Potential role of caspase-3 and -9 in arsenic trioxide-mediated apoptosis in PCI-1 head and neck cancer cells

Int J Oncol. 2001 Feb;18(2):249-55.

Abstract

Arsenic trioxide (As2O3) has been shown to inhibit the proliferation of hematologic malignant cells. Previously, we reported that As2O3 had an antitumoral effect in head and neck cancer. Here, we investigated the induction of apoptosis and its mechanism in PCI-1 head and neck squamous carcinoma cells, after treatment with As2O3. Treatment with 2 microM of As2O3 caused apoptosis in PCI-1 cells following 3 days of exposure, which was detected by the annexin V-PI and DAPI staining methods. The cell death population was markedly increased, being 88% larger than the As2O3-untreated control cells. To address the mechanism of apoptosis, a Western blot assay was performed, showing that Bax was up-regulated without a change in Bcl-2. Activation of caspase-9 during As2O3-induced apoptosis was substantiated by monitoring the proteolysis of the caspase-9, which was associated with an increase of Apaf-1 and cytochrome c protein. PCI-1 cells rapidly changed the mitochondria membrane potential (DeltaPsim) after addition of As2O3. Furthermore, activation of caspase-3 was demonstrated by monitoring the proteolysis of the caspase-3 and by measuring caspase-3 activity with a fluorogenic substrate, which was associated with the cleavage of poly(ADP-ribose) polymerase. To examine the in vivo effect of As2O3, C3H mouse inoculated with syngenic SCC7 cells was treated by intratumoral injection of As2O3 (300 microg) every day, demonstrating that tumor mass was dramatically reduced on day 4, and revealed induction of apoptosis by TUNEL assay. These results suggest that apoptosis of PCI-1 cells by As2O3 is induced by activation of caspase-3 via cytochrome c, caspase-9 and Apaf-1 complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • Apoptotic Protease-Activating Factor 1
  • Arsenic Trioxide
  • Arsenicals / pharmacology*
  • Arsenicals / therapeutic use
  • Carcinoma, Squamous Cell / drug therapy
  • Carcinoma, Squamous Cell / metabolism*
  • Caspase 3
  • Caspase 9
  • Caspases / drug effects*
  • Caspases / metabolism
  • Head and Neck Neoplasms / drug therapy
  • Head and Neck Neoplasms / metabolism*
  • Humans
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Mice
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Oxides / pharmacology*
  • Oxides / therapeutic use
  • Proteins / drug effects
  • Proteins / metabolism
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / metabolism

Substances

  • APAF1 protein, human
  • Antineoplastic Agents
  • Apaf1 protein, mouse
  • Apoptotic Protease-Activating Factor 1
  • Arsenicals
  • Oxides
  • Proteins
  • CASP3 protein, human
  • CASP9 protein, human
  • Casp3 protein, mouse
  • Casp9 protein, mouse
  • Caspase 3
  • Caspase 9
  • Caspases
  • Arsenic Trioxide