E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells.
Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.
Elliott
Mary Jane
MJ
Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, KY, USA.
Farmer
Michael R
MR
Atienza
Cesar
C
Stilwell
Ariel
A
Dong
Yan Bin
YB
Yang
Hai Liang
HL
Wong
Sandra L
SL
McMasters
Kelly M
KM
D000260:Adenoviruses, Human / Q000235:genetics; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors; D010190:Pancreatic Neoplasms / Q000473:pathology* / Q000628:therapy; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D000077546:Roscovitine; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D000964:Antimetabolites, Antineoplastic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D011687:Purines; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D000077546:Roscovitine; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases; D018844:Cyclin-Dependent Kinases; D005472:Fluorouracil