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Cellular Stress Response Induced by 5-Fluorouracil Potentiates the Efficacy of Oncolytic Herpes Viral Gene Therapy in the Treatment of Pancreas Cancer
David P. Eisenberg, Prasad S. Adusumilli, Zhenkun Yu, Mei-Ki Chan, Karen Hendershott, Ting-Chao Chou, Yuman Fong, Memorial Sloan-Kettering Cancer Center, New York, NY

BACKGROUND:
Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that selectively infect and lyse a wide variety of human cancer cells. NV1066 is an oncolytic HSV strain attenuated via single deletions in the viral replicative genes gamma-1 34.5, ICP0 and ICP4. The protein product of the deleted gamma-1 34.5 gene is highly homologous with GADD34, a host cell DNA repair protein induced by cellular stress such as chemotherapy exposure. We hypothesize that 5-fluorouracil (5-FU) potentiates the efficacy of oncolytic HSV against pancreas cancer cell lines by inducing upregulation of GADD34.

METHODS:
The human pancreatic cancer cell lines Hs 700T, MIA PaCa-2 and PANC-1 were treated in vitro with NV1066 at multiplicities of infection (MOI; ratio of the number of viral particles per tumor cell) of 0.025, 0.05 and 0.1 with or without 5-fluorouracil (1-10 uM). Cytotoxicity was measured using a lactate dehydrogenase release colorimetric assay. Synergistic efficacy was determined by the combination index and isobologram methods of Chou and Talalay. GADD34 mRNA expression was quantified using real-time RT-PCR and viral replication was quantified with a one-step viral growth curve using real-time PCR for the viral gene ICP0.

RESULTS:
Six days after combination therapy, 76% of Hs 700T cells were killed compared to 43% with NV1066 infection alone (MOI 0.1) or 1% with 5-FU alone (2 uM) (p<0.01). Combination index analysis confirmed strong synergism between both agents at all viral and drug combinations tested (LD10 LD90) in the three cell lines. Isobologram analysis demonstrated up to 6-fold and 78-fold dose reductions for NV1066 and 5-FU, respectively, without compromising cell kill. Combination treatment with 5-FU increased GADD34 mRNA expression 2.3-fold (p<0.01) and viral replication 2.2-fold (p<0.01).

CONCLUSIONS:
5-fluorouracil-induced stress response potentiates the efficacy of NV1066 in the treatment of pancreas cancer cell lines resulting in synergistic cytotoxicity. This data provides the cellular basis for the clinical investigation of combined oncolytic herpes virus therapy and chemotherapy in the treatment of pancreatic cancer to achieve maximum efficacy while minimizing dosage and toxicity.


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