Abstract
BackgroundCopper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated.MethodsSub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant.ResultsIn the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance.ConclusionOur results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn enable individualization of the chemotherapy in the early stage of treatment.
Highlights
Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin
Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro
We suggest that the reduced lysosomal compartment may reflect defects in protein recycling, which may in turn be responsible for altered localisation of the efflux transporters ATP7A and ATP7B in the A2780cis resistant cell line
Summary
Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatinresistant variant, A2780cis, was investigated. A number of studies have provided evidence that the copper homeostasis proteins ATP7A and ATP7B are involved [7,8,9,10] These Ptype ATPases have been suggested to either sequester cisplatin in vesicular structures or to mediate efflux of the drug [11,12]. Increased expression of ATP7A and ATP7B has been associated with acquired resistance to cisplatin in cancer cell line models and in clinical samples [7,8,11,13,14]
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