Effects of a non-IGF binding mutant of IGFBP-5 on cell death in human breast cancer cells

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Abstract

We have demonstrated previously that IGFBP-5 alone had no effect on cell death but modulated ceramide-induced apoptosis in Hs578T IGF non-responsive cells. To investigate if IGFBP-5 maintains its intrinsic ability to modulate apoptosis in IGF-responsive cells, we used a non-IGF binding mutant of IGFBP-5. In Hs578T cells, non-glycosylated, glycosylated or mutant IGFBP-5 alone each had no effect on cell death, whereas all forms inhibited ceramide-induced apoptosis. In IGF-responsive MCF-7 cells, each wild type form reduced ceramide-induced cell death but mutant IGFBP-5 was without effect. In the presence of mutant IGFBP-5, however, IGF-I no longer conferred survival and in the presence of wild type IGFBP-5, long R3 IGF-I was also unable to confer survival. In summary, all forms of IGFBP-5 modulated ceramide-induced apoptosis in Hs578T cells. In MCF-7 cells, IGF-I-induced survival could be facilitated by IGFBP-5, but also blocked by IGFBP-5 if association with IGFBP-5 was prevented.

Section snippets

Materials and methods

Materials. Insulin-like growth factor-I (IGF-I), long R3 IGF-I (LR3; analogue of IGF-I that has no affinity for IGFBPs) and non-glycosylated (ng) IGFBP-5 were purchased from Gropep (Adelaide, Australia). Human glycosylated (gly) and non-IGF binding mutant of IGFBP-5 (mut) were prepared as described previously [17]. The ceramide analogue (C2) was obtained from Calbiochem (Nottingham, UK). Tissue culture plastics were purchased from Greiner Labortechnik (Stonehouse, UK). The IGFBP-5 antibody was

Effects of IGFBP-5 on C2-induced apoptosis in IGF-non-responsive Hs578T cells

In the Hs578T cell line, C2 induced a significant (P<0.001) increase in cell death from 7.3% to 56.9%. As demonstrated previously [10], we confirmed that non-glycosylated IGFBP-5 was able to significantly (P<0.01) inhibit C2-induced cell death from 56.9% to 34.6%. We have now demonstrated further that gly and mut IGFBP-5 can also dramatically (P<0.01,P<0.001, respectively) inhibit C2-induced cell death from 56.9% to 26% and 30.5%, respectively (Fig. 1A). We have demonstrated previously [10]

Discussion

We have demonstrated in the Hs578T IGF non-responsive cell line that non-glycosylated, glycosylated, and mutant IGFBP-5 can all confer an identical survival effect against C2-induced cell death, clearly in an IGF-independent manner. Having established that the non-IGF binding mutant of IGFBP-5 maintained its intrinsic actions on cell death, we then examined the effects of this mutant in the IGF responsive MCF-7 cell line. We observed in this model that IGF-I acted as a potent survival factor

Acknowledgements

We thank the Association for International Cancer Research and the Needham Cooper Trust for supporting this work.

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