Targeting antiapoptotic Bcl-2 family members with cell-permeable BH3 peptides induces apoptosis signaling and death in head and neck squamous cell carcinoma cells.
Head and neck squamous cell carcinomas (HNSCC) are frequently characterized by chemotherapy and radiation resistance, and by overexpression of Bcl-XL, an antiapoptotic member of the Bcl-2 protein family. In this report we examined whether cell-permeable peptides derived from the BH3 domains of proapoptotic Bax, Bad, or Bak could be used to target Bcl-XL and/or Bcl-2 in HNSCC cells, and induce apoptotic death in these cells. To render the peptides cell permeable, Antennapedia (Ant) or polyarginine (R8) peptide transduction domains were fused to the amino termini. Fluorescence microscopy of peptide-treated HNSCC cells revealed that the BH3 peptides colocalized with mitochondria, the site of Bcl-XL and Bcl-2 expression. By contrast, a mutant peptide (BaxE BH3) which cannot bind Bcl-XL or Bcl-2 was diffusely localized throughout the cytoplasm. Treatment of three HNSCC cell lines (1483, UM-22A, UM-22B) with the wild-type BH3 peptides resulted in loss of viability and induction of apoptosis, as assessed by MTS assays and annexin V staining. In general, Ant-conjugated peptides were more potent than R8-conjugated peptides, and Bad BH3 peptide was typically more potent than Bax BH3 or Bak BH3. Treatment of purified HNSCC mitochondria with BH3 peptides resulted in robust release of cytochrome c. Thus, the relative apoptosis resistance of HNSCC cells is not due to a deficit in this step of the intrinsic, mitochondrial-mediated apoptosis pathway. We conclude that cell-permeable BH3 peptides can be used to target Bcl-XL and/or Bcl-2 in HNSCC, and targeting of these proteins may have therapeutic value in the treatment of this disease.
Li
Rongxiu
R
Department of Medicine, University of Pittsburgh School of Medicine, and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213-1863, USA.
Boehm
Amanda L
AL
Miranda
Michelle B
MB
Shangary
Sanjeev
S
Grandis
Jennifer R
JR
Johnson
Daniel E
DE
D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D005434:Flow Cytometry; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
D051943:Antennapedia Homeodomain Protein; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; C015462:polyarginine