Caspases and ER stress regulates Shikonin induced cell death in prostate cancer cells. (A) Shikonin regulated caspase dependent cell death in DU-145 and PC-3 cells. Cells were preincubated with 50 μM of caspase-3 or 9 inhibitors, for 2 h, CCK-8 assay was performed after 48 h post treatment of Shikonin in these cells. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment induces apoptotic phenotype in prostate cancer cells. DU-145 and PC-3 cells were treated with 2.5 μM for 24 h, and their nuclear morphology was examined using DAPI staining in with or without salubrinal pre-incubated prostate cancer cells. In contrast to the control group, the group treated with Shikonin showed characteristic apoptotic phenotypes including heterogeneous staining, chromatin condensation, and DNA fragmentation. However the group pretreated with Salubrinal significantly reversed the Shikonin induced effect in both cell types. (C) The proposed shikonin induced cell death pathways in prostate cancer cells. Shikonin can directly influences intracellular calcium, and ER stress and activated ROS dependent mitochondrial apoptosis. Alternatively, Shikonin induced ROS may regulated intracellular calcium, and ER stress and cell death involving mitochondria and caspases in prostate cancer cells.