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Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells

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Abstract

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

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Abbreviations

RT-PCR:

Reverse-transcription polymerase chain reaction

TRAIL:

Tumor necrosis factor-related apoptosis-inducing ligand

MTS:

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

FLIP:

FLICE-inhibitory protein

IAP:

Inhibitor of apoptosis protein

BIR:

Baculovirus IAP repeat

FACS:

Fluorescence-activated cell sorter

FITC:

Fluorescein isothiocyanate

PI:

Propidium iodide

XIAP:

X-linked inhibitor of apoptosis protein

HEK-293:

Human embryonic kidney-293

PARP:

Poly (ADP-ribose) polymerase

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Acknowledgments

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science, and Technology (NRF-2013R1A1A2A100005494) and a faculty research grant of Yonsei University College of Medicine for 2014 (6-2014-0138). Zhezhu Han, Seungha Lee and Suyeon Je were supported by the Brain Korea 21 Plus project for Medical Science (Yonsei University, College of Medicine, Seoul, Republic of Korea).

Authors’ contributions

ZZH and SHL carried out overall research, experimental studies and data acquisition. SYJ participated in the animal study and helped to western blots. CYE participated in the immunohistochemistry and data acquisition. HJC carried out the data acquisition, and helped to draft the manuscript. JJS participated in the overall study design and drafted and revised the manuscript. JHK proposed the study and participated in its design and helped to draft, and assisted writing the manuscript. All authors had already read and approved the final manuscript.

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    Authors and Affiliations

    1. Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea

      Zhezhu Han, Seungha Lee, Suyeon Je & Jae J. Song

    2. Department of Oncology, Affiliated Hospital of Yanbian University, Yanji, Jilin Province, People’s Republic of China

      Zhezhu Han

    3. STEREO Imaging Group, Seoul Center, Korea Basic Science Institute, Seoul, Republic of Korea

      Chi-Yong Eom

    4. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

      Hye Jin Choi & Joo-Hang Kim

    5. Severance Biomedical Science Institute, Yonsei University, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

      Zhezhu Han & Jae J. Song

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    Correspondence to Jae J. Song or Joo-Hang Kim.

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    Zhezhu Han and Seungha Lee contributed equally to this study.

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    10495_2015_1208_MOESM1_ESM.tif

    Supplementary figure 1. Screening of human survivin short hairpin RNAs (shRNAs), with sequences of five shRNA oligomers targeting survivin. The selected target sequence is indicated in bold (bottom of top panel). Five oligomers of the target and the positive control shRNA were transfected into HeLa cells. The knockdown efficiency of each oligomer was measured using quantitative real-time PCR to amplify survivin. Relative expression levels of survivin were plotted after normalization to the scrambled shRNA as a negative control (bottom panel) (TIFF 1082 kb)

    10495_2015_1208_MOESM2_ESM.tif

    Supplementary figure 2. The number of infectious viral particles was determined as a measure of oncolytic adenoviral replication. MiaPaCa-2 cells were infected with Ad-3484-NC or Ad-3484-shSurvivin or Ad-3484-TRAIL-shSurvivin adenovirus at an MOI of 50 for various times. After infection, the supernatants were examined for virus production. Error bars represent standard errors from three independent experiments (TIFF 419 kb)

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    Han, Z., Lee, S., Je, S. et al. Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells. Apoptosis 21, 351–364 (2016). https://doi.org/10.1007/s10495-015-1208-z

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