In vivo potential of recombinant granulysin against human melanoma.

9-kDa granulysin is a protein expressed into the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It has been shown to exert cytolysis on microbes and tumors. We showed previously that 9-kDa granulysin exerted cell death by apoptosis in vitro on hematological tumor cell lines and also on cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. In addition, we have shown the anti-tumor efficiency of granulysin as a single agent in two in vivo models of human tumor development in athymic mice, the MDA-MB-231 mammary adenocarcinoma and the NCI-H929 multiple myeloma, without signs of overt secondary effects by itself. In this work, we have tested recombinant 9-kDa granulysin in an in vivo and especially aggressive model of melanoma development, xenografted UACC62 cells in athymic mice. Recombinant granulysin was administered once UACC62-derived tumors were detectable and it substantially retarded the in vivo development of this aggressive tumor. We could also detect apoptosis induction and increased NK cell infiltration inside granulysin-treated tumor tissues. These observations are especially interesting given the possibility of treating melanoma by intra-tumor injection.

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of intratumoral injection of recombinant granulysin using in vivo models of breast cancer and multiple myeloma. In the present work we have developed a granulysin gene fusion to the anti-carcinoembryonic antigen (CEA/CEACAM5) single chain Fv antibody fragment MFE23. Both granulysin and the granulysin-based immunotoxin were expressed in Pichia pastoris. The immunotoxin specifically recognized CEA, purified or expressed on the cell surface. Moreover, the bioactivity of the immunotoxin against several CEA+ cell lines was higher than that of granulysin alone. Granulysin and the immunotoxin were tested as a treatment in in vivo xenograft models in athymic mice. When injected intratumorally, both granulysin and the immunotoxin were able to inhibit tumor growth. Furthermore, systemic administration of the immunotoxin demonstrated a decrease in tumor growth in a CEA+ tumor-bearing mouse model, whereas granulysin did not exhibit a therapeutic effect. This is the first granulysin-based immunotoxin and the present work constitutes the proof of concept of its therapeutic potential.

Mutations in the ND2 Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells.

Multiprotein complexes of the mitochondrial electron transport chain form associations to generate supercomplexes. The relationship between tumor cell ability to assemble mitochondrial supercomplexes, tumorigenesis and metastasis has not been studied thoroughly. The mitochondrial and metabolic differences between L929dt cells, which lost matrix attachment and MHC-I expression, and their parental cell line L929, were analyzed. L929dt cells have lower capacity to generate energy through OXPHOS and lower respiratory capacity than parental L929 cells. Most importantly, L929dt cells show defects in mitochondrial supercomplex assembly, especially in those that contain complex I. These defects correlate with mtDNA mutations in L929dt cells at the ND2 subunit of complex I and are accompanied by a glycolytic shift. In addition, L929dt cells show higher in vivo tumorigenic and metastatic potential than the parental cell line. Cybrids with L929dt mitochondria in L929 nuclear background reproduce all L929dt properties, demonstrating that mitochondrial mutations are responsible for the aggressive tumor phenotype. In spite of their higher tumorigenic potential, L929dt or mitochondrial L929dt cybrid cells are sensitive both in vitro and in vivo to the PDK1 inhibitor dichloroacetate, which favors OXPHOS, suggesting benefits for the use of metabolic inhibitors in the treatment of especially aggressive tumors.

In vivo potential of recombinant granulysin against human tumors.

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.

Death ligands and granulysin: mechanisms of tumor cell death induction and therapeutic opportunities.

The immune system plays a key role in cancer immune surveillance to control tumor development. The final goal is recognizing and killing transformed cells and consequently the elimination of the tumor. The main effector cell types exerting cytotoxicity against tumors are natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although the mechanism of activation of NK cells and CTLs are quite different, both cell types share common antitumor effector mechanisms of cytotoxicity which lead to induction of cell death of tumor cells by apoptosis. Among these mechanisms are the death ligand- and granulysin-mediated cell deaths. In this review, we summarize the main concepts of these effector cytotoxic mechanisms against cancer cells, how NK cells and CTLs use them to control tumor development and the therapeutic approaches currently developed based on these molecules.