Abstract
The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.
Highlights
In a previous article, we demonstrated that application of extracellular ATP stimulated the rapid uptake and accumulation of a small molecule DNA-binding cationic dye, Hoechst 33258 (H33258), into cultured cervical cancer cells in a manner dependent on both the ATP and H33258 concentrations [1]
We began by reproducing this key finding, using CXT2 cervical cancer cells cultured on glass coverslips mounted into a bath on the stage of a Nikon TE200 inverted microscope and perfused with extracellular solution. 30 μM H33258 was added to the bath at the beginning of the experiments, and fluorescence images were captured every 10 s to monitor H33258 uptake as a function of nuclear fluorescence staining over time
We report evidence that the intermediate conductance Ca2+-activated K+-ion channel KCa3.1 is functionally upregulated in cervical cancer cells relative to cells isolated from healthy cervical epithelium
Summary
We demonstrated that application of extracellular ATP stimulated the rapid uptake and accumulation of a small molecule DNA-binding cationic dye, Hoechst 33258 (H33258), into cultured cervical cancer cells in a manner dependent on both the ATP and H33258 concentrations [1]. We endeavored to test the reasonable hypothesis that lack of KCa3.1-dependent H33258 uptake in healthy cervical epithelial cells was because of a lower functional expression level of these channels in the plasma membrane compared with cancerous cells.
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