Oxidative degradation of polyamines by serum supplement causes cytotoxicity on cultured cells

Abstract

Serum is a common supplement for cell culture due to it containing the essential active components for the growth and maintenance of cells. However, the knowledges of the active components in serum are incomplete. Apart from the direct influence of serum components on cultured cells, the reaction of serum components with tested drugs cannot be ignored, which usually results in the false conclusion on the activity of the tested drugs. Here we report the toxicity effect of polyamines (spermidine and spermine) on cultured cells, especially on drug-resistant cancer cell lines, which resulted from the oxidative degradation of polyamines by amine oxidases in serum supplement. Upon adding spermidine or spermine, high concentration of H2O2, an enzyme oxidation product of polyamines, was generated in culture media containing ruminant serum, such as fetal bovine serum (FBS), calf serum, bovine serum, goat serum or horse serum, but not in the media containing human serum. Drug-resistant cancer cell lines showed much higher sensitivity to the oxidation products of polyamines (H2O2 and acrolein) than their wild cell lines, which was due to their low antioxidative capacity.

Dietary and Gut Microbiota Polyamines in Obesity- and Age-Related Diseases

“…One of these factors in in vitro studies could be the use of animal serum in the cell culture medium, which contains amino oxidases that can oxidize exogenously administrated polyamines and generate ROS, resulting in cell toxicity independently of the action of the polyamine itself. Interestingly, a recent work demonstrated that in the presence of human serum, polyamine administration to the culture medium does not increase ROS production and does not affect cell viability as in the case of the same experiment in presence of either bovine or horse serum ( 45 ). Importantly, studies showing a polyamine-dependent cell toxicity in human cell lines in presence of significant amounts of bovine/horse serum should be reevaluated with human serum to corroborate that toxicity could be due to the production of oxidized polyamine-derived products by the action of serum polyamine oxidases and not to a toxic effect of the polyamines per se .…”

A mechanism for increased sensitivity of acute myeloid leukemia to mitotoxic drugs

“…For determination of cytotoxicity, cells at a final density of 5 × 10 5 cells/ml in serum-free RPMI-1640 media were treated with one or more drugs for 24 h for each cell line. The sole exception was cytarabine, where LD50 was determined over 48 h, with a media change and fresh cytarabine added at 24 h. Drug cytotoxicity was determined in serum-free media to prevent reaction of serum components with drugs, as this has been shown to compromise the test results on cytotoxicity 12 . Survival rates were assessed using exclusion of 0.4% Trypan Blue (Gibco) using a Countess TM II FL (ThermoFisher) automated cell counter.…”

Polyamine catabolism and oxidative damage

“…Many studies have since concluded that adding Spd or Spm to mammalian cells in the presence of bovine serum results in extracellular oxidation of the PA and growth inhibition due to the oxidation products, not the exogenous PA (63-66). Studies testing the inhibitory effects of Spm, Spd, their aldehyde reac-tion products (which can convert to acrolein), and H 2 O 2 in mammalian cell lines have indicated major roles for acrolein and H 2 O 2 in the cytotoxic responses (51,(67)(68)(69), and in most systems, treatment with aldehyde dehydrogenase inhibitors and catalase together yielded protection from cytotoxicity (70,71). Therefore, caution must be used when interpreting results involving PA treatment of cells in culture, particularly with regard to cellular processes involving ROS, such as autophagy.…”