oa Bortezomib Mediated Downregulation of F-box Protein, S-phase Kinase-Associated Protein 2 (SKP2) Causes Apoptotic Cell Death in Chronic Myelogenous Leukemia Cells

Background

Chronic myeloid leukemia is characterized by the reciprocal chromosomal translocation t(9;22)(q34;q11), leading to the formation of the Philadelphia chromosome. This encodes the constitutively active Bcr-Abl tyrosine kinase, which profoundly affects proliferation, apoptosis, and cell adhesion signaling pathways. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance. Therefore, there is an urgent need for more potent and safer therapies against CML cells for the efficient management of CML.Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as bortezomib, a proteasome inhibitor that has been approved by the food and Drug Administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML.

Material and Methods

Reagents

Bortezomib and antibodies against caspase-3, caspase-9, PARP, p27, GAPDH were obtained from Santa Cruz Biotechnology Inc (USA). Antibodies against SKP2 and caspase-8 were obtained from Cell Signaling Technology, Inc USA) Arsenic trioxide (As2O3) and cyclohexamide were purchased from Sigma. Chronic myelogenous leukemia cells (CML) cell lines: K562, LAMA and AR230 cell lines were cultured in RPMI 1640 medium supplemented with 10? (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml streptomycin at 37°C in an humidified atmosphere containing 5? CO2. Proliferation assays: 104 cells were incubated in triplicate in a 96-well plate in the presence or absence of indicated test doses of Bortezomib in a final volume of 0.20 ml for 24 hour. The ability of Bortezomib to suppress cell growth was determined by MTT cell proliferation assays.

Apoptosis

CML cell lines were treated with bortezomib in different conditions as indicated in each experiment for 24 hours. Apoptosis was determined by flow cytometry using Annexin V (Molecular probes, Eugene, OR), PI staining for cell cycle analysis and DNA laddering using an apoptotic kit (Roche, Indianapolis, IN).

JC1 staining

1 × 106 cells were treated with different doses of bortezomib for 24 hours and stained with 10 mMol JC1 (Alexis corp., San Diego, CA) and measured by flow cytometry.

Western blot

Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies. Cytochrome c release from mitochondria: 20 μg of proteins from the cytosolic fraction were analyzed by immunoblotting with a specific antibody.

Results

Our data showed that proteasome inhibitor bortezomib decreased cell viability as well as induced apoptosis in a dose-dependent manner in a panel of CML cell lines. S-phase kinase-associated protein 2 (SKP2) is an F-box protein that targets cell cycle regulators including cyclin-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation. SKP2 is overexpressed in CML cells, and Bortezomib treatment of these cells resulted in down-regulation of SKP2 and stabilization of p27Kip1. Furthermore, bortezomib-treatment of CML cells led to the loss of mitochondrial membrane potential with the subsequent release of cytochrome c from mitochondria into the cytosol. Cytochrome c release caused activation of caspase-3 followed by polyadenosin-5-diphosphate-ribose polymerase (PARP) cleavage. Finally, we assessed effects on leukemic progenitors (CFU-L) using clonogenic assays in methylcellulose. The treatment of CML cells with bortezomib resulted in inhibition of CFU-L colony growth of leukemic precursors in the CML cell lines. We also provide evidence that co-treatment of chronic CML cells with bortezomib and arsenic trioxide (As2O3) potentiated the inhibition of cell viability. In addition clonogenic assays in methylcellulose demonstrate potent suppressive effects of the combination of these agents on primitive leukemic progenitors derived from CML cells.

Conclusion

Altogether, our results suggest that bortezomib-mediated downregulation SKP2-induced efficient apoptosis in CML cells and exhibited antileukemic effects. Furthermore, co-treatment of CML cells with bortezomib and As2O3 potentiated anti-cancer effects. These data suggests that proteasome-ubiquitin pathway may be a potential target for therapeutic intervention for treatment of CML.

Keywords

Proteasome Pathway, CML, Apoptosis