Conjugation of the 9-kDa Isoform of Granulysin with Liposomes Potentiates Its Cytotoxicity

Abstract

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni²⁺ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-x_L. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway.

Keywords: acute lymphoid leukemia; apoptosis; granulysin; immunotherapy; lipid nanoparticles.

Figure 1

(A) Analysis of the purification process using SDS-PAGE polyacrylamide 15% gel stained with Coomassie blue. (B) Western-blot analysis of ultracentrifugated pellets (P) and supernatant (S) fractions from LUV-GRNLY using an anti-His antibody. (C) Western blot of ultracentrifugated pellets and supernatant fractions from LUV.0-GRNLY using an anti-His antibody. (D) Schematic representation of the interaction between GRNLY-His₆ and liposome surface through DOGS-NTA-Ni.

Figure 2

In vitro cytotoxicity of GRNLY and LUV-GRNLY against Jurkat cell line. (A) Jurkat cells were incubated with increasing concentrations of GRNLY for 24 h. Cell death was determined by detection on phosphatidylserine exposure (PS) by staining with annexin-V-FITC using flow cytometry. (B) Jurkat cells were incubated with increasing concentrations of GRNLY and LUV-GRNLY (2.5, 5 and 7.5 µM) for 24 h, and cell death was determined by flow cytometry as previously described. The results are expressed as the mean ± SD of 3 independent experiments. *** p < 0.001. (C) Representative flow cytometry histograms corresponding to one of the experiments performed, showing the Annexin-V-FITC staining data (FL1-H channel) on Jurkat cells treated with control LUVs, with 7.5 µM GRNLY, or with 7.5 µM LUV-GRNLY, as indicated.

Figure 3

Analysis of the mechanism of cell death induced by GRNLY and LUV-GRNLY. (A) Jurkat cells were treated with 7.5 µM of GRNLY or LUV-GRNLY for 24 h, respectively. Then, cells were simultaneously stained with annexin-V-FITC and 7-AAD and were analyzed using flow cytometry. The dot-plots (upper, left panel) represent the evolution of treated cell population compared to the control. The values shown correspond to a percentage of cells in each quadrant. The upper right figure corresponds to a graphical representation of obtained data. Data shown are representative of four different experiments. (B,C) Jurkat and mutant cells (Jurkat-Bcl-x_L, Jurkat-plvTHM and Jurkat-shBak) were treated with 7.5 µM of GRNLY or LUV-GRNLY, respectively, for 24 h. Cell death was determined by exposure of PS by staining with annexin-V-APC and analyzing by flow cytometry. The results are expressed as the mean ± SD of 3 independent experiments. *** p < 0.001.

Figure 4

Expression levels of proteins from the Bcl-2 family upon treatment with GRNLY or LUV-GRNLY. The expression of Bim, PUMA, Bax, Bcl-x_L or Bcl-2 in Jurkat cells treated with 7.5 µM of GRNLY or LUV-GRNLY for 24 h was analyzed by Western blot using specific antibodies. Cell lysates (5 × 10⁶ cells) were separated by SDS-PAGE 12% polyacrylamide gel, transferred to PVDF membranes and analyzed by Western blot using specific antibodies. Levels of β-actin were used as a control of protein loading.

Figure 5

Toxicity of GRNLY or LUV-GRNLY towards fresh PBMC or 7-day T cell blasts obtained from healthy donors. (A) Freshly isolated PBMCs from healthy donors were treated for 24 h with increasing concentrations of LUV-GRNLY, as indicated. (B) Seven-day T cell blasts from the same healthy donors were treated for 24 h with increasing concentrations of GRNLY or LUV-GRNLY, as indicated. Then, cells were stained with annexin-V-FITC and analyzed using flow cytometry. * p < 0.5; ** p < 0.01; *** p < 0.001.