Nec-1s

Human oral squamous cell carcinoma (OSCC) represents a significant global health challenge, with conventional treatments showing limited efficacy in improving patient survival rates. To investigate the therapeutic potential of acetylshikonin on OSCC, we conducted comprehensive analyses including cell viability assays, flow cytometry, and molecular pathway investigations. Our findings demonstrate that acetylshikonin significantly inhibits OSCC cell proliferation with IC50 values of 3.81 μM and 5.87 μM in HSC3 and SCC4 cells respectively. Flow cytometry analysis revealed that acetylshikonin treatment significantly increased reactive oxygen species (ROS) production and decreased mitochondrial membrane potential in OSCC cells. Additionally, Western blot analysis showed enhanced phosphorylation of RIPK1, RIPK3, and MLKL proteins, indicating activation of the necroptotic pathway. The critical role of necroptosis was further confirmed using specific inhibitors (GSK872, Necrostatin-1, and 7-CL-O Nec-1), which significantly attenuated acetylshikonin-induced cell death. Transmission electron microscopy revealed distinct ultrastructural changes in cellular organelles, while decreased GPX4 expression suggested potential cross-activation of ferroptotic pathways. These data demonstrate that acetylshikonin suppresses OSCC growth through selective activation of oxidative stress-mediated necroptosis and mitochondrial dysfunction, identifying it as a promising natural compound for OSCC therapy through its ability to activate alternative cell death pathways and overcome traditional therapy limitations.

Due to its critical role in inflammation and necroptotic cell death, RIPK1 has been considered a prominent therapeutic drug target for managing a wide variety of diseases, including sepsis. Therefore, we aimed to investigate whether the RIPK1-driven necroptotic pathway contributes to the nitrosative stress-mediated cardiorenal inflammatory necroptotic injury and mortality using RIPK1 inhibitor, Nec-1s, in the murine sepsis model induced by LPS

Experiments were performed using mice injected intraperitoneally with DMSO or Nec-1s with saline and/or LPS. Following euthanasia and 6 hours after the injection of these agents, arteriovenous blood samples, hearts, and kidneys of the animals were collected. Serum MPO, iNOS, CKMB, creatinine, and HMGB1 levels were measured by ELISA. Associated proteins were measured by immunoblotting. H&E staining was used to evaluate histopathological changes in the tissues. In the mortality studies, the mice were monitored every 6 hours for mortality up to 96 hours after saline, LPS, DMSO, and/or Nec-1s injection.

In the LPS-injected mice, a rise in serum MPO, iNOS, CK-MB, creatinine, and HMGB1 levels was associated with the enhanced expression/activity of RIPK1/RIPK3/MLKL necrosome, HMGB1, iNOS, nitrotyrosine, gp91phox, and p47phox, in addition to scores related to histopathological changes in their tissues. Nec-1s attenuated the LPS-induced changes. Mortality rates of 10%, 50%, and 60% were observed at the 24th, 36th, and 48th hours, respectively, in the LPS-treated mice. When endotoxemic mice were treated with Nec-1s, mortality rates were 60%, 90%, and 100% at 18, 30, and 42 hours, respectively.

These findings suggest that RIPK1/RIPK3/MLKL necrosome contributes to not only LPS-induced nitrosative stress-mediated cardiorenal inflammatory necroptotic injury, but also mortality.