Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy.

A C Croce,R Supino,E Prosperi,G Bottiroli

doi:10.1038/bjc.1986.265

Abstract

The influence of anthracyclines on membrane permeability functions has been investigated in HeLa cells by monitoring the efflux of fluorescein. Release of the fluorescent dye, dependent on the metabolic energy supply, occurs after the intracellular accumulation and enzymatic hydrolysis of the non-fluorescent substrate fluorescein diacetate (FDA). Flow cytometric evaluation of the efflux kinetics showed that adriamycin (ADR), N-trifluoroacetyladriamycin-14-valerate (AD-32) and daunorubicin (DNR) inhibited the permeability process. The degree of inhibition was dependent, though to different extent, on the intracellular concentration of each drug. An increase in the efflux rate was always observed when the cells were treated with the drugs in the presence of 20 mM glucose. Relationship of these effects with energetic metabolism was supported by the finding that ATP levels were lowered by the drugs and increased by glucose. Evaluation of the cytotoxicity induced by each drug showed that the intracellular amount necessary to inhibit cell survival by 50% was of the same order of magnitude as that which decreases to 50% membrane permeability to fluorescein. These results indicate a correspondence in the concentrations of anthracyclines required for inducing cytotoxicity and for inhibiting membrane permeability functions dependent on the metabolic energy supply.

Highlights

In a previous study we showed that daunorubicin (DNR) was able to affect membrane permeability properties determining the process of fluorochromasia in living cells (Prosperi et al, 1985)
The decrease in the efflux rate induced by the drugs, was not significantly different when the cells were loaded with fluorescein by using different fluorescein diacetate (FDA) concentrations
Cells treated with 6,iM ADR, for instance, showed a rate constant of 1.82x 10-4 (±0.52) s-', when incubated with 2.4pM FDA, versus a constant of

Summary

Materials and methods

HeLa cells were grown as monolayers in Corning flasks in MEM (minimum essential medium), supplemented by non-essential aminoacids, antibiotics and 10% foetal calf serum (Flow Laboratories, Irvine, Ayrshire, U.K.). Twenty-four hours after incubation, the cells were detached following the standard trypsinization procedure and suspended in MEM for suspension cultures, supplemented by antibiotics and 10% foetal calf serum. The cells were washed from the culture medium with PBS and incubated in the drug solutions for 15min at 37°C, at a cell density of 106 ml- 1. At the end of treatments, the cell suspension was divided into aliquots for the evaluation of drug uptake, or fluorescein efflux. Milan, Italy) was added directly to the drug solutions at the final concentration of 20mM. Twenty-four hours later, the cells were treated with the drugs for 15min, washed with PBS and incubated in drug-free medium for 72 h. FDA (Sigma, St Louis, USA) was prepared as the stock solution at 5mgml-1 in acetone, and diluted prior to use in PBS. Reabsorption or fluorescence energy transfer from fluorescein to anthracyclines were already considered (Prosperi et al, 1985): these phenomena did not occur to any significant extent, given the low absorption coefficient of anthracyclines in the region where partial overlap with fluorescein emission occurs, and the low quantum yield of anthracyclines

A TP determinations

Results

Discussion