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TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions

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Abstract

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca2+ elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.

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Abbreviations

TRAIL:

Tumor necrosis factor-related apoptosis inducing ligand

VEGF:

Vascular endothelial growth factor

HUVECs:

Human umbilical vein endothelial cells

FRNK:

FAK-related non-kinase

cFLIP:

Cellular FLICE-inhibitory protein

DEVDase:

Caspase-3-like protease

IETDase:

Caspase-8-like protease

FBS:

Fetal bovine serum

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

bFGF:

Basic fibroblast growth factor

CAM:

Chick chorioallantoic membrane

eNOS:

Endothelial nitric oxide synthase

NO:

Nitric oxide

DAF-FM:

3-Amino-4-(N-methylamino)-2′,7′-difluorofluorescein

PMSF:

Phenylmethylsulfonyl fluoride

DED:

Death effector domain

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Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2011-0028790). We thank Dr. Elaine Por for helpful comments and critical reading of this manuscript.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Vascular Homeostasis Laboratory and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 200-701, Korea

    Hee-Jun Na, Kwang-Soon Lee, Yoon Kyung Choi, Jongseon Choe, Moo-Ho Won, Kwon-Soo Ha & Young-Myeong Kim

  2. Department of Obstetrics and Gynecology, Kangwon National University Hospital, Chuncheon, Gangwon-do, 200-701, Korea

    Jong-Yun Hwang

  3. School of Medicine, Ewha Womans University, Seoul, 158-710, Korea

    Ji-Yoon Kim

  4. NeuroVascular Coordination Research Center, College of Pharmacy, Seoul National University, Seoul, 151-742, Korea

    Hyo-Eun Moon & Kyu-Won Kim

  5. School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea

    Gou Young Koh

  6. Departments of Biological Sciences and Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-do, 200-701, Korea

    Hansoo Lee & Dooil Jeoung

  7. Department of Biochemistry, College of Science and Biotechnology, Yonsei University, Seoul, 120-749, Korea

    Young-Guen Kwon

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  1. Hee-Jun Na
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  2. Jong-Yun Hwang
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Correspondence to Young-Myeong Kim.

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Hee-Jun Na and Jong-Yun Hwang have contributed equally to this study.

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Na, HJ., Hwang, JY., Lee, KS. et al. TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions. Angiogenesis 17, 179–194 (2014). https://doi.org/10.1007/s10456-013-9387-0

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