Osthole Induces Apoptosis, Secondary Necrosis and Mitophagy Via NQO1-Mediated ROS Overproduction in HeLa Cells

Background: Osthole is a natural coumarin which has been proved to inhibit growth of cancer cells by inducing cancer cells death, while its mechanism of anticancer remains unclearly. In our study, we found that osthole activated multiple forms of cell death including apoptosis, secondary necrosis and mitophagy in receptor interacting protein kinase (RIP) 3-deficient cervical cancer HeLa cells. Methods: : Cell viability was detected by MTT assay. Cell membrane integrity was detected by LDH release assay and PI staining. Cell apoptosis and necrosis were detected by flow cytometry assay. Reactive oxygen species (ROS) was detected by DCFH-DA staining and mitochondrial membrane potential (MMP) was detected by JC-1 staining using flow cytometry. The expression of proteins was detected by western blotting assay and proteomics. Xenograft tumor model was used to evaluate the effect of osthole in vivo. Results: : Our study showed osthole caused HeLa cells apoptosis and secondary necrosis, which is a phenomenon of the apoptotic cells’ plasma membrane breakdown. And when Hela cells pretreatment with Z-DEVD-FMK, an irreversible caspase-3 inhibitor, not only inhibited osthole-induced apoptosis but also necrosis. Moreover, we found that Z-DEVD-FMK reversed the effect of osthole on the induction of cleaved the N-terminal fragment of GSDME in Hela cells. Furthermore, inhibition of NAD (P) H: quinone oxidoreductase 1 (NQO1) by osthole induced the overproduction of reactive oxygen species (ROS). ROS inhibitor N-Acetyl-L-cysteine (NAC) not only reduced osthole-induced apoptosis, but also reversed its effect on the necrotic induction and the GSDME N-terminal generation. It was shown that osthole decreased mitochondrial membrane potential (MMP) and increased the expression of PTEN-induced putative kinase 1 (PINK1) and Parkin, which indicated that the activation of mitophagy induced by osthole. Meanwhile, as well as apoptosis and secondary necrosis, mitophagy was also restrained by NAC. Conclusions: : In conclusion, all these data suggested that osthole induced apoptosis, secondary necrosis and mitophagy via NQO1-mediated ROS overproduction.