Monitoring incorporation, transformation and subcellular distribution of N-l-leucyl-doxorubicin in uterine sarcoma cells using capillary electrophoretic techniques

Wang, Yaohua; Arriaga, Edgar A.

doi:10.1016/j.canlet.2007.11.034

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Volume 262, Issue 1 , Pages 123-132, 8 April 2008

Monitoring incorporation, transformation and subcellular distribution of N-l-leucyl-doxorubicin in uterine sarcoma cells using capillary electrophoretic techniques

Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA

Received 6 August 2007; received in revised form 27 November 2007; accepted 27 November 2007. published online 15 January 2008.

Abstract

Previous reports have demonstrated that N-l-leucyl-doxorubicin (LeuDox) is less toxic than its parent drug, Dox, but the underlying causes of this reduced toxicity have yet to be fully elucidated. In this study, the incorporation of LeuDox into (i) the MES-SA human uterine sarcoma cell line and (ii) its Dox resistant counterpart, MES-SA/Dx5 cell line and the subsequent transformation of LeuDox into Dox and its subcellular distribution, were investigated by micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF).

In both cell lines the cellular uptakes of Dox and LeuDox were similar at equimolar doses, while the percent transformation of LeuDox into Dox in MES-SA/Dx5 cells was about twice as great as its transformation in MES-SA cells, which is beneficial for reaching Dox cytotoxic levels in this resistant cell line. When both cells lines were treated with IC₃₅ concentrations of either Dox and LeuDox, the intracellular Dox amounts were 6-fold higher in the resistant cell line than in the sensitive cell line, suggesting that other cellular processes play a role in the cytotoxicity of Dox in the resistant cell line.

The amounts and ratios of Dox and LeuDox in four subcellular fractions of LeuDox-treated MES-SA/Dx5 cells were also investigated. The highest Dox/LeuDox ratio (i.e. 2.92) was found in the nuclear fraction, followed by the ratio in the low density organelle fraction (i.e. 1.92) that contains lysosomes, organelles in which lysosomal hydrolytic enzymes, capthesins, transform LeuDox into Dox.

Abbreviations: Dox, doxorubicin, LeuDox, N-l-leucyl-doxorubicin, Doxol, doxorubicinol, CTAB, cetyl trimethyl ammonium bromide, CE, capillary electrophoresis, MEKC-LIF, micellar electrokinetic chromatography with laser-induced fluorescence detection, NF, nucleus enriched fraction, HDOF, high density organelle enriched fraction, LDOF, low density organelle enriched fraction, CF, cytosolic enriched fraction