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Oxidative Degradation of Polyamines by Serum Supplement Causes www.nature.com/scientificreports OPEN Oxidative degradation of polyamines by serum supplement causes cytotoxicity on cultured cells Received: 16 February 2018 Linlin Wang1,2, Ying Liu1,2, Cui Qi3, Luyao Shen1,2, Junyan Wang1,2, Xiangjun Liu1,2, Nan Zhang1,2, Accepted: 10 May 2018 Tao Bing 1,2 & Dihua Shangguan 1,2 Published: xx xx xxxx 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 infuence 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 efect 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. Cell culture is a widely used tool to study physiological, biological and pharmacological activities in vitro, as well as to produce biological components, such as proteins, hormones and vaccines. Tis method is fast, cheap, reproducible, and greatly reduces the use of experimental animals. Serum from animals or human is a common supplement for the culture of eukaryotic cells due to it containing a large number of active components, like growth factors, enzymes, hormones, etc., which are essential for the growth and maintenance of cells1–3. Fetal bovine serum (FBS) is the most commonly used serum in cell culture, because it is rich in fetal growth factors and hormones that stimulate cellular proliferation and maintenance. Although FBS has been used for more than 50 years, the knowledge is incomplete regarding the serum components and their infuence on cultured cells4,5. Apart from the unknown infuence of serum components on the cultured cells directly, the efects of enzymes in serum on the tested drugs should be paid much more attention, otherwise it may result in the false positive or negative results, consequently lead to misleading conclusions. In previous work, we have shown that the cytotox- icity activity of guanine-rich oligonucleotides is attributed to the cytotoxicity of guanine derivatives derived from degradation of oligonucleotides by nuclease in FBS, but not attributed to the direct action of oligonucleotides on cells as reported elsewhere6. As ubiquitous endogenous metabolites, polyamines are essential organic compounds for cell growth and pro- liferation. Putrescine, spermidine and spermine, existing normally in millimolar concentration range in nucleus7, are the mainly naturally occurring polyamines in mammalian cells8. Polyamines are involved in a wide variety of cellular processes: participating in the regulation of gene expression and enzyme activity, activating DNA synthe- sis, facilitating the interaction of DNA and protein, as well as protecting DNA molecules from putative damaging agents7,9. Among these polyamines, spermine is reported to possess the highest biological activity10. Under physi- ologic ionic and pH conditions, polyamines are multivalent cations with aliphatic hydrocarbon chains separating the charges11, and hence negatively charged nucleic acids, including DNA and RNA, are their prime targets of interaction, and consequently regulate the structures of DNA, condense DNA molecules to a liquid crystalline 1Department Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China. 2University of the Chinese Academy of Sciences, Beijing, 100049, China. 3CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190, China. Correspondence and requests for materials should be addressed to N.Z. (email: [email protected]) or D.S. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:10384 | DOI:10.1038/s41598-018-28648-8 1 www.nature.com/scientificreports/ Figure 1. Dose-dependent cytotoxicity efects of polyamines, TETA, hydrogen peroxide and acrolein on cells. A549, A549T, MCF-7, MCF-7R, HCT-8, HCT-8T, A2780 and A2780T cells were respectively incubated with diferent concentration of (A) spermine, (B) spermidine, (C) putrescine, (D) TETA, (E) H2O2, and (F) acrolein for 48 h at 37 °C. Each point represents the mean value of the results of 2 to 4 wells of three or four experiments. Te error bars indicate ± S.D., when not show, S.D. was smaller than the symbol. phase12, and enhance the activity of G-quadruplex/hemin DNAzymes in vitro13. Many evidences suggest that polyamines play important roles as modulators of nucleic acid structure14. Te intracellular polyamines are maintained at a correct level through the subtle coordination and regu- lation of biosynthesis, transport, and catabolism. In mammalian cells, polyamine catabolism occurs through two distinct pathways with the help of three kinds of enzymes: spermidine/spermine N1-acetyltransferase, N1-acetylpolyamine oxidase and spermine oxidase15 (Fig. S1). Polyamine catabolism can generate cytotoxic metabolites such as hydrogen peroxide (H2O2) and aldehydes in situ, thereafer inducing apoptosis, necrosis, inhi- bition of cell proliferation, and inhibition of DNA and protein synthesis7,16,17. Polyamines are also the substrates of various kinds of amine oxidases, such as monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Tese enzymes catalyze the oxidative deamination of polyamines to generate the reaction products H2O2 and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines18–23. Terefore, polyamines and amine oxidases are also considered to have potential in therapeutic applications15,17. Here, we describe the cytotoxicity of polyamines and their degradation products on diferent cell lines. Te generation of H2O2 (one of the degradation product of polyamines) in culture media containing diferent serums was measured afer addition of spermidine and spermine. Te degradation products of spermidine and spermine resulted from the amine oxidases in serum were found to be the main reason of the cytotoxicity of spermidine and spermine. Results and Discussion The toxicity efect of polyamines on cultured cells. Polyamines (including spermine analogues) have been reported to have cytotoxicity efect on diferent cells24–27. Diferent mechanisms of action have been reported, such as inducing gene overexpression28, telomerase inhibiting29,30 and cytotoxicity of the metabolites generated by amine oxidases in cells31,32. In order to understand the real mechanism, we tested the cytotoxicity activity of four polyamines (putrescine, spermidine, spermine and triethylenetetramine (TETA)) on eight cell lines in vitro, including four cancer cell lines (A549, MCF-7, HCT-8 and A2780) and their drug-resistant sublines (A549T, MCF-7R, HCT-8T and A2780T). As shown in Fig. 1A–D, afer treated with spermine and spermidine for 48 h, a dose-dependent cytotoxicity efect on all four drug-resistant cell lines was observed; whereas no signifcant efect was observed on four wild cell lines. Putrescine and TETA did not show signifcant cytotoxicity efect on all the tested cell lines. Since TETA, an unnatural compound with a structure similar to spermine, and putrescine are not the substrates of amine oxidases; above suggests that the cytotoxicity efects may not be caused by spermine and spermidine directly, but may be related to the metabolites of polyamines. Since spermine showed stronger cytotoxicity activity on drug-resistant cells over that of spermidine, and an taxol-resistant A549 subline (A549T) was the most sensitive cells to spermine and spermidine among these drug-resistant sublines; the apoptosis assay and cell cycle assay of A549T cells afer treatment with spermine SCIENTIFIC REPORTS | (2018) 8:10384 | DOI:10.1038/s41598-018-28648-8 2 www.nature.com/scientificreports/ Figure 2. Cell apoptosis and cell cycle assay. (A,B) Cell apoptosis assay of A549T cells afer treated with spermine at 37 °C for 6 h in RPMI1640 with 10% FBS (A) and FBS-free RPMI1640 (B) respectively. Te concentration of spermine respectively is 0, 10, 20, 30, 50 and 80 μM. Cells were double-stained by Annexin V-FITC and PI. Crossing gates divided the dot plots into four quadrants. Dots in lower right quadrants (Annexin V+ and PI−) indicate early apoptosis cells; dots in upper right quadrants (Annexin V+ and PI+) indicate late apoptosis cells or dead cells. Te numbers indicate the percentage of cells in the corresponding quadrants. (C) Te cell cycle profles analysis of A549T treated with spermine. Te concentration of spermine respectively is 0, 30 and 60 μM. Quantifcation of G1, S and G2 phase percentage was performed by FlowJo (Treestar, San Caros, USA). Results are single representative of three independent
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