Targeting Bcl-2-mediated cell death as a novel therapy in pancreatic cancer.
BACKGROUND
Bcl-2 is an essential regulator of programmed cell death (PCD). Overexpression of Bcl-2 is common in pancreatic cancer; the high levels have been shown to correlate with resistance to PCD. This resistance is mediated by binding of Bcl-2 via its BH-3 domain to diverse proteins, including the Bax/Bak family members, various protein kinases, and beclin 1, which are involved in regulation of autophagy (type II PCD). Small molecule inhibitors of BH-3-mediated binding of Bcl-2 have been recently developed, although no investigation has been conducted in pancreatic cancer, a malignancy characterized by extreme resistance to PCD.


METHODS
The effect of the Bcl-2 binding inhibitor A-779024 on PCD was assessed by fluorescence activated cell sorting; the effect on Bcl-2 and other PCD-related proteins was analyzed by immunoblotting. Induction of autophagy was determined by fluorescence microscopy using a stably transfected GFP-LC3 construct to visualize autophagosome formation. Co-localization of Bcl-2 with binding partners regulating PCD was examined by immunoprecipitation and confocal immunofluorescent microscopy.


RESULTS
A-779024 induced PCD in a dose- and time-dependent fashion. No change was seen in the protein levels of Bcl-2, Bax, Bcl-XL, or Mcl-1. Contrary to prediction, A-779024 was ineffective at inducing autophagy in these cells. Co-localization studies demonstrated that Bcl-2 was not bound to beclin 1 and, therefore, treatment with A-779024 could not induce release of beclin 1 and initiation of autophagy.


CONCLUSIONS
Disruption of Bcl-2 activity using the small molecule inhibitor A-779024 induces apoptotic but not autophagic PCD. This approach may be a novel therapy, either alone or in combination with other treatments such as chemotherapy or autophagy modulating agents in pancreatic cancer.
Muilenburg
Diego J
DJ
Department of Surgery, Division of Surgical Oncology, University of California, Davis, California, USA.
Coates
Jodi M
JM
Virudachalam
Subbulakshmi
S
Bold
Richard J
RJ
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D045744:Cell Line, Tumor; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D010446:Peptide Fragments / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; C474007:Bax protein (53-86); D000071186:Beclin-1; D008565:Membrane Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2
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