Bcl-2 resistant mitochondrial toxicity mediated by the isoquinoline carboxamide PK11195 involves de novo generation of reactive oxygen species

Abstract

SummaryResistance to apoptosis is a major obstacle preventing effective therapy for malignancy. Mitochondria localized anti-death proteins of the Bcl-2 family play a central role in inhibiting apoptosis and therefore present valid targets for novel therapy. The peripheral benzodiazepine receptor (PBR) shares a close physical association with the permeability transition pore complex (PTPC), a pivotal regulator of cell death located at mitochondrial contact sites. In this study we investigated the cytotoxicity of the PBR ligand, PK11195, in the micromolar concentration range. PK11195 induced antioxidant inhibitable collapse of the inner mitochondrial membrane potential (∆Ψ m ) and mitochondrial swelling in HL60 human leukaemia cells, but not in SUDHL4 lymphoma cells (which exhibited a higher level of reduced glutathione and relative tolerance to chemotherapy or pro-oxidant induced ∆Ψ m dissipation). PK11195 induced the production of hydrogen peroxide that was not inhibited by Bcl-2 transfection, nor depletion of mitochondrial DNA. ROS production was however blocked by protonophore, implicating a requirement for ∆Ψ m . Our findings suggest that PK11195-induced cytotoxicity relies upon Bcl-2 resistant generation of oxidative stress; a process only observed at concentrations several orders of magnitude higher that required to saturate its receptor. 1397-1404 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.1788, available online at http://www.idealibrary.com on http://www.bjcancer.com methyl-N-methyl-N-(1-methylpropyl)-isoquinoline carboxamide (PK11195), carbonylcyanide m-chlorophenylhdrazone (CCCP), propidium iodide and all cell culture reagents were purchased from Sigma-Aldrich Ltd, UK. FITC-conjugated mouse antihuman Bcl-2 monoclonal antibody, and the Dako intrastain fixation/permeabilization kit were purchased from Dako, UK. Cell culture and treatmentsHL60, KG1A, K652 cells stably transfected with the Bcl-2 gene (K562 B4) or vector only (K562 N2) (kindly provided by Dr DE Banker and Dr F Applebaum, The Fred Hutchinson Cancer Research Center, USA), and SUDHL4 lymphoma cell lines, were maintained in exponential suspension cultures in RPMI 1640 medium supplemented with 10% fetal calf serum, 5 mM glutamine, 100 µg ml -1 streptomycin and 100 U ml -1 penicillin. Cells were grown in a humidified atmosphere of 5% CO 2 /95% air at 37˚C. PK11195 was dissolved in ethanol at a stock concentration of 8.7 mg ml -1 , and added to cells at a 75 µM final concentration for 4 hours; vehicle alone was also used in medium. To test the effect of an antioxidant on PK11195 activity, cells were treated with 10 mM N-acetylcysteine for 45 minutes prior to treatment with PK11195.Cells were incubated with VP16, or ara-C at 10 µM final concentration for 24 hours, or with the thiol crosslinking prooxidant, diamide, at 100 µM concentration for 24 hours. The protonophore CCCP was used to dissipate ∆Ψ m by treating cells at a concentration of 10 µM for 15 minutes prior to treatment with PK11195. Depletion of mitochondrial DNAKG1A cells ...

Identification of a Peptide Antagonist to the Peripheral-Type Benzodiazepine Receptor That Inhibits Hormone-Stimulated Leydig Cell Steroid Formation

“…Studies using the TAT-STPH-STP peptide on the MDA-MB-231 aggressive human breast cancer cell line showed that the peptide failed to block the inhibitory effect of micromolar concentrations of Ro5-4864 on cell proliferation (data not shown). These results suggest that either this function of micromolar concentrations of highnanomolar-affinity PBR drug ligands is not related to PBR, as previously suggested by Zisterer et al (1998) andFennell et al (2001), or the concentration of the antagonist needed to block the effect of micromolar concentrations of Ro5-4864 is too high to be reached with the culture conditions used.…”

Comparative Anticancer Effects of Flavonoids and Diazepam in Cultured Cancer Cells

“…PK11195 has been reported to generate a reversal action on the chemoresistance in cancer cells, independent of the direct activation of PBR. [25][26][27] In this study, PK11195, a putative chemosensitizer, showed further enhancement of their anticancer effects.…”

Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas

“…This toxicity has been exploited as an effective strategy for treatment of various cancers (14,16,17). Several agents have been used to generate ROS, inside or around cancerous tissues, including D-amino acid oxidase, glucose oxidase, xanthine oxidase, arsenic trioxide, hydrogen peroxide, PK11195, 4-HPR, and dithiophene (18,19).…”