Isobutyrylshikonin Elicits Reactive Oxygen Species-Mediated Necroptosis, not Apoptosis

Isobutyrylshikonin (IBS), purified from the root of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), exhibits various pharmacological properties. However, the anti-cancer activity of IBS has not been fully elucidated. In the current study, we investigated whether IBS elicits cell death via necroptosis in MCF-7 and MDA-MB-231 cancer cells. Our data showed that IBS induces high propidium iodide (PI)(+) and annexin-V- populations at the early stage of cell death in both cancer cell types, and high PI+ and annexin-V+ cell populations at the late stage, suggesting that IBS promotes necroptosis. Moreover, specific inhibitors of caspases, including a caspase-3 inhibitor, Z-DEVD-FMK, a caspase-8 inhibitor, Z-IETD-FMK, a caspase-9 inhibitor, z-LEHD- FMK, and a pan caspase inhibitor, Z-VAD-FMK, did not downregulate IBS-induced cell death in the cancer cells, indicating that IBS-induced cell death is not apoptotic. Treatment with necrostatin-1 (NS-1), a potent and selective inhibitor of necroptosis, inhibited IBS-mediated cell death, and moreover, attenuated the percentage of cells at the sub-G1 phase after IBS treatment. Fluorescence analysis using 2'7'-Dichlorofluorescin diacetate (DCFDA) showed that IBS cumulatively increased reactive oxygen species (ROS) production, and demonstrated that the ROS inhibitors, N-acetylcysteine (NAC) and glutathione (GSH) completely reversed IBS-induced cell death. Taken together, these results indicate that IBS significantly induces cell death via necroptosis in MCF-7 and MDA-MB-231 cancer cells by increasing ROS generation, suggesting that IBS is a potential candidate for use in apoptosis-resistant cancers.