title: Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo.
abstract: OBJECTIVE
  Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo.
  
  
  METHODS
  Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects.
  
  
  RESULTS
  Inhibition of the Ca(2+)-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.
authors.affiliation: Department of Medicine, University of Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Room 4963B, Tucson, AZ 85724, USA.
authors.forename: Aluvia M
authors.initials: AM
authors.lastname: Escalante
authors.forename: Ryan T
authors.initials: RT
authors.lastname: McGrath
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Karolak
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dorr
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Lynch
authors.forename: Terry H
authors.initials: TH
authors.lastname: Landowski
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D001897:Boronic Acids / Q000494:pharmacology* / Q000627:therapeutic use; D000069286:Bortezomib; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D017320:HIV Protease Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009101:Multiple Myeloma / Q000473:pathology; D019888:Nelfinavir / Q000494:pharmacology* / Q000627:therapeutic use; D011719:Pyrazines / Q000494:pharmacology* / Q000627:therapeutic use; D034741:RNA, Small Interfering / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D017320:HIV Protease Inhibitors; D011719:Pyrazines; D034741:RNA, Small Interfering; D000069286:Bortezomib; D002154:Calpain; D019888:Nelfinavir