PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis

Mol Cancer Ther. 2002 Aug;1(10):841-9.

Abstract

Treatment with the proteasome inhibitor, PS-341 resulted in concentration- and time-dependent effects on Bcl-2 phosphorylation and cleavage in H460 cells that coincided with the PS-341-induced G2-M phase arrest. The observed Bcl-2 cleavage paralleled the degree of PS-341-induced apoptosis but was detected to a similar extent with comparable concentrations of two other proteasome inhibitors (MG-132 and PSI). Calpain inhibitors, ALLM and ALLN, and the caspase inhibitors, Z-VAD and AC-YVAD did not induce BcI-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional Mr 25,000 cleavage fragment of Bcl-2, whereas only a Mr 23,000 fragment was observed with other anticancer agents. The formation of the Mr 25,000 fragment was not prevented by caspase inhibitors unlike the Mr 23,000 fragment, which suggests mediation by a caspase-independent pathway. Cell fractionation studies revealed that the Bcl-2 cleaved fragments localize within membrane structures and was an early event (at approximately 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), beta-catenin, and DNA fragmentation (at approximately 36 h posttreatment). The Mr 23,000 Bcl-2 cleavage product was inhibited by the pan-caspase inhibitor and the inhibitors of capase-3, -8, -9; but the PARP cleavage was prevented only by the pan-caspase and caspase-3 inhibitors, which suggests that the Mr 23,000 Bcl-2 cleavage occurred at both the initiation and execution stages of apoptosis. The inhibition of the ubiquitin/proteasome pathway by PS-341 leads, at an early stage of apoptosis, to Bcl-2 phosphorylation and a unique proteolytic cleavage product, which are associated with G2-M phase arrest and the induction of apoptosis.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Antineoplastic Agents / pharmacology*
  • Apoptosis*
  • Blotting, Western
  • Boronic Acids / pharmacology*
  • Bortezomib
  • Cell Line
  • Cysteine Endopeptidases
  • DNA Fragmentation
  • Flow Cytometry
  • G2 Phase
  • Humans
  • Mitosis
  • Models, Biological
  • Multienzyme Complexes / antagonists & inhibitors*
  • Phosphorylation
  • Protease Inhibitors / pharmacology*
  • Proteasome Endopeptidase Complex
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • Pyrazines / pharmacology*
  • Subcellular Fractions / metabolism
  • Time Factors
  • Tosylphenylalanyl Chloromethyl Ketone / analogs & derivatives*
  • Tosylphenylalanyl Chloromethyl Ketone / pharmacology
  • Tumor Cells, Cultured

Substances

  • Amino Acid Chloromethyl Ketones
  • Antineoplastic Agents
  • Boronic Acids
  • Multienzyme Complexes
  • Protease Inhibitors
  • Proto-Oncogene Proteins c-bcl-2
  • Pyrazines
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • tyrosyl-valyl-alanyl-aspartic acid fluoromethyl ketone
  • Tosylphenylalanyl Chloromethyl Ketone
  • Bortezomib
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex