The molecular mechanisms and gene expression profiling for shikonin-induced apoptotic and necroptotic cell death in U937 cells

• Shikonin (SHK) induced apoptosis and necroptosis were studied in U937 cells.
• Intracellular oxidative stress play important roles in SHK-induced cell death.
• The effect of SHK on gene expression was studied using GeneChip® analysis.
• ATF3 and DDIT3 were identified in apoptosis induced by low-dose of SHK.
• TNF was identified in necroptosis induced by high-dose of SHK.

Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1 μM after 6 h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10 μM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1 μM SHK-induced apoptosis and converted 10 μM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1 μM and 85 genes by 10 μM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1 μM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10 μM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.