title: A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line.
abstract: The 26S proteasome is a proteolytic enzyme found in both cytoplasm and nucleus. In this study, we examined the differential expression of proteasome inhibitor bortezomib-induced proteins in p53-deficient 4T1 cells. It was found that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24h. Next, we analyzed the expression of intracellular proteins in response to treatment with 100nM bortezomib for 24h by label-free LC-MS/MS. These analyses showed that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The proteins identified by label-free LC-MS/MS were then analyzed by Ingenuity Pathway Analysis (IPA) Tool to determine biological networks affected by inhibition of the 26S proteasome. The analysis results showed that post-translational modifications, protein folding, DNA replication, energy production and nucleic acid metabolism were found to be among the top functions affected by the 26S proteasome inhibition. The biological network analysis indicated that ubiquitin may be the central regulator of the pathways modulated after bortezomib-treatment. Further investigation of the mechanism of the proteins modulated in response to the proteasomal inhibition may lead to the design of more effective and novel therapeutic strategies for cancer.
  
  
  CONCLUSIONS
  Although the proteasome inhibitor bortezomib is approved and used for the treatment of human cancer (multiple myeloma), the mechanism of action is not entirely understood. A number of studies showed that proteasome inhibitors induced apoptosis through upregulation of tumor suppressor protein p53. However, the role of tumor suppressor protein p53 in bortezomib-induced apoptosis is controversial and not well-understood. The tumor suppressor p53 is mutated in at least 50% of human cancers and is strongly induced by proteasomal inhibition. Some also reported that the proteasome inhibitor can induce apoptosis in a p53-independent manner. Also, it is reported that Noxa, a target of p53, is induced in response to proteasomal inhibition in a p53-independent manner. However, we have also previously reported that neither Puma nor Noxa are induced by proteasomal inhibition in p53-null 4T1 breast cancer cells, which is commonly used for in vivo breast cancer tumor models. The current results provided additional targets of proteasome inhibitor bortezomib and may therefore help in understanding the p53-independent mechanism of apoptosis induction by proteasome inhibitors. In addition, the results presented in this current study report for the first time that proteasomal subunit Psmd14, anti-apoptotic GRP78, anti apoptotic protein Card10, Dffb, Traf3 and Trp53bp2 are regulated and overexpressed in response to proteasome inhibitor bortezomib in p53-deficient 4T1 cells. Therefore, novel therapeutic strategies targeting these anti-apoptotic or pro-apoptotic proteins as well as inhibiting the proteasome simultaneously may be more effective against cancer cells. The proteins identified here present new avenues for the development of anti-cancer drugs.
authors.affiliation: Dumlupınar University, Faculty of Medicine, Department of Medical Biology, Kütahya, Turkey. Electronic address: azmiyerlikaya@yahoo.com.
authors.forename: Azmi
authors.initials: A
authors.lastname: Yerlikaya
authors.affiliation: Dumlupınar University, Art and Science Faculty, Department of Biology, Kütahya, Turkey.
authors.forename: Emrah
authors.initials: E
authors.lastname: Okur
authors.affiliation: İstanbul Medipol University, Medical School, Department of Medical Biochemistry, İstanbul, Turkey.
authors.forename: Ahmet Tarık
authors.initials: AT
authors.lastname: Baykal
authors.affiliation: TÜBİTAK, MAM, Genetic Engineering and Biotechnology Department, Gebze, Kocaeli, Turkey.
authors.forename: Ceyda
authors.initials: C
authors.lastname: Acılan
authors.affiliation: İstanbul Medipol University, Vatan Clinic, İstanbul, 34214, Turkey.
authors.forename: Ihsan
authors.initials: I
authors.lastname: Boyacı
authors.affiliation: Department of Medical Biochemistry, Uludağ University, Bursa, Turkey.
authors.forename: Engin
authors.initials: E
authors.lastname: Ulukaya
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001897:Boronic Acids; D000069286:Bortezomib; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis; D000091342:Endoplasmic Reticulum Chaperone BiP; D004734:Energy Metabolism / Q000187:drug effects; D005260:Female; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D011719:Pyrazines; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D004273:DNA, Neoplasm; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D020543:Proteome; D011719:Pyrazines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000069286:Bortezomib; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
keywords: Apoptosis; Bortezomib; Cancer; Proteasome; Proteomics; Ubiquitin