Extracellular ATP stimulates uptake of a topoisomerase poison into cervical cancer cells

Abstract

Most anticancer drugs disrupt cancer cells by interfering with vital intracellular components, such as the cell's DNA, and so must overcome the barrier posed by the plasma membrane to reach their targets. Extracellular adenosine 5'-triphosphate (ATP) can permeabilize the plasma membrane of certain cell types to relatively large ion species, through activation of P2X ion channels and through a less well-defined phospholipase C (PLC)-dependent mechanism. Using fluorescence imaging, we tested the hypothesis that extracellular ATP could stimulate uptake of fluorescent topoisomerase poisons into cervical cancer cells. Hoechst 33258 showed no evidence of uptake when applied alone, consistent with its primary use as a cell viability dye. However, subsequent co-application of ATP in the continued presence of Hoechst 33258 stimulated rapid intracellular accumulation (Fig 1A, B) via a mechanism dependent on activation of G protein-coupled P2Y receptors, PLC stimulation, and an elevation in cytosolic free Ca2+, but which was not due to a loss of immediate cell viability. Doxorubicin showed poor permeability whether applied alone or in combination with ATP. Cell viability analysis 48 hrs after a 20 min exposure to ATP and Hoechst 33258 showed that cells loaded with the toxin underwent significant cell death. ATP-evoked Hoechst 33258 uptake was observed in cultured cancer cells obtained from 5/6 sources. We did not observe ATP-evoked Hoechst 33258 uptake in healthy cervical epithelial cells obtained from the transformation zone and ectocervical tissue of 6/7 patients (Fig 1C). In summary, ATP can induce the uptake of a topoisomerase poison into cervical cancer cells without apparently causing substantial uptake into normal cervical epithelial cells. [Figure1]