oa Embelin-Mediated Apoptosis in Leukemic Cells via Generation of Reactive Oxygen Species

Background

The X-linked inhibitor of apoptosis (XIAP) is a promising molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Embelin, identified primarily from the Embelica ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity.

Material and Methods

Reagents

Embelin was purchased from Tocris (Cambridge, MA). TRAIL was purchased from Alexis Corporation (Lausen, Switzerland). Antibodies against Caspase 3, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, Propidium Iodide Staining Solution, Annexin V Binding Buffer, Mitochondrial Membrane Potential Detection (JC-1) Kit, Stain Buffer (FBS), Annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, Rabbit Anti- Active Caspase-3- BV605 antibody and PARP Cleaved Form-AF700 antibody were obtained from BD Biosciences (NJ, USA). CellROX Deep Red Reagent was obtained from Molecular Probes, Life Technologies (CA, USA). (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) solution (MTT) powder, DAPI, and N-Acetyl-L-Cysteine were obtained from Sigma-Aldrich (MO, USA). Apoptotic DNA ladder Kit was procured from Thermo fisher Scientific (USA).

Methodology

We used following assays and methods for this study.

Cell culture

Leukemic cell lines K562 and U937 leukemic cells were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin at 370C in humidified atmosphere containing 5% CO2. All experiments were conducted in 5% serum.

Cell viability

Experiments were performed following treatment with various doses of embelin with and without pre-treatment with NAC for 24 hours using MTT assay.

ROS Production

CellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The cell-permeable reagent is non-fluorescent or very weakly fluorescent while in a reduced state and upon oxidation exhibit strong fluorogenic signal. The signals from CellROX Deep Red Reagent are localized in the cytoplasm and measured by flow cytometry (Excitation 640 nm/Emission 665 nm). K562 cells were treated with embelin for indicated time periods and finally analyzed by flow cytometry.

Apoptosis

Apoptosis was measured using annexinV-FITC/PI staining and analyzed by flow cytometry. Cells were treated with embelin in the presence and absence of NAC for 24 hours. Following treatment, cells were harvested, washed with PBS and stained with annexin V-FITC/PI for 20 minutes at room temperature and apoptosis was measured by flow cytometry.

Western blot

Following treatment with embelin and NAC for 24 hours, cells were lysed and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies.

Results

The results from our study showed that Embelin causes a dose dependent inhibition of cell proliferation in K562 and U937 leukemic cells. Anti-proliferative activity of Embelin correlated with induction of apoptosis. In addition, Embelin treatment of K562 cells decreased the constitutive phosphorylation/activation of AKT followed by the upregulation of proapototic protein Bax. Embelin also induced loss of mitochondrial membrane potential, as determined by JC1 staining, with subsequent activation of caspase-3 and polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. Pretreatment of K562 cells with N-acetyl-L-cystein, a scavenger of reactive oxygen species (ROS) prevented Embelin mediated apoptotic effects. Embelin also suppressed K562 derived progenitor colony formation, suggesting its antileukemic effect. Finally our data also showed that co-treatment of subtoxic doses of Embelin and TRAIL potentiated anticancer activity in leukemic cells.

Conclusion

Altogether, these findings suggest that Embelin causes inhibition of cell proliferation and induction of apoptosis via generation of ROS in leukemic cells, which raises the possibility that Embelin alone or in combination of chemotherapeutic agents may have a future therapeutic role in leukemia and possibly other malignancies with up-regulated XIAP pathway.

Keywords

Apoptosis, CML, ROS