β-Elemene inhibits cell growth only at high concentrations. Previously we found that β-elemene substituted with a tryptophan methyl ester improved its antiproliferative effects and induced apoptosis in leukemia cells through a ROS-mediated pathway 
. In this study, we found that β-elemene substituted with a piperazine was even more effective than β-elemene substituted with a tryptophan methyl ester in inducing apoptosis. These compounds acted through both ROS production and c-FLIP reduction. The IG50 of these β-elemene derivatives substituted with a piperazine was decreased to less than 10 µM (). Based on the cell growth inhibition abilities of DX1 to DX5, the substitution with one (DX1) or two methyl groups (DX2) of piperazine does not evidently influence the antiproliferative and cytotoxic effects of these piperazine derivatives. However, replacement of the hydrogen of the C-4 of piperazine with an ethyl (DX3) or an isopropyl (DX4) decreases the antiproliferative and cytotoxic effects compared to those of DX1, DX2 and DX5 which have a hydrogen at C-4. DX1, DX2 and DX5 induced evident apoptosis at concentrations of 6–10 µM after treatment for 10 h (). DX3 and DX4 induced apoptosis at higher concentrations and more prolonged incubation times (data not shown). These data suggest that the apoptosis induction of these compounds is an important mechanism for their ability to induce cytotoxicity.
DX1 was used to explore the mechanism of apoptosis induction. It is known that death receptor and mitochondrial apoptotic pathways play important roles in apoptosis induction due to chemotherapeutic agents 
. Several agents have been found to induce the mitochondrial-mediated apoptotic pathway through the generation of ROS which decreases the MMP which then leads to activation of caspase-3 
. DX1 increased the production of ROS, decreased MMP levels, and activated caspase-3 in HL-60 cells (). To investigate the role of ROS in DX1-induced apoptosis, antioxidants NAC and CAT were used. Although both NAC and CAT could prevent the ROS production due to DX1 treatment in HL-60 cells, they only had minimal effects in preventing cells from undergoing DX1-induced apoptosis (). Since both NAC and CAT only partially block DX1-induced decrease in MMP, it seems that the decrease in MMP after DX1 treatment, at least in part, is mediated through an ROS-independent pathway (). HP100-1 cells, which are resistant to H2
, are responsive to DX1 treatment (). These data suggest that ROS production only plays a partial role in DX1-induced apoptosis. Thus, the action of DX1 is different from that of N-(b-elemene-13-yl)tryptophan methyl ester which induced apoptosis only by increasing ROS 
The death receptor-mediated pathway can also lead to decreases in MMP through cleavage of BID due to activated caspase-8 
. It has been found that several agents could activate caspase-8 through increasing the levels of death receptors 
. DX1 treatment activated caspase-8 based on the determination of its cleavage and decrease in the levels of Bid (). Therefore, the decrease in MMP levels by DX1 treatment could be partly due to cleaved Bid. Previously we have found that boswellic acids induced apoptosis by activated caspase-8 due to induction of DR4 and DR5 proteins 
. However, the levels of DR4, DR5 and CD95 as well as their ligands CD95L and TRAIL were not increased after DX1 treatment (). Neither CD95L neutralizing antibody NOK-1 nor human recombinant DR5 (TRAIL R2)/Fc chimera protein was able to block DX1-induced apoptosis (). These data suggest that DX1 activates caspase-8 through a pathway independent of the regulation of death receptor levels and their activation. c-FLIP has been found to inhibit the activation of caspase-8. Several other agents have been found to activate caspase-8 by decreasing the levels of c-FLIP 
. The protein levels of both c-FLIPL
were reduced by DX1 treatment in a concentration-dependent pattern that was correlated with the cleavage of both caspase-8 and PARP (). DX1 treatment induced cleavage of caspase-8 and PARP and decreased c-FLIP levels in NB4 and HP100-1 cells, but not in K562 cells (). DX2 and DX5 treatments decreased the levels of c-FLIP and induced the cleavage of caspase-8 and PARP in HL-60 cells (). DX3 and DX4 treatment did not decrease the levels of c-FLIP nor induce the cleavage of caspase-8 and PARP in HL-60 cells (). FADD-deficient and caspase-8-deficient Jurkat subclones were less sensitive to DX1-induced apoptosis (). Silencing c-FLIP augmented DX1-induced apoptosis in K562 cells (). These data suggest the activation of caspase-8 is probably mediated through downregulation of c-FLIP and that activated caspase-8 plays a partial, but pivotal, role in the apoptosis induction by these compounds.
c-FLIP is known to be regulated by a ubiquitin-proteasome mechanism, and several cancer therapeutic agents have been found to induce downregulation of c-FLIP through this mechanism 
. MG-132 has been reported to inhibit the decrease of c-FLIP levels induced by several agents in cancer cells 
. However, it also has been reported that MG-132 decreased c-FLIP levels and enhanced TRAIL-induced apoptosis in prostate cancer cells and chronic lymphocytic leukemia cells 
. The difference of MG-132 action on c-FLIP levels may be cell type- and/or agent-dependent. In HL-60 cells MG-132 alone induced apoptosis and decreased the levels of c-FLIP at a concentration higher than 1 µM (). We examined the effects of DX1 on the levels of c-FLIP and apoptosis induction in the presence and absence of MG-132 at a lower concentration. MG-132, at a concentration of 0.5 µM, weakly decreased c-FLIP levels and augmented DX1 induction of apoptosis and its ability to decrease c-FLIP levels (). Our data indicate that MG-132 enhances the effects of these DX compounds to induce apoptosis by decreasing c-FLIP levels in these leukemia cells. Since the concentrations of MG-132 used here are very low, it is possible that reduction of c-FLIP by MG-132 is independent of its ability of inhibiting proteasome activity. The mechanism of inducing downregulation of c-FLIP levels by DX and MG-132 in leukemia cell lines needs to be further studied.
In summary, the present study reports the apoptotic effects and the mechanisms of action of five novel β-elemene piperazine derivatives. They induce apoptosis through production of ROS and decrease in c-FLIP levels and, thus, activate both death receptor-mediated and mitochondrial-mediated apoptotic pathways.