Synergistic Effect Of Shikonin And Low-dose Cytarabine On AML Cell Proliferation And Apoptosis

The aim of this study is to determine the synergistic anti-cancer effect of shikonin and low-dose Cytarabine on human acute myeloid leukemia(AML) cell line HL-60 and U937. CCK8 assay was used to measure the growth of HL-60 and U937 cells, showed shikonin and Ara-C blocked their proliferation in a dose- and time-dependent manner. The 50% inhibitory concentration(IC50) of shikonin and Ara-C on HL-60 and U937 cells was determined by CKK8 assay. Cells were exposed to low-dose shikonin and Ara-C which did not influence the survival of AML cells. In this anti-proliferation process, 0.5μM shikonin and 100 nM Ara-C together rendered the cells susceptible to apoptosis, compared with single compound administration, manifested by increased number of Annexin V+/PI- and Annexin V+/PI+ cells. Shikonin was able to activate caspase-3 and cleaved poly(ADP-ribose) polymerase(PARP) in a dose-dependent manner in two cell lines. A combination of shikonin and low-dose Ara-C could activate caspase-3 significantly, compared with either of these two compounds. HL-60 cell cycle was arrested in G1 phase by shikonin and in S phase by Ara-C. U937 cell cycle progression was blocked at the G2 phase and mildly blocked by low-dose Ara-C at the S phase. Shikonin induced Reactive oxygen species generation in a dose-dependent way, which was detected to be generated in abundance when cells were treated with the two drugs together. ROS scavenger NAC, was able to reduce ROS level and inhibited cells apoptosis,indicating that ROS play an essential role in cell death initiated by combined usage of drugs. The application of the NADPH inhibitors apocynin or DPI further confirmed function of NOX in ROS generation and cell death, which were partly diminished. The changes in mitochondrial morphology, dose-dependently decreased in Δ m, were observed in different concentrations of the shikonin treatment group and combined treatment group. NAC completely inhibited the mitochondrial membrane depolarization by depleting ROS, apocynin and DPI could partly block the Δ m reduction. The migratory ability of HL-60 and U937 cells was detected by a transwell migration assay, in which low-dose Ara-C enhanced their migration ability by stimulating CD147 expression. CD147 deficiency casued by shRNA or shikonin could significantly reduce migrated cell numbers. In addition, MAPK pathways were found to involve in shikonin-induced apoptosis, in which ERK phosphorylation was suppressed and JNK and p38 were activated. Therefore shikonin should be considered as a candidate agent for the treatment of human leukemia in combination with Ara-C.