activation of caspases-1/3/4 during pyroptosis. The Cas-NP was prepared by the assembly of three different fluorophores-labeled peptides, specific response to caspases-1/3/4 on Au nanoparticles via the Au-Se bond to in situ monitor caspase-1/3/4 with high selectivity and sensitivity. Moreover, the selenopeptide specific to caspase-4 (Cyanine-5-LEVD-SeH) was synthesized for the first time to overcome the difficulty in commercial synthesis. During the pyroptosis of cancer cells induced by adenosine triphosphate (ATP), only the fluorescence of caspase-1 significantly increases. When the cells are stimulated with lipopolysaccharide (LPS), the fluorescence signals corresponding to caspases-3 and 4 first appear and then the fluorescence of caspase-1 is observed. Furthermore, the inhibitor study indicates that the activated caspase-4 can lead to the activation of caspase-1 after the LPS treatment. We first discovered that caspase-3 is activated during the pyroptosis process stimulated by LPS and further verified the activation sequence of caspases-1/3/4 via visualized fluorescence detection. The study provides an effective tool for understanding complex signaling mechanisms in pyroptosis cells and new ideas to explore useful therapeutic inhibitors based on pyroptosis.