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

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.

Lipid Systems for the Delivery of Amphotericin B in Antifungal Therapy

“…Despite several decades of clinical use, AmB mechanism of action at the molecular level remains elusive and several models have been proposed based on extensive experimental research and theoretical studies [45][46][47][48]54,55,[58][59][60][61][62][63][64][65][66]. AmB has been shown to bind sterol-containing membranes of eukaryotic cells and to insert into the lipid bilayer forming pore-like supramolecular structures that can act as transmembrane ion channels, leading to increased membrane permeability, K + leakage, and disruption of ion transport [44,45,48,53].…”

“…On the other hand, imaging of both normal epithelial and colon adenocarcinoma human cells exposed to AmB revealed a detoxifying mechanism based on the formation of AmB-containing exosomes devoid of cholesterol, suggesting that insertion of the drug within the hydrophobic membrane core is sufficient to disturb the membrane structure and lead to cytotoxic effects [61]. The fungicidal activity of AmB has also been attributed to vacuole disintegration resulting from trafficking of the drug to the vacuolar lumen via autophagy [70].…”

Selective protection of human cardiomyocytes from anthracycline cardiotoxicity by small molecule inhibitors of MAP4K4

“…The resulting cardiomyocytes were maintained for three weeks in serum-free RPMI-1640 (R8758, Sigma-Aldrich), supplemented with B27 and Antibiotic-Antimycotic (Thermo Fisher). The final concentration of amphotericin B was 270 nM, well below the threshold concentrations reported for toxicity of this compound 76,77 . The IMR90 cardiomyocytes were reseeded at a density of 12,500 cells well −1 onto half-area 96-well plates (675096, Grenier Bio-One), coated as above, for 1 week prior to treatment.…”

ABCA1 transporter reduces amphotericin B cytotoxicity in mammalian cells

“…Therefore, this ABCA1mediated mechanism can be considered as protective against AmB cell toxicity. Recent study indicated a different defense mechanism, in which human colon cell lines minimize toxic effects of AmB by extrusion of the drug molecules from their membranes via formation of AmB-rich exosomes [60]. However, the participation of any protein in this mechanism is not documented so far.…”