Radiation Therapy Potentiates Effective Oncolytic Viral Therapy in the Treatment of Lung Cancer

doi:10.1016/j.athoracsur.2005.01.048

The Annals of Thoracic Surgery

Volume 80, Issue 2, August 2005, Pages 409-417

Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.

https://doi.org/10.1016/j.athoracsur.2005.01.048 Get rights and content

Background

Replication-competent oncolytic herpes simplex viruses with deletion of the γ₁34.5 gene preferentially replicate in and kill malignant cells. The γ₁34.5 gene codes for ICP 34.5, a protein that enhances viral replication, and is homologous to growth arrest and DNA damage protein 34 (GADD34), a radiation-inducible DNA repair gene. We hypothesized that radiation therapy may potentiate efficacy of oncolytic viral therapy by upregulating GADD34 and promoting viral replication.

Methods

The A549 and H1299 lung cancer cell lines were infected with NV1066, an oncolytic herpes simplex virus, at multiplicities of infection (number of viral particles per tumor cell) of 0.1 to 0.5 in vitro with radiation (2 to 10 Gy) or without radiation. Viral replication was determined by plaque assay, cell-to-cell spread was determined by flow cytometry, cell kill was determined by lactate dehydrogenase assay, and GADD34 induction was determined by real-time reverse transcription–polymerase chain reaction and Western blot method. Evidence of synergistic cytotoxicity dependence with GADD34 induction is further confirmed by small inhibitory RNA inhibition of GADD34 expression.

Results

Using both the isobologram method and combination index method of Chou and Talalay, significant synergism was demonstrated between radiation therapy and NV1066 both in vitro and in vivo. As a result of such synergism, a dose reduction for each agent (2- to 6,000-fold) can be accomplished for a wide range of therapeutic effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of small inhibitory RNA directed against GADD34.

Conclusions

These data provide the cellular basis for the clinical investigation of combined use of radiation therapy with oncolytic herpes simplex virus therapy in the treatment of lung cancer to achieve synergistic efficacy while minimizing dosage and toxicity.

Section snippets

Cell Culture

The human non-small-cell lung cancer cell lines A549 (p53⁺), H1299 (p53⁻), and Vero cells (from the African green monkey kidney) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All cells were maintained in appropriate media as recommended by ATCC and were incubated in a humidified incubator supplied with 5% carbon dioxide.

Viruses

NV1066 is a replication-competent attenuated HSV-1 oncolytic virus with deletion of a single copy of the viral gene γ₁34.5. G207 and NV3616 are

In Vitro Cytotoxicity of Radiotherapy and NV1066

Both radiotherapy and NV1066 demonstrate dose-dependent cytotoxicity against A549 and H1299 lung cancer cells. Combination therapy killed more tumor cells than either single agent alone and showed greater efficacy than the expected additive effect by day 7 in both cell lines (p = 0.002). Cytotoxicity derived by lactate dehydrogenase release assay on each day up to day 7 is represented for A549 (Fig 1A) and H1299 (Fig 1B) cells. Synergistic cytotoxicity (p < 0.01) is demonstrated with other γ₁

Comment

The current study demonstrates that radiation-induced GADD34 upregulation greatly enhances replication and antitumor efficacy of a γ₁34.5-deficient HSV oncolytic virus. The genotoxic response to DNA damage by radiation results in complex cellular events that direct DNA repair. A series of five growth arrest and DNA damage-inducible (GADD) genes have been identified in mammalian cells in response to ionizing radiation, medium-depletion, and alkylating agents [17, 18]. As noted in other studies [

Acknowledgment

The authors thank Yuhong She, MD, and Wong Wai, MS, of the Anti-Tumor Core Facility, and Scott Tuorto of the Department of Surgery at Memorial Sloan-Kettering Cancer Center for their assistance with this project. We also thank Brian Horsburgh, PhD, and Medigene, Inc, for constructing and providing us with the NV1066 virus. This project is supported in part by AACR-AstraZeneca Cancer Research and Prevention Foundation fellowship (PSA), grants R01 CA76416 and R01 CA/DK80982 (YF) from the National

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Thoracic Surgery Directors Association AwardRadiation Therapy Potentiates Effective Oncolytic Viral Therapy in the Treatment of Lung Cancer

Background

Methods

Results

Conclusions

Section snippets

Cell Culture

Viruses

In Vitro Cytotoxicity of Radiotherapy and NV1066

Comment

Acknowledgment

Lung Cancer

Int J Radiat Oncol Biol Phys

Surgery

J Thorac Cardiovasc Surg

Surgery

Adv Enzyme Regul

Cancer Facts & Figures

A systematic overview of radiation therapy effects in non-small cell lung cancer

Acta Oncol

Long-term observations of the patterns of failure in patients with unresectable non-oat cell carcinoma of the lung treated with definitive radiotherapy. Report by the Radiation Therapy Oncology Group

Cancer

Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas

Nat Med