Panoply™ Human NOTCH3 Knockdown Stable Cell Line

Ferroptosis is a type of programmed cell death known to be associated with the development and progression of certain cancers. The Notch3 signaling pathway has been reported to be involved in the tumorigenesis of non-small cell lung cancer (NSCLC) and to regulate iron metabolism, lipid synthesis, and oxidative stress in certain tissues. However, whether the Notch3 signaling pathway regulates ferroptosis remains unclear. Here, researchers found that the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 can protect cells from Notch3 knockdown-induced cell death. Notch3 knockdown initiates ferroptosis in NSCLC cells by increasing reactive oxygen species (ROS) levels, lipid peroxidation, and Fe2+ levels, accompanied by downregulation of glutathione peroxidase 4 (GPX4) and peroxidase 6 (PRDX6). Conversely, overexpression of the Notch3 intracellular domain inhibits erastin-induced ferroptosis, and MJ33 synergistically enhances this inhibitory effect in H1299 cells by reducing ROS levels and lipid peroxidation, accompanied by upregulation of GPX4 and PRDX6. Furthermore, Notch3 knockdown reduced tumorigenesis in vivo by downregulating GPX4 and PRDX6. In summary, these studies suggest that Notch3 is a potential negative regulator of ferroptosis in NSCLC.

Here, researchers treated Notch3 knockdown cells and control cells with the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1, respectively. After 48 hours of treatment, the viability of Notch3 knockdown cells was affected, but the viability of control cells was not affected (Figure 1A-C). Compared with untreated cells, fewer Notch3 knockdown cells treated with ferroptosis inhibitors died, indicating that ferroptosis is involved in Notch3 knockdown-induced cell death (Figure 1A-C). Increased reactive oxygen species (ROS), iron, and lipid peroxidation are hallmarks and causes of ferroptosis. Here, researchers found that Notch3 knockdown significantly increased intracellular ROS levels (Figure 1D-F). Consistent with this, Notch3 knockdown also significantly increased intracellular iron content and lipid peroxidation levels (Figure 1G,H). Subsequently, researchers investigated the effects of Notch3 knockdown on the expression of GPX4 and PRDX6 proteins. GPX4 inhibits ferroptosis by reducing ROS production. PRDX6, as a dual negative regulator of ferroptosis, scavenges lipid ROS in addition to GPX activity through its iPLA2 activity. As shown in Figure 1I, Notch3 knockdown significantly reduced the expression of GPX4 and PRDX6 in NSCLC cells. In summary, these results indicate that specific inhibition of Notch3 initiates ferroptosis by increasing ROS-induced lipid peroxidation through GPX4 and PRDX6.

Figure 1. Notch3 knockdown induces ferroptosis. (Li Z, et al., 2022)