Uptake of free, calcium-bound and liposomal encapsulated nitrogen containing bisphosphonates by breast cancer cells

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• 作者：Hristo P. Zlatev^a ; ^{hristo.zlatev@uef.fi" class="auth_mail" title="E-mail the corresponding author} ; Seppo Auriola^a ; Jukka Mö ; nkkö ; nen^a ; Jorma A. Mä ; ä ; ttä ; ^a ; ^b
• 关键词：Zoledronic acid ; Risedronic acid ; Drug uptake ; Breast cancer
• 刊名：European Journal of Pharmaceutical Sciences
• 出版年：2016
• 出版时间：30 April 2016
• 年：2016
• 卷：86
• 期：Complete
• 页码：58-66
• 全文大小：1353 K

文摘

Purpose: We have examined the uptake routes by which breast cancer cells internalize different formulations of nitrogen containing bisphosphonates (N-BPs).

Methods: Cell viability was assessed with the tetrazolium colorimetric test (MTT assay) after treatment with different N-BP formulations in the presence or absence of inhibitors for different endocytosis mechanisms. Intracellular formation of isopentenyl pyrophosphate (IPP) and triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), were quantified with mass spectrometry (ES-LTQ-MS) as surrogate markers for N-BP efficacy. Direct quantification intracellular [¹⁴C]-labeled zoledronic acid was done with liquid scintillation counting.

Results: The main uptake route for all the different formulations of nitrogen containing bisphosphonates was shown to be dynamin dependent endocytosis, which was significantly enhanced with calcium. This uptake mechanism was mostly caveolin and clathrin independent in MCF7 cells, but more clathrin dependent in T47D cells. Liposome encapsulation of the drug shifted the uptake mechanism to be more dependent on caveolin in both the cell lines. The cytotoxicity of N-BPs and the concentrations of formed intracellular ApppI and IPP were significantly increased by calcium chelation and liposome encapsulation, the latter being the most potent formulation.

Conclusion: Nitrogen containing bisphosphonates require active endocytosis for cellular uptake and in the breast cancer cells the mechanism is uniformly dynamin dependent for all the formulations tested. This differs e.g. from the previous observations on macrophages, which mostly utilize macropinocytosis. Liposomal formulation was found to prolong the duration of the drug effect in cells.