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Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia

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Abstract

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.

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Abbreviations

Ad:

Adenovirus

CBT:

Canine breast tumor

CMV:

Cytomegalovirus

dCasp3:

Dog caspase 3

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

DFO:

Deferoxamine mesylate

DMEM:

Dulbecco’s modified Eagle’s medium

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

FBS:

fetal bovine serum

FITC:

fluorescein isothiocyanate

GFP:

Green fluorescent protein

HEK:

Human embryonic kidney

HIF-1:

hypoxia inducible factor-1

IFU:

Infectious unit

JAK-STAT:

Janus kinase signal transducers and activator of transcription

MAPK:

Mitogen activated protein kinase

MDCK:

Madin-Darby canine kidney

MOI:

Multiplicity of infection

ODD:

Oxygen-dependent degradation

PBS:

Phosphate buffered saline

PI3K:

Phosphatidylinositol-3 kinase

RGD:

Arginine-Glycine-Aspartic acid

RT:

Room temperature

TGF:

Transforming growth factor

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Acknowledgments

This study was partially supported by a project grant (Young Scientist Research Training Award) to MO funded by the Azabu University Research Services Division, and a Grant-in-Aid to MO from Japan Society for the Promotion of Science (No. 26450450).

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Authors and Affiliations

  1. Laboratory of Veterinary Immunology, Department of Veterinary Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan

    Mariko Okamoto, Ai Asamura, Ko Tanaka, Takefumi Soeda, Kyo Watanabe & Teruo Ikeda

  2. Laboratory of Biochemistry and Molecular Biology, Graduate School and School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Hiroyuki Mizuguchi

  3. Laboratory of Hepatocyte Regulation, Health and Nutrition, National Institutes of Biomedical Innovation, 7-6-8 Saito, Asagi, Osaka, Ibaraki, 567-0085, Japan

    Hiroyuki Mizuguchi

Authors
  1. Mariko Okamoto
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  2. Ai Asamura
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  3. Ko Tanaka
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  4. Takefumi Soeda
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  6. Hiroyuki Mizuguchi
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  7. Teruo Ikeda
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Corresponding author

Correspondence to Mariko Okamoto.

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None of the authors have any conflict of interests.

Additional information

Mariko Okamoto and Ai Asamura contributed equally to this work.

Electronic supplementary material

Supplementary Fig. 1

Quantitative PCR (qPCR) Total RNA was extracted from CBT cells transduced with AdRGD-hEGFR/dCasp3 or AdRGD-hEGFR/ODD-dCasp3 respectively using Ribozol RNA Extraction Reagent (Amresco, Solon, OH, USA). Single-stranded cDNA was synthesized from total RNA using PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio Inc., Kusatsu, Shiga, Japan). Exogenous gene expression of caspase-3 or ODD-caspase 3 was determined by real-time qPCR with DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions. The selected gene expression was calculated using ribosomal protein (RP) S18 as a normalization control according to the comparative CT method. Exogenous caspase-3 or ODD-caspase 3 expression in CBT cells transduced with hEGFR promoter driven adenovirus vector (a) CBT cells were transduced with AdRGD-hEGFR/dCasp3 or AdRGD-hEGFR/ODD-dCasp3 at 0, 5, 15, or 45 MOI respectively. After 24 h, mRNA expression was assessed by qPCR. Primers used in qPCR to detect exogenous caspase-3 or ODD-caspase 3 expression were shown by blue arrow in schematic representation of Ad-vectors. (b) CBT cells were transduced with AdRGD-hEGFR/dCasp3 or AdRGD-hEGFR/ODD-dCasp3 at 0, 5, 15, or 45 MOI respectively. After 48 h, protein expression was assessed by western blotting (PPTX 59 kb)

Supplementary Fig. 2

Reduction of mitochondrial membrane potential in CBT cells transduced with AdRGD-hEGFR/dCasp3. CBT cells were transduced with AdRGD-hEGFR/dCasp3 at 0 or 45 MOI for 24 h. Mitochondrial membrane potential was verified by flow cytometric analysis. (PPTX 24 kb)

Supplementary Fig. 3

Active caspase 3 expression in CBT cells transduced with AdRGD-hEGFR/dCasp3. CBT cells were transduced with AdRGD-hEGFR/dCasp3 at different MOI values, and active caspase 3 was detected using an antibody against active form of caspase 3 (PPTX 577 kb)

Supplementary Fig. 4

Dominant expression of ODD-caspase-3 under hypoxic conditions. (a) CBT cell were transduced with AdRGD-hEGFR/ODD- dCasp3 at 45 MOI under normoxic (Normoxia) or hypoxic (Hypoxia) conditions. ODD-caspase 3 expression was assessed by western blotting. HIF-1α expression in CBT cells under normoxic (Normoxia) or hypoxic (Hypoxia) conditions was also assessed by western blotting. (b) CBT cell were transduced with AdRGD-hEGFR/ODD- dCasp3 under the same conditions as in (A), and active caspase 3 was detected using an antibody against active form of caspase 3. (c) CBT cell were transduced with AdRGD-hEGFR/ODD- dCasp3 under the same conditions as in (A), mitochondrial membrane potential was verified by flow cytometric analysis. (PPTX 130 kb)

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Okamoto, M., Asamura, A., Tanaka, K. et al. Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia. Vet Res Commun 40, 131–139 (2016). https://doi.org/10.1007/s11259-016-9664-7

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