Conceived and designed the experiments: ZY RW JH YJ. Performed the experiments: ZY RW. Analyzed the data: ZY RW YJ. Contributed reagents/materials/analysis tools: SX LX JD. Wrote the paper: ZY YJ.
Current address: Guangdong Food and Drug Vocation College, Guangzhou, China
The authors have declared that no competing interests exist.
β-Elemene is an active component of the herb medicine
β-Elemene is one of the active components in the essential oil of
Based on these previous observations, five novel β-elemene derivatives with substitution of one different piperazinyl group, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl )-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The abilities of these compounds to inhibit cell growth and to induce apoptosis as well as their mechanisms of apoptosis induction were investigated in several human leukemia cell lines. All of the five compounds inhibited cell growth with IG50s less than 10 µM. Compounds with a secondary amino moiety (DX1, DX2 and DX5) were more potent than compounds without a secondary amino moiety (DX3 and DX4) in inducing apoptosis. Mechanism studies of apoptosis induction revealed that both the mitochondrial- and the death receptor-mediated apoptotic pathways were involved. The mitochondrial apoptotic pathway is activated due to cleavage of Bid by activated caspase-8 and by the production of reactive oxygen species (ROS). The role of ROS in the apoptosis induction by these compounds was investigated using antioxidants and a H2O2-resistant cell line. The activation of caspase-8 was investigated by assessing levels of the death receptors and the cellular FLICE-inhibitory protein (c-FLIP). Jurkat cells deficient of Fas-associated death domain protein (FADD) and caspase-8 were used to evaluate the role of caspase-8 activation in the apoptosis induction due to these compounds. Our data suggest that these novel β-elemene derivatives induce apoptosis through both the death receptor- and the mitochondrial-mediated apoptotic pathways due to down-regulation of c-FLIP protein and the production of ROS, respectively.
DX1-DX5 were synthesized using similar methods to those that we reported previously
HL-60, NB4 and K562 cells were cultured in RPMI-1640 medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 1 mmol/L
Cells were seeded at 1×105 cells/mL and incubated with various concentrations of β-elemene piperazine derivatives for 72 h. Total cell number was determined with the aid of a hemocytometer and cell viability was estimated by trypan blue exclusion
Two hundred µL of HL-60 cells at a density of 1×105/ml containing various concentrations of DX1 were plated in each well of 96-well plates. The cells were cultured for 12 and 24 h at 37°C. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (50 µl of 2 mg/ml) was added per well and the cultures were continued for an additional 4 h. The medium was aspirated after centrifugation at 1000 RPM for 10 min, the cells were dissolved in 100 µl DMSO, and the optical density (OD) at 570 nm was determined in each well with a 96-well plate reader. The cytotoxicity was calculated as ODt/ODc×100%. ODc represents the OD of the control group and ODt represents the OD of the treated group.
Apoptotic cells were determined by morphologic observation, fluorescence-activated cell sorting (FACS) analysis after staining with PI, and Annexin V/PI.
DNA fragmentation was quantified as described previously
Intracellular H2O2 was monitored by flow cytometry after staining with DCFH-DA. In the present study, cells were labeled with 5 µM DCFH-DA for 1 h and then treated with or without β-elemene piperazine derivatives at 37°C for various times. After washing with phosphate buffer saline (PBS), cells were analyzed by flow cytometry with excitation and emission wavelengths of 495 and 525 nm, respectively. Cells treated with 100 µM H2O2 for 1 h were used as a positive control
MMP was assessed by the retention of Rh123, a membrane-permeable fluorescent cationic dye that is selectively taken up by mitochondria. Its fluorescence intensity is proportional to MMP levels. Cells treated with β-elemene piperazine derivatives for various times were collected and incubated with 0.3 µg/mL Rh123 in the dark for 20 min at room temperature. After washing with PBS, the cells were analyzed by flow cytometry with excitation and emission wavelengths of 495 and 535 nm, respectively.
Protein extracts (50 µg) prepared with RIPA lysis buffer [50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 100 µmol/L leupeptin, and 2 µg/mL aprotinin, PH 8.0] were separated on an 8% or 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were stained with 0.2% Ponceau S red to assure equal protein loading and transfer. After blocking with 5% nonfat milk, the membranes were incubated with a specific antibody overnight at 4°C. Immunocomplexes were visualized by ECL Western Blotting Detection reagents (Amersahm Biosciences, UK). Protein contents in the lysate were determined by the Bradford protein binding assay
FLIPS/L siRNA (sc-35388) and a negative control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. siRNA was transfected into the K562 cells by electroporation (Amaxa, Gaithersburg, MD) following the manufacturer's instructions. Briefly, 2×106 cells were electroporated in 100 µL nucleofector solution (Amaxa Reagent V) containing 30 pmol of each siRNA using the preselected Amaxa Program T-016. siRNA transfected cells were plated in a 6-well plate with 2 mL supplemented RPMI-1640 medium with 10% FBS for 15 h and subsequently further treated with or without 10 µM DX1 for 24 h. Cells treated with or without DX1 were harvested for Western blotting analysis.
The Student's t-test (Microsoft Excel, Microsoft Corporation, Seattle, WA) was performed to determine the significance between groups. A
HL-60 cells were treated with various concentrations of β-elemene piperazine derivatives for 4 days. The levels of cell numbers and viable cells were determined. The concentrations which inhibited 50% of cell growth (IG50) and killed half of the cells (IC50) were calculated. DX1, DX2 and DX5 were more potent than DX3 and DX4 in inhibiting cell growth and in inducing cytotoxicity (
(
To determine whether the cytotoxicity of DX1 is due to induction of apoptosis, apoptotic cells were determined based on morphologic examination after staining with AO and EB in HL-60 cells. DX1 induced apoptosis in a time- and dose-dependent pattern (
(
H2O2 production was measured in HL-60 cells after DX1 treatment using a H2O2-sensitive fluorescent probe, DCFH-DA. The effect of DX1 on MMP was determined by flow cytometry using the cationic dye Rh123. DX1 induced a dose-dependent production of H2O2 and a decrease in MMP (
To evaluate the role of ROS accumulation in DX1-induced apoptosis, we investigated the effects of antioxidants NAC and CAT on DX1-induced apoptosis and on the decrease in MMP. Pretreatment with antioxidants NAC and CAT prevented DX1-induced H2O2 accumulation (
(
To explore the apoptotic machinery, the protein levels of Bid, caspase-3, caspase-8, caspase-9, CD95, CD95L, c-FLIP, DR4, DR5, PARP and TRAIL were determined in HL-60 cells after treatment with several concentrations of DX1 using Western blotting analyses. DX1 treatment induced a dose-dependent reduction in the levels of pro-caspase-3, pro-caspase-8, Bid and c-FLIP (
(
It has been shown that c-FLIP is degraded by a proteasome-mediated pathway and that the proteasome inhibitor MG-132 blocked c-FLIP degradation in cells treated with several agents
(
The abilities of DX-1 to induce apoptosis and to downregulate c-FLIP were further investigated in three additional leukemia cell lines. NB4 cells were as sensitive as HL-60 cells to DX1-induced apoptosis (
(
The apoptosis induction abilities of DX2, DX3, DX4 and DX5 were compared to that of DX1 in HL-60 cells. DX2 and DX5 had similar activities as that of DX1 in apoptosis induction. DX1, DX2 and DX5 at 12 µM induced more than 80% of HL-60 cells to undergo apoptosis after 10 h of treatment (
(
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
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
The death receptor-mediated pathway can also lead to decreases in MMP through cleavage of BID due to activated caspase-8
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
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.