Cytomodulation of interleukin-2 effect by L-2-oxothiazolidine-4-carboxylate on human malignant melanoma

Abstract

Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in the interleukin-2 (IL-2)-induced proliferative activity of normal and tumour cells expressing IL-2 receptor (IL-2R). In the present study, we investigate the effect of IL-2 on proliferation of the human melanoma A375 cell line, and the possible selective cytomodulation effect of this cytokine by L-2-oxothiazolidine-4-carboxylate (OTZ) on these melanoma cells and on human peripheral blood mononuclear cells (PBMCs). We found that recombinant IL-2 (rIL-2) significantly increased the proliferation rate of A375 melanoma cells, which was associated with an increase in GSH levels, the enhancement of IL-2Ralpha expression and the endogenous production of IL-2 in these tumour cells. In contrast, OTZ decreased GSH content and the proliferation rate of A375 cells, and abrogated the growth-promoting effects of rIL-2. Thus, compared to cells treated with rIL-2, pre-treatment with OTZ reduced IL-2Ralpha expression, and also decreased the consumption of rIL-2 and the endogenous secretion of IL-2 by these tumour cells. With regard to PBMCs, the combination of OTZ plus rIL-2 resulted in a more rapid and greater increase of IL-2Ralpha expression than rIL-2 alone, with the proliferation rate being similar in the first 24 h, but with a lower PBMC' count found thereafter compared to rIL-2 treatment alone. These results suggest that OTZ plays a crucial role in obtaining a selective cytomodulation of rIL-2, enabling it to exert its growth-promoting effect on normal cells, but not on melanoma cells, thereby possibly improving biochemotherapy with rIL-2.

Fig. 1

Effect of cysteine deliverer OTZ on GSH levels and cell proliferation in A375 melanoma cells. a Effect of several concentrations of OTZ (1, 2.5 and 5 mM) on GSH content, expressed as a percentage of controls. b Time course of GSH levels after exposure of cells to 2.5 mM OTZ. Values are expressed as percentage of controls. c Relative growth rate of A375 control cells and OTZ-treated cells (2.5 mM). Results are expressed as the mean ± SD of three experiments

Fig. 2

Effect of AC treatment on intracellular GSH levels (a) and proliferation rate (b) in A375 melanoma cells cultured with or without previous exposure to OTZ. Results are expressed as the mean ± SD of three experiments

Fig. 3

Effect of OTZ (2.5 mM) and rIL-2 (200 IU/ml) alone and in combination on intracellular GSH levels (a) and proliferation rate (b) in A375 melanoma cells. Results are expressed as the mean ± SD of three experiments

Fig. 4

Time course of GSH levels (a) and proliferation rate (b) of PBMCs exposed to OTZ or IL-2 alone and in combination. In order to determine intracellular GSH content, cell samples were incubated in the presence of fluorochrome monochlorobimane and the intensity of the fluorescence emitted was measured by flow cytometry (the values are expressed as a percentage of controls). Cell proliferation was evaluated by a colorimetric assay (see Materials and methods), using an ELISA plate reader. Results are expressed as the mean ± SD of three experiments

Fig. 5

Measure of IL-2 levels in the supernate of A375 melanoma cell culture. The figure shows the different initial consumption of exogenously administered rIL-2 (6060.6 pg/ml) and subsequent endogenous secretion of IL-2 by melanoma cells treated with rIL-2 alone or rIL-2 + OTZ. The time course of detected IL-2 levels is expressed in pg/ml. The lower detection limit of the ELISA kit used was 7 pg/ml

Fig. 6

Cytofluorimetric analysis of IL-2Rα expression on A375 melanoma cells and PBMC by labelling with anti-hIL-2R/CD25 conjugated with PE-Cy7 (C Isotype control). For each analysis, 10,000 events were recorded. One representative experiment out of four is shown

Fig. 7

Time course of IL-2Rα expression on A375 melanoma cells (a) and PBMC (b) treated with OTZ and IL-2, alone and in combination. Cells were labelled with anti-hIL-2R/CD25 conjugated with PE-Cy7, and the intensity of the fluorescence emitted was measured by flow cytometry. For each analysis, 10,000 events were recorded. Data are expressed as percentage of controls