title: Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells.
abstract: BACKGROUND
  Proteasome 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.
  
  
  METHODS
  Cell 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.
  
  
  RESULTS
  Treatment 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.
  
  
  CONCLUSIONS
  Altogether, 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.
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. AIskandarani@hamad.qa.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Iskandarani
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. ajaz.bhatt@gmail.com.
authors.forename: Ajaz A
authors.initials: AA
authors.lastname: Bhat
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. SSivaraman@hamad.qa.
authors.forename: Kodappully S
authors.initials: KS
authors.lastname: Siveen
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. KPrabhu@hamad.qa.
authors.forename: Kirti S
authors.initials: KS
authors.lastname: Prabhu
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. SKuttikrishnan@hamad.qa.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Kuttikrishnan
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. MKhan39@hamad.qa.
authors.forename: Muzammil A
authors.initials: MA
authors.lastname: Khan
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. RKishnankutty@hamad.qa.
authors.forename: Roopesh
authors.initials: R
authors.lastname: Krishnankutty
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. MKulinski@hamad.qa.
authors.forename: Michal
authors.initials: M
authors.lastname: Kulinski
authors.affiliation: Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut, Lebanon. rn03@aub.edu.lb.
authors.forename: Rihab R
authors.initials: RR
authors.lastname: Nasr
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. RMohammad2@hamad.qa.
authors.forename: Ramzi M
authors.initials: RM
authors.lastname: Mohammad
authors.affiliation: Translational Research Institute, Academic Health System, Hamad Medical Corporation, PO Box 3050, Doha, State of Qatar. SKhan34@hamad.qa.
authors.forename: Shahab
authors.initials: S
authors.lastname: Uddin
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D000069286:Bortezomib / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology / Q000473:pathology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D061988:Proteasome Inhibitors; D044786:S-Phase Kinase-Associated Proteins; D057149:Ubiquitinated Proteins; D000069286:Bortezomib; D020169:Caspases; D046988:Proteasome Endopeptidase Complex