Abstract
Abstract
Background: Lysosomal targeting has been investigated for the treatment of cancer but details of the resulting cytotoxicity remain unclear. The induced lysosomal membrane permeabilization (LMP) has been shown to induce apoptosis and cytotoxicity. The purpose of this study is to elucidate details of doxorubicin (DOX) induced LMP in breast cancer cells and identify correlations with cytotoxicity.
Methods: A gelatin – doxorubicin conjugate (GDOX) to target lysosomes was synthesized by EDC chemistry in formamide and purified by precipitation and desalting. GDox and free Dox uptake studies at 10 µM or its equivalent were conducted in MCF7 and MDA-MB-231 (triple negative breast cancer-TNBC) cells in growth media. Intracellular localization was followed by fluorescent markers. LMP was shown by cytosolic release of 10 kD Dextran-Alexa Fluor 488 previously loaded into lysosomes. Intra-lysosomal release of Dox from GDOX was examined by rupture of lysosomes and nuclear accumulation of released Dox followed by fluorescent microscopy and ImageJ. Released Dox accumulation in the nucleus without lysosome rupture was analyzed by UHPLC. Viability, growth inhibition profiles, and IC50 values were determined by the MTT procedure and GraphPad Prism 7.
Results: GDox localized substantially to the lysosomes in both MCF7 and TNBC cells while free DOX localized to the nucleus, as expected. GDOX induced LMP by 24 hrs in 100% of the TNBC cells but only 20% of the MCF7 cells even up to 48 hrs. Intra-lysosomal DOX release in the MCF7 cells was notable only by 24 hrs and was estimated by fluorescent intensity (300,000 integrated density units per cell) at one-half the amount of free DOX accumulation in the nucleus. Nuclear accumulation of released DOX escaping intact lysosomes in MCF7 cells by 24 hrs was one-half that at 48 hrs which reached 0.13 µg Dox per 106 cells by 48 hrs representing only one-tenth that achieved by free DOX. MCF7 cell viability in GDOX was reduced to 62±3% of controls by 24 hr but reached 19±4% by 48 hrs compared to 11±1% for free Dox. The IC50 values of GDOX after 48 hr were 1.3±0.7 µM and 4.5±2 µM in TNBC and MCF7 cells, respectively.
Conclusions: Taken together these results indicate that LMP is not induced by pooling of released DOX in MCF7 lysosomes nor does LMP contribute to cytotoxicity in these cells. The lysosomal accumulation of GDOX in MDA cells, in contrast to MCF7 cells, leads to extensive LMP which may contribute to the greater cytotoxicity in these cells compared to the MCF7 cells. The small amount of released DOX accumulating in the MCF7 nuclei may induce a nuclear pathway of cytotoxicity in these cells.
Acknowledgements: Financial support is appreciated from the Agnes Varis Trust for Women’s Leadership and Health, King Abdul-Aziz University, and NIH R15 CA135421.
Citation Format: Mohammed Alvi, Rachel Nicoletto, Bayan A. Eshmawi, Clyde M. Ofner. Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2091.