Cellular Internalisation of an Inositol Phosphate Visualised by Using Fluorescent InsP(5)

When applied extracellularly, myo-inositol hexakisphosphate (InsP(6)) and myo-inositol pentakisphosphate (InsP(5)) can inhibit the growth and proliferation of tumour cells. There is debate about whether these effects result from interactions of InsP(6) and InsP(5) with intracellular or extracellular targets. We synthesised FAM-InsP(5), a fluorescent conjugate of InsP(5) that allows direct visualisation of its interaction with cells. FAM-InsP(5) was internalised by H1229 tumour cells, a finding that supports earlier reports that externally applied inositol phosphates can—perhaps surprisingly—enter into cells. Close examination of the process of FAM-InsP(5) uptake suggests a mechanism of non-receptor-mediated endocytosis, which is blocked at 4 °C and probably involves interaction of the ligand with the glycocalyx. However, our results are difficult to reconcile with antiproliferative mechanisms that require direct interactions of externally applied InsP(5) or InsP(6) with cytosolic proteins, because internalised FAM-InsP(5) appears in lysosomes and apparently does not enter the cytoplasm. Studies using FAM-InsP(5) are less difficult and time-consuming than experiments using InsP(5) or InsP(6), a factor that allowed us to analyse cellular uptake across a range of human cell types, identifying strong cell-specific differences.