In vivo potential of recombinant granulysin against human tumors

Abstract

9 kDa granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work from our group demonstrated that this granulysin isoform induced apoptosis in vitro on hematological tumor cells and on primary tumor cells from B-CLL patients. In the present work, recombinant 9 kDa granulysin was used as an anti-tumoral agent to study its in vivo effect on tumor development in athymic “nude” mice models bearing human breast adenocarcinoma MDA-MB-231 or multiple myeloma NCI-H929–derived xenografts. Granulysin prevented the in vivo development of detectable MDA-MB-231-derived tumors. In addition, recombinant granulysin was able to completely eradicate NCI-H929-derived tumors. All granulysin-treated tumors exhibited signs of apoptosis induction and an increased NK cell infiltration inside the tumor tissue comparing to control ones. Moreover, no in vivo deleterious effects of the recombinant 9 kDa granulysin doses used in this study were observed on the skin or on the internal organs of the animals. In conclusion, granulysin was able to inhibit the progression of MDA-MB-231-derived xenografts and also to eradicate multiple myeloma NCI-H929-derived xenografts. This work opens the door to the initiation of preclinical and possibly clinical studies for the use of 9 kDa granulysin as a new anti-tumoral treatment.

Keywords:

• apoptosis
• athymic mice
• granulysin
• mammary adenocarcinoma
• multiple myeloma

Abbreviations:

• GNLY, granulysin
• CTL, cytotoxic T lymphocyte
• NK, natural killer
• PBS, phosphate buffered saline
• FCS, fetal calf serum
• DNA, deoxy-ribonucleic acid
• mAb, monoclonal antibody
• B-CLL, B cell chronic lymphocytic leukemia
• LPS, lipopolysaccharide
• ROS, reactive oxygen species
• AIF, apoptosis inducing factor
• PBMC, peripheral blood mononuclear cells
• LAL, Limulus amoebocyte lysate
• FITC, fluorescein isothiocianate
• PE, phycoerythrin
• 7-AAD, 7-amine-actinomycin D
• H&E, hematoxylin/eosin
• EpCAM, epithelial cell adhesion molecule
• ALT, alanine aminotransferase

Disclosure of Potential Conflicts of Interest

The use of granulysin as an anti-tumoral treatment is protected by the patent application PCT/ES2014/070086 presented to the Spanish Bureau of Patents and Brands (OEPM) on 2/6/2013 and extended to international application on 2/6/2014, with the code PCT/ES2014/070086.

Acknowledgments

We gratefully acknowledge Dr. Joan Massagué, Memorial Sloan Kettering Cancer Center, New York, USA, for MDA-MB 231-Luciferase cells and Dr. Javier Azúa for processing of mice tumor tissues.

Funding

This work was supported by grants SAF2010-15341 and SAF2013-48626-C2-1-R from Ministerio de Economía y Competitividad (Spain) and by Gobierno de Aragón/Fondo Social Europeo. SAW was supported by a pre-doctoral fellowship from the Government of Yemen.