Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells.

Ahmad Iskandarani,Kirti S Prabhu,Muzammil A Khan,Kodappully S Siveen,Ramzi M Mohammad,Michal Kulinski,Roopesh Krishnankutty,Rihab R Nasr,Ajaz A Bhat,Shahab Uddin,Shilpa Kuttikrishnan

doi:10.1186/s12967-016-0823-y

Abstract

BackgroundProteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade®, 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.MethodsCell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose.ResultsTreatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells.ConclusionsAltogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0823-y) contains supplementary material, which is available to authorized users.

Highlights

Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosisrelated proteins
S-phase kinase protein 2 (SKP2) gene silencing using small inference RNA in many cancer cell lines has led to the accumulation of p27Kip1, p21waf1 expression and inhibited cell growth and survival via cell cycle arrest and induction of apoptosis [8,9,10]
Our findings indicate that bortezomib-mediated inhibition of cell proliferation of chronic myelogenous leukemia cells (CML) cell lines is associated with down-regulation of SKP2 with concomitant up-regulation/stabilization of p27Kip1

Summary

Introduction

Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosisrelated proteins. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. We determine whether bortezomib induces cell death/apoptosis in CML. The significance of SKP2 in regulating p27Kip expression has been shown in various human malignancies, including diffuse large cell lymphoma and many solid tumors [19,20,21,22,23]. SKP2 gene silencing using small inference RNA (siRNA) in many cancer cell lines has led to the accumulation of p27Kip, p21waf expression and inhibited cell growth and survival via cell cycle arrest and induction of apoptosis [8,9,10]. Growing number of studies have shown that proteasome inhibitors suppress the growth of many human cancer cells via induction of apoptosis mediated cell cycle arrest/cell death [24,25,26,27]. Several clinical studies are currently ongoing to determine the efficiency of bortezomib for the better management of other types of human cancers [28]

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