Sensitization of interferon-gamma induced apoptosis in human osteosarcoma cells by extracellular S100A4

Abstract

Background: S100A4 is a small Ca2+-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension.

Methods: Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-gamma and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-gamma mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B.

Results: In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-gamma-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-gamma. The S100A4/IFN-gamma-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-kappa B (NF-kappa B).

Conclusion: In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-gamma-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-kappa B, but whether these events are causally related remains unknown.

Figure 1

OHS cells are more sensitive than II-11b cells to IFN-γ-mediated suppression of cell viability. Cell viability was measured in OHS cells (A) and II-11b cells (B) after treatment with the indicated concentrations of IFN-γ for 24, 48 and 72 hours. In each experiment, untreated control cells were included at all time points, and all values are given as a percentage of corresponding untreated control cells. The results are presented as mean values ± S.D. of at least three independent experiments performed in triplicate. *, p < 0.0001 and **, p < 0.05 as compared to untreated control cells.

Figure 2

DNA fragmentation, PARP and Lamin B cleavage in IFN-γ-treated OHS cells. A, Ethidium bromide stained agarose gel of DNA isolated from OHS cells grown with or without 100 u/ml IFN-γ for 72 hours. B, Western blot analysis of the expression of PARP and Lamin B in total cell lysates. OHS cells were left untreated (-) or treated with 100 u/ml IFN-γ (+) for 24, 48 and 72 hours, and lysates subjected to Western blot analysis as described in the "Materials and Methods" section.

Figure 3

OHS cells secrete S100A4. Immunoprecipitation of cell culture medium alone (control) or cell culture medium from II-11b and OHS cells cultured for 24, 48 and 72 hours as indicated. 1.5 ml of medium was immunoprecipitated using rabbit polyclonal anti-S100A4 or anti-p300, and subsequently immunoblotted with anti-S100A4.

Figure 4

Addition of recombinant S100A4 sensitizes OHS and II-11b cells to IFN-γ-induced apoptosis. Cell viability was measured in OHS and II-11b cells after addition of the indicated concentrations of recombinant S100A4 to the cell culture medium with or without 100 u/ml IFN-γ. All values are given as a percentage of viable cells relative to untreated control cells. The results are presented as mean values ± S.D. of at least three independent experiments performed in triplicate. *, p < 0.0001 and **, p < 0.05 as compared to untreated control cells.

Figure 5

DNA fragmentation, PARP and Lamin B cleavage in rS100A4/IFN-γ-treated II-11b cells. A, Ethidium bromide stained agarose gel of DNA isolated from II-11b cells. Cells were stimulated or left untreated with 100 u/ml IFN-γ for 72 hours in the presence or absence of 20 μg/ml rS100A4 as indicated. B, Western blot analysis of the expression of PARP and Lamin B in total cell lysates. II-11b cells were left untreated or treated with 100 u/ml IFN-γ and/or 20 μg/ml rS100A4 for 24, 48 and 72 hours as indicated, and lysates subjected to Western blot analysis as described in "Materials and Methods".

Figure 6

Western blot analysis of caspase-3 and caspase-9 in total cell lysates. OHS cells were left untreated (-) or treated with 100 u/ml IFN-γ (+) for 24, 48 and 72 hours, and lysates subjected to Western blot analysis as described in "Materials and Methods". As a positive control for caspase activation, an immunotoxin-treated breast cancer cell line was included. α-tubulin serves as a loading control. The results shown are representative of three independent experiments.

Figure 7

Inhibition of cell death by zVAD-fmk, zFA-fmk, DMSO and NAC. OHS cells were cultivated in the presence or absence of 100 u/ml IFN-γ and the indicated inhibitors for 72 hours, and cell viability was measured as described in "Materials and Methods". All values are given as a percentage of viable cells relative to corresponding cells treated with the indicated inhibitor, but without IFN-γ-treatment. Data are mean values ± S.D. The results represent at least three independent experiments performed in triplicate. *, p < 0.001 and **, p < 0.05 as compared to IFN-γ-treated cells without inhibitor.

Figure 8

Recombinant S100A4 induces NF-κB transactivation. A, II-11b cells were transiently transfected with an NF-κB reporter construct and incubated with or without 4 μg/ml rS100A4 and 50 μM zFA-fmk for 48 hours. Luciferase activity was expressed as fold induction of activity compared to the corresponding untreated control. Data are mean values ± S.D. The results represent at least three independent experiments performed in duplicate. *, p < 0.001 and **, p < 0.01 as compared to untreated control. ***, p < 0.005 as compared to rS100A4-treatment without zFA-fmk. B, Western blot analysis of NF-κB p65 and phospho-IκBα (Ser32/36) in total cell lysates from II-11b cells, untreated or treated with 100 u/ml IFN-γ and/or 20 μg/ml rS100A4 for 24 and 48 hours as indicated.