Evaluation of the caspases activity in shikonin treatment.

<p>(A) Hs683 and U87MG cells were treated with 2 μM of shikonin (shi) or an apoptosis positive agent, doxorubicin (doxo) for 6 h. After drugs treatment, the cells were washed once with PBS, detached by trypsinization, and collected by centrifugation. Aliquot 1×10⁶ cells were suspended in a DMEM medium, and then various homogeneous substrate reagents, Z-DEVD-R110 for caspase 3/7, FITC-IETD-FMK for caspase 8, and FITC-LEHD-FMK for caspase 9, were added to the cells, maintaining a 1∶1 ratio of reagent to cell solution. After 1 h of incubation at 37 °C, the cells were washed once with PBS, collected by centrifugation, and suspended in PBS. The substrate cleavage to release free R110 or FITC fluorescence intensity in cells was analyzed using a Becton–Dickinson FACS-Calibur flow cytometer. The values shown are mean (SD) (n = 5–8 of individual experiments). Significant differences from the untreated group (un) are P<0.05 (*). (B) Hs683 and U87MG cells were treated with 2 μM shikonin (Shi) alone for 24 h or pretreated with 100 μM of Z-VAD-FMK (a cell-permeant pan caspase inhibitor), Z-DEVD-FMK (a cell-permeable, irreversible caspase-3 inhibitor), Z-IETD-FMK (a cell-permeable, irreversible caspase-8 inhibitor) or Z-LEHD-FMK (a cell-permeable, irreversible caspase-9 inhibitor) for 1 h, followed by 2 μM shikonin treatment for 24 h. All samples were subsequently processed for cell cycle analysis to evaluate the percentage of apoptosis subG1 phase. The value shown are mean (SD) (n = 5–8 of individual experiments). Significant differences from the shikonin alone groups are P<0.05 (*).