Imaging of human cells exposed to an antifungal antibiotic amphotericin B reveals the mechanisms associated with the drug toxicity and cell defence

Ewa Grela,Rafal Luchowski,Wieslaw I Gruszecki,Wojciech Grudzinski,Roman Paduch,Mateusz Piet

doi:10.1038/s41598-018-32301-9

Ewa Grela, Rafal Luchowski + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-32301-9

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Abstract

Amphotericin B is an antibiotic used in pharmacotherapy of life-threatening mycotic infections. Unfortunately, the applicability of this antibiotic is associated with highly toxic side effects. In order to understand molecular mechanisms underlying toxicity of amphotericin B to patients, two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. The results of the cell viability assays confirm high toxicity of amphotericin B towards human cells. The images recorded demonstrate effective binding of amphotericin B to biomembranes. Analysis of the images reveals the operation of a defence mechanism based upon the elimination of molecules of the drug from living cells via formation of small amphotericin B-containing lipid vesicles. The fact that exosomes formed are devoid of cholesterol, as concluded on the basis of the results of Raman analysis, suggests that sequestration of sterols from the lipid phase of biomembranes is not a sole mechanism responsible for the toxic side effects of amphotericin B. Alternatively, the results imply that molecules of the drug present directly within the hydrophobic membrane core disturb the lipid membrane structure and affect their biological functions.

Highlights

The dramatic increase in mycotic infections, we are facing nowadays, is a challenge for researchers working on developing effective antifungal drugs[1,2]
Both CCD 841 CoTr and HT-29 cells were susceptible to Amphotericin B (AmB), but up to a concentration of 5 μg/ml the cytotoxic effect did not exceed 15% compared to the control
A level of AmB fluorescence lifetime and fluorescence emission spectra recorded based on a micro-spectroscopy approach (Fig. S4), confirm that molecules of the antibiotic accommodated into the membranes and localized in the exosomes are associated in small supramolecular structures

Summary

Introduction

The dramatic increase in mycotic infections, we are facing nowadays, is a challenge for researchers working on developing effective antifungal drugs[1,2]. An effect of AmB on structural properties of biomembranes via destabilization of their lipid phase and resulting impairment of the membrane functionality was postulated[8,9,10]. Another molecular mechanism has been proposed, which consists in impairing of biomembranes functionality via sequestration of sterols from the lipid phase of biomembranes, owing to the formation of extramembraneous AmB-sterol structures[11]. In order to understand molecular mechanisms underlying potential toxicity of AmB, to oncological patients of selected alimentary tract tumour, human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy

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Results

Conclusion